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United States Depai (nient of Jusi

Drug Enforcement Administratio


Otfice of Science and Tecliiiology
Foiensic Sciences Division
Special Testing anti Reseaicli DibonUo

Proceedings of the
International Symposium,
May 29-30, 1978
Instrumental Applications
In Forensic Drug Chemistry
Proceedings of the International
Symposium, May 29-30, 1978

Edited by:
Michael Klein
Alice V. Kruegel
Stanley P. Sobol

For sab by tho Supcrintondoiitof Documonls, U.S, Qovermnont Printing Ofilco

.
Washington, D.G, 2CH02

Stock Number 027-000-00770-8


Table of Contents

Letter from the Administrator


P. B. Bensinger 1

Foreword
J.'W.GlinUyJv %

Preface
The Editors 4

U.S. International Narcotics Policy


C. O’Keeffe 6

Spectroscopy Session M. Klein, chairman


I. Mass Spectrometry
Development of Mass Spectrometry as a Tool in Forensic Dmg Analysis— Review
M. Klein 14

The Use of Stable Isotopes in the Quantification of Dmgs by GC-MS-COM


M.G. FlorningiJ. Nowlin, K, Lertratanangkoon and J-P,Thenot ........ 41

Negative Ion Mass Spectrometry — a New Tool for the Forensic Toxicologist
H. Brandenherger 48

II. Infrared and Nuclear Magnetic Resonance

Applications of Fourier Transform Infrared Spectrometry in Forensic Analysis


P,R, Griffiths, D.Kuehi and M.P. Fuller 60

Nuclear Magnetic Resonance; Recent Developments in Techniques and Their Po-


tentialApplication to Forensic Research
E. D. Becker 70

Computer Applications Session c. f. Hammer, chairman


I. Drug Identification
A Brief Review of the Computerized Identification of Known Compounds and the
Elucidation of Unknown Structures
C. F, Hammer 82

The Use of Mass Spectrometry for Drug Identification


G. W. A Milne, H. M. Faies and N. C. Law
. 91
The Identification of Drugs and Related Substances by Computer-aided Mass
Spectrometry
K. Biemann, J.E. Biller and C. E. Costello

U. Computer Systems in Government Facilities


Development of a National Criminalistics Laboiatory Information System
C.G.McWright 114

Development and Use of Computer Systems in the Virginia Bureau of Forensic Science
P.li, Fen ara, M, P. McGee and R. J, McGann 118

The Development and Applications of the Computer System of the HOCRE


Pearson and C. Brown
E. F. 126

Chromatographic Advances Session a. v. Kruegei, chairperson

I. Instrumentation
Applications of HPLC to the Analysis of Drugs
B, B. Wheats and R. L. Williams 136

On-line Liquid Chromatography-Mass Spectrometry; The Monitoring of HPLC Ef-


fluents by a Quadrupoie Mass Spectrometer and a Direct Liquid Inlet Interface (DLI)
P.J.Arpino . . 151

II. Methodology
A New Approach to the Optimization of Chromatographic Systems and the Use of
a Generally Accessible Data Bank in Systematic Toxicological Analysis
R. A, deZeeim, P. Schepers, J, E. Greving andJ. P. Franke 167

The Application of Derivatization Techniques in Forensic Drug Analysis


J, M. Moore 180

lU, Drug Analysis

Advances in Chemical Signature Analyses of Drags


L. StrombergandA.C.Maehly 202

Special Problems in TLC, GC and GC-MS Analysis of Heroin


G. Paulig 210

Sheet Drugs and the Forensic Toxicologist


J. Wellsand G. Cimbura 214

Special Topics Session r. Sager, Chairman

Impact of Instruments on Analysis of Drug Purity and Reference Standards


L, T. Grady 224
Drug Analysis by Immunoassays
I. Sunshine 230

Light Microscopy and Forensic Diug Analysis


W. C. McCroite 263
Evolution of SEM Utilization in the Crime Laboratory
V. R. Matricardi 270
Indistinguishable from Magic — the Threat and the Promise of Laboratory Utopia
B.S.Finkle 274

List of Attendees 280

V.
Letter From the Administrator

The Drug Enforcement Administration hosted a two-day International Symposium on Instru-


mental Applications in Forensic Drug Chemistry in Arlington, Virginia on May 29 and 30, 1978.
The Symposium focused on the increasing reliance of the forensic scientist on modern instrumen-
tation and computers which make possible the rapid accumulation of accurate data.
The Symposium consisted of presentations fiom internationally known invited speakers who
covered current methodology and approaches, and attempted to project future needs and
developments.
The purpose of the Symposium was to provide significant contributions to the forensic sciences
in the field of Forensic Drug Chemistry, highlighting advances in instrumental and computer
applications to drug analysis. Leading scientists in both Europe and the USA presented papers
to an international audience of scientists from government, university, and law enforcement
laboratories.
The Drug Enforcement Administration was honored to be able to sponsor this Symposium and
provide a forum for the sharing of scientific information which can be applied to forensic work on
a worldwide scale. This Symposium should expand knowledge in the forensic sciences and
support international law enforcement.
This volume presents the papers at the International Symposium. By publishing the proceed-
ings, it is our hope that the widest possible international audience will be reached. Thus, it will
contribute to one of DEA’s primary responsibilities

“coordination and cooperation with other Federal,


State, and local agenciesand foreign governments
in programs designed to the tiaining of foreign
officials, and the encouragement of knowledge and
commitment against drug abuse.”
I wish to express my appreciation to members of the DEA staff who were involved in preparing
thisSymposium. Also, a warm word of praise and thanks is offered to those most responsible for
the success of the Symposium —the speakers. The interest they aroused is a testimony to their
professional competence.

Drug Enforcement Administration

1 .
Foreword

The Di ug Enforcement Administration of the U.S. Department of Justice was created in 1973
by Presidential i eotganization to enforce the U.S. Drug Laws and to bring to justice those organi-
zations and principal members of organizations involved in illicit drug activities. DEA has
recognized science and technology as an integral part of its law enforcement efforts. Further-
more, oiii policy from the beginning has been to share our knowledge with our colleagues in police
agencies and laboratories on an international basis. The Symposium and its proceedings were
one more attempt by DEA to provide a means to share scientific information which will help meet
not only today’s needs but also to address advances in new instrumental techniques to help solve
future problems,
The 1973 reoiganization which led to the formation of DEA combined the function and assets of
the Bureau of Narcotics and Dangerous Drugs, the narcotic enforcement segment of the U.S.
Customs Service, the Office of Di ug Abuse Law Enforcement (ODALE), the Office of National
Narcotics Intelligence (ONNIl and a segment of the White House Office of Science and
Technology.
We aie concerned with illicit activities which involve the cultivation, manufacture, or distribu-
tion of di ugs appearing in or destined for the U.S. illicit market. DEA also supports non-enforce-
ment programs aimed at combating the dnig traffic at home and abroad.
To accomplish this mission we have a total of 4,200 employees. Half of these employees are
criminal investigators who carry the title “Special Agent” As of October 1 1978 we will consol-
. ,

idate our present 12 domestic regions into 5 legions. Overseas we have 3 regions, with 64 offices
in 39 countries manned by 200 Special Agents.

Control of narcotics and dangerous diugs is an international and multifaceted endeavor and
requires not only the full effoits of DEA, but the cooperation of many agencies including our

foreign police colleagues, the United Nations, Interpol, Department of State, U.S. Customs Ser-
vice, Federal Bureau of Investigation, Immigration and Naturalization Service, Internal Revenue
Service, U.S. Coast Guard, and State, County and Local Police.
Besides investigative and enfoicement drug control requires the application of regula-
efforts,
tory efforts and the effective use of science and technology. In our Office of Science and Tech-
nology we have a staff of over 250, with specialists in the fields of chemistry, mathematics,
engineering, electronics, psychology and other disciplines.
The office has three divisions. The Research and Engineering Division conducts feasibility
studies on various aspects of technology, produces actual equipment for use by our Special
Agents in the field and conducts behavioral and social research such as the study of the effects of
drugs on crime.

The Technical Opeiation Division manages the National UHF Radio System, the Single Side
Band Radio System, the National Secure Teletype System and the investigative equipment
program.
Our managed by the Forensic Sciences Division. As in other
eight forensic laboratories are
crime labs, DEA labs examine evidence and present evidence in court when necessary. They
also conduct research, method development, training of other foiensic chemists and distribute
various scientific and technical publications.

2 .
I want to personally thank each of the Symposium participants for contributing to the Sympo-

sium and helping us provide a worthwhile scientific sharing experience. It takes many people to
stage an event such as this, and I would like to recognize some of these now for their outstanding
work: Drs. Michael Klein and Alice V. Kruegei the individuals who first proposed the idea of
holding the Symposium. I would also like to thank Clayton McNeill, Irene Armstrong, Lani
Hidalgo and, of course, Stanley P. Sobol, the General Chairman.

John W. Gunn, Jr.

Director
Office of Science and Technology
Drug Enforcement Administration

3 .
Preface

The papers collected in this volume were presented at the International Symposium on Instru-
mental Applications in Forensic Drug Chemistry held in Arlington, Virginia on May 29 and 30,
1978.
The Symposium consisted of four sessions: Spectioscopy, Computer Applications, Chromato-
graphic Advances, and Special Topics. It covered current methodology and approaches and also
projected future needs and developments.
The Symposium presented 24 experts from 8 countries highlighting current state-of-the-art of
instrumental advances encompassing spectrometry, computers, chiomatography, and such
topics as drug standards, scanning electron and light microscopy, immunoassays and toxicol-
ogy. We heard review papers in mass spectrometry and chemical identification processes. We
examined the advantages of using stable isotopes in the quantification of diugs by GC-MS-COM
systems, as well as the current interest in negative ion mass spectrometry. Techniques of FT-IR
and NMR
were discussed, and their possible future applications to the forensic sciences. The
use of mass spectral computer systems in drug identification, and some computer programs in
Federal, State and Overseas facilities were described.
Many and varied applications of high pressure liquid chromatography and gas chromatog-
raphy, as well as improvements and capabilities of the instmments used in these techniques were
covered. Also, derivatization techniques, detection systems and a new appioach to the opti-
mization of chromatographic systems were discussed.
We wish to express our appreciation to the staff of the Drug Enforcement Administration Spe-
cial Testing and Research Laboratory, Office of Science and Technology and Administrative Ser-
vices for their contributions to theSymposium and publication of the proceedings. It was the
consensus of the participants and attendants that forensic drug chemistry is a unique, continually
developing and intellectually stimulating applied science. We hope that those who read the fol-
lowing manuscripts will agree.

The Editors
Washington, D.C.

4 .
U.S. International by

Narcotics Policy Charles O’Keeffe

The White House


Washington, D.C.

It is a pleasure to be here today to talk about Atlanta, and a drugstore in Los Angeles.
the international narcotics policy of the United The programs that we have in these three
States. Before I move specifically to our inter- —
arenas our international narcotics control
national policy, let me say that this Administi a- program, domestic drug law enforcement, and
tion has two broad policy objectives in the field domestic treatment and prevention efforts, are
of drug abuse prevention and control: first, to the three important components of our entire
reduce the health and social consequences of drug abuse prevention and control program.
drug abuse; and second, to reduce all illicit con- Each is important, and the international pio-
sumption of psychoactive drugs on the as- gram is especially so, as it contributes to this
sumption that, regardless of the presence or Administration’s emphasis on international co-
absence of health hazaids, the interests of so- operation to address the problems of basic
ciety are not served by the widespread use of human needs worldwide.
illicit drugs. I’d like to take just a few minutes to describe
Drug abuse is a reality of modern life, and we our international program which has four major
cannot promise to make it disappear. In the parts: 1 efforts to reduce supplies of illegal nar-
.

United States in 1978, drug abuse has evolved cotic souice materials worldwide; 2. coopera-
from an epidemic to a chronic problem af- tion with international organizations in a
fecting every segment of our society. variety of endeavors; 3. U.S. cooperation
There are three important arenas for Federal with foreign narcotics enforcement agencies;
action to deal with the drug abuse phenomenon and, 4. international drug abuse prevention
as it exists today. initiatives.
The first is the world of international agricul-
The U.S. role in reducing supplies of illegal
ture, economics, and human needs — ancient narcotics source materials is complicated, and
traditions often colliding abruptly with modern
we hope will produce long-term lasting results.
values and ciises.
The second is the world of crime and ciimi- The plants that produce some of the drugs we
nals, people who build networks of illicit traffic are most concerned with (heroin, cocaine, and

to reap huge profits and thwart the laws and to a large degree marijuana) generally grow
custom.-; of many societies. outside of U.S. borders. The laboratories for

And the third is the world of millions of peo- processing drugs are likewise beyond our
ple, young people and working people, affluent boundaries. The flow of drugs into this coun-

and poor, for whom life is a complex of frustra- try passes through many nations, and increas-
tions too often compounded by easily available ingly, people of other countries are
the

drugs. experiencing drug problems of their own.


These worlds seem remote and unrelated to There are ten countries in the world which
the daily lives of most Americans, and that is an we view as top priority targets for diplomatic,
unfortunate misconception. The quality of economic, and technical initiatives designed to
our lives and the promise of our future is af- reduce the cultivation and production of
fected by what goes on in a Burmese village, a drugs. More than a hundred other nations fig-
Mexican border town, a clinic in Portland, a ure significantly in our international drug initia-
shooting gallery in New York, a schoolroom in tives, but these ten are the major sources of
.

“problem drugs” and we intend to concentrate Mexico provides an example of a success-


our energies there: ful eradication progiam. Opium is not an
indigenous crop in Mexico; it is not part of
• Burma produces some 400 tons of opium
Mexican history or culture. In 1972 Turkey ef-
every year.
fectively banned opium cultivation, and crimi-
• Thailand and Laos each grow about 50 tons nal entrepreneurs had turned to Mexico to
of opium a year. plant and process their poppies. This action
concerned both the governments of Mexico
• Mexico grows about 40 tons of opium, which
and the United States, The Mexicans were
is converted to about 4 tons of heroin, most
concerned for two reasons. First, the vast
of which makes its way into the United
profits in the illegal enterprise were undermin-
States.
ing their legitimate market economy in favor of
• The Afghanistan-Pakistan region of South a drug-based economy. Second, the Mexican
Asia is now the world’s largest producer of government was fearful that, given a ready sup-
illicit opium with production far in excess of ply of heroin, use would spread in Mexico with
500 tons annually. enormous health and social consequences.
The United States was concerned over the
• Bolivia and Peru have the potential to pro-
amount of heroin flooding across the 2,000-mile
duce over 50,000 tons of coca leaf, making 15
border, bringing violence, crime and death to
-20 tons of cocaine available to the illict U.S
our cities.
market in the course of a year.
The opium eradication program was started
• Ecuador and Colombia are major processing
in late 1973 as a cooperative U.S. -Mexican ef-
and trafficking countries for the cocaine flow
fort. The U.S. provided technical assistance
to the United States.
and equipment for the opium eradication. The
In most of'these countries, the growth and program slowly began to have results. In
consumption of opium or coca leaves is an old 1975, almost 1,800 people died of heroin over-

an integral part of the culture and ec-


tradition,
dose. Two years later in 1977, 584 died, and

onomics of the nation. Only Mexico, among this year, if projections bear out, only about

the opium-growing nations, has no history of 400 people will die, a decrease since 1975 of
opium use. For the poor farmers of the remote 78%, and a prevention of 1 ,400 deaths. Heroin
availability today is at the lowest level in seven
regions of these countries these crops are the
years, with a national average retail purity of
only cash crops they know, providing them
with a bare livelihood. 5% and a price of $1 .65 per milligram pure.
The United States supports a broad range of But the program remains centrally and oper-
approaches in partnership with other countries ationally a Mexican government initiative. In
and multilateral institutions. Of these ap- matters of how the government carries out its
proaches, crop eradication has proven to be the crop eradication programs, U.S. policy will be
most successful way to reduce bulk cultivation to encourage effective alternatives where we
of illegal drugs. Eradication is unfortunately find a trouble spot and continue to support the
only appropriate in some countries, and in primary role of the Mexican authorities in deal-
every instance must be a cooperative effort, ing with that country’s illicit drug production.
supported by the host government, Crop eradication is only the first phase of a

7 .
program reduce supplies of illicit drugs at
to Department of State.
their source. It must be followed by efforts to
* The United Nations Fund for Drug Abuse
provide alternative sources of income for the
Control has projects underway in Thailand
farmers who are engaged in cultivating illicit
and Pakistan, and is developing one in
drugs.
Burma.
Rural development is a long-term strategy to
reduce the cultivation of illegal drugs, but it is • U.S. Department of Agriculture research
likely to have lasting results. Farmers will not projectsare underway in Thailand and
stop growing opium or coca until they have an Pakistan.
alternate income source to support themselves
The U.S. Government is prepared to provide
and their families;
funds and technical assistance foi some of
This is a difficult and long-range proposition,
these and other well-designed projects in rural
far more than simply
telling a farmer, “Yester-
development. But the initiative for carrying
day you grew poppies, tomorrow it will be len-
out the long-term economic renaissance in re-
tils.” The growers are generally cut off from
mote farming regions must come from the host
central governments; agriculture is often primi-
government and the people of the country.
tive: lines of communication and transporta-
Coordinated development assistance is one
tion are much more secure between the
way we can help put these forces in motion.
individual farmer and the buyer of his crop than
between the farmer and the government. And The difficulty in carrying out crop substitu-
the tradition of growing and using both the tion programs is sharply drawn in analyses pre-
poppy and coca is an ancient one in many pared by the leading international financial
areas, and the use of these plant-drags is inte- institutions. They point to high incomes from

grated into local culture and medicine. poppy cultivation; margins of profit which will
Instead of single-crop substitution efforts, allow the price of opium to go up without dis-
we are attempting to work out rural and agricul- rupting flow; and the political isolation of many
tural development programs for the regions of of the poppy-growing regions.
the countries where the crops are produced. Nevertheless, U.S. policy is to encourage
This will have to include attention to such mat- rural development loans to drug-producing
ters as land-use planning; irrigation and har- countries, and to influence the actions of global
vesting; transportation; economic projections; financial agencies in their lending. Institutions

political liaisonbetween and among the United like the World Bank, Monetary
International
States, the host government, and the individual Fund, and the Regional Development Banks all
farmer and his community. have an important role to play in strengthening
Programs of this scope and complexity will the economies of countries where new crops
require the full-scale cooperation of the host might be substituted for narcotic-producing
government, various international agencies, crops.
and our government. Some modest experi- It is very important to remember that the pri-
ments are already underway in selected mary responsibility for these efforts to reduce
countries^ the supply of illegal drugs rests with the gov-
ernments of other countries. We are asking
• Income and crop substitution projects in Bo- our allies and neighbors to join us as partners,
livia and Pakistan are being assisted by the and our role thus becomes one of assisting and

8 .
supporting the leadership those governments sistance; to cooperate with governments
first,

show. Drug abuse and the drug problem is not in identifying and shutting down major pro-
just an “American disease” as it has been ducers and traffickers of illegal drugs; and sec-
styled; but there is a world of difference be- ond, to strengthen the enforcement capabilities
tween our perceptions of heroin, for example, of host country police and narcotics-related
and those of a Thai farmer or a Mexican campe- agencies. We have long believed that success-
sino. Our strategy will be to respect those dif- ful control of trafficking and use of illicit drugs
ferences, close the gap, and seek international in this country depends on the cooperation and
cooperation. In summary, we have three assistance of other countries. Our strategy is

strategies to reduce the supply of illegal nar- to emphasize efforts to apprehend traffickers
cotic drugs; eradication, rural development, and reduce drug supplies as close to their base
and international cooperation. of operation and place of origin as possible.
The United States has long advocated multi- This strategy maximizes our effectiveness by
national cooperation to solve truly global prob- identifying bulk traffickers and large amounts
lems . no area is this emphasis more impor-
In of drugs before they are dispersed in a more dif-

tant, or more urgent, than in health and drug fused distribution system. There is continuing
abuse prevention, treatment, rehabilitation, evidence that this strategy works, such as the
and control. While there are many immediate 1,123 pounds of heroin seized domestically vs.
gains from one-to-one partnerships with other 2,695 seized overseas by foreign officials, and
countries, as with crop eradication or income the recent seizure of 674 tons of marijuana near
substitution programs, the long-term resolu- an airport in Colombia.
tion to international drug problems requires Our role in overseas enforcement is chang-
systematic, shared collaboration with many ing, as is our perception of the comprehensive
partners —
and world organizations are the mission of cultural, economic, and technical
proper forum for these efforts. planning in assisting victim countries deal with
The United States is a participant in nearly a drugs. In 1976, the “Mansfield Amendment”
dozen international organizations, groups and specifically prohibited involvement of U.S. law

networks dedicated to world health, narcotics enforcement officers in direct police actions or
control, and human needs. These include the arrests in foreign countries. This has meant
U.N. family of agencies that deal with drug a continual evolution in the role of the U.S.
abuse or health issues: Interpol for the sharing agencies assigned to strengthen foreign
of enforcement information, the Customs Co- enforcement.
operation Council for the sharing of smuggling There are several activities in which U.S.
information and a variety of regional and inter- agencies are now involved in support of inter-

regional collaborations. national narcotics enforcement. Intelligence

Our cooperation with foreign narcotics agen- isone of the most critical activities.
cies is another major part of our international The Federal Government cannot carry out
narcotics program. an effective international narcotics program
By 1978, the United States will have been in- without adequate intelligence about producers,
volved in cooperative overseas narcotics en- crop production, financing, trafficking and the
forcement assistance for 66 years, dating from other elements of the networks involved in
the Hague Opium Convention of 1912. This drug distribution. But the government is
equally concerned that the intelligence-
country has tried to provide two kinds of as-

9 .
gathering functions be performed sjensitively, projects is to strengthen the capabilities of host
thoughtfully, and with specific purpose. This country police institutions and build a self-de-
Administration emphasize the importance
will velopment potential in their enforcement
of evaluating and sharing sensitive and timely systems.
information which helps to support policy and In a single year, 272 foreign enforcement of-
pinpoint targets for enforcement. ficers from approximately 50 counti ies came to
Although U.S. enforcement personnel take the United States for narcotics enforcement
no active part in foreign police actions, U.S. training. The course work ranged from man-
agents overseas do carry out undercover and agement and supervisory techniques, to foren-
other intelligence-gathering activities where sic chemistry foi lab technicians who examine
force will not be a factor. They handle and de- drugs as evidence, to the use of dogs to detect
velop informants, evaluate intelligence gen- narcotics in shipments of cargo.
erated by several sources, and cooperate with Abroad, the Drug Enfoicement Administra-
foreign police and enfoi cement agencies in tion concentrates on joint training with officers
handling special surveillance assignments. from the host country, offering work in nar-
The flow of illicit diugs into the United States cotics intelligence, specialized subjects, and
has been significantly reduced as a result of manpower development. Customs foreign
U.S. assistance to foreign law enforcement training emphasizes border control procedures
agencies. Other governments are as in- and techniques, search and seizure ap-
terested as we aie in shutting down criminal proaches, and drug concealment. In a year,
networks and conspiracies and preventing the the two programs combined reached 1 ,794 for-
distortion of their economies by huge illegal eign narcotics officers.
profits Our participation means that they will
. is the important area of finan-
Finally, there
be able to multiply the impact of limited en- and compliance.
cial disclosures
forcement personnel and resoui’ces. In Thai- Narcotics production and trafficking is a
land and Ecuador, for example, U.S. Customs multi-billion dollar industry. Enormous sums
narcotics advisors are helping the local govern- of money are routinely generated by the sale of
ments improve border control procedures. illicit drugs. The economies of many small
This will mean better enforcement of anti- countries are seriously distorted by the ebb and
smuggling laws at the border, and improved flow of illicit drug-related funds: the economies
overall customs procedures in those countries of many urban centers in the United States and
— which translates into more revenue for the other large countries are seriously weakened
host countries and more resources available for by diug profiteering.
narcotics control. A first initiative in this area is the enforce-
In general, U.S. Government personnel as- ment of the provisions of the Bank Secrecy
sist foreign law enforcement agencies with sup- Act, which enable U.S. officials to monitor
port services aimed at identifying and stopping more closely the transactions in foreign banks
criminal networks and majoi narcotics viola- that might be serving as cover for large nar-
tors as close to their base of operations as cotics financingand profiteering.
possible. U.S. agencies can, and do, share narcotics fi-
Training is also an important part of our as- nancial information between and among them-
sistance to foreign enforcement. selves. DEA, IRS, Customs, and the Office of
The immediate goal for these training Law Enforcement in the Department of the

10 .
Treasury all collect and monitor certain kinds We could not, it was claimed, inteivene in
of financial information, dealing with currency treatment or prevention in other countries, on
tiansactions and apparent violations of finan- at least two bases: (1) since we had not
cial records-keeping requirements. The agen- “solved” our drug problem, we had no right to
cies cooperate as appi opriate in the sharing and tell others what to do; and (2) we needed all our

collaboi ative analysis of this information. A money and resources to apply to domestic pro-
special financial intelligence working group has grams and approaches. A thiid factor was in-
been formed by the Stiategy Council to facili- ternational indifference to U.S. assistance;
tate this cooperation. many governments categorically denied they
There are a number of ways the U.S. can ex- had a drug problem in the first place. (Some still
change financial data with foreign countries do.)

relative to narcotics trafficking. Mutual But the world has changed in the last several
Assistance Treaties between nations allow years. Abroad, the leaders of many nations
sharing of data and information to help enforce have identified their own drug problems. At
the domestic laws of each countiy. Similar in- home, we have begun to realize the importance
come tax treaties allow exchanges of tax data. of trying to cope with drug misuse and abuse
The Single Convention on Narcotic Drugs of wherever it appears, and of addressing the
1961 allows governments
exchange financial
to global realities of an international crisis.
data on traffickers. And a
United Nations There are several important reasons for U.S.
Resolution in 1976 urged all governments to Government support of global initiatives to
make narcotics financing a crime, and to ex- treat and prevent drug misuse and abuse:
change information on all such criminals.
The Government of the United States can a. Millions of people around the world are
underscore the importance of financial disclo- suffering from drug involvement, and
sures by aggressively applying these statutes we cannot ignore the health and stability
and provisions, and by seeking full penalties of the world’s people.
for violations.
The final part of our international policy in- b. Our help with another nation’s drug
volves cooperation in drug abuse prevention problem can lead to that country’s par-
initiatives. ticipation in broader programs of inter-
Americans are not the only people in the national narcotics control.
world who have a drug problem. Misuse and
abuse of licit and illicit drugs have made serious
c. A viableand consistentU.S. foreign pol-
icy jeopardized by erosion of the qual-
is
inroads in the health of several nations, partic-
ularly countries with rapidly changing social ity of life in other countries and —
serious drug abuse clearly erodes that
customs or emerging technological/industrial
quality.
crises. There are few, if any, areas of the
world that have managed to escape involve- The continued presence
d. of a market for
ment with the drug problem. drugs in any country confounds
illicit

It was once argued that the United States had our attempts to reduce or eliminate pro-
no role, and no business, in trying to deal with duction; the illegal flow of drugs will
addiction and abuse outside our own borders. cross any border and follow any flag.

11 .
These and other factors encourage US to par- overseas drug initiatives, and we place high
ticipate in international drug abuse programs, on continuing to build trust, confi-
priority
Our goal is to assist host governments in identi- dence, and support for this international
fying and carrying out useful prevention and endeavor,
treatment programs, in the context of overall
social health policy for each nation with which
we are associated.

In summary, the international programs of


the United States Government will, more and

more, our emphasis on developing a


reflect
>• —
host country’s own capacity and motivation
— to take effective action against narcotics
production and trafficking.

In rural and economic development pro-


grams, our intention is to encouiage narcotics-
'

producing nations to take the initiative in


planning and financing the agricultural and cul-
tural transition from narcotics to non-narcotics
as principal cash crops.
In prevention and treatment, our goal is to
strengthen what the host countiies themselves
are prepared to do to begin to bring about the
lifestyle changes that will alter the ways drugs
affect the people of the country.

In the multi-national arena of global organi-


zations, the U.S. will play an active participa-
tory role, not seeking to dominate the agenda of
any organization but seeking, instead, to con-
tribute to all meaningful international efforts.
And cooperation with narcotics enforce-
in
ment agencies abroad, U.S. personnel and re-
sources will be used to buttress what host
country police and enforcement agencies are
already doing, and to strengthen the country’s
capacity to deal with its own enforcement
priorities.
We will emphasize the importance of the
world arena as a critical threshold in our at-
tempts to deal with illegal drugs, and cut down
the flow of drugs to our streets. We depend on
mutual respect and cooperation for all our

12 ,
I. Mass Spectrometry
II. Infrared and Nuclear
Magnetic Resonance

by
Development of Mass
Michael Klein
Spectrometry as a Tool in
Drug Enforcement Administration
Forensic Drug Special Testing and Research Laboratory

Analysis —Review 7704 Old Springhouse Road


McLean, Virginia 22101
Present Address;
Office of Compliance and Regulatory Affairs
Regulatory Control Division
Washington, D.C. 20537

I. INTRODUCTION GC and MS has made it possible to use these


com-
tools separately or in conjunction with a
A , General puter(COMP). The methodology can be im-
proved by the incorporation of digital
Although this Symposium is organized ac-
computers into the system to allow automatic
cording to the individual disciplines Spec-
collection and analysis of mass spectra. (4)
trometry, Computer Applications, Chromato-
graphic Advances, and Special Topics — these The detection by GC-MS-COMP of specific
categories are, in fact, interrelated. Develop- compounds, whether introduced exogenously
ment of improved analytical techniques in the
or arising from aknown disease state, has been
determination of potential or established
developed to the point such that the system is
“drugs of abuse’’ requires the fast, accurate relied upon as a routine method in the labora-

and precise, highly sensitive and specific, qual- tory. The advantages of speed, reliability, and

itative and quantitative determination of in- dependability of this technique for the analysis

creasingly lower levels of drugs, drug of volatile and non-volatile materials will be
impurities and drug metabolites. The com- emphasized in this review.
pound being analyzed may be in the medium of Most common instrumental methods do not
the drug sample itself, or in extracts of biologi- provide the necessary levels of sensitivity, as
cal fluids (blood, urine, sweat, saliva, milk, well as specificity, for the identification and
cerebrospinal and synovial) fiom individuals quantification of drug components. Fluoro-
who have received the drug. Optimum utiliza- metry, i-adioimmiinoassays, thin layer chroma-
tion of the spectrometric analysis may be de- tography (TLC) and many spectrophotometric
pendent upon interactive computer systems, methods often may not provide the adequate
chromatographic analysis, and chemical tech- sensitivity and/or necessary specificity. When
niques, e.g., derivatization and extraction high pressure liquid chromatography (HPLC)
procedures. is limited because of low molai' absorptivity of
By introduction of compounds eluting from a a chromophore group with ultraviolet (UV) de-
gas chromatographic (GC) column into the tection, the required sensitivity for analysis
mass spectrometer (MS), spectral data col- would be lacking! The hazards and ethical re-
lected on each peak makes possible their posi- sponsibilities associated with
measurement of
tive identifications. GC is the most suitable radiolabeled drugs limit this application. Col-
method currently available for resolving into orimetric procedures provide adequate sensi-
individual components the highly complex tivity, but lack the specificity of mass
mixtures of compounds encountered in drug or spectrometric analysis. (69)
biological specimens. (57) Of course, recent Eveti gas chromatographic methods with the
articles describing the development of the liq- flame ionization detector (FID) seldom meet
uid chroniatogiaph (LC-MS) interface point the sensitivity requirements. The maximum
out advantages over GC-MS in the analysis of usable sensitivity of GC (FID) would be around
theimally unstable molecules. (9) (28) De- 0.05 /ig with biological samples. Under these
velopment of sophisticated instrumentation for conditions, the flame tends to be somewhat

J4.
noisy, however, and interfering peaks limit These include the following:
sensitivity. In addition, peaks of interest may
1. Identification of the Illicit Drug Sample
be masked (or only partially resolved) by bio-
logical components present in the sample. (49) a. Routine analysis of samples of drugs,
Nitrogen detectors or electron capture de- diluents, and other major constituents.
tectors (ECD) meet sensitivity requirements, GC-MS techniques are increasingly used
but adequate chromatographic separation of as a tool for dnig identification. Standard
structurally similar drugs is difficult. Another techniques for the analysis of street drugs
disadvantage of ECD-GC is that no structural are based upon infrared (IR) and UV
still

information would be associated with a re- in conjunctionwith specific chemical


sponse, and slight differences in conditions tests. These methods often do not pro-
may effect that response. These disadvan- vide conclusive results in analyses of drug
tages have led in the past to mistakes both in mixtures. Methaqualone, phendimetra-
identity and quantification of material. (31) zine and phenmetrazine found by GC-MS
Even mass fragmentographic methods cause in a confiscated illicit street drug was dis-
problems when ions of sufficient intensity but cussed. (7) The incorrect identification of
of low specificity are measured, i.e., when the phendimetrazine and phenmetrazine, pos-
most intense fragment ions are of low mass or sible by other techniques, could be dan-
formed from structurally similar molecules, gerous due to the toxic nature of the latter.
However, the advantage of using GC-MS
b. Drugs difficult to analyze. Lysergic
assays with chemical ionization (Cl) is that the
acid diethylamide (LSD) is generally con-
most characteristic ion of the compound, i.e.,
sidered to be one of the more difficult illicit
the parent ion, would be available for
preparations to analyze. because
This is
quantification. (70)
of the numerous forms and sizes of the
Only the GC-MS methoct has been shown
preparations, and the presence of highly
overall to be sensitive and specific enough for
colored excipients. Therefore, the de-
monitoring concentrations of various drugs in
velopment of a single standardized extrac-
whole blood or other media, even after a single
tion sequence has been difficult. In
oral dose of 50 or even 25 mg. (15) Inaddition,
since serum constituents can interfere with
addition to the different forms of LSD
preparations, differences in the chemical
many photometric determinations, often quite
composition of the ergot content occur,
tedious extraction procedures are necessary to
including small amounts of the stereo-
minimize these effects. By using mass frag-
mentography, losses in extraction or chroma-
isomeric iso-LSD, LSD degradation
products and other ergot alkaloids.
tographic absorption can be allowed for by use
Recently, an analytical scheme with the
of the appropriate deuterium-labeled standard.
use of MS for the unequivocal confirma-
(5)
tion of LSD in forensic samples was de-
B. Applicalions of Mass Spectrometry in scribed. (66)
Forensic Drug Analysis
c. Identification of impurities and dilu-
In forensic analysis, several specific exam- ents for evidence comparison. The com-
ples can be cited to illustrate modern applica- parison of evidence is an important
tions of mass spectrometry in the laboratory. function of the forensic laboratory. The

15 .
examinations are based upon the compari- supporting MS data.
son of an exhibit with either a refeience The stimulant methamphetamine has
collection oi another specific exhibit. If been obtained both by diversion of
correlations among diiig exhibits can be legitimately manufactured material and
made, then specific infoimation legarding by synthesis in illicit or clandes-
the history of the drug samples can be tine laborator ies, Methamphetamine pro-
developed. The usual means of com- duced in clandestine laboratories often
paring di'iig exhibits is to identify the im- contains impurities arising from incom-
pui'ities which ai'e present in the material plete reaction and inadequate purification
and then determine their relative concen- of inter mediates and the final product of
trations. (63) the synthesis. As the synthesis pr-oceeds,
Identification of heroin and its diluents various impurities (reactants, by-prod-
by chemical ionization mass spectr oscopy ucts, and intermediates, as well as con-
has been shown. (12) The procedure re- taminants within reagents themselves) are
quired no sample ptepaiation or prior produced. The identification of these im-
chromatographic treatment. Its sensitiv- purities are elucidated with the aid of mass
ity permitted a dii*ect and rapid identifica- specti’ometiy (GC-MS) as well as other in-
tion of miciogram quantities of illicit strumental metliods, to further support the
pieparations, by solid probe. Isobutane structural assignments, e.g., nuclear mag-
was the reagent gas of choice, since it has netic resonance spectroscopy (NMR), IR,
been demonstrated as yielding relatively etc., as well as
chemical synthesis. In this
simple Cl mass spectra. case, identifying impurities may provide
Leaders in the area of field ionization significant information about the manufac-
(FI) mass spectrometry emphasize that its turing process, distinguishing between
unique characteristics make it a poten- samples of licit and illicit manufacture.
tially powerful tool in such diverse fields (3) (45)
as medicine, criminalistics, and environ-
2. Verification of the Analytical Approach
mental I'esearch. (I) Recent work has
described two impoifant aieas of The partial thermal decomposition of
application — analysisof complex multi- drugs in the iruection port of a gas chromato-
component mixtures without pi'esepara- graph can affect the accuracy of analysis.
tion and isotope dilution analysis by use of The decomposition products of methylphen-
multilabeled molecular tracers. idate were identified by GC-MS as methyl
Impurities in commei-cially available phenylacetate and a tetrahydropyridine.
chlorphenti amine were analyzed by a The extent of decomposition was found to be
combination of separation techniques, a function primarily of the injector tempera-
followed by electron impact, chemical ture, and this resulted in considerable vari-
ionization and field desorption mass ability. After identification by MS and
spectrometr y to determine whether the subsequent determination of the problem, an
drug was safe for human consumption. improved analytical method, eliminating this
(72) Several methods of ionization wer e thermal decomposition, was accomplished
required for detei'mi nation of various im- by derivatization with trifluoroacetic anhy-
purities, and each furnished unique and dride. (18)

16 .
3. Identification of Drugs in B ody Fluids by compounds, drugs, drug metabolites, biolog-
Mass Spectrometry ical samples, pesticides, and environmental

a. Rapid identification of a drug in emer-


pollutants.The ion-detecting device of the
mass spectrometer is set to monitor, as a
gency overdoses. Phencyclidine (PCP)
function of time, a fragment ion of relatively
concentrations in blood, when associated
high abundance in the spectrum of the com-
with intoxication are generally low. In
pound of interest. The resulting record is
addition, the limited quantity of blood
chromatogram performed with a
that of a gas
available from living individuals necessi-
selective detector. Depending on the ions
tated the development of specific and sen-
chosen, a limited number of peaks will be ob-
sitive assays.Sensitive and specific
served at the retention time of each com-
GC-MS methods have been developed for
pound being analyzed. Therefore, the
the rapid determination of PCP. (56)
chromatogram is relatively free of interfer-
b. Rapididentification of drug metabo- ences from other compounds in the sample,
lites. A
combination of GC and MS has and the method often offers sensitivity
been shown to be useful in the identifica- greater than that obtainable with conven-
tion of small amounts of mecloqualone tional GC detectors.
metabolites in cases of poisoning, particu- Due to monitoring of specific mass ions of
larly when several other drugs have been the compound being analyzed, a preceding
taken at the same time. Identification of separation of other drugs is not required.
these metabolites was performed by com- Quantitative GC requires that the compound
paring physico-chemical and mass spec- being analyzed and the internal standard be
tral data of synthetically prepared completely resolved but this is not needed in
,

compounds with those of the isolated mass fragmentography. (54)


metabolites, (67) By focusing on a fixed m/e ratio, the mass
spectrometer behaves as a highly specific gas
4. Quantitative Methods —Mass chromatography detector, only responding
Fragmentography
to compounds that, on fragmentation, yield
Combined GC-MS has become an estab- ions at the m/e ratio upon which it is focused.
lished method for detecting and quantifying a. Single ion monitoring {SIM). Single
very low levels of compounds in complex ion monitoring usually requires selection
mixtures. The technique of mass frag- of an appropriate derivative of a compo-
mentography is one of the most sensitive nent which has at least one unique ion of
detection systems known.
. Mass frag- high abundance at a reasonably high m/e
mentography, also known as single or multi- ratio so that interference is minimized.
ple ion mass detection ,
is the simultaneous Calibration curves must be obtained to de-
monitoring of one or more fragment ions termine the losses in extraction, purifica-

rather than the scanning of the total ion spec- tionand derivatization, as well as losses in
trum as in conventional mass spectrometry. column absorption and variation in instru-
(20) (23) The use of mass fragmentography mental response. This is often time-con-
'

as a single or multiple ion monitoring for GC suming and unreliable.


has been applied in the qualitative and quan- The sensitivity of detection over that of
titative analysis of low levels of endogenous conventional GC methods, e.g., electron

17 .
capture, flame detection and thermal con- extremely small. However, the nat-
‘*C is

ductivity, is lO'* to 10*. (23) Hiish resolu- uralabundance of '^C is much greater than
tion can be used when more than one that of and this would decrease the sen-
fragment ion with a similar nominal m/e sitivity of the method. (71) In addition,
ratio is pieseiit. When a number of com- the deuterated compounds are less

pounds in a sample and an internal stand- expensive.


ard produce on ionization an ion fragment Since the chemical propeities of labeled
with the identical m/e ratio, but have dif- drugs aie veiy similar to those of the par-
ferent GC retention times, quantitation of ent compound, this procedure overcomes
more than one compound is performed. problems caused by incomplete extraction
The mass spectrometer is acting as spe- and derivatization and also minimizes ab-
cific detectors of certain stiuctural fea- sorptive losses encountered on GC. (24)
tures in a mixture and this is referred to as In addition, they show similar fragmenta-
functional group analysis. tion patterns to the unlabeled compound.
Due to its extra mass, the resultant frag-
/}.Multiple ion monitoring {MIM), The ments will have different m/e ratios, distin-
mass spectrometer focuses on several guishing them from the unlabeled
unique prominent fragment ions. MIM is fragments. Suitably labeled compounds
a powerful method for the simultaneous are theiefore useful for quantifying the
quantitation of drugs and their metabo- compound . One disadvantage is that mul-
lites. Maximum sensitivity results from tiply-labeled standards can show GC or
monitoring a single ion. As more ions are HPLC properties slightly different from
selected, each will be monitored for a those of the compound of interest. This
shorter period with subsequent decrease approach will be discussed in greater de-
in signal-to-noise ratio and increased con- tailby Dr. Horning later in this
tribution from random electrical noise. Symposium. (35)
However, MIM has the added advantage When compounds other than stable-iso-
of greater specificity, by focusing on more tope-labeled analogs are used as internal
than one characteristic fragment. One ap- standards, it is necessary to determine a
plication of MIM can be for detection of response factor, which is the ratio of the
drugs and metabolites containing elements peak area per microgram of drug divided
with more than one naturally-occurring by the peak area per microgram of internal
stable isotope (e.g., Cl and Br). standard. The response factors should be
In carrying out quantitative estimations, constant over the range of concentrations
the ideal standard should be as chemically measured. In the case of unavailable
similar to, but in some way distinguishable standards, a hydrocarbon or drug with
from the compound under investigation. suitable GC properties can be added as an
®H-, '®C- and i*N-labeled compounds are internal standard. (34)
the best suited, and should be added prior
c. Therapeutic drug monitoring.
to extractionand analysis. Generally, la-
beling with '®C would be preferred over i. Pharmacokinetic determination — de-
deuteration since it would be likely to ex- termination of proper drug dose. Drug
change; in addition, the isotope effect of monitoring is useful in the management of

18 .
patients undei going chronic therapy, could provide sufficiently accurate mea-
especially if the therapeutic concentration surements in patients. Therefore, indi-
range of the drug is Determina-
narrow. vidual pharmacokinetic parameters were
tion of plasma levels of several drugs not previously determined. (69)
is important for rational therapy. Pa-
ii. Limited sample size. Mass fragmen-
tients treated for grand mal epilepsy have
tography was useful for monitoring theo-
been significantly improved by adjust-
phylline, a bronchodilator, in newborns
ing plasma levels of diphenylhydantoin
and pediatric cases. There are risks of
to the therapeutic level of 10-20 ju,g/ml.
toxicity associated with the use of the drug
The method applied must be sensitive
and, therefore, it is generallyrecom-
since the therapeutic dose is low. Speci-
mended that its concentrations be moni-
ficity is especially important because of
tored in the blood. Chromatographic
the multiple drug nature of anticonvulsant
from other drugs, metabolites
interference
therapy and extensive metabolism of the
and organic material found in serum or
drug. (51) Carbamazepine is used for the
blood usually occurs. In addition, several
treatment of convulsive disorders and tri-
such procedures require large volumes of
geminal neuralgia. In order for its
blood or serum for analysis and therefore
pharmacokinetics to be determined in
cannot be used loutinely to monitor theo-
man, a specific and sensitive analytical
phylline concentrations in newborns or
method was needed. (54) A variety of
small children. (62) Mass fragmen-
techniques was used to eliminate endoge-
tography has been used to study the trans-
nous material which may interfere with the
fer of drug from the mother’ s circulation to
assay. (60)
the fetus and to amniotic fluid and breast
Preliminary studies on the metabolism
milk. (33)
and pharmacology of chemotherapeutic
Etorphine, 6 14-cndo-etheno-7- [ I -(7?)-
,

drugs have been carried out using radioac-


hydroxy-l-methylbiityl]-6,7,8,14-tetrahy-
tively labeled compounds. Investigations
droripavine, an analgesic approximately
in humans over extended periods of time
1000 times as potent as morphine, and
during long-term chemotherapy have been
which can be taken sublingually, may be-
difficult because the concentration at
come a future threat in the drug abuse
which toxicity occurs and the minimum
field. Its study in biological systems in
concentration required for therapy have
hindered since it cannot be detected in
not been determined It is also not known
.

urine by the usual analytical approaches


if the concentration of the drug within indi-
(GC), after administration of pharmaco-
viduals decreases with chronic therapy as
logically effective doses. The SIM
a consequence of enhanced drug metabo-
method was developed for the urine analy-
lism. The CI-MS assay currently is being
sis with (tritiated) '*H 2 -etorpliine as the in-
used to measure concentrations of 1,3-
ternal standard. (39)
bis(2-chloroethyl)-l -nitrosourea (BCNU)
in the plasma of patients who are undergo- iii. Determination of Alternate Clinical
ing a schedule of BCNU treatment for Methods. Although blood is the usual
brain tumor. With the exception of mass sample for drug monitoring, saliva sam-
fragmentography, no analytical method ples have also been used The concentra-
.

19 .
tion of mostdnigs in saliva corresponds to and in newborn infants of mothers who are
unbound plasma dmg concentrations, and maintained on methadone. This method
that value may be a more meaningful value will also permit studies in animals in which
for considerations of pharmacological ac- plasma methadone levels are lower and
tivityor toxicity than a value that reflects turnover rates are much faster than in
both bound and unbound drugs. In addi- humans. (24)
tion, saliva can be obtained by noninva-
sive techniques and this is helpful when vi. Drug assays. Mass fragmento-
multiple serial samples are needed and in graphic assays are among the most selec-
monitoring drug concentrations in chil- tive analytical methods available to date.
dren. Furthermore, most therapeutic The elucidationof the biochemical
agents transfer rapidly from plasma to sa- pharmacology of a drug has been predi-
liva, and the concentration of drugs in sa- cated upon the availability of assays capa-
liva is proportional to the concentration in ble of measuring the drug in plasma and/or
plasma. (33) serum. (60) (61)

iv. Potential overdoses. Methods have


vii. Indirect measurement of enzyme ac-
been developed for monitoring the con-
tivity (inborn errors of metabolism). In
centration of tricyclic antidepressants in
plasma to be able to successfully manage
phenylketonuria, one of the most common
inborn errors of human metabolism,
patients with depression. There is an in-
phenyl-4-monooxygenase, the liver en-
creased prevalence of toxic overdose as a
zyme which converts phenylalanine into
result of the ready availability of the tricy-
tyrosine, shows no, or only partial, activ-
clic drugs to the depressed patient. Over-
ity. In order to evaluate the remaining en-
dose symptoms may include grand mal
zyme activity or to determine an increase
seizures, cardiac failure, coma, and even-
in the enzyme activity after therapeutic
tual death. It is important to monitor low
treatment, an indirect in vivo measure-
therapeutic concentrations in cases where
ment of phenyl-4-monooxygenase was
the drug identity is known, and to be able
done by SIM MS The specificity and reli-
.

to screen for toxic concentrations in cases


ability of other determinations are disput-
wheie the drug identity is not known. (70)
able. (71) FD-MS has also
Quantitative
V. Monitoring patients on methadone been applied medical research studies.
in
maintenance. Recently, chemical-ioniza- When phenylalanine-dy was administered
tion mass spectrometry and deuterium-la- to patients suffering from phenylketo-
beled moiphine were used to monitor the nuria, the determination of the ratio of
possible abuse of narcotics by methadone tyrosine-do to tyrosine-dg also gave a mea-
maintenance patients, as well as low level sure of the enzymatic reactivity still avail-
quantitation of methadone in biological able. (47)
•samples. The latter method permits more
accurate measurement of low methadone viii. Amino acid analysis. GC-MS has
levels present in adolescent heroin addicts been used for amino acid analysis because
on methadone maintenance therapy who of the high sensitivity and specificity in
are receiving lower dosages (5-40 mg/day) mass spectrometric detection. (19)

20 .
//. ANALYTICAL METHODS BY prove the compatibility of various chromato-
IONIZATION TECHNIQUE graphic and mass spectral techniques. With
respect to improvement of MS techniques, our
A. General desire is for greater structural information from
Often the success of a mass spectrometric mass spectrometry and improved detectability

analysis depends upon the type of ion source with GC detectors or with GC-MS selected ion
chosen. Often, electron impact mass spec- detection.
trometry (EI-MS) of the compounds does not
1. Applications of Improved Derivatization
provide the necessary level of confidence
Procedures in El Mass Spectral Analysis
needed to make molecular assignments.
Therefore, alternative (Cl, FI and FD) ioniza- The gas chromatographic separation of
tion techniques can be performed to further major and minor components of marihuana
substantiate the results and thereby obtain a and hashish extract has been described.
high degree of confidence. (72) These alter- Mass spectrometric data have been pro-
native methods of ionization most often duced for these components. Some of the
provide simpler mass spectra dominated by minor components of cannabis resin are in
fact isomers and homologues of the major
afew highly intense ions. In addition, FD ioni-
cannabinoids: cannabidiol, A''Metrahydro-
zation processes allow the analysis of non-
cannabinol and cannabinol. (65) These
volatile compounds to be achieved without
derivatization.
components include cannabidlvarol (the
cannabidiol-Cg homologue), cannabicyclol,
The mass spectra of some biologically im-
and the Cj-homologue of A '’“-and A'-Metra-
portant compounds, e.g., amino acids, tri-
hydrocannabinol, etc. More recently, the
glycerides, have been measured using field
use of improved derivatization techniques
ionization (FI) and field desorption (FD) means
has provided better gas chromatographic
of ionization and comparing the results to the
separation of additional cannabinoids and, in
more common approaches of electron impact
some cases, has increased mass spectral ca-
(El) and chemical ionization (Cl). (17) The
pabilities in structural elucidation of minor
mass spectra obtained suggested that neither of
components. Fairly good separations have
these techniques is superior to the others, but
been obtained using trimethylsilyl (TMS) de-
that the method of choice in any analytical
rivatives of cannabis extracts and a 3% SE-
problem depends on the type of compound
30 column. However, some poorly resolved
involved.
peaks were present. These corresponded to
B . Electron Impact (El) Ionization mixtures of mono- and dihydroxy com-
Several derivatization techniques have been pounds and separation of them was achieved
studied with respect to their effect on gas chro- by variation of the derivatives— i.e., substi-
matography and resultant electron impact tution of the TMS group with silyl moieties
mass spectral characteristics. These studies containing higher alkyl substituents. This
have been critical with respect to analysis of resulted in the production of longer retention
cannabinoids. Since the major function of times for cannabis diols, thus shifting their
derivatization is to enhance volatility and ther- respective signals away from the other con-
mal stability, efforts have been devoted to the still retaining good gas chro-
stituents while
development of new derivatives which also im- matographic properties and being stable.

21 .
The tri-zj-alkylsilanes included triethyl-, tri- exhibited molecular ions as the most abun-
H-propyl-, tri-/!-butyl, and tri-n-hexylsilanes. dant in the spectia, a marked improvement
(26) Derivatives with alkyl groups greater over the TMS derivatives.
than Cj required, however, undesirably high Additional new derivatives examined for
elution teinpeiatuies. By increasing the GC analytical chemistry (steroids and nu-
molecular weight of the silyl moiety, how- cleosides) included r<?//-butyldimethylsilyl
ever, SIM and MIM work was improved due (TBDMS) ,
cyclotetramethyleneisopropylsi-
to lack of background ions at the higher lyl (TMIPS), and cyclotetramethylene-Zcrt-
masses. In general, the mass spectral char- butylsily] (TMTBS) derivatives. (59) These
acteristics of the higher alkylsilyl derivatives
derivatives offered several advantages over
were similar to the TMS derivatives. TMS, including greater stability for TLC and
Fresh samples of Cannabis sativa L. isolation of standards, better separations by
usually contain cannabinoids in the form of GC and structural information by EI-MS.
their carboxylic acid derivatives (A'-tetrahy-
Silylation has proved to be one of the most
drocannabinolic acid). These acids cannot
effective derivatization methods for a wide
be examined directly by gas chromatography
variety of compounds. Due to steric crowd-
since they decarboxylate upon heating, even
ing, these bulky groups have decreased sus-
to some extent, as the TMS ethers. Prepara-
of cyclic alkylboronate
ceptibility to nucleophilic attack. The large
tion derivatives
(alkyl =methyl or butyl) has been shown to
silyl ethers have greater stability towards hy-
drolysis than do TMS ethers, and, therefore,
be suitable for GC-MS studies on these types
of compounds. In addition, isomeric acids
offer much greater utility as protecting

in which the phenolic hydroxyl was para to


groups in synthesis. Like TMS ethers, the
the carboxylic acid have been reported to be
bulky silyl ethers offer good volatility and
thermal stability and some other characteris-
present in cannabis. Since they are incapa-
ble of forming cyclic alkylboronates, this
tic properties make them valuable for identi-
fying fragment ion types and deducing
method of deiivatization provided an ideal
fragmentation pathways in mass spectrom-
means of distinguishing between the two iso-
mers. (25) The methylboronates had reten-
etry. The high mass of the bulky ether

tion times comparable with those of the TMS derivatives in the case of polyhydroxy
derivatives, whereas the /j-butylboronates
compounds may be a disadvantage when
using some models of mass spectrometers
had longer retention times. A major advan-
with low mass limits. However, it may be.
tage over TMS derivatization was the reduc-
tion in molecular weight obtained for
an additional advantage in GC-MS-SIM,
all of
the alkylboronate derivatives as the result of
where the increased mass will place impor-
replacing two TMS
groups with the rela-
tant fragment ions in a region free from GC
tively low mass boronate moiety.
bleed peaks.
In addi-
tion, the mass spectral characteristics The mass spectra of these ethers are
observed for the alkylboronate derivatives usually dominated by peaks arising from ini-
included positive charge localization away tial siliconium formation, rationalized by
from the boronate ring systems onto the elimination of a stable, branched alkyl radi-
heterocyclic oxygen atom. The spectra of cal, thus relieving steric crowding in the silyl
the A*-THC-acid boronate derivatives group. Ions of these derivatives are much

22 .
more prominent in the important high mass or opiate addicts) who take massive doses
region than those in the spectrum of the TMS of morphine have been confirmed by Gl-
ethers. and EI-MS (approximately 5% normor-
Siliconium ion centers can interact with phine and >0.1% norcodeine relative to
sterically accessible electron dense groups in the administered morphine dose). The
a molecule, often resulting in cyclic transi- difficulty in detecting normorphine was at-
tion states which subsequently rearrange. tributed to its instability in acidic and alka-
These fragmentation processes have pro- line media, its poor solubility in water
vided important structural information use- immiscible solvent systems and/or to its
ful in distinguishing between isomers, limited sensitivity to potassium iodo-
specifically between derivatives of deoxynu- platinate, the spray reagent commonly
cleosides and of ribonucJeosides. Even used for alkaloids in TLC examination.
when the molecule does not contain the steri- The presence of normorphine and norco-
cally accessible electron dense function, the deine in urine of individuals who are
derivative may be useful for isomer differen- chronic users or have received a single
tiation. One example of this is the differ- large dose of morphine suggested that
ence between the mass spectra of the morphine metabolizes to a limited extent
epimeric steroids androsterone and epian- to normorphine and to codeine and norco-
drosterone, where the intensity of the [M- deine. Codeine formation and conse-
(R-HXaSiOH)]^ ion depends upon the quent norcodeine formation seem to
stereochemistry of ring A. appear after prolonged heroin abuse, and
is possibly attributed to changes in hepatic
2. Mass Spectral Analysis of Compounds by functions frequently observed in opiate
EI-MS addicts. (6)
a. Massspectral analysis of opiate de- A
sensitive assay for the determination
rivatives. Several recent articles have uti- of etorphine in urine was developed. The
lized GC-MS, high resolution MS, along sensitivity of the method was about 5
with supporting instrumental and chemi- ng/ml, which compared favorably with a
cal methods to identify atypical or unusual sensitivity of 100 ng/ml by GC analysis.

morphine or heroin trace impurities. The procedure included repeatedly basify-


These have included /J-chloromoiphide, ing the urine of animals, extraction into
(29) 3,6,17 -triacetylnormorphine, (42) butyl chloride, and reacidiflcation. A
and 0"-monoacetylmorphine (43) (52) as — final organic extract was derivatized with
well as acetylcodeine and 0*^-monoacetyl- N,0-bis(trimethyIsiIyI)trifluoroacetamide
morphine. (42) In addition, synthesis of to form the TMS derivatives. •’Hu-Etor-

deuterated heroin analogs and mass spec- phine was added to control urine and
tral analysis has allowed differentiation treated by the identical procedure. The
between the isomers, 0®- and 0®-monoace- mass spectrum of etorphine showed a mo-
tylmorphines, which led to their identifica- lecular ion at m/e 483. The spectrum of
tion and confirmation in illicit heroin tritiated etorphine showed a molecular ion

samples. (41) at m/e 487(®H2) and ions at m/e 483 (®Ho)

The levels of normorphine and norco- and 485(®Hi), Samples were extracted,
deine in the urine of individuals (patients derivatized and analyzed on the GC-MS

23 .
focused m/e 483 and 487. The method
at LSD (mol. wt. = 323). (66)

could possibly be twice as sensitive with a


c. Analysis of methainphetoniine hn
fully deuteiated standard, although this
purities. The impurities identified in illicii
was not available. Using a radioisotope,
methamphetamine include N-formylnicth-
however, enabled each step of the analyti-
amphetamine (N-methyl-N-fa-nicthyl-
cal procedure to be monitored. Using
phenethyl)formamidc), N-a,a;'-tritnct]iyl-
tritiated compounds in the mass spectrom-
diphenethylamine and a-benzyl-N-mcthyl-
eter posed no problem with safety proce-
phenethylamine. Other laboratory exhib-
dures, e.g., proper exhaustion from the
its have been shown to contain formic
vacuum lines and proper precautions
acid, methylamine, N-fonnylamphet-
when oil was changed in the vacuum
amine, and dibenzylketone. They were
pumps. (39)
identified by UV, IR, NMR
and combined
b. Determination of LSD. The analysis GC-MS techniques. The methods used to
began by prior extraction of an ammo- isolate and identify each compound as
niated LSD sample with 1 ,2-dichloroeth- well as the manner in which each can
ane, followed by re-extraction into 0.5N occur in methamphetamine samples were
HuS 04 . This latter aqueous solution was described. (3) (45) An impurity detected
then divided into two portions. The LSD in illicit amphetamine has been identified

inone half was again basihed, le-exti acted as ' a-benzylphenethylamine. (44) The
with 1 ,2-dichloroethane, and recovered. above combined instrumental approaches
The second half was scanned from 210- were used in determining the substance.
360 nm to give a broad absorption band This contaminant was cited for its reported
with the maximum at approximately 313 toxicity (CNS stimulation, increase in
nm. After scanning, the solution in dilute blood pressure, hypotensive action, res-
sulfuric acid was irradiated with long wave piratory difficulties).
ultraviolet light for
minutes, re-
five d. Identijication of mecloqtialone me-
scanned to show a shift to the broad ab- tabolites. Mecloqualone (2-methyl-3-(2-
sorption maximum to approximately 294
chlorophenyl)-4-(3H)-quinazolinone) is a
nm. The reaction product was then re- non-barbituric hypnotic structurally simi-
extracted by the usual method. The shift lar tomethaqualone (2-methyI-3-o-tolyl-4-
in UV absorption maximum, due to a pho- (3H)-quinazolinone). After oral Inges-
tochemically induced hydration reaction
tion, .unmetabolized mecloqualone is
at the Cg jj double bond, was previously excreted in very small amounts (2-3%),
too non-specific to provide unequivocal
and the majority (95%) undergoes hydrox-
confirmation of the presence of LSD.
ylation, followed by glucuronide forma-
The El mass spectral analyses of the non- tion. Hydrolysis with 20% hydrochloric
irradiated and the irradiated products acid or i3-glucuronidase yields the free
provided
hy-
the specificity required for droxy compounds.
forensic purposes, as well as proving
that Eight synthetic monohydroxy
the hypothesized hydration product
com-
was pounds (including four with a hydroxy
formed. Observation of a molecular ion
at group on the chlorophenyl moiety and four
m/e 341 confirmed the addition of water to
others with a hydroxy group on the
quin-

24 .
azoHnone moiety) were examined mass compound in the metabolite mixture.
spectrometrically, as were their TMS de- After acid hydrolysis, four metabolites
rivatives. Depending on the site of hy- could be isolated and Identified in urine
droxylation, distinct differences between (GC). Combined GC-MS enabled deter-
the mass spectra of these compounds were mination of the chemical structures of
observed. Compounds hydroxylated on all four metabolites: 2-methyl-3-(2-chloro-
the quinazolinone moiety exhibited frag- 3 -hydroxypheny l)-4(3 H)-quinazolinone:
ment ions at m/e 111 and 152 (both con- 2-methyl-3-(2-chloro-4-hydroxyphenyl)-
taining the chlorine atom). M/e 152 ion 4{3H)-quinazolinone; 2-methyl-3(2-chloro-
shifted to m/e 168 in the mass spectra from phenyl)-7-hydroxy-4(3H)-quinazolinone;
the compounds hydroxylated at the and 2-methyl-3(2-chlorophenyl)8-hydroxy-
chlorophenyl moiety. However, no frag- 4(3H)-quinazolinone. The other four
ment at m/e 127 resulting from a shift of the hydroxy isomers were not detected in the
m/e 1 1 1 fragment ion was observed. This isolated metabolite mixtures. (67)
difference provided a means of differentia-
e. Measurement of enzyme activity. A
tion between the two groups. However,
specific method for quantitation of deu-
differences among the individual members
terated and non-deuterated phenylalanine
of the same group were less clear. A frag-
and tyrosine in human plasma by the
ment at m/e 160, present in the MS of only
GC-MS-SIM technique was developed to
the 8-hydroxy compound, was used to
measure phenylalanine-4-monooxygenase
identify one of the major metabolites.
activity. (71) The N- and N,0-trifluoioa-
The mass spectra of the compounds sil-
cetyl methyl ester derivatives provided
ylated in the position 5, 6, 7 or 8 (quinazo-
sensitivity measurements as small as
linone) exhibitedm/e 111 and 152. These
ca. 2.5 ng/ml; coefficient of variation ca.
fragment ions were not present in the mass
1,6% (phenylalanine) and 3.0% (tyro-
spectra of the compounds substituted in
sine). The chosen derivatives show char-
the chlorophenyl nucleus (3', 4', 5' or 6')
acteristic and intensive signals typical for
which, however, showed characteristic
tyrosine, or phenylalanine. Subjects
fragments at m/e 143, 1 17 and 1 16. Again
were administered deuterated L-phenyl-
the presence or absence of the chlorine
alanine-ds and resulting deuterated L-
isotopes facilitated identification of some
tyrosine-d., and lesidual L-phenylalanine-
typical fragments.
dg were measured.
No distinct differences could be ob-
served among the compounds silylated on /. Analysis of amino acids. A method
positions 3', 4', 5' or 6'. There were, for GC-MS analysis of amino acids hydro-
however, characteristic differences in the lysates containing low picomole quantities
mass spectra of compounds silylated in of oligopeptides was described. (19) The
positions 5, 6, 7, or 8. The 6-TMS com- method utilized GC-MS-SIM of their tri-

pound produced a molecular ion. The fluoroacetyl n-butyl ester derivatives.


base peak mass spectrum of the 7-
in the Analysis of amino acids released by hy-
TMS compound was at m/e 323, and for drolysis of picomole quantities of oligo-
the other compounds at m/e 343 This al-. peptides generally requires stringent
lowed for identification of the 7-hydroxy precautions to prevent introduction of

25 .
amino acid or peptide containing contami- was 5-(p-methylphenyl)-5-phenyIhydan-
nants into the sample. This has been toin (MPPH). Both compounds were
avoided by use of vacuum line and micro- chromatographed as their 1 ,3-dimethyl de-
techniques involving solution and reaction rivatives following quantitative on-col-
mixture volumes of ca, 1 jA. The GC col- umn methylation with trimethylanilium
umn was protected by means of a sol- hydroxide. MPPH reacted similarly, in
ventyreagent vent valve from reactive both methyl derivative formation and in
reagents present in the amino acid deriva- recovery from plasma, because of its
tization reaction mixtuie. Techniques chemical similar-ity to DPH.
were developed which permitted analysis The most abundant ion common to both
of 25-50 pmol of a decapeptide based on DPH and the inter nal standard was at m/e
GC-MS, hydrolysis, derivatization and 118. Although monitoring at m/e 1 18 pro-
amino acid analysis. Two critical prob- duced the greatest sensitivity, the rela-
lems were overcome: (1) avoiding intro- tively low m/e ratio of that fragment
duction of amino acids or proteins into the increased the possibility of interference
sample during processing, and (2) main- from other components. Larger ions
taining high performance chaiacter- common to both compounds are at m/e 203
i sties of the column despite the necessity and m/e 194. M/e 194 was chosen to be
of injecting highly reactive reagents monitored due to its greater intensity in
(trifluoroacetic acid, anhydride). The in- the MPPH spectrum. The retention times
strumental techniques used to avoid col- of the 1,3-dimethyl derivatives of DPH
umn degradation involved use of a bypass and MPPH were 6.0 and 7.7 min. by GC-
valve and a precolumn to remove solvent MS. DPH was quantified down to 0.2 ng
and reagents prior to adsorption onto the (with 3:1 signal- to-rioise I'atio), The de-
chromatographic column. To avoid con- tection limit was 0.05 ng.
tamination with extraneous amino acids, A mass fragmentographic method for
specialized microtechniques were devel- the quantitative determination of small
oped including vacuum line reagent and amounts of carbamazepine in plasma,
solvent transfer methods. using 10,11-dihydrocarbamazepine as the
Methodology for hydrolysis of oligo- internal standard, was accomplished by
peptides and derivatization of amino acid mo!jitofi,tjg the Intensities of the molecular
mixtures using reaction solution volumes ion? of carbamazepine (m/e 236) and dihy-
of 1 /xl was developed. Vacuum line drocarb,arnazepine (rrt/e 238). (54) When
transfer of solvents and reagents and carbamazepine is injected into a GC, de-
sealed capillaries as reaction vessels sig- composition to iminostilbene occurs.
reduced amino acid (or protein)
nificantly The decomposition is not reproducible,
contamination and allowed amino acid depending on several factors. To com-
analysis of picomole quantities of pensate for the variability of decomposi-
oligopeptides. tion, yield of extraction, and injection
g. Mass fragiiienlography. Mass fiag- volume, choice of an internal standard as
mentogiaphy was applied to the analysis chemically similar to carbamazepine was
of the anti-epileptic drug, diphenylhydan- critical. 10,11 -Dihydrocarbamazepine
toin (DPH), (49) The internal standard has properties almost identical to carba-

26 .
mazepine with respect to decomposition, majority of GC-MS work is performed on
i.e.,loss of HNCO. It was possible, low resolution instruments. The use of
therefore, to quantitate carbamazepine high resolution GC-MS-SIM would allow
down to 50 ng/ml using 0.5 ml plasma. for quantification of a specific fragment in
The illegitimate use of phencyclidine the presence of other fragments with the
(PCP), l-(l-phenylcyclohexyl)-piperidine, identical nomipal mass.
has increased drastically throughout the The application has been demonstrated
USA, partially due to the relative ease in SIM quantitation of dimethylnitrosa-
with which it may be synthesized. PCP mine, a potential carcinogen in tobacco
preparations are self-administered by smoke condensates, by monitoring the
smoking, insufflation, oral ingestion and molecular ion at m/e 74.0480. (14) The
iryection. samples, of course, are extremely com-
A procedure was developed for iden- plex and some of the ions with identical
tifying and measuring PCP in blood with nominal masses which interfered with the
internal standard, l-Cl-phenyl-pHjJ-cy- component being analyzed included
clohexyl) piperidine (pentadeuterophen- C3 H 0 O 2+ (74.0368), '••’CC2H502+ (74.0401),
cyclidine). (56) A known quantity of this CH4N3O+ (74.0354) and C3H8NO+
was added to a measured
internal standard (74.0606). However, the use of high reso-
amount of blood, and to aqueous PCP so- lution avoided interference by other
lutions of appropriate concentrations. components.
Each sample was then carried through a
three-step separation. The resulting ex-
C. Chemical Ionization {Cl).

tract was taken to dryness, reconstituted CI-MS, a “softer” method of ionization,


and two aliquots subjected to analysis, one often provides a larger fraction of ions related
for quantitation and the other for positive to the molecular weight of the sample under
identiflcation. The PCP was quantitated analysis. Other advantages are that the ion
by monitoring the ions at m/e 205 (pH,]- source can take most or all of the column efflu-
phencyclidine) and 200 (phencyclidine). ent, and the carrier gas can be used as the re-
The concentration of PCP was calculated agent gas. CI-MS is best utilized in
by normalizing the area of the response at conjunction with EI-MS, in order to comple-
m/e 205 to 100%. Applying the factor ob- ment molecular weight information with mass
tained to the area of the response at m/e spectral structural information,. (22) The oc-
200 yielded the concentration of PCP in currence of both proton transfer and addition
ftg/liter. A second injection of a similar reactions on the same compound can often pro-
volume was used to confirm the presence vide further evidence for the molecular weight
of PCP. The ions at m/e 243 (molecular assigned. This results in the quasimolecular
ion), 242, 200 and 186 were monitored and ion, (MH)'^, an even electron species which, if
the calculated ratios of the responses com- unstable, can further fragment to lose a neutral
pared to that of a standard sample. molecule. One would expect more fragmenta-
The use of SIM can be limited by contri- tion as the proton affinity of the reagent gas de-
bution of unresolved components which creases. Proton abstraction (M-H)+ can
give ions at the same nominal mass as occur mostly with compounds of low proton af-
those compounds being analyzed. The finity, e.g., alkanes. Addition reactions be-

27 .
tween functional groups and the reagent gas 1. Reagent Gases
can also be observed,
Methane and isobutane have been use
SIM analysis using Cl for various com- reagent gases since their “reactive aci
pounds (e.g,, morphine) has been reported to ties” cover a wide range. A reagent gas
achieve comparable sensitivity with a specific- be used to identify basic groups in an
ity not equalled by immunoassay or hemagglu- known molecule. In addition, if the ft
tination analyses. Because of its high tional groups present in a molecule
sensitivity and specificity, SIM analyses allow known, a reagent gas can be selected wh
quantitation of nanogram orpicogram amounts will selectively protonate one of these, a
of several drugs and drug metabolites in a sin- therefore, provide fragmentations which
gle run. Chemical ionization is now freouentlv initiated by and are specific for that parti
used for the quantitation of drugs and drug iar functional group, Hydrogen (Hj), me
metabolites. Compounds which can not be ane (CH^) and isobutane (i-C^Hjo)
derivatized easily or which show poor chroma- enhance the MH"*" ion and can be used in <

tographic properties can be quantitated using analyses. In general, more fragmentatioi


the CI-MS direct insertion-SIM method. observed with H
2 and less with i-C 4 l-

The degree of interference depends on the Water has been shown useful in direct det
uniqueness of the ion masses monitored, and, mination of organic compounds in aquec
therefore. Cl offers significant advantages over solution down to 1 ppm, producing typii

the more commonly used electron impact ioni-


ions, MH'*' and M-t-HgO"''. Deuterium oxi
zation due to the high abundance of quasi-
{D2 O) can additionally be used in the det
mination of the number of active hydroge
molecular ions. The Ion current from the
compound being analyzed is concentrated in a
in a compound. Amide hydrogens can
exchanged, whereas hydrogens a- to a
relatively few high m/e values where back-
bonyl groups are exchanged only in amour
ground contributions are low. Cl has the addi-
less than 15%. (22)
tional advantage of eliminating the need for a
separator between the gas chromatograph and Ammonia (NH,,) and alkylamines (R
the mass spectrometer. The separator can be NHg) selectively protonate amines, amide
and a, /3-unsaturated ketones and form adc
am^or source oi sample loss, owing to adsorp-
tion products (M-t-NHR)'’' from ketone's, i
tion, decomposition, or diffusion. CI-MS
dehydes, acids and esters. When ammon
offers the advantage in determining isotope
was used as the reagent gas, the CI-MS 1

ratios on compounds obtained from biological


triglycerides produced peaks which corn
sources by improving the uniqueness of a par-
sponded to quasimolecular ions. When is<
ticular ion and thus minimizing interferences
butane was the reagent gas, quasimolecuh
from ions due to natural biological contami- ions were usually not recorded. With an
nants. (24) Several disadvantages of CI-MS
monia as the reagent gas, the (M+NH^)^ io
are possible; these include sensitivity to tem-
was the base peak. (53) An alternative t
perature changes, which affect the extent of CI-MS with ammonia is isobutane modifie
fragmentation, and as the ion source gets con- with ethanolamine and ethyienediamlne, i
taminated, gradual deterioration of linearity which Jess fragmentation is obtained tha
and precision can be expected, (70) with isobutane used alone.
Another method of selective detection by clonazepam, and in the plasma of thirteen
CI-MS is the reactant ion monitoring tech- subjects on a clonazepam oral dosing regi-
nique. (27) As each component in a sample men. The assay developed involves a
elutes from the GC into the source of the simple extraction of plasma or blood, fol-
mass spectrometer, there is a decrease in the lowed by GC-MS analysis of the residue
ion current of the reactant ions, because of after evaporation of the extraction sol-
the reactions of these ions with the added vent. Specificity was obtained by moni-
sample. Following this GC trace is called toring the MH+ ions of clonazepam and its
reactant ion monitoring and it is equivalent 7-amino metabolite, generated by ammo-
to a plot of total sample ion current vs. time. nia Cl. Assay accuracy was insured by
Selective detection of various classes of the use of stable isotope analogs of clona-
compounds is obtained by CI-MS, because zepam and the metabolite as internal
each class should react at a different rate standards. The ions monitored were m/e
with a given reactant ion. An example is 286 (MH+ of 7-amino metabolite), m/e 287
that f-C^H/ reacts with alcohols at a much (MH'*' of *®N-7-amino metabolite), m/e 3 16
greater rate than with hydrocarbons. In ad- (MH+ of clonazepam) and m/e 317 (MH’’*
dition, it reacts much more slowly with pri- of '*N-clonazepam). The limit of detec-
mary alcohols than with secondary or tion of the method ,
I ng/ml for clonazepam
tertiary alcohols; therefore, structural infor- and 2 ng/ml for the 7-araino metabolite,
mation may be obtained from relative rate was sufficient for measuring clonazepam
constants for reactions, This method can and its metabolite in most subjects on the
help eliminate large peaks on a GG-FID trace chronic dosing regimen, and in many sub-
which cover up minor peaks. Another ex- jects following administration of a single
ample of applicability is the qualitative anal- dose of clonazepam.
ysis of complex mixtures Also, protonated
.

2. Charge Exchange Gases


acetone can distinguish among primary, sec-
ondary, and tertiary alcohols. Proton trans- Charge exchange (CE) reactions arise

fer from CaHyO*'' to ketones should be rapid when the ionized reagent gas cannot donate a
and proton transfer from C 3H 7 O+ to aromatic proton. The ionization potential of the mol-
hydrocarbons should be slow. Therefore, ecule must be less than the recombination
ketones and aromatic hydrocarbons can be energy of the ionized gas. The degree of
distinguished by reactant ion monitoring fragmentation will depend on the energy dif-
with C H 7O+.
3
ference between the two parameters. Com-
bined charge exchange-chemical ionization
a. Clonazepam and its 7-amino metabo- occurs when a second gas containing hydro-
lite. A sensitive GC, ammonia chemical gen is used with the CE gas. The second
ionization mass spectrometry, **N isotope gas once ionized, can then ionize the sample
,

dilution assay was developed to measure molecule. The spectra of molecules ionized
the antiepileptic drug clonazepam and its in this way will present a situation between
7 -amino metabolite in blood or plasma. that obtained for true El and Cl spectra. (22)
(51) The method was used to measure Generally, CE gases (He, Ar, Nj) yield
both compounds in the blood of one sub- spectra which do not differ significantly from
ject administered a single 2 -mg dose of those obtained by El. Mass spectra ob-

29 .
tained with nitric oxide, however, show rela- lecular ions, Therefore, this method of
tively abundant (M+NO)'*^ ions with direct analysis is useful in cases of drug over-
molecules containing ^/-electrons. There- dose in which rapid identification is critical.
fore, alkenes can be distinguished from al- The disadvantages, however, are the inabil-
kanes and cycloalkanes since only the ity to differentiate between some pairs of
former give (M+NO)^ ions. drugs, relatively large quantities of material
Nitric oxide Cl can also differentiate be- are needed, and, of course, the piesence of
tween primary, secondary, and tertiary alco- non-drug components could be falsely
hols. Primary alcohols give spectra in interpreted.
which (M— 2), (M-3) and (M— 2+NO) ions
a. Illicit heroin. The Cl-isobutane spec-
are present. Secondary alcohols give major
trum of illicit heroin and other samples
ions (M- 1 ), (M-17) and (M— 2H-NO),
clearly indicated the presence of a number
whereas M-17 ions only are produced from
of common drugs by this approach. ( 12)
tertiary alcohols. The mass spectra of
Heioin, acetylcodeine and 0®-monoace-
twelve morphine and tropane alkaloids ob-
tylmorphine were easily identified, al-
tained by using Nj/NO as the reagent gas all
though O'^-monoacetylmorphine was not
showed the M"*" as the base peak. (37) In
distinguished from the latter compound.
general, acetoxy, hydroxy, carbonyl and ar-
Diluents which were easily distinguished
omatic groups will not be involved in CE
by this method included caffeine, metha-
since their ionization potentials are greater
pyrilene, quinine and procaine, owing to
than the recombination energy of the NO+
relatively strongand characteristic molec-
ion. Therefore, fragmentations characteris-
ular and fragment ions. The isomeric hex-
tic of these groups will not occur to the same
ahydric alcohols of mannitol and sorbitol
extent as in the El spectra,
weie not differentiated by this method,
El was shown to be four times more sensi-
nor were the monosaccharides, glucose,
tive thanCt and twenty times more sensitive
fructose, galactose and mannose. The
than CE by measuring the relative intensity
di saccharides, suciose and lactose, ex-
of the MH+ molecular ion of heroin by SIM.
hibited spectra similar to those of the mon-
The use of mixtures of Cl and CE gases also
osaccharides; this was attributed to their
appears to offer some advantages. Mixtures
decomposition in the Cl source prior to
of Ar/H^and Ar/CH^have been shown to dis-
ionization. In addition, identical Cl spec-
tinguish between the two isomeric com-
tra were produced by a compound in both
pounds amobaibital and pentobarbital mass
salt and fiee-base form. No attempt was
spectrometrically. The major CE gases are
made to quantitate the constituents based
Ar (combined with NO, H^O or CH 4), He
on mass fragmentation other than provid-
(combined with H 2O or NO), Nj (combined
ing a potentially useful fingerprint.
with NO), NO and Oj. (22)
h. Quantitation of cjuinicline. Direct
3, Direct CI-MS Analysis
analysis has been shown improved to GC
Multi-drug mixtures have been applied method for quantitation of quinidine (m/e
directly to the probe of the Cl mass spec- 325) and the impurity dihydroqiiinidine
trometer, in order to identify compounds (m/e 327). The ion doublet observed for
rapidly by abundant and unique quasimo- each compound allows ready identifica-

30.
tion and quantitation with incorporation of ratiomea.surements of pure BCNU and
the internal standard, a deuterated dihy- BCNU-''*H8 mixture was 10“" mole.
droquinidine, m/e 329. (32)
4. —
Gas Chromatography Cl Mass
c. Quantitation of 1 ,3-bis{2-chloio~ Spectrometry (GC-CI-MS)
ethyiyi -nitrosourea (BCNU). BCNU is
The Cl mass spectrometer is more easily
an effective chemotherapeutic agent used
combined than its El counterpait with the
in the treatment of brain tumors. A direct
gas chromatograph. The ability of the Cl
insertion CI-MS method for the analysis of
source to handle high gas loads decreases the
BCNU in biological samples, with the use pressure differential between the two instru-
of a stable octadeuterium-labeled BCNU
ments. When reagent gases (HjO, NO, Oj)
internal standard was used for determining
that may adversely affect the GC stationary
drug concentrations in the plasma of ex-
phase are used, these should be introduced
perimental animals and in humans under-
as far as possible from the GC interface.
going chemotherapy. (69) The direct
insertion method for determination of
a. Phencyclidine (PCP). PCP has been
BCNU pharmacokinetics was required quantitated in body fluids and the struc-
tural elucidations of two of its metabolites
because of the drug’s labile chemical na-
were achieved by GC-Cl-MS. Blood and
ture. The internal standard was added to
urine samples from individuals intoxicated
blood, plasma, water, or enzyme prepara-
with PCP on extinction gave only un-
tions, after which hexane/ether extraction
and mass spectrometric analysis of the ex-
changed drug. The metabolites were
present as conjugates and could be ex-
tract are performed at low temperatures,
tracted after enzymatic hydrolysis with /J-
with isobutane as the carrier gas. SIM of
glucuronidase.
the protonated molecular ions of the drug
Quantitation of PCP was achieved by
and internal standard yielded ion intensity
SIM using l-(]-phenyl-(“H 5)-cyclohexyl)-
ratios, from which the concentration of the
piperidine as the internal standard.
drug was calculated . The protonated mo-
Levels of I ng/ml in body fluid could be
lecular ions of BCNU occurred at m/e 214,
measured. It was established that 1-phen-
216, and 218, with a ratio close to
ylcyclohexane, previously reported as a
9.3/6.2/1.0, (characteristic of a molecule
metabolite, actually resulted from the
containing two chlorine atoms in the ratio
thermal degradation in the injection port of
of their natural abundance). A similar
the GC even at tempeiatures as low as
pattern was observed from BCNU-*Hg at
ISO^C. Molecular weight (Cl methane
m/e 222, 224, and 226. The ions 214, 216,
MS), data from El spectra, and compari-
222 and 224 were monitored in the course
son with reference standard allowed con-
of each sample analysis. The ratios re-
firmation of the structures of the PCP
mained constant during the probe evapo-
metabolites. (38)
ration. Both the 214/222 and the 216/224
ratio gave satisfactory quantitative re- b. Methadone. A CI-MS-SIM method
sults. The mass
sensitivity limit for the for monitoring methadone maintenance
spectrometer used in these analyses was individuals was an improvement over pre-
about 10~i^ mole; the lower limit for peak vious GC analyses that did not provide re-

31 .
liable quantitation less than 10 ng/ml with 0*-monoacety]morphine. Prior to th<
high precision. The internal standard was GC-MS run of the plasma samples, thi
*Hj-methadone, and body fluids analyzed standards, trideuteromoiphine and trideu
included plasma and uiine. Isotope ratios tero-0®-monoacetylmorphine were addec
were obtained using isobutane Cl and by to the sample, and therefore subjected tc
monitoring the protonated molecular ions identical chemical treatment as the sub-
at m/e3]0and3l5 for methadone and strate being analyzed. In the other mor-
methadone, lespectively. Thisprocedure phine determination, in which the TMS
was an improvement over previous El derivatives were analyzed, four masses
ion monitoring methods, in which the were monitored: m/e 340, 343 414 and ,

(M-15)+ m/e 294 ion was used for quanti' 417. Although it was only necessary to
tation and its relative abundance was monitor one pair of masses, monitoring an
low. This procedure used one of the most additional pair provided convenient cor-
intense ions in Cl mass spectrum of metha- roborative information.
done (m/e 310, (MH)+). The sensitivity
was also about a factor of 15-20 better ci. Secondary and tertiary tricyclic anti-
than GC procedures which also required a depressants, The tricyclic antidepres-
larger volume of plasma. (24) sants are used extensively in the treatment
c. Heroin, Several CI-MS-MIM meth- of depression. The common tricyclic ter-
ods are available for monitoring heroin in tiary amines include amitriptyline, imipia-
patients on methadone maintenance. mine and doxepin, and the common
This is accomplished by determining mor- secondary amines include nortriptyline,
phine (the heroin metabolite) in blood and desipramine and desmethyldoxepin. In
urine. Two methods leported utilized a order to correlate laboratory results with
stable isotope internal standard — N-tri- the clinical effect, both the primary

deuteromethyl-morphine which was de- drug and demethylated metabolite were
rivatized in one case with trifluoroacetic measured.
anhydride (TFA) (16) and in the other, tri- The assay is performed in two parts: (a)
methylsilylated with N,0-bis(trimethyI- extraction and direct iqjection of the ex-
silyl)acetaniide. (13) The previous case tract, for analysis of the tertiary amines,
was a study of heroin hydrolysis in blood and (b) derivatization and re-iiyection. for
plasma and therefore also included deter- analysis of the secondary amines. To de-
mination of 0®-nionoacetylmoiphine with termine the tertiary tricyclic antidepres-
its respective deuteio-TFA derivative as sant amines by GC-MS, amitriptyline was
internal standard. (16) With the TFA de- measured at m/e 278, imipramine at m/e
rivative, m/e 364 (protio compound) and 281, and doxepin at m/e 280; clomipra-
m/e 367 (deutero compound) were chosen mine, the internal standard, was measured
to be monitored by MIM because they at m/e 317, the (M-h2)+ isotope peak. To
were high molecular weight fragments to determine the derivatized (TFA) second-
insure specificity, and showed a high rela- ary tricyclic antidepressants by GC-MS,
tive total intensity to insure sensitivity. desmethyltrimipramine-TFA (internal
The same fragments, m/e 364 and m/e 367, standard) was monitored at m/e 377, desi-
were monitored for determination of pramine-TFA at m/e 363, desmethyldoxe-
pin-TFA at m/e 363 and nortriptyline-TFA nations have been determined by FI on
and protriptyline-TFA at m/e 360. (70) a high-resolution mass spectrometer on
compounds which do not produce electron
D. Other Ionization Techniques
impact moleculai ions. (11)
1. Field Ionization (FI) It has also been shown that alcoholic

FI techniques minimize fragmentation and dehydration is not necessarily thermal in


also provide molecular ions of high inten- nature; it occurs with the FI source,
sity. Therefore, their mass spectra are producing the (M — 18)+ intense fragment.
potentially useful in the analysis of mixtures (17) Dual field ionization-electron impact,
that are often encountered in biological
and dual field ionization-field desorption
samples. (17) Field ionization of gaseous sources have been constructed. (10) (68) (2)
sample molecules occurs when the molecule The advantages of FI and Cl over El are
is brought near a metal surface in the vicinity
obvious in that there will be an indication of
of a high electric field (a field anode held at a the resulting molecular ions. However, at

highly positive potential), higher source temperatures (owing to re-


Quantum mechanical tunneling ot an duced volatilities), protonated molecular
electron from a molecule into the metal ions are not observed.

surface provides a positive ion. Since the


2. Field Desorption (FD)
ion has very little internal energy in the form
of electronic or vibrational excitation, it is The utility of EI-, FI- and CI-MS for
usually detected as a highly stable molecular identification or structural elucidation of
ion. However, the
efficiency and the salts and highly polar, thermally labile,

FI is much less than that of El


sensitivity of organic molecules is restricted because
or Cl methods (Vio to Vioo), and ionization samples must be in the gaseous s.tate prior to
components are fragile. (10) (46) ionization and analysis. For these types of
In many cases, El does not produce an compounds, the energy required to disrupt
intense (or any) molecular ion, even in the intermolecular bonds is often greater than
case of derivatization. Many compounds the required energy to break bonds within
(e.g.,amino acids) do not possess an the molecule. Therefore, heating of the
adequate vapor pressure and therefore sample to volatilize decompose it. (36)
it will
produce only the quasimolecular ions, FD-MS does however, have the
not,
(M+H) or (M-H), in their mass spectra. disadvantage that the substance be volatile,
This is sufficient for indicating their and many non-volatile solids have been
molecular weights, but due to the absence of analyzed by this technique. In addition, its
any fragment peaks in the spectra, structural resulting mass spectra can be expected to
information must be obtained by another lead to minimal fragmentation. (17) In
method. FI, on the other hand, may show FD-MS, the sample is ionized from the
an intermediate degree of fragmentation, as adsorbed state from the field anode on which
well as an intense molecular ion. (17) it is deposited. These anodes consist of 10
Field ionization, in general, may be used fim tungsten wires covered with a large
with the same types of samples and inlet number of carbon microneedles approxi-
systems as electron impact ionization. In mately 30 /zm in length. Application of an
addition, elemental composition determi- external field lowers the potential for

33 .
migration of an electron from the sample in solvents, including deionized water,
molecule towire electrode.
the By methanol, ethanol, or dimethylsulfoxide
increasing temperature of the wire
the (DMSO).
anode, ionization of the sample occurs under Many of the thermally labile and
mild conditions and repulsion between the nonvolatile compounds which have been
resulting positive ion and the emitter drives analyzed by F'D-MS include the alkali metal
the ion into the gas phase. It is estimated saltsof acetic acid, nucleotides, amino acids,
that the total thermal energy involved in this sodium salts of glucose phosphates and

process may be two or three times smaller glucuronides and quaternary ammonium
than that required for direct thermal salts, potassium salts of some alkyl sulfates
vaporization. (36) and cyclohexylphenyl sulfate, sodium salts

As the excess thermal energy is veiy of stilbestrol glucuronide, cortisone sulfate,


small, highlypolar compounds of low deoxycholic acid, taurodeoxycholic acid,
volatility can be analyzed without the dehydrocholic acid, glycocholic acid, tau-
decomposition expected on thermal volatil- rocholic acid, taurochenodeoxycholic acid
ization. Although FD has been shown and cyclic adenosine monophosphate, meth-
to give spectra of many physiological com- otrexate and folic acid analogs, gentamicins,
pounds (without prior derivatization), the ionic dyes, and alkali elements. The spectra
nature of the method prohibits GC were obtained from solutions of the salts in
introduction. (46) This may not, however, water, with the addition of some methanol.
necessarily limit its general use to pure Ions were produced which not only
compounds. identified the molecular ions, but, in some
Other problems with the method include cases, structurally useful fragment ions were
an uncertainty in the precise moment of also obtained. (21)
volatilization, irregular volatilization proc- FD-MS has been shown to be uniquely
esses, instrument dependent heating rates, useful for identifying sub-millipam quan-
sample decomposition prior to ionization tities of ionic dyes. Identification of ionic
and excessive ion source contamination. dyes is generally difficult because molecular
(64) Since FD allows formation of weights of the dye salts cannot be obtained
molecular ions of molecules with low by ionization methods which require
due to better control of emitter
volatility, vaporization of the dyes. Molecular weight
current, many times will produce mass
it information and characteristic fragment
spectra which contain intense protonated ions, for some dyes, were observed. (50)
molecular ions at these lower temperatures. In most cases, the potassium salts all
FD may give comparable results to FI, showed quasimolecular (M+K)+, and the so-
although differences in the intensity of the dium salts showed (M-I-Na)+ quasimolecular
molecular ion by this latter technique may be ions. Other prominent ions could include
dependent upon the solubility of the sample the doubly-charged ion (M+2Na)‘*+ and in
in the solvent on which it is adsorbed on the the sodium salts of alkyl hydroxy-carboxylic
anode, and the resulting temperature at acids (M+Na-18)+ was produced. In the
which field desorption will occur. Sample case of compounds containing a glycosidic
loading can be by the emitter dipping linkage, Cl spectra exhibit some fragmenta-
technique, or by use of an ultra-microsyringe tion, although FD conditions produce the

34 .
(M+H)+ ions with little glycosidic cleav- tected are already present as positive ions on
age. In addition, FD
has been shown to be the emitter surface. Field desorption of
useful in studies on underivatized sugars, as these monovalent ions is much more effi-
well as dissaccharides. (55) Related com- com-
cient than field desorption of organic
pounds present in mixtures as trace impuri- pounds. In the case of organic compounds,
ties may also be identified by their (M+H)+ the processes of thermal decomposition and
ions. In typical FD mass spectra, the molec- evaporation of neutral molecules that do not
ular ion or quasimolecuiar ion (M+H)'*' was undergo ionization compete effectively with
usually the base peak. The most frequent the ionization. However, this does not
fragment ions of alcohols were due to occur for field desorption of alkali cations.
(M-OH)'*' or (M-HaO)^. Many times, Levels of cesium were estimated in a vari-
acids produced peaks at (M-l-23)+ due to ca- ety of media; in spectrograde solvents, in
tionization by sodium present as an impurity
body fluids such as saliva and blood, and
in either the sample or the solvent. Diso-
in environmental samples, e.g., drinking
dium salts of dicarboxylic acids generally water, seawater and a natural aerosol. The
produced the more prominent ion due to determination of cesium in sample sizes of
(M-HNa)+, although other ions produced 0.2 to 1 ^1 containing 0.3 to 100 pg of the ele-
were attributed to (M+2Na)*'*', (M- ment was achieved with precision and accu-
(ONa)^)'- and (2M-Na+H)*+. (40) racy of ± 10% and ±20%, respectively. A
FD, as well as FI, techniques have also
linear emitter heating currem piogidinmer
been used for SIM in drug analysis. (20)
was used for the desorption of the samples,
It had been proposed that ions from each
and the evaporation profiles for (Cs)-* were
major component in a sample mixture could
obtained . From the peak areas of the evapo-
be used to afford a more rapid and precise
ration profiles obtained in a standard, the un-
method of quantification than those available
known amount of the alkali element present
for thermally labile compounds. Ions from
each mqjor component at (M+H)*- could be
in the sample was calculated. The results
showed the by FD-
potential of estimation
monitored and related compounds as trace
impurities could be identified by their highly
MS of alkali elements in very small amounts
of untreated biological and environmental
characteristic (M^-H)' ions. (55) Until re-
samples. (48)
cently, however, no analytical application of
FD-MS had been reported Experiments by.
The quantitative FD data was in good
H. R. Schulten, el al., (48) explored the sen- agreement with the El measurements and
sitivity and precision of FD-MS in the SIM demonstrated the utility of FD-MS as a quan-
mode for quantitative studies of alkali ions to titative technique in biomedical research.

show the potential of this technique for trace In selecting internal standards for quantita-

analysis. The sensitivity of the FD method tive FD-MS, homologous compounds and
for alkali ions exceeded the sensitivity for or- extensively deuieiaied compounds are poor
ganic compounds. The sensitivity was of choices since their desorption behavior dif-
the same order of magnitude as the sensitiv- fers strongly. However, substances labeled
ity of EI-MS for the detection of organic with only a few deuterium atoms or
One of the reasons for this ‘*0 are good internal standards since no frac-
compounds.
phenomenon is that the particles that are de- tionated desorption has been observed. (47)

35 .
3. Atmospheric Pressure Ionization (API) compounds (with halogen-containing deriva-

The sample is introduced in a solvent and


tizing agents —pentafluoropropionic anhy-
dride, heptafiuorobutyric anhydride,
swept along by nitrogen carrier gas. The
trifluoroacetic anhydride, etc.), substituted
sample undergoes a complex series of ion
phenols, barbituric acid derivatives, di-
molecule reactions in a chamber containing a
phenylhydantoin, biphenyls, polycyclic
®*Ni foil or a corona discharge, as a source of
aromatics and heteroaromatics. (31) Addi-
electrons .Positive or negative sample ions
are produced. These pass through a small
oxygen as a CE gas appears to be
tionally,

aperture (25 /:im diameter) to be analyzed in


Cl studies on
particularly useful for negative

the mass analyzer. The reported limits of


polyaromatic and com-
polyhalogenated
pounds. A negative ion mass spectrum can
detection are in the femlogram range
provide direct knowledge of the chemical
(10"'®g). Although no structural informa-
tion is available, the simplicity of such spec-
events occurring under BCD conditions.
It also provides far less structural informa-
tra permits detection of known compounds
with a separation stage. (45) tion than a positive ion mass spectioim.
However, in quantitative procedures for se-
The system is very versatile; the source is
lected molecules, electron capture currents
able to accept a variety of gases and sol-
can exceed their corresponding positive ion
vents The sample may be introduced in the
.

currents by two or three orders of magni-


gas phase without solvents, by probe injec-
tion, or in the effluent stream from a gas
tude. (58) The basic route of formation of a

chromatograph. Samples have been intro-


negative ion may be
either by direct attach-

duced in the liquid phase in solvents by injec- ment of an electron (electron capture) or
by reaction with a negative reactant ion.
tion, or in the effluent stream by HPLC, or in
solution by syringe Further applications will be discussed in
injection. (30)
greater detail by Dr. Brandenberger in this
4. Negative Ion Formation Symposium. (8)

In conventional ion sources, negative ions


which are produced are normally trapped.
A reversal of the potentials of the ion repeller ///. CONCLUSION
and accelerator voltage plates can result in
the acceleration and focusing of these nega- Within the past few years, many reviews
tive ions. have been written describing various aspects of
Many compounds which show a response mass spectral applications. These summaries
(BCD) can also
to electron capture detection have included applications in clinical chemis-
be determined mass spectrometrically by the try, analytical systems based on mass spec-
resultant negative ions formed which lead to trometry, mass fragmentography in biological
that response. The use of ECD-GC In the and drug research, and chemical ionization
analysis of compounds present in low con- methods in metabolite studies. This is the re-
centrations in highly complex samples is ef- sult of the increasing interest in modern scien-
fective as a selective detector. Typical tific methods in many
of today’s
solving
compounds which respond to BCD are problems. As such,review has attempted
this
halogen-containing drugs, derivatized to cover applications in forensic drug analysis.

36 .
as well as related areas in which the techniques 6. Boerner, U., Roe, R. L., and Becker,
applied may make a significant future contribu- C. E., J. Pharm. Phannac., 26, 393-98
tion to the science. (1973).
For any review to be of value to the reader, it
7. Boyd, P. M., Moses, P. J., and Bowman,
should do more than merely compile, abstract
Spectroscopy Letter.^, 7, (4 & 5),223-
1^.,
and organize past accomplishments of re-
27(1974).
searchers. should attempt to
In addition, it

project future developments in the “state-of- 8. Brandenberger, H., presented at the In-
the-art." The more common applications of ternational Symposium on Instrumental
El and Cl mass spectrometry have been cov- Applications in Forensic Drug Chemis-
ered. In addition, one can anticipate increased try, Arlington, VA May 29-30, 1978.
usage of FI, FD, and API methods, and instru-
9. Carroll, D.I., Dzidic, I., Stillwell, R. N.,
mental development of multiple ionization
Haegele, K. D., and Horning, E. C.,
sources. With respect to sample handling Anal. Chem., 47 (14), 2369-73 (1975).
prior to ionization in the mass spectrometer,
more selective derivatizing techniques, the de- 10. Chait, E. M., A/iai. Chem., 44 (3), 77A-
velopment of more applications of stable iso- 91A(1972).
tope internal standards in the quantification of 11. Chait, E. M., Shannon, T. W., Amy,
drugs, and increased usage of the liquid chro- J. W., and McLafferty, F. W., Anal.
matograph-mass spectrometer interface can be C/jcm.. 40 (4), 835-37 (1968).
expected. Other methods of improvement in
12. Chao, J.-M., Saferstein, R., and Manura,
selective detection of sample molecules can be
J.,Anal. Chem., 46 (2), 296-98 (1974).
anticipated by increased application of SIM
with high resolution mass spectrometers, fur- 13. Clarke, P. A, and Foltz, R. L., Clin.
ther development of reagent gas mixtures in Chem., 20 (4), 465-69 (1974).
Cl, and more common use of negative ion MS.
14. Compson, K, R., Evans, S.,Hazelby,D.,
and Moore, L. E., presented at the
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70. Wilson, J. M., Williamson, L. J., and


Raisys, V. A,, Clin. Chem., 23 (6), 1012-
17(1977).

71. Zagalak, M. J., Curtins, H.-Ch., Leim-


bacher, W„ and Redweik, U,, J, Chro-
maiogr., 142,523-31 (1977),

72. Zarembo, J. E., Killmer, L. B., Berkoff,


C, E., Rivard, D., and Kirkpatrick, D.,
presented at the Twenty- Fifth Annual
Conference on Mass Spectrometry and
Allied Topics, Washington, DC, May 29-
June3, 1977.

ACKNOWLEDGMENT
The author wishes to expres.t his appreciation to Mrs. Jean
Nolan for her assistance in the preparation of this
manuscript.

40 .
The Use of Stable Isotopes
in the Quantification of G. Horning, Jean Nowtin, K.
Lertratanangkoon and J'P.
Thenot
Institute for Lipid Research
Baylor College of Medicine
Houston, Texas 77030

I. INTRODUCTION ble and preferably before isolation is initiated.


Recently, interest in monitoring blood levels It isimportant that the initial measurements of
of drugs, particularly those drugs which are ad- the biological sample and the internal standard
ministered chronically, has increased signifi- be done very caretiilly because any error in ei-
ther measurement will decrease the precision
cantly. Many methods for quantifying drugs
in plasma, urine, and saliva are available based and accuracy of the final determination. Pipet-
either on gas chromatography (GC), high per-
ting mistakes can be a major source of error.
If the labeled internal standard is added to
formance liquid chromatography (HPLC),
competitive protein binding (RIA and EMIT), the plasma, urine, or saliva prior to isolation, it
is necessary to carry out only one extraction
spectrophotometric methods or gas chromato-
graphic-mass spectroraetric computer (GC-
with an organic solvent, thus saving time. Al-
MS-COM) though subsequent extractions will increase
procedures. From an analytical
point of view, two types of procedures are the total amount of drug extracted, the ratio of
needed; (a) simple, rapid, and reasonably pre- the drug to internal standard will remain un-
cise methods for clinical laboratories and (b)
changed and in the GC-MS-COM analyses, it is
rcfei'ence procedures with high precision and
the ratio of drug to internal standard that is
accuracy that can be used to validate the clini-
measured. If the internal standards are homo-
logs or analogs, it may be necessary to carry
cal procedures. The reference methods fre-
quently involve
out several extractions, depending on differ-
several complicated and
ences in the partition coefficients of the drug
time-consuming steps and consequently are
not suitable for routine use in clinical chemistry
and internal standard.
laboratories.
Adding the internal standard to the biological
The most sample before isolation provides an additional
satisfactory reference procedures
advantage. Subsequent loss of part of the
at the present time are those based on the use of
sample, for whatever reason, will not affect the
GC-MS-COM systems. The analyses are car-
precision or the accuracy of the analysis; the
ried out by selected ion monitoring using mag-
ratio of drug to internal standard will not be al-
netic or electrical field instruments operated in
tered by the loss.
either the electron impact (El) or chemical ioni-
zation (Cl) mode. These reference methods
The stability of solutions of labeled internal
standards (and also unlabeled internal stan-
are usually based on the use of stable isotope-
dards) must be checked frequently. This is ac-
labeled internal standards which aie essential
complished most readily by carrying out
for high precision and accuracy.
quantitative mass spectrometric comparisons
of the old solution and a freshly prepared solu-
II. SAMPLE PREPARATION tion of labeled internal standard with a refer-
ence solution of the unlabeled drug.
For GC-MS-COM analysis sample prepara-
tion involves the transfer of thedrug(s) from an III. DERIVATIZATION
aqueous an organic matrix. The stable
into
isotope-labeled internal standard should be In our studies, the extracted drugs and inter-
added to the biological sample as early as possi- nal standards were converted into derivatives

41 .
for GC-MS-COM analysis. Methylation with be a characteristic fragment ion; under thes
diazomethane or ethylation with diazoethane conditions the fragment ion or the fragment io
followed by silylation with bis-trimethylsilyl- and MH+ can be monitored. If Cl condition
acetamide and pyridine was the preferred pro- are used with nitrogen, argon, or helium as thi
cedure (1). However, many other types of carrier gas, the resulting spectra resemble E
derivatives can be used (2), The chief consid- spectra taken under 15-20 eV conditions
erations governing the choice of derivatives for This form of ionization may be useful in some
quantitative work are adsorption behavior and drug analyses.
stability. When adsorption is minimal, sym-
metrical GC peaks are obtained resulting in im-
proved and losses in the
quantification, V. INTERNAL STANDARDS
analytical system are reduced resulting in in-
creased sensitivity of detection. Most deriva- In our studies, internal standards labeled
tives used in gas phase analytical methods have with '*C, *®N and (deuterium) have been
sufficient stability for quantitative work. Pre- used, Many labeled drugs are available from
cautions may be needed for relatively labile commercial sources.' However, if the drugs
compounds; for example, N-silyl derivatives under study contain methyl, ethyl, or isopropyl
are silylating agents and trimethylsilyl esters of groups, it may be easier and cheaper to synthe-
carboxylic acids are easily hydrolyzed. size the deuterated analog of the drug in the lab-
oratory by the use of the appropriate
IV. GC-MS-COM ANALYSIS deuterated alkyl iodide. It is advisable to have
at least three heavy atoms in the labeled inter-

When quantification is carried out with a GC- nal standard to avoid any overlap with ions at

MS-COM mass spectrometer


system, the is
M -I- 1 and M-h2 due to the natural abundance of
used as a specific ion detector. This technique heavy isotopes of carbon, hydrogen, sulfur,

has been called “mass fragmentography” (3) chlorine, bromine, or silicon which may be
but the term "selected ion detection" (SID) Is present in the drug or its derivative. For ex-

used more frequently today. The detection ample, in Figure 1 ions at M+ 1 in the spectrum
process involves monitoring the appearance of of unlabeled Dilantin are due to the natural
preselected ions which are characteristic of the abundance of Regardless of the source of
drugs under study and the labeled internal the labeled internal standard, it is necessary to
standards. The mass spectrometer can be check its isotopic composition and purity.

operated in either the electron impact (El) or The isotopic composition can be checked fay
the chemical ionization (Cl) mode. A com- mass spectrometric analysis. An example is
parison of the El spectrum of diphenylhydan- shown in 'Figure 2; the chemical ionization
toin (N-methyl derivative) (Figure 1) with the spectrum of diphenylhydantoin-2,4,5-'®C
Cl (methane spectmm) of the same compound (MH^=270) is compared to the chemical ioni-
(Figure 2) shows why Cl techniques are useful zation spectrum of the unlabeled diphenylhy-
in quantitative Only a few ions are ob-
work. dantoin (MH'''=267) obtained as an analytical
tained under Cl conditions and the major ionic
product usually corresponds to MH+, the pro- ' Stable isotope labeled drugs can be obtained from
Merck, Sharp and Dohme, Canada, Ltd.; Pointe Clalre-
tonated molecular Ion. For some drugs and Dorvat, Quebec, Canada; and Kor Isotopes, Cambridge,
drug derivatives, however, the major ion may Massachusetts,

42 .

I

DIPHENYLHYDANTOIN M=266

180 (M-86)
100 12.9
>
f— +
HH NH
g 7&
LU
104
z
I


I
(M)
266
5CH (M-29) he
UJ 77 237
(M-57)
o
209 Wi
165
5
LtJ
^5^

CO

iL,J.jl|Jllllli,llliLu|l
,
|
. .III
.illllllLiull..,
..1.11 L4
50 lOp 150 200 250
m/e
Figure 1 Electron impact Ionization mass spectrum of the N-methyl derivative of diphen-
.

ylhydantoln; molecular Ion (M) is present at m/e 266.

standard from Applied Science Laboratories. can be used to check the purity. The quantitn
From the chemical ionization spectrum it is ob- live response of the labeled internal standard
vious that the labeled internal standard con- and the analytical standard should be the same
tains an appreciable amount (20%) of (miciogram/microgram) after correction for
diphenylhydantoin containing oniy two '*C the isotopic composition of the labeled internal
atoms in the molecule (MH+=269). Thus for standard has been made. In our experience,
every milligram of the labeled standard added the commercial stable isotope-labeled stand-
only 0.8 milligram is present as the species ards have been very satisfactory.
quantified by mass spectrometry at If the internal standard has been labeled with

MH^ =270. This correction factor must be in- deuterium, an additional source of error in
cluded in the calculations of plasma quantification may be introduced because the
concentrations. properties of the deuterated internal standard
purity of the labeled standard must also
The may differ significantly from those of the unla-
be evaluated. The Cl spectrum of the labeled beled drug. For example, the solubility, parti-
diphenylhydantoin is identical with the corre- tion coefficients and hydrogen bonding of the
sponding spectrum of analytical standard of di- two species may not be identical. These ef-
phenylhydantoin (Figure 2). However, in fects are illustrated in Figures 3 and 4.

some samples impurities eluting with a differ- in figure 3, caffeine and r/v’-trideuterometh-
ent retention time in a GC-MS analysis or non- ylxanthine (do-caffeine) apparently were eluted
volatile substances (salts) may contaminate la- as one symmetrical gas chromatographic
beled standards. Mass spectral analysis (SID) peak. In Figure 4, the same sample was ana-

43 .
?0

Figure 2. Chemical ionization (methane) mass spectra of the N-methyl derivatives of di-
phenyihydantoin (Diiantin) and the stable isotope-labeled standard, 2,4,5,-'^C-diphenyl-
hydantoin. The major ions at 267 and 270 correspond to the protonated molecuiar ions,
MH+

44 .
Figure 3. Qas chromatographic analyses
of a mixture of caffeine and 1,3,7 -tr/-trideu-
teromethylxanthine (dg>caffelne). The
analysis was carried out by temperature
Figure 4. Selected ion detection chart for
programming at 27min from ISO^’C using a the analysis of caffeine and dg-caffeine;
3.7 m glass column packed with 3% PZ<179
using a GC-MS-COM system operated in
(Poly-S 179, Applied Science Labors*
the Cl mode with methane as the carrier
tories).
gas. The ions monitored were m/e 204
lyzed by selected ion monitoring with a GC- (MH+, dg-caffeine) and mie 195 (IVIH+,

MS-COM system operated in the Cl mode. caffeine).


The protonated molecular ions at mfe 195 a GC-MS-COM system using stable isotope-
(MH+, d„-caffeine) and 204 (MH+, do-caffeine) labeled internal standards should be 2-6% for
were monitored. It is obvious that dg-caffeine the overall analysis. If the biological sample
is eluted from the column (scan 77) before do-
(plasma, saliva, urine) and the internal stand-
caffeine (scan 79). If the caffeine had been
ard are measured by weight rather than by
quantified on the basis of the amount of dj-caf-
volume, the precision and accuracy are im-
feine present in scan 79 rather than scan 77, a
proved significantly. The overall precision of
considerable error would have been intro-
GC-MS methods under these conditions can be
duced. Similar results have been obtained
as low as 0.33% and the precision of the mass
With several other deuterium-labeled drugs,
spectrometric analysis as low as 0, 17% (4),
Wehave compared the precision and accu-
VI. PRECISION AND ACCURACY racy of GC-MS-COM procedures for monitor-
ing anticonvulsant drugs with methods based
The precision and accuracy of quantitative on HPLC and EMIT, Plasma standards sup-
analyses of biological samples carried out with plied by the National Bureau of Standards con-

45 .
taining several anticonvulsant drugs were methods was 7.3 (Ug/ml; the value supplied by
used. The GC-MS-COM procedure involved NBS was 7.3 /itg/ml. In Table 11, the results of
four steps: extraction, concentration of the HPLC and GC-MS-COM analyses are com-
sample, derivatization, and instrumental anal- pared using another NBS plasma sample. The
ysis; the HPLC procedure involved three SD for the HPLC analyses of phenytoin was
steps: extraction, concentration of the sample, 0.76 /ug/ml and the SD for the GC-MS-COM
and instrumental analysis; EMIT involved only analyses was 0,51 /xg/ml. The mean of the
instnimental analysis. Ten analyses were HPLC analyses was 22 8 fig/ml and the mean of
.

made of each sample by each procedure. The theGC-MS'COM analyses was 22.3 ju&^ml.
mean and standard deviation were calculated The value provided by NBS for this sample
by the use of a computerprogram. The results was 23.8 /tg/ml. The precision and accuracy
are summarized in Tables T-ITI. of the thiee methods for these samples were
In Table I quantification of phenytoin by excellent.
EMIT and HPLC are compared. The standard In Table III the analyses of phenobarbital by
deviation (SD) for the EMIT analyses was 0.28 GC-MS-COM, EMIT, and HPLC are com-
/ig/ml and for HPLC the standard deviation pared, The SD was 1.5 /x.g/ml, 1.4 /.tg/ml, and
was 1,12 figlmX. The mean found by both 1.5 jxg/ial for the GC-MS-COM, HPLC, and
EMIT analyses, respectively. The mean of
Table I
the GC-MS-COM determinations was 33.7
jitg/ml; the mean obtained by HPLC was 37.2
PHENYTOPN-7.3 (NBS VALUE) jug/ml; and the mean obtained by EMIT was
37.9 /ug/ml. The NBS value for this sample
METHOD MEAN* SD AC was 36.3 |Ltg/ml. The precision of the three
/ag/ml methods was essentially the same but the re-
sults found by the GC-MS-COM method were
EMIT 7.3 0.26 0.0
less accurate than the HPLC and EMIT deter-
HPLC** 7.3 1.12 0.0 minations . Since the precision of the GC-MS-

*ren determinations
*^Based art peak area
Table 111

PHENOBARBITAL-3G.3 /ug/ml
Table 11
(NBS VALUE)
PHENYTOIN-23.8 /ng/ml
(NBS VALUE) METHOD MEAN* SD AC
/ag/ml
METHOD MEAN* SD AC GC-MS-COM 33.7 1.5 -2.6
Mg/ml
EMIT 37.9 1.4 +1.6
GC-MS-COM 22.3 0.S1 -1.5
HPLC** 37.2 1.5 +0.9
HPLC 22.8 0.76 -1.0
“Jen determlnaVotta
*Ten determinations “Based on peak area

46 .
COM analyses was excellent and since the should approach the precision. This assumes
precision and accuracy of the GC-MS-COM that appropriate precautions have been taken
determinations for the phenytoin set in Table II in sample preparation and that sources of error
was excellent, these results suggest that a slight have been identified and eliminated. The sen-
error in the addition of the internal standard sitivity and selectivity of detection coupled
and/or the sample occurred. with the precision and accuracy that GC-MS-
Table IV illustrates one of the difficulties en- COM methods provide when stable isotope-
countered with the EMIT procedure. Two dif- labeled internal standards are used, make these
ferent sets of plasma samples with low, the method of choice for validation of other an-
medium, and high concentrations of phenobar- alytical procedures.
bital were analyzed by GC-MS and EMIT. At

high plasma concentrations, the EMIT method


was not very reliable and dilution of the sample ACKNOWLEDGMENT
usually did not improve the accuracy. The This work was stipponed by NIH Grants GM-14092 and
GC-MS-COM method was accuiate at the high GM-13901.

concentrations; the GC-MS method was also


precise and accurate at concentrations much
lower than can be measured by EMIT.
It is not possible to define the accuracy to be
REFERENCES
expected from GC-MS-COM methods in all ap-
plications, but in most woik the accuracy
1. Harvey, D. J., Glazener, L., Johnson,
D. G., Butler, C. M., and Horning, M. G,,
Drug Me tab. Dispos., 5, 527 (1977).
Table IV
2. Horning, E. C., Thenot, J-P., and Horn-
PHENOBARBITAL ing, M. G., “Quantitative Mass Spectro-
metry in Life Sciences,’’ p. 1. Elsevier
Scientific Publishing Company, Amster-
NBS- dam, 1977.
GC-MS* EMIT* VALUE
SET 1 pglml pglml pglml 3. Hammar, C-G., Holmstedt, B., and Ry-
hage, R.f Anal. Biochem., 25,532 (1968).
1 (Low) 8.65 9.0 8.5
4. Schaffer, R., 'White, E., Sniegoski, L, T.,
(Medium) 22.0 22.2 22.7
II
and Cohen, A., “Ti’ace Organic Analysis:
III (High) 110.6 100.0 112.2 A New Frontier in Analytical Chemistry,”
presented at the 9th Materials Research
SET 2 Symposium, Gaithersburg, MD, April
10-13, 1978; Special Publication 591,
(Low) 8.6 9.0 8.3
1
National Bureau of Standards,
II (Medium) 35.4 37.8 36.3

III (High) 110.6 95.0 107.0

*Average ofduplloale determinations

47 .
f>y
Negative Ion Mass
Spectrometry a New — Hans Brandenberger
Department of Forensic Chemistry,
Tool for the Forensic University of Zurich, Switzerland
Toxicologist (Ziirichbergstr.S, CH>8032
Zurich)

/. INTRODUCTION to negative ion formation. In all cases, reagent


gases such as argon, nitrogen, oxygen and
A . Negative Ions by Electron Impact others are used to transmit the negative charge
to the substrate molecules. The chamber
Conventional mass spectrometry by elec-
pressure is usually kept high, at 0.1 to 1 torr
tron impact (EI-MS) is essentially a positive
(analogous to positive Cl) (2, 5) or even at at-
ion method. Table I lists a selection of nega-
mospheric pressure (3,4). Good yields are re-
tive ions which are produced. Most of them
ported, but in most cases, the investments
belong to the low mass range and have little an-
were high, both with respect to instrumenta-
alytical value. Among few exceptions are
the
tion and time. Special ion sources or even
some molecular range anions yielded by alco-
custom-made instruments were constructed
hols and acids. But even those have not much
for the negative ion work: a Duoplasmatron
significance since the anion yields are more
mass spectrometer by v. Ardenne (1), two ex-
than 4 magnitudes of 10 lower than the cation
ternal atmospheric pressure chambers fay
yields.
Horning, one with a radio-Ni-foil (3), the other
with a Corona source (4), a Townsend dis-
B. Negative Ions by Methods related to
charge source by Hunt, who has also built an
Chemical Ionization
instrument for simultaneous positive and nega-
Table 11 lists the approaches of some re- tive ion recording (5).
search groups which have successfully applied
techniques related to chemical ioniVatinn fCI'* II. OUR STUDIES WITH ANION
MASS SPECTROMETRY
Table J

A, Instrumentation
Negative Ions by electron Impact
(at 70 eV and near 10~® torr) When we decided to start with negative mass
spectrometry by Cl, we chose a very simple ap-
Allphatfcs: [H]", [C*]-, [C,.}S proach. In collaboration with Dr. R. Ryhage
[CxH]~ from the Karolinska Institute for Mass Spec-
tiometry, 2 units of commercially available
Aromatics: [C.H]-, [CoH]- magnetic sector instruments with combined
Oxygen compounds: [O^]', IQH]~, EI/CI source (LKB-2091) were equipped with
[CjHO]- new power supplies which permit reversion of
— alcohols little and [M-3]" magnet current, accelerating voltage and re-
— acids some [M-1 r pelJer voltage by shifting a single switch. The
modified instruments are capable of recording
Nitrogen compounds: [CNr, [C^N]" anion or cation spectra. The switch-over time
is less than a minute (6, 7, 8).
Halogen compounds:
w~ B. General Aspects of Anion .spectra
Negative Ions yield ^ 10~^ of positive
We have now dealt with anion MS by Cl for
ion yield
about one year. We have tried over 10 differ-

48 .
Table li

Negative Ion formation by methods related to chemicaf ionization

Research instrumentation Chamber First


group and principle pressure paper Ref.

V. Ardenne Argon Duoplasmatron, electron 10-2 1958 (1 )

attachment torr

Dougherty CI-MS with CH 4 and CHjClj, clus- 1 1968 (2)


ter formation torr

Horning External reaction chamber with atm. 1973 (3)


radlo-Ni-foll (API)

Horning External reaction chamber with atm. 1974 (4)

Corona source (API)

Hunt Townsend discharge source, si- 1 1976 (5)

multaneous positive and negative torr


Ion recording

ent reagent gases (6,7), optimized some of the weeks (12). Anion MS would have been use-
conditions and made a few systematical studies ful, especially in combination with GC. It

(9,10). Even though the field is still new and a shows the moleculai- mass 396 at first sight, it

great deal of development work remains to be indicates the presence of a nitro group by the
done, anion CI-MS has already become in — anions with mass 46 and (M- 16]”, and it points
our forensic laboratory in Zurich — one of the to the loss of a side chain with 100 mass units
most powerful analytical tools in the daily toxi- (side chain abstraction is typical for anion MS’
cological service work (8, 11). The following by Cl).
examples are chosen in order to outline point — Figure 2 shows that the molecular anion may

by point the advantages of the technique as
lose hydrogen, often one (as in the case of co-
we use it. two (as in the case of mor-
deine), sometimes
Figure 1 gives the cation El and the anion Cl phine), and in. some cases even more. It is
mass spectra of etonitazene, which is probably interesting to note that dihydrocodeinone,
the most potent analgetic ever synthesized. which does not possess a hydroxy group, gives
We realize that anion CI-MS is a soft ionization a stable molecular anion.
technique which quite often permits recogni-
tion of the molecular mass. In 1966, a batch of Figure 3 contains the El-spectrum and the
this substance was sold on the European black cation and anion Cl-spectra of a saturated bar-
market, and we had to identify it. It took us biturate. The anion in the molecular range is

with the help of UV, IR, NMR


and MS, and [M-ir, an even electron ion. It is also the
with all the necessary purification steps— base anion, just like the quasimolecular ion

49 .
Fig. 1. EJ and anion Cl spectra of the narcotic Etonltazene.

[M+ IJ^ in cation Cl. Two additional facts are method. Itdoes not duplicate the El-
important: results; it supplements them. That is
one of the most important points.
1 . Anion mass spectra are often simple, but
still highly characteristic. This helps in
Figure 4 shows the corresponding spectra of an
the interpretation. Barbiturates show,
allyl barbituric acid. Allyl abstraction is fa-
besides proton abstraction, sidechain
vored over proton abstraction; is the
abstraction (anions M-29 and M-57) base anion. This holds tor ail allyl oarbituric
and an anion with mass 42.
acids. Again, we would like to point out that
2. —
While cation Cl apart from pointing cation El and anion Cl are complementary,
out the molecular mass —
does not fur- Often, the sum of the base ions of the 2 methods
nish much more information than EI- gives the molecular mass of the compound in-
MS, anion Cl is a complementary vestigated. For identification work we recom-

50 .
Fig, 2. Anion Cl spectra of Morphine, Co Fig. 3. El, cation Cl and anion Cl spectra of
deine and Dlhydrocodelnone a saturated barbiturate

mend using — side by side —cation El and can convert substrate molecules to anions by
anion Cl mass spectrometry. electron attachment and dissociative electron
attachment.
C. Reagent Gases and Flexibility For the analysis of compounds with low elec-
tron affinity, reagents which readily form

Anion MS by Cl is an extremely flexible negatively-charged ions are often better


method. It is possible to guide the fragmenta- choices. The negative charge is transmitted
tion process by proper choice of the reagent from the reagent anion to the substrate mole-
gas. So far, we have worked with over 10 dif- cule by charge exchange or by anion cluster
ferent reagents. Figure 5 gives the anion mass formation. Methylene chloride has been used
spectra for 8 of them. for this purpose (2). We prefer Freons. Since
Noble gases, nitrogen, and essentially also they are gases, dosage is easier. The chloride
methane do not yield negative ions on electron and fluoride ions are good cluster-forming
impact. They act primarily as moderators for agents. SFg" was initially our choice as a
the production of |ow energy electrons which charge exchange agent, since it is formed easily
.

Fig. 4. El, cation Cl and anion Cl spectra of Fig. 5. Anion spectra of 8 reagent gases
an allyl barbituric acid used for anion Ci

on electron impact; however, it also produces molecular masses by 15 or 30 mass units arc ox-
fluoride cluster anions. ygen substitution products. By using deu-
Nitrous oxide yields 0~ on electron impact terated compounds, we could verify that I or 2
and permits oxidations in the ion source, in hydrogens aie replaced by oxygen.
presence of the heated filament. Other au- Figure 7 illustrates the importance of the
thors have used [Ojr, which has to be pjo- choice of reagent gas. For a large number of
duccd from oxygen for this purpose (3, 4). tricyclic psychoactive drugs such as Desipra-

This necessitates a special ion source. mine, the reagent methane yields matnly
The anion spectra in Figures 1 to4 have been [M-1]" with only a small contribution of an
obtained by electron attachment and dissocia- anion representing the tricyclic ring system.
tive electron attachment, using methane as re- With nitrous oxide, [M- 1]~ disappears and the
agent. Figure 6 shows the anion spectra of 3 ring anion (R") becomes the base peak
hypnotics obtained with nitrous oxide. The
strong base 0~ is a powerful charge exchange D. Sensitivity
agent. Proton abstraction leads to the base
ions The anions which exceed the Table III gives an idea of the sensitivity of

52 .
Fig. 6. Anion Cl spectra of 3 hypnotics with
plperldlne-structure with N^O as reagent Anion Cl spectra of Desipramine
Flg. 7.
gas with CH4 ana n^o as reagents

anion MS by Cl. If measure, in GC-MS


we
ment leads to an abundant molecular anion.
combination, the positive and the negative
Figure 8 shows such an example, flunitraze-
total ion current of a peak (we have to run iden-
pam which is on its way to becoming one bf the
tical chromatograms for this purpose) a fairly
,

most dangerous drugs of abuse. The anion


good comparison results. With saturated bar-
current 40 times larger than the cation cur-
is
biturates, the total ion currents are not too dif-
rent. The molecular ion dominates the anion
ferent, the sensitivities are comparable (see
spectrum. The fluoro group (m/e 19) and the
With allyl barbiturates,
figures for barbital).
nitro group (m/e 46, M— 16, M— 32) are visible.
however, anion Cl can be over a factor of 10
Figure 9 contains the corresponding spectra
more sensitive than cation El and cation Cl
(see figures for allobarbital).
of a similar benzodiazepine, this time chloro-
substituted diazepam. Again the anion cur-
Compounds with fluoro or nitro groups give
rent much larger than the cation current.
very intense anion spectra. Electron attach-
is

i
Table III

(Barbital,
Relative total ion current and base peaks of 4 barbiturates
Phenobarbital, Hexobarbital, Allobarbital) using 5 different ionization
techniques.

base
Ri R2 mode reagent rel.TIC peak % of TIC

El pos. [M-28P 21
Cl pos. CH 4 80 tM+l]+ 49
-CH2 -CH 3 •CH 2 -CH 3 Cl neg. CH 4 45 rM-r 33
Cl neg. CH4+SI^ 1100 [M-r 50
Cl neg. N2 O 460 [M-1]- 61

pos. - 190 21
El [M-28f
Cl pos. CH 4 70 [M+ir 52
CH 2 *CH 3 Cl neg. CH 4 360 [M-773- 55
Cl neg. CH4+SI% 120 [M-r 60
Cl neg. N2O 360 (M-77] 30
El pos. - 440 16
(M-16f
-0 Cl pos.
Cl neg.
CH 4
CH 4
530
180
[m-79]*
m/e 42
32
26
Cl neg. 0 (A 520 [M-r 67
BHH
'

o> 1

Cl neg. N2O 390 [M-r 51

-
El pos. 430 m/e 41 10
Cl pos. CH4 310 [M.ir 19
•CH2-CH*CH2 •CH -CI+CH Cl neg. CH 4 6700 [M -41]‘ 89
2 2
Cl neg. cH^+SFe 2000 [M-4lj" 84
Cl neg, N20 1250 [M -41]" 88

But the molecular anion is not stable. Chlo- show high intensity anion spectra. The quali-
ride is the base anion. The chloride ions make tative information may be somewhat one-
up the largest part of the total anion current.
sided, since most of the total ion current results
Bromo-substituted compounds give very simi- from one or a few anions. Identification must
lar results.
be carried out in conjunction with cation mass
spectrometry using El. For trace analysis by
E. Mass Specific Trace Detection in mass specific detection,
however, the situation
GC
is ideal, An anion mass
spectrometer can be a
All compounds with high electron affinity more sensitive GC detector than a conven-

54 ,
Fig. 8. El and anion Cl spectra of Fiunitrazepam, the active constituent of Rohypnol

tional elcclroii-ciipture system. It is, of tion which is possible with our detection sys-
course, much more specific and flexible. We tem. Sub-pg-detection is feasible, as long as

believe that it will become the ECD of the adsorption effects and background problems
future. can be handled.
Figure 10 gives an example. It shows a
chromatogram with bromide anion detection. F. The Source Pressure
Each peak repi’csents about 20 pg of a bromo
sedative; one is bromadal and the other biom- In Table IV, the anion intensities of the hyp-
isoval. Both are among the most heavily notic methaqualone are tabulated along with
Their identification the corresponding ion source pressures. Ni-
abused drugs in Europe. is

assured by the retention times and the intensity trous oxide is the reagent gas.

ratio of the 2 isotopic bromide anion signals. A In contrast to the U.S. investigators (2-5),

packed column was used. The base lines of we work with much lower pressure, in the
range of 10”® to 10"® torr. That is quite an ad-
the GC tracings would permit higher amplifica-

55 ..
vantage. The ion source stays clean. spectra are produced. Even in the usual
In our working range, the pressure depen-
working range between 0. 1 and 1 torr the pres-
dence of the anion Cl-spectra is tolerable.
sure dependence is quite pronounced.
[M- IJ- and the fragment anions are not much
affected by the pressure change, nor is the oxy-
pn substitution product [M-H5]-. [M-l-30]- ///, TEN POSITIVE ASPECTS OF
is probably the cluster ion [M+NO]“, since it is NEGATIVE ION MS BY Cl
influenced by the pressure. In our recent
work, we therefore tend to avoid cluster ion
What have we learned from our present work
formation.
with anion CI-MS?
Table V shows that in positive Cl it is not
possible to work with as low chamber
pres- 1. It can be carried out at much lower
sures as in the negative mode, since
El-like source-pressure than cation CI-MS.

56 .
Pig. 1 0. Trace analysis of bromo-sedatives by QC with bromide anion detection

2. 'ri)c pressure dependence in our work- 7. Oxygen substitution in the conven-


ing range (10”^ to 10“** torr) is moderate. tional ion source is possible by using ni-
10.
trous oxide as reagent which produces
3. It a soft ionization method which
is 0 -.
usually permits the recognition of the
molecular mass. 8. It is often more sensitive than cation
CI-MS and EI-MS.
4. The anion Cl-spectra are often simple
which makes interpreta,tion relatively
9. It is specifically suited for trace detec-
easy.
tion of substances with high electron af-

5. Anion Cl-spectra are complementary finity GC with selected anion


by
recording (the ECD of the future).
to cation El-spectra.

6. The method is flexible. By proper Compounds with similar structures can

choice of reagent gases we can favor be detected simultaneously by GC with


electron attachment, negative charge specific anion recording of a common

exchange, or negative, cluster ion structural unit such as a halogen or a

formation. ring anion.

57 .
Table IV
intensities at different pressures of the
Anion Ci o f Methaq ualone. Relative anion
reagent gas NaO. —

233 247 248 249 261 265 280


135 159
M-17 M-3 M-2 M~1 M+11 M+15 M+30’
m Torr H-91

63.6 “
1 6.6 3.6 26.1 17.7 51.1 100 0.9

3 7.9 4.4 25.4 19.7 55.7 4.4 67.2 0.7

5 8.4 4.3 25.1 18.9 56.8 ^^9 6.2 67.6 0.8

7 ,8.1 2.7 25.7 21.6 59.5 100 9. 67 ,6 1.4

9 7.7 4.6 26.2 20,0 58.5 100 13.8 67.7 3.1 ,

REFERENCES 4. Horning, E. C., et al., ./. Chromatogr.,

99, 13 (1974); Anal. Chem., 47, 2369


1. V. Ardenne, M., Kernenergie, 1, 1029 (1975).
(1958); V. Ardenne, M., and Steinfelder,
K., Kernenergie, 3, 717 (1960); v. Ar- 5. Hunt, D. F.,etal., Aha/. Chem., 48,2098
denne, M., Steinfelder, K., and (1976).
Tilmmler, R., “Elektronenanlagerungs-
6. Brandenberger, H., and Ryhage, R., “4th
Massenspektrographie organischer Sub-
Intern. Symp. on Mass Spectrometry in
stanzen". Springer, Berlin, 1971,
Biochemistry & Medicine”, June 1977,
2. Dougherty, R. C., and Weisenberger, Riva del Gaida; A. Frigeiio (Ed.), “Re-
C. R., J. Am. Chem. Soc., 90, 6570 cent Developments in Mass Spectrom-
(1968); Dougherty, R. C., Dalton, J., and etry in Biochemistry & Medicine",
Biros, F. J., Org. Mass Specttometry, 6, Plenum Publ. & Co. New York, 1978.
,

1171(1972).
7. Brandehberger, H., and Ryhage, R., “In-
3. Homing, E. C„ et al.. Anal. Chem., 45, tern; Bio'analytical Forum”, September
036 (1973); J. ofChrom., 142,481 (1977). iW"/, (iulldford; E. Reid (Ed.), “Assay of

58 .
Table V

Cation C^of Methaqualone. Relative cation intensities at different pressures of the


reagent gas CH4.

91 : 233 235 250 251 265 279 291 307


Torr

27.0;
27. l'
M-17

26.5,
24. 5|
m
M-15 M

69.8'
68.4
M+1

27.0
38.1
M+15

3.7
4.7 i

'
M+29

1.3
2,5
M+41

-
M+57

21. 23.61 100 92.9 96.4 6.6 - -


7|
9.3
-
12.
j

0,180 3.0' 12.9 18,1 100 1.9 17.7 5.2 0.6


\

0.220 3.0' 11.3' 17.2 100 2.2 18.8 5.8 0.8


'

0,300 2.5 8.0 14,3 100 2.3 20.5 7.2 1.0

Drugs and other Trace Compounds in Bi- posium on the Analytical Chemistry of
ological Fluids”, Vol. II, North-Holland Pollutants”, Geneva, 1978.
Publ. Co., Amsterdam, in press.
Brandenberger, H., Deutsche Lebens-
8, Brandenberger, H., and Ryhage, R., miitel-Rundschau, 70, 31 (1974).
^ass Fragments, 1, 1(1978).
9. Fraugi-Schnyder, D., and Branden-
berger, H., Z, Anal. Chem,, 290, 153
(1978).

10. Ryhage, R. and Brandenberger, H. sub-


, ,

mitted for publication in Biom, Mass


Spectrometry.

11. Brandenberger, H., Amsler, U., and


Frangi-Schnyder, D., ‘*8th Annual Sym-

59 .
by
Applications of Fourier
Peter R. Griffiths, Donald Kuehl
Transform Infrared and Michael P. Fulier
Spectrometry in Forensic
Department of Chemistry
Analysis Ohio University
Athens, Ohio 45701

/. INTRODUCTION Since the commercial development of


Fourier transform infl ated (FT-IR) spectrome-
Right at the outset of this paper it can safely ters, detection limits for samples measured by
be said that, in spite of its popularity for general infrared spectrometry have been substantially
analytical work, infrared (IR) spectrophoto- reduced. These instruments show a practical
metry has not been accepted as widely as other Improvement in sensitivity of at least an order
instrumental techniques of forensic drug analy- of magnitude when compared with conven-
sis. The very paper is bejng
fact that this tional IR spectrometers (I), and this improved
given by workers in an academic laboratory sensitivity can be traded off for even greater re-
which does not even have a license to handle ductions in the time required to measure high
drugs of abuse testifies to the truth of this quality spectra. (The scan time required for
statement! Most analyses of abused drugs are the measurement of medium resolution spectra
currently made using some type of chromato- is typically less than 1 second). When a liquid
graphy, in particular thin-layer chromato- nitrogen cooled mercury cadmium telluride
graphy (TLC), gas chromatography (GC), and (MCT) photodetector is substituted for the
high performance liquid chromatography more commonly used room temperature trigly-
(HPLC). However the retention character- cine sulfate (TGS) pyroelectric bolometer, a
isticsof chromatographic separations do not further increase in sensitivity of up to an order
provide completely unambiguous identifica- of magnitude is obtained. Additionally, all
tions of each “peak”, and subsequent coiv modern FT-IR spectrometers are equipped
firmation of the identity is often necessary with a general purpose data system which en-
through the use of some ancillary technique, ables the analyst to perform arithmetic opera-
e.g., (he interface of a mass spectrometer with tions on digitally stored spectra, thereby
a gas chromatograph (GC-MS). further increasing the versatility of these
in-
Where (hen should IR spectrophotometry fit struments. The theory, instrumentation and
into the overall picture of forensic drug
analy- chemical applications of FT-IR spectrometry
sis? Certainly the identification
of materials have been summarized in several books and re-
through their IR absorption spectrum
gives an view articles (2-7), and will not be discussed in
unambiguous identification of the material In . detail in this paper.
addition, IR spectrometry hds the
important
property of allowing great versatility
in the
type of samples which can be investigated,
spectra of samples in the solid,
and II. ABSORBANCE SUBTRACTION
liquid,and
vapor phases can all be readily
measured. I’erhaps the most important
However, conventional IR grating application of
spectrome- FT-IR spectrometers in the analytical
ters are generally less labora-
sensitive than many tory is one where the high
sensitivity of the in-
other instmments used in the
forensic labora- strument
tory , and it is probably this
is secondary to the presence of a
lack of sensitivity versatile data system.
rather than any other single Spectra can be added,
factor which has subti acted, divided, raised to
ied to the unpopularity a power, logged
of IR spectrometry in and antilogged.
this context. The most commonly used
arithmetic operation is the so-called “absorb-

60 .
ance subtraction” routine. In this procedure, shampoo on hair several days after application.
the absorbance spectrum of a mixture and the Attenuated total reflection (ATR) spectta
spectra of one oi more of the individual compo- of hair three days after shampooing (Fig. lA)
nents are measured. The absorbance spec- and six days after the shampoo (Fig. IB) were
trum of the component is multiplied by a measured, and the result of subtracting the two
suitable scaling factor which is chosen so that, spectra is shown in Fig. 2. Measurement of
on subtraction of the scaled spectrum from the the ATR spectra of fibers requires that several
original spectrum of the mixture, all the bands fibers are wrapped around the internal reflec-
due to the component disappear. This proce- tion element, but another technique has been
dure may be repeated several times for multi- developed for a similar analysis using only a
component mixtures so that intense bands due fraction of a single hair. The hair is placed be-
to major components may be eliminated from tween two diamond anvils of a high pressure
the spectra of mixtures, enabling weaker bands cell (8) and compressed until the thickness is
caused by minor components to become suitable for infrared analysis. Although dia-
evident. monds usually totally absorb in the region be-
One example of absorbance subtraction pro- tween 2250 and 1850 cm~‘, little significant
cedures to the analysis of samples of forensic chemical information is found in this region and
interest is the detection and identification of a a useful spectrum can be measured (see Fig. 3).

61 .
III. DIFFUSE REFLECTANCE
SPECTROSCOPY

The examples discussed above are certainly


rather nonroutine analyses, and we believe that
it is in the areas which are not leadily studied

by other analytical methods that FT-IR spec-


troscopy will prove to have the gieatest appli-
cation. For example, no instrumental method
may currently be used for the direct identifi-
cation of street drugs, controlled substances,
prescription pharmaceuticals, and over-the-
counter (OTC) pharmaceuticals. Therefore
any technique which permits the rapid, un-
equivocal identification of the active mevior in-
gredient and other active trace components in
samples believed to contain physiologically ac-
tive materials should find extensive application
in the forensic drug laboratory.
At two infrared techniques have been
least
previously proposed for this pur pose. In one
method, the active component must be ex-
Fig. 2: Difference spectrum resulting from tracted from the tablet and prepared as a KBr
the scaled subtraction of the spectrum disc prior to measurement of its spectr-um (9).
shown in Fig. IB from that In Fig. 1 A, show- After the spectrum is measur-cd, the sample can
ing the nature of the shampoo. be classified into one of nine groups of con-
trolled substances (such as baibitur'ates, am-
phetamines, etc.) by an easily followed flow
chart based on the pr esence or absence of char-
acteristic absorption band.s. After classifica-
tion, the active component may be identified by
comparison with the small number of reference
spectra of drugs in its class. This method is
simple and elegant but not fast, since the active
ittgredient(s) must be separated from the ex-
cipients prior to the preparation of the
KBr
disc. In addition, all the measur ements
in this
study were performed using a grating spectro-
photometer, so that at least two hours wer'C re-
quired between receipt of the sample
and
identification of the active ingrerlient,
Sadtler Research Laboratories, Inc.,
devel-
oped a method for reducing sample pr'eparation

62 .
time. Tablets were subjected to extractions in
acidic and basic chloroform, after which a film
of the extracted diug was cast on a salt win-
dow, and its absorption spectrum was then

measured. Once again in this work a grating


spectrophotometer was used for the spectral
measurement, although it may be noted that
Sadder now uses an FT-IR spectrometer for
the measurement of all their infraied reference
spectra. The extracted drugs could then be
identified by comparison with a compilation of
reference spectra of samples prepared in the

same way Although this method cer-


(10).
tainly reduced sample preparation time com-
pared to the previous method, over an hour
expires between receipt of the sample and iden-
tification of the drug. For drug overdose cases
where several tablets may have to be rapidly
identified, this lime lag may prove to be too
long and an even more rapid technique is
needed.
We have developed a novel method for iden-
tifying drug tablets in which the time lag be-
tween receipt of the sample and initiation of the
spectral measurement can be less than one
minute. The method is based on the measure-
ment of the diffuse reflectance spectrum of the
ground sample, either as received or diluted
(but not pressed) with an alkali halide, usually
size to approximate a perfect reference
ma-
KCl (11), Diffuse reflectance infrared spec-
Diffuse reflectance spectra of tablets
trometry has not been considered to be a useful terial,

(either whole or crushed) can be measured


analytical technique because of the low energy
directly (see Fig. 4). However, we often
dilute
of the diffusely reflected radiation, but we have 20
one part of the ground sample with about
found that a combination of an FT-IR spec-
parts of KCl to increase the information con-
trometer with an MCT detector and efficient el-
tent of the spectrum. Provided that
the pro-
lipsoidal collecting mirror gives such intense
high,
portion of excipient in the tablets is not
signals that we have to screen out much of the
diffuse reflectance spectra measured in this
radiation passing through the interferometer to
wqy may generally be used to identify the ac-
avoid digitization noise. The spectra that are On a
tive ingredient very rapidly (see Fig.
5).
measured are single-beam traces, and must be current state-of-
from spectrometer operating at the
ratioed against the single-beam spectrum speed, less
We
generally use the-art in sensitivity and computing
a ‘ 100%’ diffuse reflector.
'

receipt
to less than 10/u,m particle
than 3 min, would be required between
KCl powder ground

63
, ,

Kubelka and Munk (12, 13):

_ ka c
2/?„ s

where a is the absorptivity, ^ is a scattering fac-


tor, and A: is a constant of proportionality.

Spectra of neat, finely ground powders plotted


asjlR„) are closely similar to the absorbance
spectra of KBrdiscs of the same materials (11).

We have found that in practice/(/?„) is accu-


rately proportional to c provided R® is fairly
high typically greater than about 30%. Thus
,

provided that no strong bands are present in the

Fig. S: Kubelka-Munk plots of the diffuse


reflectance spectra of Tigan (a capsule to
counteract nausea, especially morning
sickness) and Urlspas (a tablet to counter-
act bladder infections), after grinding each
for one minute.

of the sample and display of the final spectrum


on an oscilloscope. Small libraries of spectra
(typically less than 1000 curves) could be
stored on 10 megabyte moving head discs, fa-
cilitating a rapid limited spectral search to pro-
vide the identity of the tablet.
In the same way that absorption bands in

absorbance spectra can be related to concen-


tration through Beer’s Law, the diffuse reflec- Fig. 6: Kubelka-Munk plots of the diffuse
tance of a sample at infinite depth (about 3 mm reflectance spectra of (A) a crushed tablet
or more), H « can be related to sample concen-
,
of Vallum containing 1% Valium and 99%
tration, c by the expression first developed by lactose and (B) pure lactose.

64 .
spectrum, one should be able to perform sub-
traction operations on diffuse reflectance spec-
tra stored as /(R„) in the same way that
absorbance subtraction routines can be applied
to absorption spectra. For example, the spec-
trum of lactose may be subtracted from the
spectrum of a tablet of Valium, which contains
about 99% lactose, to yield several weak bands
of Valium (see Figs, 6 and 7).
Another example of difference measure-
ments may be seen for the/(i?o.) spectra of two

500
Fig. 7: Spectrum computed after subtract-
ing 99% of the iactose spectrum in Fig. 6B
from the spectrum of the Vallum tablet. All

the bands in this spectrum may be as-


signed to Valium. reflectance In the
The
region between 1500 and 1100 cm"‘ is so Fig. 8:Kubelka-Munk plots of the diffuse
low that useful analytical information can- reflectance spectra of two APC tablets
not be obtained In this region. from different manufacturers.

65 .
Fig. 10A: Kubelka-Munk plot of the diffuse
reflectance spectrum of a dried leaf after
spraying with a commercial Paraquat
spray. The was ground with KCI and
leaf
the spectrum was ratloed against a KCI ref-
erence spectrum.

Fig. 9: Spectrum computed when the spec-


trum 8B is subtracted from the spec-
In Fig.
trum In Fig. 8A. Upward-going bands are
primarily due to phenacetin and down-
ward-going bands are primarily due to
aspirin.

aspirin-phenacetin-caffeine (APC) tablets with


the same specified composition (3.5 gr aspirin,
2.5 gr phenacetin, 0.5 gr caffeine) but from dif-
ferent manufacturers. The two spectra are
shown and the unsealed difference
in Fig. 8,
spectrum is shown in Fig.
9. It can be seen that
the tablet from manufacturer A contains more
phenacetin, while the one from manufacturers
contains more aspirin. Although this example lowed to dry naturally. The sample was
uses OTC pharmaceuticals, the application of prepared Identically to the sample In
difference techniques to the analysis of foren-
Fig.lOA.

66 .
Fig. IOC: Unsealed difference spectrum Fig. 10D: Spectrum of the evaporated
produced when the spectrum in Fig. 10B water extract of the Paraquat spray used In
was subtracted from the spectrum in this work.
Fig.fOA.
sponded to the frequencies of the bands in the
spectrum of a sample prepared by spraying the
presence of strychnine
sic samples, e.g. for the
contents of the can directly into water, separa-
in heroin, isimmediately apparent.
ting off the aqueous phase, evaporating off the
In the past few weeks a crisis has arisen on all
water from the extract, and mixing the residue
university campuses with the discovery that
with ground KCl (see Fig. lOD) Although our
.

much of the marijuana being purchased is con- primary goal of developing an analytical
taminated with the herbicide. Paraquat. We
method for the in situ determination of Para-
attempted to prepare simulated samples by
quat was not reached, this method did provide
spraying a common local broadleaf weed,
an indirect indication of the presence, and the
Polygonaceae Rumex, with a commercially
experiment again gives an indication of the tre-
available Paraquat spray (“Ortho” Spot Weed
mendous versatility of FT-IR spectrometry.
and Grass Killer)which contained only 0.44%
Paraquat. The weed was allowed to die, at
which time it was ground with powdered KCl CHROMATOGRAPHIC
/V.
and its diffuse reflectance spectrum was mea-
APPLICATIONS
sured (see Fig. lOA). An untreated plant was
cut and also allowed to die after which it was
A. GC-IR
treated in an identical fashion (see Fig. lOB).
On subtraction of the spectr a of the treated and Another common application of FT-IR spec-
the untreated plants, several bands were ap- trometry in general organic analysis is the
parent in the difference plot (see Fig. IOC) Al-
. interface of the spectrometer to a gas chroma-
though none of the bands in the difference tograph for the on-line identification of eluting
spectrum may be assigned directly to Paraquat, samples (GC-IR) (14-16). This experiment
frequencies of the mor-e intense bands corre- makes use of the short scan time of a lapid-
scanning interferometer, which typically re- fluent from the chromatograph on powdered
quires less than I second per scan to generate KCl. After each peak elutes, the diffuse re-
the information for a medium resolution infra- flectance spectrum of the solute may be rapidly
red spectrum. The GC effluent is passed measured. This work is currently at a very
through a long, narrow gas cell, light-pipe, early stage, but feasibility studies indicate that
through which the infrared beam is also submicrogram detection limits are possible.
passed, Data is collected while each compo-
nent is in the light-pipe, and after each peak C. TLC-IR
elutes the signal-averaged interferograms are
Fourier spectrometers may also be used to
stored in the spectrometer memory until the
measure the infrared spectra of separated
end of the chromatogram, at which time all the
“spots" directly on TLC plates (TLC-IR).
spectra are calculated.
One method involves depositing the adsorbent
We
have not encountered any examples of
layer on an infrared transmitting substrate,
GC-IR in forensic drug analysis, and to our
such as a sheet of AgCl, and measuring the
knowledge GC-IR is not being used in a routine
transmittance spectrum through the adsorbent
fashion in any forensic laboratory. In view of
layer either without treatment (20) or after
GC-IR detection limits (between 25 ng and 1 (ig
treatment with an infrared mulling oil to de-
of sample injected into the chromatograph), it
crease scattering (21). TLC-IR measurements
is not likely that GC-IR will supersede GC-MS
are optimized by combining the high sensitivity
in the forensic laboratory. However, GC-IR
of FT-IR with the high efficiency of pro-
can produce useful confirmatory evidence in
grammed multiple development TLC. In this
cases where mass spectrometry does not pro-
case detection limits of 10 ng of sample may be
vide a definite identification.
obtained (22). These methods have the disad-
vantage that the analyst must prepare his own
B. LC-m TLC plates, since AgCl plates are not available
commercially. To circumvent this problem
While GC-IR is, to a certain extent, a com-
we have studied the possibility of using diffuse
petitive technique to GC-MS, no such well ac-
reflectance spectroscopy to identify materials
cepted instrument as a mass spectrometer is
on aluminum foil backed TLC plates (which are
routinely used for the on-line identification of
commercially available) and, although the
peaks eluting from a liquid chromatograph.
early results are promising (11), more work
Some work has been published showing how
is required before the usefulness of this tech-
an FT-IR spectrometer can be used to measure
nique for TLC separations of drugs may
the spectra of HPLC peaks (LC-IR) as they
be determined.
pass through a flow-cell (17-19), but in general
submicrogram detection limits are not ob-
tained, primarily because of strong absorption
by the solvent in some frequency regions. We V. SUMMARY
believe that the best way to perform LC-IR
measurements is to eliminate the solvent prior The increased sensitivity of FT-IR over con-
to measuring the spectrum. Preliminary work ventional IR spectrometers not only permits
in this area has led us to believe that the best the detection limits of conventional infrared
way to achieve this end is to evaporate the ef- sampling methods to be decreased, but also

68 .
A

allows an even greater variety of samples to be 10. Infraied and Ultraviolet Spectra of Com-
investigated. The biggest advantage of FT-IR monly Abused Drugs, Sadtler Research
spectrometry over other non-infrared tech- Laboratories, Inc., Philadelphia, PA
niques used in the forensic laboratory is proba- (1972).
bly due to its versatility and specificity rather
11. Fuller, M. P., and Griffiths, P. R.,Anal.
than any inherent sensitivity advantage over
Chern. (submitted for publication, 1978).
other instrumental methods.
12. Kubelka, P., and Munk, F., Z. Tech.
Phys., 12,593(1931).

REFERENCES 13. Kubelka, P., J. Opt. Soc. Am., 38, 448


(1948).
1. Griffiths, P. and Han-
R. Sloane, H. J.,
14. Kizer,K.L.,A/Jier. Lab., 5(6), 40 (1973).
nah, R. W., Appl. Spectrosc., 31, 485
(1977). 15. Coffey, P., Mattson, D. R., and Wright,
J. C.,Amer. Lab., 10(5), 126 (1978).
2. Bell, R. J., Introductory Fourier Trans-
form Spectroscopy, Academic Press, 16. Griffiths, P. R. , in Fourier Transform In-
New York (1972). frared Spectroscopy-Applications to
Chemical Systems (J, R. Ferraro and L. J.
3. Griffiths, P.R., Chemical Infrared
Basile, Eds.), p. 143, Academic Press,
Fourier Transform Spectroscopy, Wiley-
New York (1978).
Interscience, New York (1975).
17. Kizer, K. L., Mantz, A. W., and Bonar,
4. Ferraro, J. R., and Basile, L. J. (eds.),
L. C.,Amer. Lab., 7(6), 85 (1975).
Fourier Transform Infrared Spectro-
scopy-Applications to Chemical Sys- 18. Vidrine, R. W., and Mattson, D. R.,
tems, Academic Press, New York (1978). Appl. Spectrosc. (in press, 1978).

5. Griffiths, P. R. (ed.), Transform Tech- 19. Gomez-Taylor, M. M., Kuehl, D,, and
niques inChemistry, Plenum Publishing Griffiths, P. R.,Eiiv. Analyt. Chem., 5,

Corp . ,
Y
New ork (1978). 103 (1978).

6. Griffiths, P. R,, Anal. Chen}., 46, 645 20. Percival, C. J., and Griffiths, P. R.,
(1974). Chem.. 47, 154 (1975).

7. Koenig, J. L., Appl. Spectrosp., 29, 293 21. Gomez-Taylor, M. M., Kuehl, D., and
(1975). Griffiths,?. R.,Appl. Spectrosc., 30,447
(1976).
8. Lippincott, E. R., Weir, C. E., Van Val-
kenburg. A., and Bunting, E. N., Spec- 22. Gomez-Taylor, M. M., and Griffiths,

trochimActa, 16,58(1960). P. R.,Appl, Spectrosc., 31, 528 (1977).

9. Hannah, R. W., and Pattacini, S. C., Per-


kin-Elmer Infrared Applications Study
No. 11, Perkin-Elmer Corp., Norwalk,
CT(I972).

69 .
by
Nuclear Magnetic
Edwin D. Becker
Resonance: Recent
National Institutes of Health
Developments in
Bethesda, Maryland 20D14
Techniques and their
Potential Application to
Forensic Research

I. INTRODUCTION tronic components. A more thorough


undei standing has been developed of the
Nuclear magnetic resonance (NMR) has various parameters that govern the mag-
long been recognized as an extremely valuable nitudes of both the signal and the noise
technique for the structure elucidation and emanating from the radio frequency re-
analysis of organic compounds. For more ceiver coil of an NMRspectrometer.
than 25 years NMR has played a major lole in This has spawned improvements in the

many types of organic chemistry. Yet, there geometry of NMR probes and has led
are other areas of chemistry — most forensic re- also to the design of more sophisticated
search and analysis included — where NMR electronic circuits that amplify the weak
has had little impact. Probably the principal NMR signal while discriminating against
reason for the failure of NMR
to make these in- unwanted noise from many sources.
roads is its limited sensitivity relative to that of Coupled with design improvements has
other spectroscopic methods. But NMR sen- been the availability of the markedly bet-
sitivity has been steadily improving over the ter electronic circuit components found
years. While it still cannot compete in sensi- in today’s integrated circuit technology.
tivity with mass spectroscopy, fluorescence,
and neutron activation analysis, NMR is now 2. Use of higher magnetic fields. During
applicable to many types of problems where it the 20 years the magnetic field
last
was once deemed useless. Thus, the advan- strengths used for high resolution NMR
tages of NMR in terms of a readily understood have increased by a factor of six. The
theory, rather easily interpretable spectra, and principal impetus for development of
remaikable versatility can be more widely ever higher field spectrometers is the in-
exploited. creased dispersion available, since
we shall examine the present
In this paper chemical shift differences expressed in
stateof the NMR art regarding sensitivity, frequency units are directly proportional
sample size, and quantitative analytical capa- to the strength of the applied field.
bilities. We shall not discuss specific applica- However, an important secondary ad-
tions to forensic problems nor shall we go into vantage is the increased sensitivity that
the interpretation of NMR spectra, information arises from the more favorable Boltz-
on which is widely available elsewhere (1). mann distribution of nuclei among the
magnetic energy levels . The fundamen-
tal limitation on NMR signal stiength
//. IMPROVEMENTS IN SENSITIVITY
comes from the fact that radio frequency
absorption is unavoidably accompanied
During the last decade there have been major
by induced emission, Since we can
advances in NMR sensitivity. Four separate
measure only the difference between
factors can be identified as the principal con-
these two processes, it is actually only
tributors, as follows;
the difference between the populations
1 . Better electronic design and better elec- of the magnetic energy levels that con-

70 .
tributes to the net absorption signal. widely-used NMR instrument. Introduced in
For protons a magnetic field of 1.4
in 1961 had a typical signal/noise ratio of about
,
it

tesla (a frequency of 60 MHz) the differ- 6. By the mid-1960’s improvements in probe


ence is only about 1 out of 200,000 nu- design and the availability of higher magnetic
clei; for a field of 8.5 tesla (360 MHz) it is fields led to substantially better sensitivity, as
six times as great. indicated for the Model HA- 100. The HR-220
was the spectrometer to employ a super-
first
3. Use of Fourier transform methods. In
conducting magnet, rather than an electromag-
the following section we shall describe
net. The greatly increased field strength
the rationale for the use ot Fourier trans-
provided another significant increase in S/N,
form (FF) methods and illustrate the
which ran about 80 for the HR-220 that we pur-
very significant gains in sensitivity
chased at NIH in 1969. Note, however, how
derived thereby.
much further improvement there has been in
4. Use of Microcells. This, too, is a topic the last decade; the XL-200 (with essentially
that we shall take up in detail in a later the same field strength as our old HR'220) has
section. just been introduced with a specified S/N that
equals or exceeds 300. Finally, the highest
Table I illustrates the remarkable improve- field high resolution NMR spectrometer
ments in sensitivity that have resulted from fac- operating today has a proton frequency of 360
tors (1) and (2) above. It is conventional to MHz. With recent improvements in probe de-
express the sensitivity of proton spec- NMR sign, that instrument provides a signal/noise of
trometers by specifying the signal/noise ratio about 800 (in one case a value of 1200 has been
obtained in a single scan (or for FT, in a single obtained).
pulse) for a particular resonance line in a sam- Thus, during the last 15 years or so, the
ple of ethylbenzene at a concentration of 1% "state of the art" in NMR sensitivity has im-
(by volume) in a suitable solvent. The Varian proved by at least a factor of 100, just on the

Model A-60 spectrometer was the first really basis of the use of higher magnetic field
strengths and of improvements in electronics
and probe design This dramatic enhancement
.

Table I
in sensitivity has opened up many new areas to
NMR, especially those in the biological field,
Signal/Noise for Various where sample size and concentration are often
Spectrometers* severely limited. However, another concur-
rent but independent development has been
Year Spectrometer Model S/N equally important in improving the effective
sensitivity of NMR. That development is the
1961 A’60 6 introduction of Fourier transform methods,
1965 HA-100 30 coupled with the principles of time averaging.
1969 HR-220 80
1978 XL-200 300
1978 WH-360 800 III. FOURIER TRANSFORM METHODS
‘Proton specification — 1% ethylbenzene,
single scanisingle pulse
Whatever the sensitivity of a particular spec-

71 .
iroscopic method, there are occasions when it netization corresponding to each different
must be pushed to the limit. It has long been resonant frequency to tip in turn through a
recognized that the effective sensitivity of the small angle, The component thus generated in

method can be improved by spending more the xy plane induces a small current in the re-
time in acquiring the data. Infrared, Raman, ceiver coil placed along the y axis, which we
visible, and ultraviolet spectra are usually ob- detect and display as an NMR
line. In a pulse

tained by rather slow scans, with suitable elec- experiment, on the other hand, a large amount
tronic filtering to reduce noise. In it has NMR of radio frequency power (larger by a factor of
usually been more feasible, for several rea- at least lO'* than that typically used in cw NMR)
sons, to scan spectra more rapidly but to repeat is applied for a short time— only a few micro-
the scan several times and coherently add the seconds — and at a single, fixed frequency. As
data in a digital computer. Such “time averag- shown in Figure 1 , the pulse causes the magne-
ing” of n spectra provides a signal n times as
great as in a single scan, but the noise (which is

not coherent) is only n as large, so that S/N is

improved by/ri'*. With typical scan times of


5-10 minutes, it is feasible to add the data from
about 100 scans in order to increase S/N by a
factor of 10, but beyond that the expenditure of
time rapidly becomes prohibitive. Clearly, the
time spent on each scan must be drastically re-
duced if very large numbers of repetitions are
tobe employed Fortunately that is just what
.
,

can be done with Fourier transform (FT)


methods.
There are several different techniques for
obtaining NMR spectra rapidly, all of which
employ a Fourier transformation some stage
at
in processing the data. The most commonly
used method at present is the use of a short
radio frequency pulse to excite a spectrum, and
it is only this pulse-FT method that we shall dis- Figure 1. Behavior of the macroscopic
cuss here. magnetization on application of a radio fre-
In an NMR
experiment the macroscopic nu- quency pulse, and resultant signal, (a)
clear magnetization can be thought of as a vec-
The radio frequency field H,, applied along
tor that resides initially along the axis parallel
z
to the applied magnetic field, and it is tipped to-
the X’ axis rotates the magnetization M
from its equilibrium position to they’ axis,
ward or into the xy plane by the action of the
(b) M decreases as Individual magnetic
radio frequency magnetic field that is applied.
moments dephase due to spin-spin relaxa-
In a conventional, continuous wave
NMR ex- tion and magnetic field inhomogeneity ef-
periment, a small amount of radio frequency
fects. Input signal, a short pulse, (d)
(c)
power is applied continuously, but at a grad-
Exponential free Induction decay resulting
ually changing frequency, to cause
the mag- from process (b).

72 .
tization to tip into the xy plane, where it in-
duces a large signal in the receiver coil. The
signal decays exponentially with a time con-
stant the magnitude of which is deter-
mined by the nuclear spin-spin relaxation time
and the magnetic field inhomogeneity. Be-
cause the radio frequency power is so high, nu-
clei over a range of resonance frequencies of
several kilohertz are affected, so that the mag-
netization vectois of nuclei at all chemical
shifts tip simultaneously. Each provides an
exponentially decaying signal at its own reso-
nance frequency. In the detection process all
of these signals beat with each other and with
the reference frequency of the pulse itself.
The resultant time response, the “free induc-
tion decay,” typically looks like that in Figure
2. Although it is not interpretable by inspec-
tion in the same way as a normal NMR spec-
trum, it does indeed contain all the same
information and requires only a second or so
for data acquisition, rather than several min- Figure 2. Free induction decay (top) and
utes for a typical slow scan. Fortunately, the Fourier-transformed spectrum (bottom) of
information is easily converted to a recogniz- '^C In 3-ethylpyridlne.
able form by carrying out a Fourier transforma-
tion and some other simple mathematical tubes at concentiations as low as 10 p,M with
processing. These operations are rapidly and overnight runs. But the area in which FT
easily carried out in a small computer, which methods become really essential is the study of
normally forms an integral part of the NMR nuclei other than hydrogen, which have lower
spectrometer. With the pulse-FT method it is inherent sensitivity and often lower isotopic
possible to make many pulse repetitions, some- abundance. The most significant in oiganic
times tens or hundreds of thousands, co- chemical problems is '®C, with a sensitivity
herently add the free induction decays, and only about ’/64 as great as that of and a natu-
then carry out the mathematical processing on ral abundance of only 1.1%. To overcome the
the co-added data with its enhanced S/N. In combined adverse factor of nearly 6000 has re-
later sections we shall see some examples of quired the development of superior instru-
FT spectra and also explore the limitations on ments and experimental techniques, but as we
the pulse repetition rate resulting from spin- shall see in the next section it is possible to ob-
lattice relaxation. tain good •*€ spectra with remarkably small
Fourier transform methods often improve amounts of sample. Figure 3 illustrates a
the sensitivity in a given time by a factor of rough but very useful guide to the relative sig-
about 10-20 and permit the study of NMR nals obtained for ’H and ’®C —
if a good W
of samples in standard 5 mm
diameter sample spectrum can be obtained with a single pulse

73 .
Figure 3. NMR spironolactone, bottom: ‘H specjrum, taken with a single
spectra of
pulse. Middle; spectrum resulting^^ from 62,000 puls^(17hoiiKexperiment)^jrqp;^-
panslon of part of the spectrum. (R.J. HIghet, private communication.)

for a particular sample, an acceptable muchjarger tubes (up to at least 30-mm diame-
spectrum can be obtained in an overnight run. ter) for studies of very dilute solutions of stud-
ies of nuclei of very low sensitivity. On the
other hand, when concentration is not a prob-
IV. MICROCELLS lem but the total amount of sample is limited,
there is nothing to be gained
by using a large
Since the earliest days of high resolution sample tube and a correspondingly large
NMR, such spectra have been obtained with amount of solvent. In fact, studies have
samples in 5-mm diameter sample tubes. Ini- shown that it is far more effective to restrict the
tially thiswas the maximum size sample tube volume of solution that is used. This can be
that could be accommodated in the magnets done in two ways: (1) With probes and inserts
and probes that were available. During the designed for use with 5-mm diameter sample
last few years it has become possible to utilize tubes, the total length of the sample solution it-

74 .
self can be limited to that just needed to fill the pie volumes as small as 5-10 lA. Resolution is
receiver coil. A volume as small as 25-50 /itl usually excellent.
can be attained, but there may be some degra-
The use of 1.7-mm diameter capillary tubes
dation of resolution. (2) Microprobes have
and a microprobe for obtaining 'H and '’C
been designed with very small diameter re-
spectra has recently been reviewed by Shoo-
ceiver coils that accommodate 1 .7-2 mm
diam-
lery (2). We present here two examples from
eter sample tubes (similar to conventional
that review. Figure 4 shows the 'H spectrum
melting point capillaries). This approach is
of gelsemine, a natural product of molecular
generally superior and permits the use of sam-
weight 322. The upper spectrum was obtained
with 100 ixg of sample in 10 fil of CeDg solvent.
With this amount of material a good spectrum,
showing all essential features, was obtained
with only 400 sec. of data acquisition. The
lower spectrum comes from only 4 /xg of gelse-
mine. It required 8000 pulses and took 2.2
hours to acquire the data. While the noise
level is higher than that of the upper spectrum,
all important features show up clearly except

those obscured by peaks due to solvent impuri-


ties. The 0.2% CeDsH isotopic impurity in the
CqDj gives by far the largest line in the spec-
trum, with a small amount of water providing
the second strongest line. For proton NMR
the real limitation in sample size now comes,
not from instrument sensitivity, but from prob-
lems in handling samples and in avoiding pro-
ton-containing impurities in the solvent.

Let us turn now to the '^C spectrum of this


same substance. As we have seen, '®C sensi-
tivity is expected to be more than three orders
of magnitude poorer than that of proton, so a
correspondingly larger sample is needed Fig- .

ure 5 shows the proton-decoupled '•*C spec-


trum of 1.0 mg of gelsemine in 6.5 ix\ of CeDg.
In this case 76.000 pulses were used for a total
experimental time of 17 hours. This spectrum
is of rather good quality. Accumulation of
data for a longer period than ovei night is
usually not worthwhile, since there is relatively
little improvement in S/N In some cases sub-
Flgure 4. Proton NMR speefiitf of gelse-
.

Shoo- milligram quantities have provided good


mlne in capillary-type miorooells.
spectra, but with current technology the limit
lery(2}.

75 .
Figure 5. NMR spectrum_pf gelsemlne (proton decoupled) in a capillary-type micrO'
cell.Shooiery (2).

lies somewhere in the vicinity of a milligram of tinction coefficients that enter into optical and
a typical organic sample of molecular weight infrared spectroscopy. Lines are usually
300.-400. sharp and well-separated, so that overlap prob-
lems are' minimized. Line assignments are
usually made rather easily, so that identifica-
V, QUANTITATIVE NMR tions are facilitated. On the other hand, there
MEASUREMENTS are some serious drawbacks in the quantitative
use of NMR, as we discuss in the next para-
NMR is, in some respects, a nearly ideal graph. For proton NMR
the difficulties have
quantitative technique. In principle the area been largely overcome (3), but NMR
has
under a line is proportional to the number of nu- been widely regarded as being almost incapa-
clei contributing to it, without differential ex- ble of use for quantitative analysis. Only re-

76 .
cently has work in a few laboratories begun to nuclei vary by a factor of at least 50, as indi-
dispel that myth. cated in Figure 6. The spectrum at the top of
The principal problems in the use of “C Figure 6 is a proton decoupled '®C spectrum
NMR for quantitative purposes stem from the taken under customary conditions, in which
following factors:

1, Because of inherently low sensitivity IC»0Ht


B
KOO
and natural abundance, S/N is often XOO I

r23*i)

lower than desired These properties of


.
T 157 4S4
cannot really be overcome com-
pletely, but instrumental improvements,
as discussed previously, have alleviated 1

JnsoUfltroi
the problem. Clearly, the poorer the
0 i\
signal/noise, the larger the random error L
in the quantitative determination.

2, Long spin-lattice relaxation times re- il


duce signals below the expected levels
when pulses are repeated too quickly
{the analog of “saturation” in contin-
uous wave NMR). Although this prob- KMKI
IMO 12

lem affects NMR also, it is usually


more severe for NMR because of the
wide disparity in Tj’s for carbon atoms
in different environments. We com-
ment below on some means for over-
coming the difficulty.

3, The nuclear Overhauser effect (NOE)


that accompanies the proton decoupling
usually carried out while nieasuring '®C
spectra is, like relaxation times, depend-
ent on the environments of the individ-
ual carbon atoms. Thus, the areas Figure 6. NMR spectra of acenaphth-
under the various do not necessar-
lines
(proton decoupled). Top: Obtained
ene
ily reflect the numbers of carbon atoms under routine conditions for optimization of
giving rise to the lines. Fortunately, as overall signal/nolse (20° flip angle, 1.0 sec
we shall see, it is possible to suppress the repetition time, 640 pulses). Measured
NOE, but at some loss in signalMoise. values of Ti are given in parentheses on
the structural formula. Bottom; Condi-
Probably the best systematic treatment of tions for quantitative measurements (90°

‘“C NMR problems is contained in a recent re- flip angle, 400 sec repetition time, de-

view by Shoolery (4) Acenaphthene was cho-


.
coupler gated off except during data acqui-
sen as a test compound, since Tj’s of its carbon sition). Shoolery (4).

77 .
their expected values, with the remaining dis-
crepancies attributable to the very long T/s,
for which even a 400-sec. delay is insufficient.
Unfortunately, such long waiting periods in-

crease the length of the experiment to a nearly


intolerable level. An alternative procedure, il-
lustrated in Figure 7, is to shorten the T,’s by
adding to the solution a paramagnetic “relax-
ation reagent.” The most commonly used
reagent is chromium acetyiacetonate; as
indicated in Figure 7, a low concentration of
this is sufficient to bring all areas to
substance
within 3%of theoretical values with only a 2-
sec, pulse delay. When such adulteration of
the sample can be tolerated, the use of a relaxa-
tion reagent is by far the most effective way of
Figure 7. '°C NMR spectrum of acenaphth- obtaining quantitative *®C data.
ene. Solution is also 0.1 M in chromium
acetyiacetonate. Conditions for quantita-
tive measurements (20° flip angle, 1.0 sec
VI. FUTURE TRENDS IN NMR
acquisition time, decoupler gated off dur-
ing 2.0 sec delay time following the acquisi- NMR is a dynamic field, with versatile new
tion time). Shoolery (4).
methods constantly being developed. One of
the most exciting areas is the study of “high
tlie magnetization flip angle and pulse repeti- resolution” spectra in solids. The techniques
tion rate are selected so as to optimize overall and instrumentation needed are, so far, rather
S/N in the minimum time. If the area under the specialized but within a few years such studies
,

line due to C-1 is arbitrarily set equal to2.00 (to should be rather widespread. Meanwhile, for
reflect the two equivalent carbons, 1 and 2), conventional liquid samples, we can expect im-
then the other lines should have areas of either provements in higher field systems, in sample
1.00 or 2.00. As indicated in Figure 6 those handling capabilities and in computer support
due and 5
to the singly-protonated carbons 3 , 4, for NMR spectrometers. Without a m^'or
are low by about 20%, while those due to the conceptual breakthrough it seems unlikely that
three non-protonated carbons are 75-80% low sensitivity, the major subject of this paper, can
The effects of long T,’s can be eliminated by be improved really substantially, but further
simply waiting long enough between pulse rep- small increments will doubtless occur.
etitions, while the NOE can be suppressed by
gating the decoupler off during the long waiting
periods and turning it on only during the brief
periods of data acquisition following each
pulse. The spectrum at the bottom of Figure 6
shows the results of incorporating both of these
features. The areas are now much closer to

78 .
HEfERENCES

1. See, for example, Becker, E, D., "High


Resolution NMR.” Academic Press,
New York, 1969.

2. Shoolery, J. N,, "Microsample Tech-


niques in 'H and ‘“C NMR
Spectros-
copy.” Varian Associates, Palo Alto,
Calif., 1977.

3. Kasler, F., "Quantitative Analysis by


NMR Spectroscopy.” Academic Press,
New York, 1973.

4. Shoolery, J, N., jProgr. NMR Spectry. 11,


79(1977).
Computer Applications
Session

I. Drug Identification
II. Computer Systems in
Government Facilities
A Brief Review of the -fev

Charles F. Hammer
Computerized
Identification of Known Department of Chemistry
Georgetown University
Compounds and the Washington, D.C. 200S7

Elucidation of Unknown
Structures

I. INTRODUCTION 11. RETRIEVAL SYSTEMS FOR


KNOWN COMPOUNDS
Topresent a thorough review of the litera-
ture in this field is impossible in the time avail- A. The NIH-BPA Chemical Information
able. In the past six years alone, there have System
been twenty-five on the search and re-
articles
The largest system in use at present is the
trieval of infrared (ir) spectra using comparison
NIH-EPA CIS, whose name has recently been
methods by many authors. There have been
changed to Structure and Nomenclature
thirteen articles using pattern recognition tech-
Search System (SANSS). It is available for
niques, mainly from two groups of workers,
use on a commercial system, the ADP-Cypher-
Jurs at Penn State (1) and Isenhour at North
netics Computer network. The system was
Carolina (2), In addition, twelve interpreta-
developed by Heller, Milne and Feldmann and
tion programs have been i eported The use of
.

a description was published mScience last year


computerized methods for mass spectra (ms)
(7, 6c). The latest version is shown in Fig-
(3) is even larger, while proton magnetic reso-
ure 1. SANSS contains 42 files of data that can
nance (pmr) (4) and carbon-13 magnetic reso-
be searched for names, structures or substruc-
nance (cmr) (5) spectra have only recently been
tures, Chemical Abstracts Service (CAS) regis-
computerized. Thus, I must restrict tl)is dis-
try number and molecular weight. The system
cussion to systems that use combined data.
also includes the enormous bibliographic files
The ACS Symposium series # 54 on “Com-
of the National Library of Medicine (NLM)
puter-Assisted Structuie Elucidation” pub-
and the LOCKHEED literature system that in-
lished last year (6) is a useful starting point for
cludes over 15,000,000 records.
the beginner.
A of developing a search or in-
difficult part The oldest part (1971) of CIS is the Mass
terpretation system is obtaining the data files. Spectral Search System (MSSS), which has a
This is especially true of the older types of carefully checked file of 31,700 mass spectra.
spectia, ir and ms. Often the file entries are As Dr. Milne will discuss the system later, I

miscoded or redundant; perhaps they do not will not describe it further, except to point out

have some of the now useful data coded at all. that MSSS includes more than searching and
It would cost too much to go back and extract
identification facilities. One program, LAB-
the intensity data for the old ir files. The NIH- DET, which was written in my laboratory (8),

EPA Chemical Information System (CIS) (6c)



calculates the mole fractions and % isotopic

has cleaned up its ms files and is now collecting C-13 or N- 15


label incorporation of deuterated,

a new file of vapor phase ir spectra that are ob- labeled compounds using the experimental

tained under the strict conditions recom- mass spectra of the labeled and unlabeled
mended by the Coblentz Society. We shall see compounds.
how these files affect the retrieval process CIS powder dif-
also includes a file of x-ray
later. fraction data (PDAS), but perhaps more useful
' NIH = National Institutes
of Health, EPA = Environ- is the Cambridge Crystal File (CRYST) (9), a
mental Protection Agency. data base of about 15,000 entries containing

82 .
published crystallographic data, mostly on or- tion of empiiical and quantum mechanical
ganic compounds. These can be searched by techniques.
CAS number, name, formula, molecular For those interested in toxicities, the files of
weight, space group and unit cell parameters. NIOSH’s® Registry of Toxic Effects of Chemi-
CAMSEQ is a program developed at Case- cal Substances (RTECS) can be searched An .

Western that uses the CRYST stnictural data


as a starting point to calculate the conforma- ^ NtOSH = Ndhona! fn.\Ulnie of OcctfpcUhtwl S({fefy
tions of molecules in solution by a combina- anef Heahh.

CIS COMPONENTS
MAY 1978

A
/ \
^
/\ /\ //\ /\ /\
/AQUATIC S
/
/ AMES
TE8T_
\
\
\ /
/0HM-TADS_S^
\
/
/ WATER
DROP
S /
/
A SIHOLE \
/\
/PARTITHJKi
CHy STAl\ /tOWFICjE«1>j

FUTURE; LEGEND: under DEVELOPMENT


UNDER development
THERMODYNAICS dD ONDER TEST
TSCA PLANT t PRODUCTION
O OPERATIONAL
DIRECTLY LINKED ON THE SAME COMPUTER
LINKED TOGETHER VIA A NETWORK S CAS REQN
L
Figure 1. The NIH-EPA Chemical Information System.

83 .
example of a RTECS search for para-dichloro- #300 was requested, Figure 4 and the entire as-
benzene is shown in Figure 2. signed spectrum was piinted for acetophe-
none. Note that the structure is given with
Finally I would like to illustiate the carbon-
13 nuclear magnetic resonance (CNMR) file carbon numbers as well as other particulars of
can be The the compound and conditions that weie used to
that contains 3800 spectra. file

specific peaks obtain the spectium. The file can be searched


searched, as in Figure 3, for
± an error and for a particular type of substructural carbon,
at a specific chemical shift
given multiplicity of the nondecoupled cmr e.g., a methyl group y to a nitrogen atom has a
spectrum. CNMR found 37 entries at

6=197.5±.5(S). The operator decided to


add different peaks at 26.0±.5(Q) and at

]37.0±.5(S). This time only 1 match was


found and was then listed. Then compound
NfH/EPA
SUBSTflUCTURE SEARCH SYSTEM

LATEST NEWS GENERATED ON 4/1S/J7

COMPLETE DATA BASE SELECTED


ENTER NEWSELECTIDN (H FOR HELP) ^2

DATABASE SELECTED 32

OPTION? RING
OPTION? ALTBO \ 2
OPTION? ABRAN I AT I 1 AT 4
OPTION? SATOM 7 0
SPECIFY ELEMENT SYMBOL = CL
OPTION? lOENT
TOTAL PROTON COUNT FOR THIS 8 TRUCTURE IS
IPFDR PROGRAM ESTIMATE) 4
FILE I, THIS STRUCTURE IS CONTAINED IN 1 COMPOUNDS
STRUCTURE I CAS REGISTRY NUMBER 1DSM6>7
NiOSH RTECS CZ4SSOO
C6H4CI2
Figure 3. NIH-EPA C-13 NMR Search Sys-
^C^
tem —
Version 3.3.
C C-CL

Lslesl NEWS daied 14 JULY 1077


CL.'c C To list options, type OPT

*C* Option: SH
6 tn 2 ine, 1,4>d}chloro>(9CI)
Bin tint, p-di:hloro- {8CJ) Enter SHIFT. DEVIATION, MULT 197 B, .5, S
P'CMoropIttny] chlotitfi 37 matches using 197 6
p-DichlotObiniene
Di'^hlonctdfl
Evolt
L(lst), Q(ult),or next SHI FT, DEVIATION, MULT. -
26,0, .6, Q
Piradt
3 matches using 197,5 26.0
Pindow
L(lst), Q(uit). or next SHIFT, DEVIATION. MULT.. 137.0, 6. S
OPTION? TSHOW 106*48-7 1 matches using 197.6 26.0 137 0
YOU ARE NOW IN THE RTECS RETRIEVAL SYSTEM
LAST NEWS UPDATE 24-MAR-7S L{lst),Q(ult), or next SHIFT, DEVIATION, MULT.: L
CAS NUMBER > 106467 NIOSH NUMBER > CZ4B50D
ORL-HMN TOLO 30QMG/K TFX UNS PCOC** -,051r ID# NAME
ORL-BAT LOSfl 50OMG/K TFX WRPCA2 9,119,70
IPR-RAT L050 2800 MQ/K TFX JAPMAS 38,124,49
ORL-MUS LD80 2980 MG/K TFX* OUCHAZ 6,103,73
300 Ethanone, 1-phenyl-
ORL-GPG LOLO 2800 MG/K TFX t4CYAT -.1338,63 CASREG# 98862 MF: C8 H8 01
Btniint, 1,4-dichlaro>{9CI}
CSH4CI2

Figure 4. Spectrum Search for Acetophe


Figure 2. A RTECS Search on the CIS. none (from Figure 3).

84 .
mean shift of 1 1 .7±0.2 ppm based on 7 entries being searched is not a part of the data base,
in the file (10).
then correct matches are only those relevant to
the structure of the unknown. Thus a search
B . The Yugoslavian COSMOSS System system must be judged by both the correct or
near-correct matches and the misleading false
Zupan e( aL (11) have recently published positives found.
data on a combined retrieval system containing
ir, ms and cmr data. Their concept was that
C. Other Retrieval Systems
“it should work as a black box; using only the
spectra as input data.” The data base consists
Since the advent of gas chromatography-
of 92,000 ir spectra based mainly on the ASTM-
WY ANDOTTE collection (12), which includes mass spectrometry instruments, there have
been a large number of search routines devel-
the 35,000 spectra of Sadtler (13) and 13,000 of
the DMS collection (14), The ras file is made oped. As Drs. Milne and Biemann will de-

up of the 16,000 spectra of the Aldermaston scribe the use of their systems, I will move on
collection (15), While many of the spectra of to mention briefly two other groups concerned
these collections are doubles and are wrongly with the problem. Clerc of Switzerland has
coded, no attempt was made to weed out the separately published search routines for ms
data base, as was done on the CIS system. (16), ir (17) and cmr (18). It is interesting that
The cmr file contained about 1600 good spec- he has not yet put them all together.
tra. The search can be made on a single or Clerc’s ms-search algorithmis used in the
multiple file basis. The authors reported data Associated Electronics Industries (A.E.I.) ms-
.

for 100 test searches for each of the data data systems.
bases Accuracy was defined as the fraction of
,
Finally, McLafferty’ s (6a) Probability Based
examples in which the correct compound was
Matching System (PBM) and Self-Training In-
found in the top five choices listed. Searches
terpretive and Retrieval System (STIRS) rep-
of this type invariably give several possibili-
lesent fairly successful attempts to identify
ties. However, a user is more interested in the
complete structures or 179 substructural parts
number of correct first choices. For ir, only
of an unknown system. A quick review of
77% first choices were correct, for ms, 91%,
and cmr, 95%. These results are probably a re- both given in the ACS Symposium volume
is

flection of the quality of the various data bases. (6a). Both of these programs are available for
use on the Cornell-TYMNET computer sys-
The user hopes that when a spectrum noi
tem. A recent comparison on the same data
pi'esent in the data base is searched, the re-
base of STIRS with Isenhour’s K-Nearest
trieved examples will be close to the correct
Neighbor (KNN) algorithm (19), whichjs apat-
structure. This can be true only when the
tern recognition procedure, has shown STIRS
choices picked are reliable. Interpretive
to be generally superior (20).
methods are actually better for the cases of true
unknowns. McLafferty (6a) has suggested While several individual spectral systems
that the performance of a search system should have been devised, it seems to me that all possi-
include a recall factor, a reliability factor and a ble data should be used. This leads us to the
false positive factor. When the spectrum next major topic.

85 .
III. COMBINED SYSTEMS FOR THE used up, or from spectral data. The CONGEN
ELUCIDATION OF UNKNOWNS approach allows the practicing chemist to be
USING INTERPRETIVE intimately involved in the process. Much
TECHNIQUES more difficult structures can thus be studied
and the likelihood of missing possible struc-
Four systems using combined data for the tures is reduced. When no constraints are
structure determination of unknown com- used, the program is similar to that used by
pounds are curiently being developed: Sasaki.
The Arizona State group (6g) has also devel-
1 . CHEMICS-F by Sasaki et al. in Japan
oped a highly sophisticated interactive pro-
(6h)
gram, CASE, that attempts to parallel the
2. CASE by Munk et al. at Arizona State natural product chemist’s reasoning process.
University (6g) The molecule assembler uses both operator
data and automatically interpreted ir and cmr
3. DENDRAL and CONGEN by Smith and
data. Possible structures are then checked by
Carhart et al. at Stanford University
calculation of thecmr spectrum. CASE has
(6i.21)
been shown to be quite effective on real-world
4 . STREC by Gribov and Elyashbeig et al. problems.
in Russia (22). Finally, let us look at the Russian system
(22), which follows a somewhat different ap-
Since the ACS Symposium volume (6) dis-
proach. Figure 5 shows a simplified and com-
cusses the first three of these systems, I will
only mention some of their characteristics and
then describe the Russian system in a little
more detail.

One of the major problems in any system is

deriving all the possible structures without re-


dundancies. The Japanese system derives all
possible structures that fit a given molecular
formula, then eliminates the various structures
on the basis of automatically acquired and in-
terpreted ir, pmr, cmr, ultraviolet (uv) and ms
data. The original work was done only on C, H
and O compounds, but more recently nitrogen
has been added (23b).
The Stanford Group (6i , 2 1 c) has developed a
highly operator-interactive heuristic program,
CONGEN, that derives the possible nonredun-
dant structures that are consistent with a mo-
lecular formula and any constraints the
operator wants to impose. These constraints
may be derived from known chemical reac- Rgure 5. Condensed Block Diagram of
tions, such as the number of moles of periodate STREC. (22).

86 .
pressed block diagram of STREC. STREC I have been unable to determine how much
sequentially identifies various structural interaction between the various interpretive
groups present in the vibrational spectrum (ir routines is allowed. In real life, the approach
and Raman) that are consistent with the ele- one uses to interpret spectral data depends
ments present in the empirical formula. These completely on the type of spectia available.
are combined into sets of fragments and then Generally, the spectral data are highly comple-
assembled into structural formulas under the mentary and not specific to only one type. As
constraints of the identified fragments plus any far as I can determine, the systems described

chemical information provided by the opera- above consider each type separately In addi-
.

tor. The structures are then double checked tion, all these systems use a molecular fotmula
against the vibrational data, previously identi- or empirical formula as input data, but do not
fied fragments and forbidden fragments (the describe how it is obtained.
latter corresponds to the BAD LIST used in
CONGEN [21c]). The spectral filter process
IV. MOFO, A PROGRAM FOR THE
is then repeated using pmr, ms and uv data.
DETERMINATION OF
The various fragment libraries include the indi-
MOLECULAR FORMULAS
cated number of fragment possibilities. Only
the 20 most intense peaks are used from their
Several years ago, we devised a general pro-
low-resolution mass spectral data. The vibra-
gram, MOFO, to assist us in determining the
tional spectrum of each compatible stru c ture i s
molecular formula of an unknown using what-
then calculated and compared to the unknown ever data were available (24 ). As we have only
spectrum. The result, along with the perform- a medium-resolution mass spectrometer, we
ance of chemical functional analysis (which could make only crude mass measurements.
was not identified in the paper), provides a The block diagram of MOFO is shown in Fig-
measure of the reliability of the answers. Sev- ure 6. Data inputs include the mass and accu-
eral examples of C, H, N and 0 containing racy of measurement of the parent molecular
compounds with molecular weights up to 202 ion and the intensities of the molecular ion clus-
were given. ter including any P-H, or H3 ions. The pro-
All of these approaches are promising. The gram has a subroutine, MSMS, that can
Japanese and Russian systems cannot yet at- calculate the mass of an ion using distance or
tempt highly complex structures, but the oper- time measurements from known standard ions,
ator also does not have to be an expert, such as perfluorokerosene or perfluorotri-/i-
versatile, natural products chemist. All the butylamine. The operator then enters the
data interpretation is done by the computer. upper and lower limits for the number of atoms
The two American systems, on the other hand, ofC, H, N, 0, S, P, Si, F, Cl, Br, I and any one
require qualified operators. Both, however, additional heteroatom. The heteroatom can
have been used successfully on quite complex be any element; we have used Se, Mg, Cd and
structures and are indeed an aid to the struc- Fe. Subroutine CHECK then generates all

tural chemist. Both are also expandable possible formulas within the limits specified
to include new algorithms that will even- and eliminates those corresponding to frag-
tually reduce the knowledge required by the ment species (points of unsaturation=n-t- 0 5 ) ,

operator. and computes the exact mass for each If pro- .

87 .
high resolution ms data are used, MOFO rarely
misses. Both our undergraduate and graduate
students have been using MOFO for several
years; thus the operator does not have to be
highly experienced.

ACKNOWLEDGMENTS
1 wish to thank Dr, G. W.A. MUne for the first four figures
used here and Prof. Sasaki ofMiyagi University ofEducO’
tioii in Sendai, Japan, for his hospitality when part {cmr)of
MOFO was being developed.

REFERENCES

1 . Jurs, P, C., et al,, Appl. Spectrosc., 27,


371 (1973); Chem., 46, 520 (1974);
ibid., 46,2126.

2. Isenhour, T. L.,ei al., Anal, Chem., 46,


150 (1974); ibid., 47, 1126 (1975); ibid.,
Figure 6. Block Diagram of MOFO. 47 2027; Appl. Spectrosc., 29, 226
,

(1975); y, Chem. Ir\f. Comput. Sci., 15,


ton integration data are available, then the for- 212 (1975); Anal. Chem., 48, 1027
mulas are further pruned to conform to the (\916)\ibid., 48, ISIZ', Appl. Spectrosc.,
correct ratio of protons; if cmr data are avail- 30,213 (1977); iWd., 31, 18(1977).
able, a further pruning is done. In either case,
3a. To mid-seventies, Pesyna, G. M., and
no interpretation by the operator is required.
McLafferty, F. W., in “Determination
The molecular ion cluster of each remaining of Organic Structures by Physical
formula is then computed with the associated Methods,” Vol. 6, F. C. Nachod, J. J.
P-H, Ha, H3 effects found in the experimental
Ziickerman, and E. W, Randall, Eds.
spectrum. The calculated spectrum is then
Academic Press, New York, 1976, pp,
compared to the M+l, 2, ions of the ex-
. . .
91-155.
'

perimental spectrum, as well as the


(M+l)/(M+2) ratio and exact mass, if avail- b, Burlingame, A. L., Kimble, B. J., and
able. These comparisons are then ranked and Derrick, P. J.,Anal. Chem., 48(5), 374R
the ten (or fewer) most likely formulas are (1976).
with the best formula first.
listed
c. Burlingame, A, L., Shackleton, C. H.
When only unit resolution ms data are avail- L., Howe, I., Chizhov, 0. S., Ibid.,
able, the coirect formula is chosen first about
50(5), 352R (1978).
75% of the time. The addition of pmr or cmr
data improves the accuracy to about 95%. 4a. Jones, R. T., Proc. Soc, Anal. Chem.,
When both cmr and pmr data are used, or when 10, 165(1973).

88 .
b. Wasson, J. R., and Johnson, D. K., e. Schwenzer, G. M., and Mitchell, T. M.,
Anal. Chem., 46(5), 317R (1974). “Computer Assisted Structure Elucida-
tion Using Automatically Acquired ^^C
c. Wasson, J. R., and Lorenz, D. R.Jbid., NMR Rules,” pp. 58-76.
48(5), 248R (1976).
f. Surprenant, H. L., and Reilley, C. N.,
d. Yamasaki, T., and Sasaki, S., Jpn.
“Computerized Structural Predictions
Anal., 213(1975).
from '®C NMR Spectra,” pp. 77-91.
5a. Ochiai, S., Hirota, Y., Kudo, Y., and
g. Munk, M. E., et al., “Interactive Struc-
Sasaki, S.,Jpn. Anal., 22, 399(1973).
ture Elucidation,” pp. 92-107.
b. Brunner, T. R., Williams, R. C., Wil-
and McCombie, P. S.,Anal.
h. Sasaki, S.,et al., “CHEMICS: A Com-
kins, C. L.,
puter Program System for Structure
C/wn., 46, 1978(1974).
Elucidation of Organic Compounds,”
c. Jezl, B. A., and Dalrymple,D. L.,Anal, pp. 108-125.
C/jem., 47, 203 (1975).
i. Carhart, R. E.,e/fl/., “Computer Assis-
d. Schwarzenbach, R., Meili, J., Konitzer, tance for the Structural Chemist,” pp.
H., and Clerc, J. T.,Org. Mag. Res., 8, 126-145.
11,(1976).
7a. Heller, S. R., Milne, G. W. A., and Feld-
e. References 6e, 6f, 6g, and 6h, below. mann, R. J., Science, 195, 253 (1977).

f. Dalrymple, D. L., Wilkins, C. L., b. Reference 6c.


Milne, G. W. A., and Heller, S. R.,Org.
8. Hammer, C. F., and Vlietstra, A. J., un-
Mag. Res., in press (1978).
published results; see references 6c (p.

6. Smith, D. H., Ed., “Computer-Assisted 42) and 7a for brief descriptions.


Stmcture Elucidation,” ACS Sympo-
9. Kennard, 0., Watson, D. G., and Town,
sium Series # 54, American Chemical
W.'G.,y. Chem. Doc., 12, 14(1972).
Society, Washington, D.C.V 1974,

McLafferty, F. W., et al., “Computer- 10. Milne, G. W. A., Zupan, J., Heller,
a.
S. R., and Miller, J. A,, Spectra-Struc-
Assisted Identification of Unknown
ture Relationships in Carbon-13 Nuclear
Mass Spectra,” pp. 1-17.
Magnetic Resonance Spectroscopy.
b. Beimann, K., et al., “Identification of
Results from a Large Data Base, Org,
the Components of Complex Mixtures
Mag. Res., submitted (1978).
by GC-MS,”pp. 18-25.
11. Zupan, J., Penca, M., Hadil, D., and
c. Milne, G. W.
A., and Heller, S. R.,
Marsel, J., Anal. Chem., 49, 2141
“The NIH-EPA Chemical Information
(1977).
System,” pp, 26-45.
d. DeHaseth, A., and Isenhour, T. L.,
J. 12. Codes and Instructions for WYAN-
“An Information Approach to the De- DOTTE- ASTM, American Society for

termination of the Secondai'y Structures Testing Materials, 1916 Race St., Phila-

of Globular Proteins,” pp. 46-57. delphia, Pa. (1964).

89 .
13. Sadtler Standard Spectra, Sadtier Re- b. Miyashita, Y., Ochiai, S., and Sasaki,
search Laboratories, Philadelphia, Pa. S.,7. Chem. Inf. Comput. Sci., 17, 228
(1977).
14. DMS, Documentation of Molecular
Spectroscopy. Veilag Chemie, Wein- 24. Hammer, C. F., Vlietstra, A. J., and
heim, and Butterworth, London. Ochiai, S., unpublished results,

15. Aldermaston Mass Spectrometry Data


Collection, Aldermaston, Reading,
U.K.
16. Clerc, J. T., and Emi, F., Top. Curr.
C;!em.,39,91(1973).

17. Clerc, J. T., Knutti, R., Koenitzer, H.,


and Zupan, J., Fresenim’ Z. Anal.
Ciiem., 283, 177(1977).

18. Schwarzenbach, R., Meili, J., Koen-


H., and Clerc,
itzer, J. T., Org. Mag.
Res., 8, II (1976).

19. Justice, J. B. Jr., and Isenhour, T. L.,


Anal. Chem., 46, 223 (1974).

20. Lowry, S. R., Isenhour, T. L., Justice,


J. B. Jr., McLafferty, F. W. Dayringer,
,

H. E., and Venkataraghavan, R., ibid.,


49, 1720(1977).

21a. Masinter, L. M., Sridharan, N. S., Le-


derberg, J., and Smith, D. H., J. Am.

Chem. Soc., 96,7702(1974).

b. Masinter, L. M., Sridharan, N. S., Car-


hart, R. E.,and Smith, D. H.,ibid., 96,
7714,(1974).

c. Carhart, R. E., Smith, D. H., Blown,


H., and Djerassie, C., ibid., 97, 5755
(1975).

22. Gribov, L. A., Elyashberg, M. E., and


Serov, V. Y.,Anal. C/tim. Ada, 95, 75
(1977) and references therein.

23a. Sasaki, S.. Abe, H., Ouki, T., Saka-


moto, M., and Ochai, S., Anal. Chem.,
40,2220(1968).

90 .
by
The Use of Mass
G. W. M.
Spectrometry for Drug A. Milne and H. Fales

National Heart, Lung, and Blood Institute


Identification
National Institutes of Health
Bethesda, MD., 20014
and

N. C. Law
Suburban Hospital,
Bcthesda, MD., 20014

/. INTRODUCTION rately identified by gas chromatography-mass


spectrometry and that, in the view of a majority
In 1969, using facilities of the (then) National of the attending physicians, the resulting infor-
Heart and Lung Institute, of the National Insti- mation was of value in the clinical handling
tutes of Health, the Suburban Hospital of Be- of drug overdoses. Accordingly, in 1971,
thesda, MD., in collaboration with NIH support was sought from NIH by Suburban
scientists, undertook a pilot study designed to Hospital for the pui chase of a gas chro-
investigate the possible utility of mass spec- matograph-mass spectrometer system which
trometry in problems of drug identification. was to be installed at the hospital and which, it
As a typical suburban community hospital. was hoped, would permit the hospital to con-
Suburban Hospital receives a small, but con- tinue to offer this service independently of the
tinuing number of patients who have consumed National Heart and Lung Institute’s labora-
excessive amounts of drugs, and it was felt that tories. A grant from NIH was secured, the gas
rapid and accurate identification of the drug or chromatograph-mass spectrometer was in-
drugs involved in each case would facilitate stalled at Suburban Hospital in 1972, and the
treatment. service'has been in use since then.
The pilot study (1) permitted the conclusions The purpose of this paper is to desci ibe the
that drugs could in fact be rapidly and accu- results that have been obtained in the period
since then.

//. DISCUSSION

A. Service Area

The major user of the drug identification ser-


vice is, of course. Suburban Hospital itself.

During the seven-year period however, in-

creasing numbers of samples from other hos-


pitals have been received at Suburban.
Currently, thirteen of the approximately fifteen
major hospitals in the Greater Washington
Area, shown in Figure 1, send samples to
Suburban on a regular basis.
In 1976, Suburban Hospital instituted, for
the first time, a charge of approximately $75 for

Fi]^ur« 1. Hospitals Using Suburban Hos- each gas chromatography-mass spectrometry


pitcil's Emergency Drug Indentiflcation (GC-MS) analysis completed. At the same
ScfVidb. time, major health insurance schemes agreed

91 .
to accept this expense. As a result of these or techniques such as thin-layer or gas chroma-
two events, usage of the seivice offered by tography. A substantial number of samples re-
Suburban had dropped slightly and at the pres- ceived contain no drugs, and in many others,
ent time, about 580 cases are handled per alcohol is the only drug found. In many of

year. This averages to about 1.5 cases per day these cases, the suspicion that drugs was in-
which i.s a load that can be carried by the Spe- volved was ill-founded and this result is of
cialChemistry Laboratory at the hospital with- value in treatment. Where drugs are involved,
out any major re-staffing. The $75 charge their identity is determined and reported and

covers the cost to Suburban of the service. this information is also of value in treatment.

The cases that are submitted to the service In this way, the service permits the physician
are usually those in which drug involvement is to reduce the number of variables that must be
suspected, but the drug or dings in question considered in diagnosis and treatment and con-
cannot be identified by other, simpler means sequently GC-MS appears to have a legitimate
such as information from the patient or others, role in the functions of a Clinical Chemistry

Figure 2. Protocol For Drug Identification by GC-MS.

92 .
Laboratory. cation . In the other method which is used as a
,

A member of the laboratory staff is on call at second-string approach, the standard electron
all times to handle drug identification sam- ionization (El) mass spectrometric technique is
ples. Those from other hospitals are usually used. This is also known ( 1) to be quite appli-
delivered to Suburban by car or taxi. The han- cable to the problem, but, because it gives
dling time, once the sample is received at more complex spectra (4), is not used
Suburban is between one and three hours. routinely.
The result is then relayed by telephone and a The Cl GC-MS analysis is carried out using
written record follows. the system that is shown in Figure 3. The sam-
ple, after having been processed as shown in
B. Methodology Figure 2, is injected into the gas chromato-
graph shown in Figure 3. As compounds are
The most readily available samples that can eluted from the chromatograph, they are ad-
be obtained for drug identification are blood, mixed with a reagent gas, typically methane,
urine, and gastric contents. Our experience and the methane/drug mixture passes into the
with blood has indicated that it is the least use- source of the mass spectrometer. Here, chem-
ful of the three for a number of reasons and it is ical ionization takes place and the Cl mass
not used unless no other sample can be ob- spectrum of each component of the mixture
tained. Gastric contents frequently contain is recorded as the substance emerges from
detectable amounts of orally ingested drugs, the chromatograph, which is temperature
unless too much time has elapsed since inges- programmed.
tion. A number of drugs, ingested in large The mass spectra that are obtained in this
amounts, can be detected unchanged in urine way are very simple. The spectrum of chlor-
many hours after ingestion. Consequently, it diazepoxide (Librium) is shown in Figure 4,
is requested that, if possible, both gastric con- and from this it can be seen that the Cl mass
tents and urine be submitted. When these are spectrum consists mainly of the protonated
received at Suburban, they are combined, and
the mixture is worked up according to the pro-
cedure shown in Figure 2. This procedure is
designed to extract from the sample all organic
acids bases and neutrals. No effort is made to
,

separate these classes, because the gas chro-


matographic step makes this unnecessary An .

internal standard of dioctyl phthalate is added


to the extract before gas chromatography.
This is used to standardize the temperature
program of the gas chromatograph.
Two mass spectrometric approaches have
been used in this work. The method of choice,
which is used routinely at Suburban, employs
chemical ionization (Cl) mass spectrometry
(2), a method that has been shown (3) to be
FIgur* 3. Gas Chromatograph-CI Mass
readily applicable to problems in drug identifi- Spectrometer Combination.

93 .
molecular ion at an m/e value of (M+ 1), where spectra.
M is the molecular weight of the substance. The second inethod employs El mass spec-
The molecular weight of the substance can thus trometry and can be accomplished using any of
be determined directly from such a spectrum, a variety of commercial GC-MS systems.
and the identity of the compound usually fol- These from the system shown in Figure 3
differ
lows immediately. A secondary check on this principally in that the sourcemust be operated
identification is afforded by the retention time, at high vacuum, and so, rather than adding a re-
or elution temperature of the compound in the agent gas to the stream emerging from the gas
gas chromatograph. The only important drugs chromatograph, a separator is built into the
with the same molecular weight as each other system to remove much of the helium carrier
are pentobarbital and amobarbital (M=226). gas before the stream enters the mass spec-
Distinction between these, when necessary, trometer source. The mass spectrometer need
is made possible by examination of either the not be a quadrupole, of course; magnetic sector
gas chromatogram or the respective El mass jnachines are used very commonly.

94 .
The El mass spectra of dmgs are usually System. An example of the use of this system
more complex than the corresponding Cl mass is given in Figure
6. The computer uses the
spectra. A typical El mass spectrum, that of two major peaks of the El mass spectrum (m/e
phenobarbital, is shown in Figure 5. From 204, intensity between 100%, and m/e 232, in-
this, it can be seen that a great deal of fragmen- tensity between 20% and 30%) to identify phe-
tation isoccurring, and the molecular ion, at nobarbital unequivocally fiom the data base of
m/e 232, is not the most intense ion in the spec- 30,000 mass spectra.
trum. Identification of substances from such Using either Cl or El mass spectrometry, in
spectra is therefore not always easy and may conjunction with gas chromatography, identifi-

require the use of a computer search system cation of drugs contained in urine or gastric
such as the NIH-EPA Mass Spectral Search contents has become a standard procedure and

95 .
In the same Figure, it can be seen that the
OPTlON7p<8li

P£A< AND INTENSITY SEARCH


number of drugs identified in each case has re-
TYPE PEAK, MEN INT MAX INT mained very close to 2 on average. The num-
CR TO E X IT J FO ft iO MW MF AND NAME
ber of cases in which no drugs were found has
USER 2e4 100 teo
been decreasing steadily for three years, in
<rREFS UvE PEAKS
spite of the increase in the total number of
94 ?04
cases. This is in part because of minor
NEXTREOUEST 232 20 30

4 refs m^e peaks


changes in reporting; alcohol, for example, has
1 204 232 been reported as a drug only since 1975. The
NEXT REQUEST 1 decrease is also due in part to the fact that the
iO» REG 01 MW MF NAME laboratory has been steadily gaining experi-
13t48 500^980 232 CI2H12N203 2 4,6111(3
H,6H|*Pyi)m»c)in(tfJo~e B-eihyr
ence and now recognizes a larger number of
1S-phenyl' (6CI|
Barbitunc ictd. 5 eihyl-6-phpnyN {8CII
drugs than was the case in earlier years.
Adonal
AyvP«>'
Amy to fen e
Barbenyl D. Drugs Encountered
Beibiphenyl
Barbipii
Barbu a
Barb VI t
>
A total of approximately 90 different drugs
Blu-pheo
CratectI
have been encountered in the seven years that
the service has been in existence. As can be
Figure 6. Identification of Phenobarbital
From its Ei Mass Spectrum By Computer
Library Searching.

has led to the results that are discussed in the


next section.

Ill, RESULTS

A. Case load

The number of cases handled by the drug


identification service can be seen in Figure 7.
These data suggest that the rapid growth in the
use of the service during 1973- 1976 has ceased
and perhaps that a steady state may soon be
reached in which between 500 and 600 cases
per year are involved. This presumably may
be taken as a rough approximation to the total
number of serious drug overdoses per year in
the Greater Washington area and, if coiTect,
suggests that a single GC-MS facility is easily
adequate to deal with the medical drug idem
tificationproblems that arise in such a
community.

96 .
seen from Figure 8, the most commonly en- probamate (Miltown).
countered substance, by far, is caffeine, which The drugs in Figure 8 are those that have
is found in about 25% of all cases. Nicotine, been found most frequently and it can be seen
though somewhat less common, is also found at a glance that the classes represented are but
very frequently. The most commonly found three; sedatives, analgesics, and tranquiliz-
‘drug’ is aspirin, which is found in perhaps 15% ers. This comes as little surprise to students of
of all cases. Aspirin alone is rarely responsible the abuse of legitimate drugs, and eliminates
for admission to a hospital, except where small the possibility that stimulants, for example, are
children are involved. The most commonly more abused than other drugs.
abused lethal drug appears to be secobarbital, The stimulants that have been found are
which has held this dubious position through- cited in Figure 9, and it can be seen that, ne-
out the entire seven years of this work. In con- glecting caffeine and cotinine, which is a nico-
trast to aspirin, secobarbital is found both tine metabolite, the use of stimulants does not
alone and in admixture with other drugs, such appear to contribute to the case load substan-
as aspirin. tiallyIn fact, of the 24 positive identifications
.

Valium, or diazepam, has been the most of amphetamine or methamphetamine that


commonly implicated tranquilizer, being found have been made in seven years, probably half
consistently more frequently than chlordiaze- were not associated with an overdosed patient,
poxide (Librium), amitriptyline(Elavil), or me- but were submitted as material samples by

DRUG 7 YEAR TOTAL 1977

CAFFEINE 605 165


ACETYL SALICYLIC ACID 391 116

NICOTINE 282 112

SECOBARBITAL 206 31

DIAZEPAM 186 32

PHENOBARBITAL 179 46

DEXTROPROPOXYPHENE 178 36

AMOBARBITAL 139 20
15
METHAQU ALONE 135
119 19
CHLORDIAZEPOXIDE
119 29
PENTOBARBITAL
118 22
MEPROBAMATF
107 15
ACETOPHENETIDIN
Figure 8. Most Commonly Encountered Drugs.

97 .
legal authorities. ethchlorvynol and glutethimide. As was men-
In Figure 10 are shown the data concerning tioned above, sedatives, particularly barbitu-
sedatives, certainly the most lethal group from rates, frequently are the only drug involved iji
the perspective of drug ovei doses. It is clear an overdose. Mixtures of barbiturates are,
from FiguEe 10 that barbiturates dominate the however, noted and a very common occur-
class with only occasional appearances of non- rence is the identification in a patient of a bar-
barbiturate sedatives such as methaqualone, biturate sedative and a tranquilizer, for

Stimifr'ant 1972 1973 1974 1976 1976 1977 1978 7 Year Total

Caffeine 15 17 49 116 162 16S 81 60S


Cotinine (Nicotine metabolite) - — - 1 13 14 16 44
Amphetamine, methamphatamine 0 3 S 1 2 8 6 24
Mescaline 0 0 1 2 0 1 1 5

Figure 9. Stimulants Encountered In Drug Identification Program.

Drug 1972 1973 1974 1975 1976 1977 1978 7 Year Total

Secobarbital 23 37 30 29 40 31 16 206
Pheno barbital 11 22 20 17 45 46 18 179
Amobarbital (Amytal) 11 30 29 16 29 20 4 139
Methaqualone (Quaalucfe) 19 38 24 19 15 IS 5 135
Pentobarbital 12 14 19 19 19 29 7 119
Ethchlorvynol (Placfdyf) 5 4 19 14 27 20 7 96
Methyprylon (Noludar) 7 7 13 16 16 18 9 86
Glutethimide (Doriden) 9 4 25 12 10 10 6 76
Chlorproinazine (Thorazine) 2 4 2 2 16 13 7 46
Meperidine (Oemeral) 0 3 5 3 17 11 6 45
Butalbital (Sandoptal) 0 3 1 10 9 11 2 36
Carbromal (Nyctal) 0 14 2 2 2 11 3 34
Butabarbital (Butisot) 0 2 4 2 5 8 2 23
Mephobarbital (Mebaral) 0 2 1 1 5 9 4 22
Vinbarbital (Oelvinal) 0 0 0 0 10 10 2 22
Barbital 0 1 1 2 3 5 2 14
Aprobarbital (Afurate) 0 0 0 0 1 5 3 9
Mexobarhital (Evipal) 0 0 1 2 1 1 0 5
AllobarbitaJ 0 0 0 1 2 1 0 4
Talbutal (Lotusate) 0 0 0 0 1 1 1 3
Alphenal 0 0 0 0 0 2 0 2
Cyclobarbftal 0 0 0 0 1 1 0 2
Benzocaine (Et Ammobenzoate) 0 0 0 0 0 1 0 1

Methohexital (Brevital) 0 0 0 0 0 0 1 1

Figure 10. Sedatives Encountered Irt Drug Identification Program.

98 .
example, secobarbital and diazepam. A seda- The tranquilizers that have been encoun-
tive which has caused great concern in the past tered in overdose cases are identified in Figure
is glutethimide. Overdoses of this diug are dif- 12. The first three —
diazepam, chlordiaze-
ficult to handle because the compound is sus- poxide, and meprobamate were, between —
ceptible to enterohepatic re-circulation. In 1972 and 1976, consistently the most com-
1 973 and 1974, it was clear from trends such as monly found drugs in this class. Since 1976,
those in Figure 1 1 that abuse of glutethimide
, however, abuse of chlordiazepoxide seems to
was threatening to become a serious problem, have been diminishing and at the same time,
and so the stricter controls that were placed on there has been a sharp increase in the appear-
sale of the drug in 1974 seem, in retrospect, to ance of amitriptyline (Elavil) and fiurazepam
have been appropriate. It is not clear that (Dalmane). As a result, it is likely that there
steady decline in abuse of glutethimide noted will be basic re-ordering of the drugs in this cat-
since then has resulted only from these con- egory during 1978.
trols, but the decline is clear and is appreciated
It can be noted from Figure 12 that phency-
by physicians. Interestingly, abuse of metha- clidine (PCP), which is widely regarded to be a
qualone (quaalude) has experienced a quite drug that is abused by the young, is not encoun-
similar history in the same time period. The tered frequently in life-threatening overdose
reasons for this are less clear, but methaqua- situations. Moreover, the frequency with
lone has received increasing attention from which this substance is found in such a setting

legal authorities in the last four years and, per- seems not to be changing much from year to
haps as a result, has become less popular with year.
young people.
Some of the trends involving tranquilizers
can be seen in Figure 13. In this Figure, it is

interesting that tne cm ve for Valium is the only


one that seems related to the curve for the total
number of cases, shown in the inset. The de-
crease in the occurrence of Librium in over-
doses has been noted, as has the increase for
Elavil The recent, sharp drop in the curve for
.

Dalmane more
defies explanation and clearly
data are needed before any commentary on
such trends is justified.

Beyondthe major classes of sedatives, tran-


quilizers, and analgesics and the trivial cases of
alcohol, nicotine, and cotinine, other classes of
drugs that are involved in overdoses are relax-
ants and anticonvulsants, of which diphenylhy-
dantoin is most frequently observed, and
antihistaminics, a group headed by diphenhy-
dramine. In addition to these a number of mis-
Figure 11. Trends Amongst Sedatives En- cellaneous compounds have been found in the

course of work. These include nicotine


countered In Drug Identification Program. this

99 ,
Drug 1972 1973 1974 1975 1976 1977 1978 7 Year Total

Diazepam (Valium) 12 21 25 39 35 32 22 186


Chlordiazepoxide (Librium) 14 12 30 15 21 19 8 119

Meprobamate (Miltown) 10 9 21 17 29 22 10 118

Amitriptyline (Elavil) 2 4 5 13 21 25 18 88
Flurazepam (Oalmane) 3 7 6 6 17 30 to 79

Phencyclidine (PCP) 2 1 6 7 7 5 4 32
Imipramine (Tofranil) 0 0 1 B 3 9 4 25
Doxepin (Sinequan) 1 1 2 4 S S 6 24
Tranxene 0 0 0 0 2 10 6 18

Thioridazine (Mellaril} 0 5 1 2 1 2 1 12

Qesipramine (Norpramin) 0 2 4 0 2 2 1 It

Trifluoperazine (Selazine) 0 0 0 0 1 2 1 4
Haloperidol (Haldol) 0 0 0 0 0 1 0 1

Mesoridazine (Serentil) 0 0 0 0 0 0 1 1

Prochloroperazine (Compazine) 0 0 0 0 0 0 1 1

Figure 12. Tranquilizers Encountered In Drug Identification Program.


and alcohol, as well as the local anesthetic xy-
locaine, the preservatives, methyl, ethyl and
propyl p-benzoate, the anorexics phenmetra-
zine and phendimetrazine and the narcotic an-
algesic,methadone. Apart from nicotine and
alcohol however, substances in this general
group are found rather rarely,, i.e. less fre-
quently than four times per year.

IV. SUMMARY
The system described in this paper handles
work connected with
the drug identiflcation
overdoses that occur in the Greater Washing-
ton area. The use of mass spectrometry in this
sense can be regarded as an example of ‘high
technology' but such a designation is not sup-
ported by the cost of the service, which is $75 Figure 13. Trends Amongst Tranquil-
per sample. izers Encountered In Drug Identification
Rather, in a regional setting, it represents a Program.

100 .
piactical solution to the problems of identifying
not only the well-known drugs of abuse, but
also less fiequently-encountered drugs in-
volved in oveidose situations.

REFERENCES

1. Law,N. C., Aandahl, V. A., Pales, H. M.,


and Milne, G., W. A,, Clin. Chim. Acta,
32, 221, (1971); Law, N. C., Pales, H. M.,
and Milne, G. W. A., Clin. Tax., 5, 17,
(1972).

2. Munson, M. S. B., and Field, P. H., J.

Amer. Chem. Sac., 88, 2621 (1966).


,

3. Pales, H. M., Milne, G. W. A., and Ax-


enrod, T.,An(tl. Chem., 42, 1432, (1970).
Milne, G. W. A., Pales, H. M., and Ax-
enrod, T., Anal. Chem., 43, 1815, (1971).
Chao, J.-M., Saferstein, R., and Manura,
J.,Anal. Chem. ,46, 296, (1974).

4. Complex mass spectra, such as El spectia


can be useil for identification of com-
pounds by library searching techniques.
See, for example, Heller, S. R., Anal.
Chem., 44, 1951, (1972); Heller, S. R.,
Pales, H. M., and Milne, G. W. A., Org.
Mass Spectrom., 7, 107, (1973).
The Identification of Drugs by

Kiaus Biemann, Janies E.


and Related Substances by and Catherine E. Costello
Biller,

Computer-Aided Mass Department of Chemistry


Spectrometry Massachusetts Institute of Technology
Cambridge, Massachusetts 02139

The combination of a gas chromatograph chromatograph can be characterized. In ear-


which is capable of separating very small quan- lier days the identification was attempted by
tities of rather complex mixtures with a mass scanning the mass spectrometer at the maxi-
spectrometer, an instrument of high sensitivity mum of each gas chromatographic peak or
producing data which are overabundantly cor- shoulder. The tediousness of this approach,
related to the structure of oiganic compounds, particularly the transformation of the raw data
has emerged as one of the most powerful tools to an interpretable format, as well as the need
in organic analytical chemistry. The two to record mass spectra at many sections of the
methods are highly compatible with respect to gas chromatogram to avoid missing a crucial
sample properties, both being very sensitive but perhaps minor component, led to the auto-
and operating with samples in the gas phase, al- mation of the recording step through digital
beit at quite different pressures. The chief lim- computers (2), Continuous recording of mass
itations, volatility and thermal stability, can spectral data during the entire gas chromato-
often be overcome by suitable chemical gram assured that no information remained
derivatization. unrecorded. This, in turn, caused a complex
It is therefore no wonder that the gas chro- data handling problem because one was sud-
matograph-naass spectrometer has found wide denly faced with hundreds of mass spectra gen-
application in organic, bio- and clinical chemis- erated within the time span of a gas
try, particularly for the identification and quan- chromatogiam, a period of perhaps thirty min-
tification of drugs and their metabolites. This utes. A number of computer approaches were
group is well suited because most drugs are or- developed to efficiently digest the information
ganic molecules of a size {generally ranging in content of these data and G. W. A. Milne has
molecular weight between mass 150 and 500) summarized these (3). Once the techniques
which present no problem for even relatively for the automatic recording of the data and
simple mass spectrometers and they are computer-based identification of the resulting
usually sufficiently volatile, especially if their mass spectra had been developed, it became
polar functional groups are properly deriva- the logical next step to fully automate this proc-
tized, to be amenable to gas chromatography ess, namely to generate a gas chromatogram in
and therefore also to mass spectrometry.. which all fractions are automatically identified
In a previous paper, M. G. Horning (1) has and the information available at the end of the
outlined the use of this technique for the quan- experiment.
tification of various components of body fluids We were prompted to develop this system in
for clinical diagnosis, chiefly in the newborn the course of a program which involved the col-
and infants. In contrast to the quantitation of laboration of our laboratory with various hos-
compounds known to be present
system,
in a pitals in the Greater Boston area to determine
the identification of unknown substances has drugs and related substances in the body fluids
somewhat different requirements. For that of comatose patients who had ingested, acci-
purpose, one has to record complete mass dentally or on purpose, a large amount of one
spectra during the entire gas chromatogram in a or more drugs or other toxic substances. In
continuous, fast, and repetitive mode to insure these cases the results had to be available in as
that every compound emerging from the gas short a time as possible and the frequency of

102 .
these cases (sometimes a few a day) made auto- question. Since this parameter is characteris-
mation mandatory. To improve our own effi- tic and reproducible for each compound under
ciency, and for use in laboratories where very identical conditions or,
if converted to the re-
experienced mass spectrometrists were not tention index (5), even under non-identical
available, it was desirable to automate the pro- conditions, one can eliminate from the com-
cedures so as to requirenominal decision mak-
ing by the analyst. Figure 1 shows a simplified
flow diagram of the steps involved.
The operation and function of the gas chro-
matograph-mass spectrometer have been out-
lined previously by M. G. Horning (1) and the
comparison technique of unknown mass spec-
tra with the collection of known spectra (“Li-
brary Search”) has also been discussed by
G. W. A. Milne (3). In our laboratory we use a
simple matching routine which compares the
relative intensities of the two most intense
peaks consecutive 14 amu sections of the
in
mass' spectrum of the unknown with the same
set of data from each one of the known sub-
stances in the library. A weighted average of
the intensity ratios of the matching pairs (or its
inverse, if the value is larger than 1) is com-
puted and serves as an indication of the degree
of identity of the mass spectnim of the un-
known and each particular known spectrum
(this is the “Similarity Index”) (4). If this
comparison had to be done for all of the pres-
ently available 20,000 to 30,000 known mass
spectra with each of the few hundied mass
spectra recorded during the gas chromatogram
the process would be too slow to allow com-
parison for each one of these spectra. Two
factors help to reduce this time to a tolerably
short period in the case of the identification of
drugs in body fluids. First, one needs to iden-
tify only compounds that are likely to be pres- Plgure 1. Flow diagram for automatic iden-
ent, particularly drugs and their metabolites components of complex mix-
tification of
and other toxic substances. The number of tures.
these is well below one thousand. Secondly,
fiep/inted with permission of authors and publisher.
one can make use of the one piece of informa- Original appeared In paper by C. £. Coste/to, H. S.
tion which the gas chromatograph provides, Hertz, T. Sakai and K. Blemann, Clinical Chemistry 20,
namely the retention time of the substance in 2SS (1974).

103 .
parison those which differ in retention index by emerging from the gas chromatograph at inter-
a preselected value and therefore cannot possi- vals corresponding to the scan time of the mass
bly be identical, To make use of this concept spectrometer, in our case 4.0 seconds (7). As
in real-time applications, however, it is neces- an illustration Figuie 2 shows the gas chroma-
sary to automatically and concurrently deter- togram of a methylene chloride extract of a
mine the retention index of the unknown blood sample from a patient brought to Massa-
material emerging from the gas chromatogiaph chusetts General Hospital after first being ad-
in order to have this information available for
7S NQ FINDS
use comparison process. Such
in the spectral 76 NO FINOS
77 NO FINOS
a technique was developed (6) based on the au- 76 ND FINOS
79 0*466 147 HEPEftlDINE
tomatic, mass spectrometric identification of 60 0*694 147
61 0,687 147 MSPERIOINE
three standard hydrocarbons co-injected with 82 0*662 147 MEPERIDINE
63 0*343 147 MEPERIDINE
64 0*614 meperidine
the sample. From the position of these hydro- OS 0*522
147
147 MEPERIDINE
66 0*462 147 MEPERIDINE
carbons in the gas chromatogram a retention 67 0*442 147 meperidine
86 0*428 147 meperidine
index scale is established and a retention index 69 0*444 147 meperidine
90 0*376 147 MEPERIOI NE
is associated with each consecutively recorded 91 0*332 147 MEPERIDINE
92 0*338 147 MEPERIDI NE
mass spectaim. 93
94
0*353
0*298
147
147
MEPERIDINE
MEPERIDINE
Using two restrictions, a limited library con- 95 0*263 147 meperidine
96 NO finds
97 NO finds
taining only the known spectra of compounds 96 0*362 129 n~alkanc
99 0*270 129 N-ALKANE
of possible interest and the correlation of reten- too 0*178 129 N-ALKANE
|0| 0*OS9 147 meperidine
tion indices, the time required for the compari- 102 0*107 147 meperidine
103 0*122 147 meperidine
son of each consecutive mass spectrum is less 104 0*144 147 MgPERIOlNE
tos ND finds
than it takes to print the name of the most simi- 106
107
NO finds
NO FINDS
108 NO FINOS
lar compound on a line printer. Thus one NO finds
109
110 NO finds
achieves an identification of the materials 1 I I 0*027 130 stfaric acid
112 0*031 92 EMYI CAMATE
U3 0*042 Q2 EHYLCAMATE
114 NO FINDS
115 NO PINOS
116 NO FINDS
117 NO FINOS
na NO FINDS
119 NO FINDS
1?0 NO PINOS
121 NO FINDS
122 NO FINDS
123 NO FINDS
l?4 NO FINDS
125 0,270 225 methaouauone
126 0*554 225 hetkaoualone
127 0*305 225 methaouaudne
128 0, 187 225 mbthaqualdne
129 NO FINOS
130 NO FINOS

Pigure 3. Section of printout of the results


of the automatic identification of each con-
secutive mass spectrum recorded during
togram) of a methylena chloride extract of the gas chromatogram shown in Figure 2.
a blood sample. For details see text.
Heprtnted with permission of authors and publisher. Reprinted with permission of authors and publisher.
Original appeared In paper by C. E. Costello, H. S, Original appeared In paper by C. E. Costello, H, S.
Hertz, T, Sakai and K, Blemann, Clinical Chemistry 20, Hertz, T, Sakai and K, Blemann, Clinical Chemistry 20,
255(1874), 255(1974).

104 .
.

mitteci to a suburban hospital. Figure 3 shows trum itself and the computer analog of the gas
a portion of the above mentioned printout of chromatogram (the total ionization plot, which
the results of the automated identification is generated by summing all intensities in each

program. one of the consecutive mass spectra and plot-


In order to render this listing visually more ting that summed intensity versus time) were
comparable to the gas chromatogram, the utilized. Alternatively, one can plot the rela-
printing of thename of the identified substance tive abundance of a particular mass in the
is displaced to the right by a number of blank course of the gas chromatogram, thus generat-
spaces related to the intensity of the spec- ing a plot that is identical to the one which
trum. Thus a profile resembling that of the would have been obtained if the mass spec-
original gas chromatogram is obtained, along trometer had remained focused at that particu-
which the name of the emerging compound is lar mass rather than having scanned the entire
,

printed . Below a certain level of intensity and spectrum. This plot can be generated after the
similarity with any of the substances in the li- experiment from the complete set of the mass
bi’ary , the comment “no finds’ ’
is printed. The spectra by selecting any particular mass and
values below each line represents the consecu- looking at its intensity veisus time. Since all
tive scan index number, the similarity index, data have been recorded, one can geneiate a
and the code number of the compound in the li- plot of this type for any and therefore all masses
brary. In this section of 55 consecutive spec- Figure 4 shows a graphic representation of
tra, meperidine elutes as the m^or component this principle: The x, y plot at the origin of the z
followed by one of the hydrocarbon standards, axis represents time (x) versus summed inten-

a trace of stearic acid, and finally methaqua- .sity (y), i.e. the gaschromatogram. The same
lone .The next peak in the gas chromatogram X, y coordinate along the z axis (mass scale)
(notshown in this figure) was identified in the represents the relative intensity of that particu-
same manner as diazepam. It is worth noting lar mass during the course of the gas chromato-

that this patienthad apparently a blood level of gram (a so-called mass chromatogram) (8).
meperidine of twice the lethal dose and his sur- Finally the z, y axis represents mass (z) versus
prising survival was due to the efforts and ex- 'intensity (y), i.e. the mass spectrum at any

pertise of the attending personnel at MGH. given point along thex axis.
This example also prints out one of the major Such mass chromatograms have turned out
problems in the analysis of body fluids, namely to be extremely useful for a vaiiety of pur-

that the ingestion of multiple drugs is becoming poses, For example, they can be used to in-
the apparent resolution of a gas
more and more common. Their unambiguous crease
chromatogram and at the same time to remove
detection and identification require methods
that are very specific and free from cross the contributions of adjacent or unresolved gas
interference. chromatographic fractions to the spectrum of
the component of interest (9) The
principle of
recording of mass spectra at repetitive
.

The
this approach is shown in Figure 5 which shows
fixed time intervals during the gas chromato-
gram not only assures that one or more mass a section of a poorly resolved gas chromato-
spectra are taken from each compound present gram (heavy solid line) and superimposed, the
in the mixture but it also adds a third dimension mass chromatograms of m/e 179, 310, and 256,
of which maximized in that general area.
It
the
to the data accumulated, namely time. In all

mass spec- immediately obvious that the component


examples discussed above, only the is

105 .
that gives rise to a sharp peak in the mass chro- tograms of masses associated with the mass
matogiamof mass3J0 is not tesolved from the spectrum of one of those three compounds
front of the very broad peak due to a substance must have exactly the same shape and one can
exhibiting an ion of m/e 256, Fui thermore, the therefore generate the “pure” mass spectrum
component that shows a peak at mass 179 trails by plotting only those masses which maximize
into the one responsible for the maximum of at the same scan index number versus relative
m/e 310. It is clear that all other mass chroma- intensity.

AT SCAN NaSTS

Mgure 4. Representation of the three dimensional nature of the Information content of


•C-MS'data.
intervals.
Figuie 6 compares the conventional total
ionization plot (gas chromatogram) with this
“mass resolved gas chromatogram". It

should also be noted that along the x axis is

plotted not only the spectium index number


representing a time scale (time = spectrum
index numbei times 4.0 seconds) but also the
retention index associated with each scan,
derived from the three internal hydrocarbon
standards as mentioned previously. This
treatment of the data not only leads to an ap-
parent improvement in the gas chromato-
graphic resolution but, as described earlier,
also generates “cleaner" mass spectra. Need-
less to say, the reliability of the comparison of
such spectra with the libiary of authentic mass
spectra leads to a better correlation because
the contributions of incompletely resolved
components have been eliminated. As an ex-
Figure 5. Overplot of three mass chromato- ample, the results of the library seaich of scan
grams characteristic of three partially re- 181 of Figure 6 is shown (Figure 7) both for the

solved components to illustrate the chromatogram where there is a barely


original

pri nciple of ‘‘reconstruction” of mass spec- mass resolved gas


discernible shoulder and the

tra (see text). chromatogram wheie this shoulder has been


converted to a discernible small peak. Coni-
Tills pioccdurc automatically eliminates the
parisOT of the original mass spectium with the
contribution of leading or tailing edges of unre- and related substances a
library of drugs lists
solved peaks to the central component. For number of methaqualone metabolites as well as
example, mass 179 will not show up in the “re-
two diphenylhydantoin derivatives, The simi-
constructed’' mass spectrum of the component
larity index, shown in the right hand column, to
giving rise to the peak at mass 3 10 and mass 256
those authentic spectia is extremely low, indi-
will also be eliminated. This approach repre-
cating a very questionable assignment. How-
sents a very sophisticated method of “back-
than ever, compaiison of the reconstructed mass
ground" subtraction, far more efficient
spectrum at this point in the gas chromatogram
that which one would have obtained by sub-
shows a significantly high similarity to a meta-
tracting the background spectrum obtained at a
bolite of diazepam, the correct identification
place in the gas chromatogram where the signal
(Figure 7).
approaches the base line Equally important is
,

the fact that plotting the sum of the maximizing One of the most unique aspects of our data
processing system is the method of display and
intensities rather than all intensities results in a
permanent storage of all data. Rather than the
plot in which the peaks appear to be extremely
experimenter using display terminals on the
sharp, their widths being one or two scan

107 .
Figure6. Top: OrIgIn aJ total Ionization plot (gas chromatogram); Bottom: “mass resolved
gas chromatogram” generated from reconstructed mass spectra, illustrating the ap-
parent Improvement in resolution.
computer to inspect the data in a dialogue mode microfilm leadei and copies of any image can
by requesting the computer to display the perti- be obtained from a hard copy unit. The mass
nent set of data such as a mass spectrum, a resolved gas chromatogram and the recon-
mass chromatogram, etc., all data are immedi- structed mass spectra are part of the micro-
ately and consecutively displayed on a cathode filmed data set if so desired. In this way the
ray tube and the image permanently recorded experimenter can interpret and inspect all the
on 16 mm microfilm (10). The entire operation data at his or her leisure independent of the
is under computer control and is carried out computer which therefore is available to others
while the raw data recorded during the experi- for the accumulation of the next data set.
ment are converted to their final form, namely This microfilming technique has made it pos-
mass spectra and mass chromatograms. In sible to treata much larger volume of data in a
this manner, the experimenter has all the pri- comprehensive manner than otheiwise possi-
mary data available on microfilm within about ble. One of these involves the simultaneous
15 minutes of the completion of the experiment inspection and interpretation of more than one
because the film is developed in an automatic set of GC-MS data. Generally, it is extremely
film developer (Prostar, Eastman Kodak and cumbersome to compare the data of one such
Company) The microfilm is inspected using a
. experiment with that of another because they

MAINE 6ftAt NOH l«l RUN NO* > 2ARI

ffeSULTS fOe StMe

METHAOUALONE MTn 2 216 0e0 20


StS-OtPHCNVLHynANTOIN t»3>OieTHVt. DEAIV. 426 0e026
METHAOUALONE HTR 4 30 7 0e0 20
01AHeNV|.HV0ANTQ|N l«3-niHETNVI. DEAIV* 372 OeOlB
Mi»THAOUAtat« MTB 3 306 Oe0l3
MET<HquALONE NTS s 3oa OeOlO
MBTHAOUALONE MTS 8 (80 309 deOOS

**«A<

uniHE MAt HQH < RKCONSTHtUCTAD ) lAl AMN NO* « 2601

MAULTA lOe SIM*

•OlAZCAAM MTA 1 440 0*454


neOAZCPAM 242 Oel32

Figure 7. Library search results of scan 181 in Figure 6 before (top) and after (bottom)
“reconstruction” of the mass spectrum free of interference from adjacent components
and background.

109 .
have to be inspected consecutively. The mi- five emerged GC-MS runs are shown in Figure
crofilming system permits one, however, to in- 9 which dramatically reveals that four of the
terleave or merge a number of experiments in five samples do contain the drug as indicated by
such a fashion that the mass chromotograms of the sharp maximum at the same spot in the gas
the same mass number of the various experi- chromatogram (normalized scan number 103)
ments are displayed together on one microfilm at the retention time appropriate for PCP while
frame for easier comparative inspection. It is the fifth one does not show this maximum.
useful to examine a number of GC-MS data sets Since all are plotted on the same scale, one can
for the presence of certain compounds, ex- quickly make an estimate of the relative
pected or unexpected, which could serve to dif- amounts of the drug present. Not shown are
ferentiate one family of samples (for example the mass chromatograms at other characteris-
patients, drugs of various origins, etc.) from tic mass numbers which show the same pat-

another. The identity of the pattern of the tern, thus supporting the conclusion.
mass chromatograms of certain m/e values Another example is the search for codeine in
within each group but differing from the other the same five samples. This substance shows
group which in turn is characterized by a family an abundant peak for the molecular ion m/e 299
of similar mass chromatograms serves this (Figure 11). Its mass chromatogram for the
purpose. same five merged sets is shown in Figure 10. It
As a very simplified example, the survey of is clear that two of the specimens do contain
urine specimens containing phencyclidine and
codeine while the other three are free of it. In
codeine and their differentiation from those these expet iments mass chromatograms (for
all
free of the drug will be discussed. Figure 8
mass 30 to mass 450) were merged and micro-
shows the mass spectrum of phencyclidine filmed for visual inspection. One could, how-
which is characterized by an abundant ion of ever, just as well merge only those mass
m/e 200, as well as others at m/e 86, 91, 243, chromatograms of masses which are expected
etc. The mass chromatograms of m/e 200 of
to be significant.
This approach is very useful for the inter-
comparison of large numbers of GC-MS data
sets by convenient visual inspection. Presen-
tation of the data on microfilm takes the load off
the computer because the merging of all the
data requires a large amount of primary and
secondary data storage capability. The num-
ber of data sets that can be merged is almost un-
limited because the microfilm reader displays
consecutive frames continuously and at any
desired speed. Even though only five or six
mass chromatograms are visible at one time
on the screen, inspection of ten to twenty con-
secutive mass chromatograms is still quite
convenient.
Figure 8. Mass spectrum of phencyclidine. The data processing and evaluation tech-

110 .
M/E SCO URINE 43a 0 = MIN SBO = MAX aisB

Figure 9. mass chromatograms of m/e 200 (characteristic for phencyclidine)


Set of ob-
tained by merging the data from five GC-MS analyses of urine samples showing that four
contain phencyclidine but one does not.

111.
WE SEB LRI^C 4aS 0 K MIN i3SX4 s Sias

Figure 10. Setof mass chromatograms of m/e 299 (charac teristi c of codeine) obtained by
merging the data from the same set of five sa mpie s shown In Fi gures, indicatinj that the
one free of phencyclidine contains codeine and one of them contains both.

112,
REFERENCES

1. M. G. Horning, Proceedings of the Inter-


national Symposium on Instrumental Ap-
plications in Forensic Drug Chemistry,
Arlington, Virginia, May 29-30, 1978.

2. R. A. Hites and K. Biemann, Anal.


Chem., 40, 1217 (1968).

3. G. W. A. Milne, Proceedings of the Inter-


national Symposium on Instrumental Ap-
plications in Forensic Drag Chemistry,
Arlington, Virginia, May 29- 30, 1978.
Figure 11. Mass spectrum of codeine.
4. H. S. Hertz, R. A. Hites and K, Biemann,
niques which we have developed in our labora- Anff/.C/iew.,43,681(1971).
tory and are describing in this paper have
5. E. Kovats, Helv. Chim, Acta, 41, 1915
proven very useful for the handling of the very
(1958).
large amount of data which are generated by a
gas chromatograph-mass spectrometer system 6.
10. H. Nau and K. Biemann, Anflf.Le/rer.v 6,
coupled to a computer and used for specific 1071 (1973).
identifications or general surveys of complex
7. C. E. Costello, H, S. Hertz, T. Sakai and
mixtures of biological origin.
K. Biemann, Clin, Chem., 20, 255 (1974).
The of all original data on 16
availability mm
microfilm, one of the most widely used infor- 8. R. A. Hites and K. Biemann, Anal.
mation storage media, lends itself well to distri- C;je/n.,42,855(1970).
bution and interchange of data. For example,
9. J. E. Biller and K. Biemann, Anal. Let-
the entire set of results can be mailed to the lab-
ters, 7.515(1974).
oratory where the sample originated and used
and interpreted there on a microfilm reader J. E. Biller, H. S. Hertz and K. Biemann,
computer compatible to
rather than requiring a Paper F2, Presented at the Nineteenth
the one which generated the data. Annual Conference of Mass Spectrom-
The permanency, completeness, and au- etry and Allied Topics, Atlanta, Georgia,
thenticity of the microfilmed data should make May, 1971.
ita particularly useful record for forensic prob-
lems where the result may become part of legal
proceedings.

113 .
by
Development of a National
Cornelius G. McWright
Criminalistics Laboratory
FBI Laboratory
Information System Washington, D.C. 20535

I. INTRODUCTION AND BA CKGROUND by a functional crime laboratory with wide for-


ensic experience and operations.
A Criminalistics Laboratory Information On September 8, 1976, the Attorney General
System (CLIS) is being established as a-service of the United States granted permission to the
to all law enforcement agencies — local, state, FBI implement a prototype and full CLIS
to
and Federal . C LIS will be a computerized lab- system. The FBI Laboratory will host the
oratory infoimation system for the collection data base and FBI National Crime Infoimation
and dissemination of foiensic science material Center (NCIC) telecommunications lines will
for law enforcement laboratories. Through it, be used to access it.

forensic science data will be identified, col-


lected, and stored for on-line retrieval by foren-
sic scientists across the country. 11. NCIC TELECOMMUNICATIONS
Late in 1969 and during 1970, the Law En-
forcement Assistance Administration spon- The idea of the National Crime Information
sored Project Search (System for Electronic Center was conceived in the early 1960’s, when
Analysis and Retrieval of Criminal Histories). itappeared that advances in computer technol-
The purpose of this project was to demonstrate ogy could answer law enforcement’s growing
the feasibility of an interstate exchange of crim- need for rapid dissemination of vital informa-
inal history data by means of a computerized tion. The FBI, in conjunction with the Advi-
system. sory Group to the Committee on Uniform
(now Search
In July, 1973, Project Seaich Crime Records and International Association
Group, Inc.) as part of its ongoing program of of Chiefs of Police (which group is made up of
facilitating the application of advanced tech- law enforcement representatives from local,
nology to the administration of criminal justice, state, and Federal agencies) recognized the ad-
formed a committee consisting of 1 6 authorities vantages a computerized index of Information
in the forensic sciences to address itself to could offer criminal justice agencies. The offi-
three topics: (1) information needs of criminal- cer on the street
would have a wealth of infor-
istics laboratories throughout the United mation concerning crime and criminals at his
States; (2) conceptual design of an automated command at all times.
information storage and retrieval system; and In January, 1967, NCIC became opera-
(3) creation of a plan for implementating the tional. This Center serves as a nucleus of a
system. This study was completed in Decem- vast communications network which includes
ber, 1974.
and Federal criminal justice agen-
local, state,
Two primary questions emerged from the cies throughout the United States, Canada, and
study: (1) What agency will host CLIS? (2) Puerto Rico. Designed to complement the de-
What telecommunications network will pro- velopment of metropolitan and state-wide in-
vide communications services for CLIS? The
formation systems, the NCIC in a matter of
study concluded that responsiveness of CHS seconds makes available information essential
to its users and high priority development at the
to the effective performance of law enforce-
national level are more likely if the national ment personnel.
data bases and the processing are maintained
The NCIC computer equipment is located at

114 ,
FBI Headquarters in Washington, D.C. The fullCLIS system; iiiut (4) make determinations as to the
be implemented m CLIS. The Boaid fur-
flies that will
equipment includes rapid access storage units
ihei resolves that the CLIS Opeialing Committee should
with a capability of accommodating records be composed of seven membeis, four to he nominated
representing an index of fugitives, missing per- by the ASCLD Goveining Boaid from among ASCLD
membet taboiaioiies for approval by the Dircctot, FBI;
sons, and stolen property. In a matter of sec-
and ihiee to be appointed by the Diiector. FBI; one
onds, stored information can be retrieved each from the FBI, Drug Enfoi cement Administra-
through the telecommunications network. tion and Bureau of Alcohol, Tobacco, and Flreaims
Lahoratoiies."
Connecting terminals are located throughout
tlie country in police departments, sheriffs of- Although the CLIS Operating Committee has
fices, state police facilities. Federal law en- overall responsibility for developing policy for
forcement agencies, and other ciiminal justice CLIS, the FBI Laboiatory has direct responsi-
agencies. The NCIC computers are linked to bility for the implementation of the system
many state-wide and metropolitan area com- which includes file development and quality
puter systems, thus providing a large number control. In addition, a representative of the
of criminal justice agencies with access to FBI Laboratory is required to furnish a semian-
NCIC files. nual status report on CLIS to the NCIC Advi-
sory Policy Board.
CLIS will function as one file of NCIC.
CLIS messages will be received via telecom-
munications lines by the NCIC computer. Im-
mediately upon receiving the message the
IV. SELECTION OF DATA FILES FOR
NCIC computer will acknowledge receipt of PROTOTYPE S YSTEM
the message. The CLIS message then will be
written on an input queue for later processing. In October, 1976, the CLIS Operating Com-
mittee held its first meeting. The primary issue
resolved was the selection of data files for the
III. MANAGEMENT OF CLIS prototype system. First priority was a recom-
mendation to establish a General Rifling Char-
In developing management guidelines for acteristic File consistent with the ability to
CLIS, provision for maximum input by the determine the possible make and model of a
American Society of Crime Laboratory Direc- firearm from the general rifling characteristics
tors (ASCLD) on all matters has been made present on a fited bullet and the possible make
since the Society is representative of the crime of a firearm on the basis of markings present on
community in the United States.
laboratory fired cartridge cases. The recommendation
The ASCLD Board of Governors had adopted was based on the perception of the widespread
the following resolution; need for this file with respect to crimes against
persons, the unavailability of the file from com-
The Goveimng Board of I he American Society of mercial sources, and the laige number of law
Crime Laboiatory Diieciors (ASCLD) resovles lhal hi enforcement agencies requiring this service for
the implementation of a Critniiialistics Laboratory In-
firearms-related matters.
formation System (CLIS) that a CLIS Opeialing Com-
mittee be established to (I) review and consider rules, Second priority was a recommendation to
regulations and procedures for opeialiott of CLIS; (2)
set slandai ds for participation by crime laboiatories; (3) establish effective standard procedmes for
select laboratories fot participation in the piototype and identification of unknown compounds by the

115 .
use of infrared spectrophotometry. The data caseload, availability of high speed circuits tc

file should be capable of being referenced NCIC and utilization of infrared spectropho-

through a manually coded data format of the in- tometry in compound identification.
ftared spectnjm. This recommendation was Laboratories which appeared to meet the
based on usage by the largest proportion of necessary criteria were sent a questionnaire tc
laboratories since the forensic community uti- obtain additional information and to verify that
technique fora wide variety
lizes this universal they did, in fact, meet the required criteria.
of physical evidence examinations. Infrared On July II, 1977, the CLIS Operating Com-
spectrophotometry is a versatile method of mittee selected 43 laboratories to participate in
analysis and is generally used in laboratories the prototype development of CLIS.
ranging from the highest to the lowest level of
sophistication.
Third priority was a recommendation that in VI. CURRENT DEVELOPMENTAL
the event of successful implementation of the STATUS OF CLIS
first and second priorities, consideration be
given to establishing a Mass Spectral Data File. The present objectives are to implement a
The priorities were developed based upon in- prototype CLIS consisting of the participating
formation available from the initial study by the laboratories, to train personnel who will be in-
CLIS Committee of Search Group, Inc. Addi- volved in the CLIS and to provide user operat-
tionally, input was provided in the areas of ing and training manuals. The prototype
firearms, instrumental analysis, computer sci- system is expected to become completely oper-
ence, and data management by advisors to the ational in 1978 with a General Rifling Charac-
CLIS Operating Committee. teristics File and an Infrared Spectral Data
File. Currently our scientific personnel have
accumulated and encoded data from over
V. SELECTION OF PR OTOTYPE 15,000 bullets and cartridge cases which is the
LABORATORIES basis of the General Rifling Characteristics
File.
A profile of potential CLIS user laboratories
was developed by the CLIS Committee of The General Rifling Characteristics File has
Search Group, Inc. in its 1974 study. This pro- been brought on-line in the FBI Laboratory.
file was developed based upon responses to de-
This prototype laboratory is serving to debug

tailed questionnaires received from 168 the system. It was originally planned to bring

laboratories supplemented by on-site staff in- each prototype laboratory on-line on an indi-
terviews with a representative sampling of vidual basis over a period of time However, it
.

is now felt that the training of a firearms exam-


these laboratories. This material was made
available to the CLIS Operating Committee to iner who will use the system from each of the

assist in the selection process of the prototype prototype laboratories can most effectively be
laboratories . In order to be considered for se- provided if all are trained as a group.
lection as a prototype laboratory, a laboratory Accordingly, a CLIS Users Meeting is sche-
must have responded to the questionnaire in duled for May 30-3 1 , 1978.
the 1974 study. Additionally, other selection Following the development of the prototype
criteria included geography, laboratory size. CLIS, implementation of the complete system
will begin consisting of an expanded data base
and additional participating laboratories.

Vn. SUMMARY
The FBI has been designated by the Attor-
ney General of the United States to develop
and implement a nationwide Criminalistics
Laboratory Information System (CLIS). This
will be a computerized laboratoi^ information
system for the collection and dissemination of
forensic science material for law enforcement
throughout the United States. Through it, fo-
rensic science data will be identified, collected
and stored for on-line retrieval by forensic sci-

enlists across the country.
The forensic science data will be stored cen-
'
trally atFBI Headquarters and transmitted
I
over the National Crime Information Center
(NCIC) telecommunications lines. The FBI
'

Laboratory will maintain the files and perform


related quality control tasks.
The model for a CLIS was developed by
Search Group, Incorporated. This portion of
the project which included the conceptual de-
sign was completed in December, 1974. The
prototype CLIS and complete system will be
developed by the FBI.
Forty-three laboratories will participate in
the prototype CLIS. The objectives of this
phase are to implement a Rifling Class Charac-
teristics File and an Infrared Spectral Data File
and train personnel who will be involved in
CLIS.
Following the development of the prototype
CLIS, implementation of the complete system
will begin consisting of an expanded data
^

;
base and increased number of participating
;
laboratories.
Development and Use of Paul B. Ferrara and Michael P. McGee

Computer Systems in the Division of Consolidated Laboratory Services


Bureau of Forensic Science
Virginia Bureau of P.O. Box 999
Forensic Science Richmond, Virginia 23208

and

Robert J. McGann
Division of Consolidated Laboratory Services
1 North 14th Street
Richmond, Virginia 23219

/. HISTORY This task determined the work load


for the laboratory data acquisition
In 1972, the Virginia General Assembly en- system. A document was prepared
acted legislation to establish the Division of that identified each instrument to be
Consolidated Laboratory Services. This Divi- scanned, scan frequency, foreground
sion consists of four Bureaus —
Forensic Sci- and background processing require-
ence, Environmental Science, Microbiological ments, type and number of reports to
Science, and Product Testing. The Bureau of be geneiated, working and bulk mem-
Forensic Science was designed to provide ory requirements, and operator inter-
comprehensive laboratory services from more face. Schematics were prepared, as
than 350 law enforcement agencies. Pres- required, to show the various hard-
ently, the Bureau of Forensic Science consists ware elements in the system including
of a central laboratory in Richmond and re- instrument detector and internal con-
gional laboratoiies in Northern Virginia, Roan- trols, electrical signal conversion and
oke, and Noi folk. conditioning elements, and computer
On April 13, 1973, Computer Sciences Cor- input/output (I/O) conversion equip-
poration was awarded a contract by Common- ment.
wealth of Virginia, Division of Automated Data
Processing, Richmond, Virginia, to perform an 3. Define Functional Capability of Com-
engineering study to develop a configuration, puter Hardware and Software.
cost, and implementation schedule for a Labo- The functional capability of com-
ratory Data Acquisition System for the Con- puter hardware and software needed
solidated Laboratory in Richmond, Virginia. for the laboratory application was de-
scribed., The various software mod-
A . The Objectives of the Study. ules were identified, including
1 . Gather Detailed Information on Labo- systems, applications, and support
ratory Instrumentation and Opeia-
routines The computer hardware re-
,

tion.
quirements were identified, covering
This includes type and number of Central Processing Unit (CPU) speed,

instruments, instrument characteris- working memory type and size, bulk


tics,number of analyses, problems as- memory type and size, analog/digital
sociated with each analysis, location input/output subsystem characteris-

of instruments, etc. In this regard, tics, telecommunications, operator


discussions were held with cognizant input/output devices, and program-
laboratory personnel. mer station.

2. Define Data Acauisition, Computa- 4. Develop Alternative System Configu-


tion, Control, Reporting, and Display rations.
Requirements. Certain design differences were in-

118 .
corporated into each system includ- The anticipated performance of each
ing: system was evaluated.
a. Technique used to gather and re- Performance factors included the fol-
duce analytical data (e.g., peak lowing:
picking vs. peak integration). a. Reliability.
b. Dedicated minicomputers to han- b. Developmental risks.
dle the same type of lab instru- c. Ease of operator use.
ment vs, general purpose (large) d. Ease of maintenance.
computer to handle all the instru- e. Future expandability.
ments.
Using the cost estimates and perform-
c. Data acquisition and reduction
ance ratings developed above, the opti-
performed on minicomputer-
based system and subsequent re-
mum system configuration was
selected.
port generation, statistical analy-
sis, etc., performed on a larger
central computer system. //. CURRENT
The reason for investigating alter-
CONFIGURATION
native system configurations was to
AND
insure that the system eventually
CAPABILITIES
selected will have the best cost-to-
performance ratio consistent with The system configuration for the Laboratory
the input/output processing require-
Data Acquisition System for the Bureau of Fo-
rensic Science in Richmond (Fig. 1) is com-
ments.
prised of two Hewlett-Packard 2 100 A
5. Conduct Survey of Vendor Hard- minicomputers. System No. 1, with a 32K
ware/Software Packages. core memory and Real Time Executive (RTE)-
A survey of existing computer hard- II operating system is interfaced to Ibw-data-
ware/software packages that included rate instruments such as gas chromatographs.
an instrument data collection program Itacquires data from each instrument in real-
was conducted. Major computer time, stores and processes the data, generates
vendors with proven hardware/soft- analysis reports, provides “background” ca-
ware were included in the survey. pability to enable compilation and debugging of
new programs and nontime critical analysis of
6, Determine Cost and Performance for
each Alternative System. reduced data on-line, and permits communica-
A cost estimate was developed for tion with System No. 2 computer and telecom-

each alternative system. The cost munications with a computer located at the

was broken down into several cate- State Data Processing Center.

gories, including: System No. 2, with 16K core memory and


a. Computer hardware and soft- Disk Operating System (DOS)-III operating
system, interfaced to the high-data-rate in-
is
ware.
struments, such as amass spectrometer. It ac-
b. System design and engineering.
installation, start-up and quires data from these instruments in real-time,
c. Field
generates analysis reports, plots analysis re-
training.

119 .
CtHtnAl C&HPUfCft ftOJM
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KA^VcTlC ^PflQCESSlHO CtHtEP

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• 68 WORDS 06 eil> UAGnE tiE • 16,384 WORDS (l6 BIT) MAGNETIC
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y
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^
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Figure 1. Computer configuration for laboratory automation system.

suits on a cathode ray tube (CRT), and pro- 3. Background to Enable


Capability
vides storage for a libraiy of spectral data and Compilation, Debug, or Non-Time
the capability to search the library for qualita- Critical Analysis of Reduced Data.
tive identification of unknowns. Both sys-
4 . Provides Storage for a Library of Data
tems, with disk storage capacity of five million
for Use in Searches.
bytes, have been operational for approxi-
mately three years. 5. Communicates with the “High-
Data-Rate” Computer System (CPU
#2).
A. Functions Performed by Processor
No.l. 6. Telecommunicates Reduced Data and
Results to IBM-370 Located at State
Data Processing Center.
1. Real-time Data Acquisition for
Low-Data-Rate Instruments with 7. Programmer’s Station Includes a Line
Scan Rates Up to 10 Points per Printer and a CRT to Permit Chem-
Second per Instrument. ist/Scientist to Print Out/Display Raw
Data or Reduced Data and to
2. Analysis Reports Generated Locally Efficiently Develop Analytical and
for Gas Chromatographs. Statistical Routines.

120 .
B. Fiiiictioiis Performed by Processor
5. Communicates Data to the Low-
No. 2,
Data-Rate Computer System (CPU
1. Real-time IData Acquisition for # 1 ).
High- Data- Rate Instruments with
Scan Rales up to 3,000 Points/Sec. per 6. CPU #2 Includes a Graphics CRT
Instrument. with a Hard-Copy Unit to Assist the
Scientist to Quickly Analyze Raw
2. Analysis Reports Generated and Data and Reduced Data.
Analysis Results Plotted for the Gas
Chromatograph/Mass Spectrometer
(GCVMS). C. Gas Chromatograph Data Reduction.

3. Provides Storage for a Library of Time constraints preclude a detailed dis-

Spectral Data for Use in Searches. cussion of all of the capabilities of this
computer system. However, one of the
4. Provides Search Capability. most widely used functions of the computer

Figure 2. Algorithm for chromatogram waveform Interpretation.


system is its data reduction capability used processor’s core memory. The an-
with gas chromatographs. alytical method is the instruction that
the chemist gives to the computer to
I. Chromatograph Signal Monitoring.
tailor it to a particular analysis.
During the period that the injected
The first step in the analytical
sample’s constituents are eluting
procedure is creation of analytical
through the column and passing over
methods which consists of inputting
the detector, the computer monitors
information to indicate the following:
the detector output signal. It

processes the signal “on the fly”, a. Duration of run.


recognizing baseline, peaks, and b. Concentration of internal or
“shoulders” and stores data lelating external standard.
to the sample for further processing at c. Names to apply to identified
the end of the ran. peaks.
Chiomatograph signals are am- d. Response factor to be applied to
plified and digitized at the in- convert peak area ratios to
strument and transmitted serially to concentration.
the computer’s central processor.
Methods are established, altered, deleted, or
Scan rate is up to 10 samples/second.
listed via on-line console terminal (e.g,,
Both hardware and softwai'e filtering
ASR-33 teletybewriter) without affecting the
are provided to control noisy signals.
scanning of detector output signals. The
The algorithm for detection of base-
chemist performs these tasks in a con-
line, peak, and shoulders operates on
versational mode wherein the computer
the absolute value of signal rate of
prompts and checks all conversational mode
change (Fig. 2).
entries, assuring that none destroy system
For each detected peak or shoulder, functioning.
the following data are stored for
Initialization of analysis and calibrate mode
processing by the analysis report
are also pei formed via the console keyboard.
routine;
Lights and pushbuttons at the interface unit
a. Peak Area. serve to indicate the chromatograph’s state and
b. Time ofpeak start, crest, and end. enable the operator to start, abort, or end an
c. Height of peak start, crest, and analysis.
end. The second
step of the analytical piocedure
d. Flags indicating the character of isthe caliblation lun. A standard of known
the peak shape (e.g., peak starts
concentration is injected with the method
on baseline and ends on interfaced in the calibrate mode. At the end of
shoulder.) the run, a new response factor is calculated
2 . Analytical Procedure
fiom the peak area ratios (supplied by the
computer) and the standard concentration
On-line analyses are performed ac- (supplied by the analyst). This new response
cording to analytical method spec- factor replaces the old response factor in the
ifications retained in the central method, ready to be used for the analysis of

122 .
samples of unknown concentration. samples are analyzed. In the library building
The third step of the analytical procedure is mode, a known compound is injected, and the
the analysis of actual samples. The method is method gathers the retention time relative to
interfaced in the analytical mode, and the run some starting time (usually the onset of the
is started. At the end of the run, the solvent front). The analyst enters a name to
concentration is calculated from the peak area correspond to this retention time. This is
ratios (supplied by the computer), and the repeated until a libraiy of up to 99 compounds
response factor (supplied by the method). The per gas chromatographic column is created.
resulting concentration, along with other Each new entry is automatically inserted into
information, is printed out on the teletype- the library according to increasing retention
writer for the analyst. time. A “window” of plus-or-minus X
When more than one component is present number of seconds is defined for the library.
in the sample, the relative retention time is After the library is completed, analysis of
used to distinguish between peaks. In the cali- unknown samples can begin.
or mixtures
bration mode, a response factor is calculated In the library search mode, the method
for each component the analyst includes in the gathers the retention time and the peak area of
method, and this response factor is stored back each peak, and, at the end of the run, gives this
into the method in association with the relative data to the library search program. The search
retention time of the peak. In the analysis program lists all matches within the limits
mode, the relative retention time of each peak defined by the “window” for each peak in the
is compared to the relative retention time of run, printing out the name, retention time, and
each component included in the method, and, peak area foi each match. If no matches are
when a naatch occurs, the corresponding found, the retention time and area of each
response factor is used to calculate the unmatched peak is printed out.
concentration of that compound. An update mode is also available, which will
A special case occurs when the gas recalculate the retention time for each

chromatograph is used to identify an unknown compound in the library, based on the change
substance, rather than to calculate the in retention time for any specified compound in

unknown concentration of a known sub- the library. Additions and deletions to the
stance. Here, the objective is to measure the libraiy can be easily made at almost any time.
retention time of an unknown substance and
compare it to the retention time of known D. Information and Retrieval Functions
compounds. A match in the retention time This computer system is also used for sev-
indicates a possible identity for the unknown. eral information storage and retrieval func-
It would be desirable to have the computer
tions. These information storage programs
search a library of known compounds to see if run in the background partition of CPU No. 1,
there are any matches to help identify unknown in a non-real-time mode. Such background
substances, and such an option is available.
programs appear to be running simultaneously
Again, the procedure involves three steps. with the real-time data acquisition programs,
a method is entered to obtain raw data for
First, but actually run in between cycles of data ac-
a set time. Second, a library of known quisition. For example, test results from some
compounds is constructed, and third, unknown 26 centers that send samples for urinalysis are

123 .

entered into the computer each month. Ap- ,//. ANTICIPATED CAPABILITIES
proximately 3 ,000 samples per month are ana-
lyzed, resulting in an equal number of entries in In conclusion, the Division utilizes a central
the data-flle. From this data a monthly report computer to perform real-time data processing
is generated, which contains valuable informa- in support of various scientific instmments
tion for planning, monitoring trends, and even used in chemical analysis of compounds as a
billing some centers. Data from several part of the Division’s major task of processing
months, or even a whole year, can be retrieved cases and samples. For a variety of reasons it
to make quarterly, semiannual, and yearly re- is desirable to accomplish this real-time data

ports. A man-hours
considerable savings in processing with a distributed network of mini-
was realized in switching from a
manual data computers (Fig. 3) each closer to the laboratory
base to a computerized data base in this pro- than a central computer can be; it is also very
gram, and the reduction in computational desirable to accomplish a new type of data
errors alone was significant. processing within the Division’s capability

puter.

124 .
that of the stoi age and retrieval of management
information related to the operation of the
Division.
Initially an information system centered
around the Division’s prime operation of proc-
essing cases and samples is anticipated; addi-
tional information systems may well be added
later based on other operational functions of
the Division. This information system aspect
of our future data processing requirements rep-
resents an entirely new area of data processing
from those previously done on the existing
equipment. This part of the Division’s data
processing must meet the formal information
requirements of operations and all levels of
management and provide scheduled reports
and inquiry capability for decision making.
Some areas will require on-line access to data;
other areas will need only batch oriented
scheduled and ad hoc reports.
The Laboratory Automation System is being
completed by the Virginia Poly technical Insti-
tute and State University (VPI&SU) under
contract. As designed, this portion of the Lab-
oratory Automation and Data Management
System (LADMS) consists of Texas Instru-
ment 980B minicomputers connected in a dis-
tributive network, Each minicomputer will
support up to sixteen instruments near its loca-
tion and is known as a Remote Instrument Ser-
vice Unit (RISU). The RISUs in the network
will be able to communicate (transmit data)
with all RISUs on the network and a cen-
other
tral control unit (CCU) computer located in the

Richmond laboratories. The use of a distribu-


tive network of minicomputers will allow for
future expansion of the number of instruments
supported, the addition of different types of au-
tomation applications, enhanced capabilities,
and the automation of the three regional Foren-
sic laboratories.
by
The Development and
E. F. Pearson and C, Brown
Applications of the
Home onice Central Research
Computer System of the Establishment, Baughurst Road,
Home Office Central Aldermaston, Reading,
Berkshire, England, RG7 4PN
Research Establishment
Crown Copyright Reserved, 1978

I. INTRODUCTION computer storage and retrieval. Most of the


applications that will be described will be in
The Home Office Central Research Estab- the field of drug chemistry and toxicology but
lishment (HOCRE) got its first computer in the HOCRE computer is used for stoiage and
1972. It was bought with the idea of using it for retrieval of data on most of the major evidence
instillment control, data processing and infor- types encountered in forensic science.
mation storage. It was soon realized that it

was unsatisfactory to use the same computer


and other computers have been
for all purposes //. EQUIPMENT
introduced for instrument control and some of
the data processing requirements. These com- The main computer at HOCRE, which was
puters are usually referred to as dedicated com- installed in 1972, isa Hewlett-Packard 2100A.
puters. Now, the aim is to dedicate small The original specification was 8K core, a paper
process units to each sophisticated piece of sci- tape reader, a magnetic tape drive and a tele-
entific equipment and to develop separate com- type. This has been enhanced almost yearly
puter units for use in storage and retrieval of and the equipment configuration now consists
literature and analytical data. of 16K core, a magnetic tape drive, a cartridge
Manufacturers of scientific apparatus often disc drive, a teletype, a
paper tape reader and
selectone type of computer for use with their punch, a line a visual display unit
printer,
equipment and they develop software pack- (VDU) and an “optical mark-card” reader.
ages for particular applications. In these cir- There are also two terminals, one in an opera-
cumstances it is often convenient to
buy the tional forensic science laboratory, the other in
pieces of scientific equipment, the computer the Enquiry Centre at HOCRE. Programming
hardware, and the software as a single pack- is normally done in Fortran IV.
age, or, if this is too costly, to buy these in
stages. In our experience, the disadvantage of
this approach is that it lacks flexibility for the in. COMP UTER APPLICA TIONS A T
development of new ideas and for applications HOCRE
not connected with large equipment. Never-
theless, it is costly for an organization to de- The main applications of the computer can
velop its own software and in modem forensic conveniently be divided into live recognizable
science laboratories there is a place for both
areas:
dedicated and non-dedicated computers.
Curry and Kazyak and Kazyak (2) re-
(i) storage and retrieval of forensic sci-
(1)
ported a number of computer applications in ence literature:
toxicology and several authors have described
(ii) data banks on evidence types;
applications to other branches of forensic sci-
ence (3-6). (lii) analytical and reference data;
In this paper we shall describe how a variety
of information files of forensic significance
(iv) statistics and data processing;
have been transcribed into a form suitable
for (v) general “housekeeping” progiams.

126 .
.

A. Storage and Retrieval of Forensic Science annual index of the monthly ATI is being pre-
Literature pared each January.
With both the ATI file and the keyword file
The computer is now an integral part of the
the accent is on the need to get the information
total information system provided for forensic
coded and stored very quickly. The articles
scientists in the UK. The computer is particu-
for storing are chosen by the appropriate spe-
larly useful for storage and retrieval of informa-
cialist within HOCRE. Once the items have
tion relating to papers, reports and books.
been selected the bibliographic details can be
The papers for indexing are obtained in four
coded and recorded immediately using clerical
main ways:
grade staff. The keywording takes longer
(i) by careful screening of about 100 se- since the keywords are chosen by Information
lected journals at HOCRE; Division scientific staff after careful reading of
(ii) by running tailored profiles on the selected material. Typically, material appear-
UK Chemical Information Service ing in the literature ina particu lar month will go
(UKCIS) computer (which uses a com- into the ATI same month or the next;
file in that
puter readable version of Chemical it will be entered in the keyword file three to six


Abstracts Chemical Condensates); months after it first appeared. Ways of speed-
(iii) by scanning Current Contents (Life ing up the keyword process are being
Sciences); researched.
(iv) by contact with other Government Es-
1. The ATI file
tablishments.
Each paper accessed by HOCRE for com-
In this way about 300 papers each month are puter given a five figure reference number
is
collected and indexed. ’
or ‘accession number. The information is

Since 1971 subject retrieval from the accu- entered into the first of five available lines of
mulated store of literature has been by a the ATI record entry as shown in Figure 1
keyword system on a computer. By 1975 there The remainder of the first line is then avail-
was a growing awareness that improved able for author details. The title of the arti-
methods of retrieval were necessary for the cle is entered on the second line, and also the
names of authors and other bibliographic de- third and fourth if necessary. The last line is
tailsand this led to the addition of a further re- used to code the article by discipline (two
trieval system known as the “Author Title character spaces available); to indicate
Index” (ATI). whether the item has been sent out in hard
The present literature system utilizes the
keyword and ATI retrieval systems as comple-
mentary search procedures. The keyword 26453 Drayton J V
system contains over 27,000 records which can
be handled through about 20 different pro- Mass Spectrometry In Forensic Science
grams. The ATI system comprises some 14
different computer programs handling a file of . C Chem Br 1975, 11, 439
about 7000 bibliographic references dating
from 1975. The ATI is prepared each month Figure 1. Sample of the Author Title Index
from the records added to the computer. An (ATI)

127 .
copy form to llie operational laboratories in tain the required information and the process
the UK (thiid character position); to present can be time consuming and costly. Addi-
bibliographic refeience details of the paper tionally, the same or similar information is
(the rest of the last line). often required by several laboratories so that
The ATI produced at HOCRE is sent to obtaining information from an outside orga-
centres in several countries where it is nization can become unnecessarily costly.
copied and distributed to authorized forensic In the UK it is preferable to maintain the in-
science laboiatories. formation at a centralized facility. For
many evidence types, of course, no commer-
2. The key word file
cial systems exist for the provision of
The keyword file can be searched using a the- information.
saurus of about 4000 keywords and is useful The main advantage of the keyword sys-
because many papers contain important tem is the speed of use of theand the file

work which is not sufficiently well described small amount of file storage space needed.
by the title alone. For example a paper enti- But the keywording process itself is time
tled “The mass spectrometry of drug me- consuming. The HOCRE is investigating
tabolites” gives relatively little away about the possibility of replacing the keyword sys-
the content. Indeed there are 1
1 papers with tem by title enrichment, i.e., by the addition
this or a vei y similar title in the last 7 ,000 rec- of selected words to the title in the ATI sys-
ords . In the 27 ,000 forensic science records tem. It is also possible that by the end of
on the keyword file, there are almost 1 ,000 1979 or 1980, brief paper summary details
records on mass spectrometry. Not all of will be added to the bibliographic file as a
these deal with drugs, of course. Most aie separate feature. All the papers are stored
keyworded under mass spectrometry and a in hard copy form and on microfilm.
particular drugname and are easily retriev-
able, although others, which deal with ge-
B, Data Banks on Evidence Types
neric classes of drugs, the benzodiazepines
from Casework
for example, present difficulties for the
keyworder. Ten operational forensic science labora-
Apait from drugs, the other references on tories in the UK
submit details to of HOCRE
the keyword file at HOCRE deal with the cases that have been examined. Information
other evidence types encountered in forensic is submitted about evidence types such as tires,

science: glass, blood, paint and so on. glass (household and vehicle), paint (house-
As will be described later, apart from liter- hold and vehicle),fiber’s, blood, and toxic sub-
ature, details on analytical data, and infor- stances. The information is usually recorded
mation from casework are stored on the on optical cards but sometimes on forms, de-
computer. The advantage to the operational pending on the evidence type. At HOCRE the
forensic scientist is that he has a wealth of in- information is stored and indexed in the
formation of direct relevance to him at his computer.
fingertips. For drugs, some of this informa- To take one example, the collection of case
tion is available via commercial information details from toxicological examinations carried
systems but it is often necessary to make en- out in the UK Forensic Science Service was
quiries of several commercial systems to ob- initiated by information scientists at HOCRE

128 .
in 1967.Operational toxicologists provided EXTRACTION TECHNIQUE
HOCRE with details of levels, type, and com- IDENTIFICATION TECHNIQUE
binations of drugs together with some general LEVEL
background information by filling in prepared
The data are presented in tabular form to the
forms and sending them to HOCRE. The data
operational scientists at six-month intervals
were compiled to form the ‘Registry of Human

and specific queries are run at any time on re-


Toxicology”. Because of the complexity of
quest (7).
the steps in the prepaiation of the document it
was only available at infrequent intervals.
C. Analytical and Reference Data Maintained
Not surprisingly, therefore, other methods
of data collection, collation and retrieval were
at HOCRE
sought as the collection of such data was con- Since the inception of HOCRE, the Estab-
sidered to be important and toxicologists were lishment has obtained and stored selected ana-
anxious for it to be continued. and reference data of interest to forensic
lytical
In 1974 it was decided to store the data on the scientists.Data are available on analytical and
HOCRE computer, and a specially designed reference information such as:
optical card, tailored tomeet the needs of the
Analytical Data
UK Forensic Science Service, was designed so
that the relevant data could be accessed. In
IR SPECTRA
addition to duplicating the earlier function of
UV SPECTRA
the “Register of Human Toxicology” the opti-
MASS SPECTRA
cal card enables more detailed information to
TLQGLC
be used to:
X-RAY DATA
Reference Data
(i) monitor extraction and identification
HEADLAMPS
techniques used by toxicologists in an
SIDE LAMPS
attempt to highlight areas of difficulty;
TYPESTYLES
(ii) survey cases involving drugs and driv-
ing; 1. Mass spectrometry
(iii) follow changes in the pattern of dnig
One of our main areas of compound identi-
abuse; and
ficationby the computer is from a collection
(iv) monitor and interpret trends in cases of
of mass spectrometric data. The steps we
multiple drug intoxication.
take in attempting to identify a mass spec-
The project to collect and store these data trum are shown in Figure 2.
from casework began in 1977 and, to date, The njain collection used contains details
there are about 1 ,400 records on file. These of names, molecular weight, and the eight
records contain information such as: largest peaks for each compound. (There
are 1,900 compounds in the collection at
CASE DETAILS The collection is used for com-
present.)
DRIVING, NOT DRIVING puter searching using a discrepancy index
COMPOUND(S) FOUND (DI) (Figure 3), and also for the production of
INGESl'ED preparation manual indexes, indexed by name, molecu-
SAMPLE (BLOOD, LIVER, ETC) and base peak, which are distrib-
lar weight,

129 .
copy foim to the operational laboiatories in tain the required information and the proces:
the UK (third character position); to present can be time corrsuming and costly. Addi
bibliographic reference details of the paper tionally, the same oi similar information i;

(the rest of the last line). often required by several laboratories so (ha
The ATI produced at HOCRE is sent to obtaining information from an outside orga
centres in several countries where it is nization can become unnecessarily costly
copied and distiibuted to authorized forensic In the UK it is preferable to maintain the in
science laboratories. formation at a centralized facility. Fo
many evidence types, of course, nocommei
2. The keyword file
cial systems exist for the provision o
The keyword file can be searched using a the- information.
saurus of about 4000 keywords and is useful The main advantage of the keyworxl sys
because many papers contain important speed of use of the file and th
tern is the
work which is not sufficiently well described small amount of file storage space needct
by the title alone . For example a paper enti- But the key wording process itself is lim
tled “The mass spectrometry of drug me- consuming. The HOCRE is investigalitt
tabolites’’ gives relatively little away about the possibility of replacing the keyword sys
the content. 1 ndeed there are 1
1 papers with tern by title enrichment, i.e., by the additio
this ora very similar title in the last 7,000 rec- of selected words to the title in the ATI syr

ords. In the 27,000 forensic science records tern. It is also possible that by the end i

on the keyword file, there are almost 1 ,000 1979 or 1980, brief paper summary detai
records on mass spectrometry. Not all of will be added to the bibliographic file as

these deal with drugs, of course. Most are separate feature. All the papers are store
keyworded under mass spectrometry and a in hard copy form and on microfilm.
particular drug name and are easily retriev-
able, although others, which deal with ge-
B. Data Banks on Evidence Types
neric classes of drugs, the benzodiazepines
from Casework
for example, present difficulties for the
keyworder. Ten operational forensic science labor
Apart from drugs, the other references on tories in the UK submit details to HOCRE
the keyword file at HOCRE
deal with the cases that have been examined. Informatii
other evidence types encountered in forensic is submitted about evidence types such as tire

science: glass, blood, paint and so on. glass (household and vehicle), paint (hous
As will be described later, apart from liter- hold and vehicle), fibers, blood, and toxic su
ature, details on analytical data, and infor- stances. The information is usually recordi
mation fi'om casework are stored on the on optical cards but sometimes on forms, d
computer. The advantage to the operational pending on the evidence type At HOCRE t .

forensic scientist is that he has a wealth of in- information is stored and indexed in t
formation of direct relevance to him at his computer.
fingertips. For drugs, some of this infoma- To take one example, the collection of ca
tion is available via commercial information details from toxicological examinations carri
systems but it is often necessary to make en- out in the UK Forensic Science Service w
quiries of several commercial systems to ob- initiated by information scientists at HOCF

128 .
in 1967. Operational toxicologists provided EXTRACTION TECHNIQUE
HOCRE with details of levels, type, and com- IDENTIFICATION TECHNIQUE
binations of drugs together with some general LEVEL
background information by filling in prepared
The data are presented in tabular form to the
forms and sending them to HOCRE. The data
operational scientists at six-month intervals
were compiled to form the “Registry of Human
and specific queries are run at any time on re-
Toxicology”. Because of the complexity of
quest (7).
the steps in the preparation of the document it
was only available at infrequent intervals.
C. Analytical and Reference Data Maintained
Not surprisingly, therefore, other methods
of data collection, collation and retrieval were
at HOCRE
sought as the collection of such data was con- Since the inception of HOCRE, the Estab-
sidered to be important and toxicologists were lishment has obtained and stored selected ana-
anxious for it to be continued. lytical and reference data of interest to forensic
In 1974 it was decided to store the data on the scientists . Data are available on analytical and
HOCRE computer, and a specially designed reference information such as:
optical card, tailored to meet the needs of the
Analytical Data
UK Forensic Science Service, was designed so IRSPECTRA
that the relevant data could be accessed. In
addition to duplicating the earlier function of
UV SPECTRA
the “Register of Human Toxicology” the opti-
MASS SPECTRA
cal card enables more detailed information to
TLC;GLC
be used to:
X-RAY DATA
Reference Data
monitor extraction and identification
(i)
HEADLAMPS
techniques used by toxicologists in an
SIDE LAMPS
attempt to highlight areas of difficulty;
TYPESTYLES
(ii) survey cases involving drugs and driv-
ing; 1 . Mass spectrometry
(iii) follow changes in the pattern of dmg One of our main areas of compound identi-
abuse; and
ficationby the computer is from a collection
(iv) monitor and interpret trends in cases of
of mass spectrometric data. The steps we
multiple drug intoxication.
take in attempting to identify a mass spec-
The project to collect and store these data trum are shown in Figure 2.
from casework began in 1977 and, to date, The nyain collection used contains details
there are about 1,400 records on file. These of names, molecular weight, and the eight
records contain information such as: largest peaks for each compound. (There
are 1,900 compounds in the collection at
CASE DETAILS present.) The collection is used for com-
DRIVING,NOT DRIVING puter searching using a discrepancy index
COMPOUND(S) FOUND (DI) (Figure 3), and also for the production of
INGESTED PREPARATION manual indexes, indexed by name, molecu-
SAMPLE (BLOOD, LIVER, ETC) lar weight, and base peak, which are distrib-

129 .
-
4). This enables us to attempt to identify
Laboratory compounds with a computer search (9) on
combinations of
1 Check HOCRE Index
(i) substi uctural fragments
(ii) molecular weight range;
2 Manual MSDC Search (iii) molecular formula;
x (iv) atoms present;
1

Enquiry Sent To HOCRE (v) numbers of atoms of specific


1
1
elements, i.e.. Cl, Br, F, S. P.

3 MS Spectral Search As with mass spectral collection, man-


the

f ual indexes of our Wiswesser file (molecular


4 Search Wiswesser File weight and molecular formula) are produced
t for the UK forensic science laboratories. If

5 Spectral Interpretatation theunknown is still unidentified, then, if the


case demands it, spectral interpretation may
have to be carried out (Step 5). Spectral
Figure 2. A model procedure for the identi- processing on the Micromass 16F low reso-
fication of a mass spectrum in a UK Home lution mass spectrometer at HOCRE is done
Office Forensic Science Laboratory. on the computer via a Garrick interface and
(MSDC « Mass Spectrometry Data Center) an off-line magnetic-tape deck. The pro-
grams for this have been developed at
Peaks 44 91 65
HOCRE.
Amphetamine
MW D|
D. Statistics and Data Processing
44 91 45 65 42 43 41 51 135 0. 274
Amphetamine The discriminating power of a chromato-
graphic system is defined as the probability that
44 103 91 65 42 104 45 102 135 0,648
two compounds selected at random would be
Ethyl Amphetamine
separated by that system (10). Two com-
72 44 91 73 65 42 56 0 163 1644 pounds are regarded as having been separated
if the difference between their chromato-
Figure 3. Part of a mass spectral search graphic values exceeds a certain critical limit
using peaks at 44, 91 and 65. (D/=D/screp- termed the error factor. Compounds that are
ancy Index.) not separated are assumed to be chromato-

uted to the UK forensic science laboratories graphically similar, i.e., they form a matched
(8). These actions are summarized in step 1
pair. Discriminating power (DP) can be ex-
of Figure 2. Steps 2 and 3 involve searching
pressed by the equation
the available large commercial collections of
DF=1
2M
spectra. If we still have not identified the N{N-\)
compound, we have, on our computer, a file Where M«the number of matched pairs from
of about 8,000 compounds each coded with all compounds recorded;
name, molecular formula, molecular weight, A^=the number of chromatographic
and Wiswesser line notation (WLN) (Step values recorded.

130 .
Discriminating power measurements can be The comparison is repeated for each of the

applied to any chromatographic system or three TLC systems used and the index is com-
combination of systems. At HOCRE this ap- puted between the data tor the unknown drug
pioach has been used, for example, to select and the data for each drug held on file. The
Thin-Layer Chromatography (TLC) systems computer then prints out the names of the
for the separation of basic, neutral, and acidic drugs producing the smallest DI. The ap-
drugs (11). proach can be extended to include other pa-
The number of paired comparisons is ex- rameters besides TLC data, for example other
tremely large when many systems are investi- chromatographic properties, and spectro-
gated and these comparisons and the DP metric data (Figure 4).

calculation are carried out on the HOCRE The approach is now being tried on an inter-
computer. The best system (or combination of laboratory basis for the identification of drugs.

systems) is the one which produces the highest The level of success has been such that data for
DP value, For example, for basic drugs, three other types of drugs are to be added to the file

(A. C. Moffat, personal communication).


TLC systems were chosen as most suitable for
the separation of the 100 basic drugs most com- Similarly, data processing progfams are

monly encountered in forensic science (12). being used for comparison of bullet striation
patterns, handwriting characteristics, color
The systems are: cyclohexane-toluene-dieth-
ylamine; chloroform-methanol; acetone. data, as well as for multi-element data from an-
alyticalmeasurements on paint, glass, and
Unknown basic drugs can be identified by
hair. Programs are run for the processing of
running them on the three TLC systems to-
radioimmunoassay data.
gether with reference compounds (13). The
compounds are
data obtained for the reference E. General
used to correct those for the unknown drug.
The remaining computer applications are
The data for the unknown are then compared
quite simply of the “housekeeping” type
against data held on the computer for the 100
and small individual and non-specific
most commonly encountered basic drugs.
requirements.
Clearly, exact agreement between the data for
the unknown drug and any of the data on file for
the three systems run is most unlikely and a sta- T1 T2 T3 GC UV SH Dl

tistical approach is used to produce a Discrep-


Benzphotamlne 67 70 70 1850 257 PPT 2.6
ancy Index.

Meclozine 68 79 70 3050 230 0 9.6

Diothylproplon 62 63 64 1485 263 -6 184


Xt=Rf for the unknown drug on .system /;

Yf=Rf fov the any one of the 100 drugs on sys-


Figure 4. Part of a computer output using
tem i ;
the Compound Identification program.
\j=the standard deviation of the system as de- (T1, T2, T3ssThln Layer Chromatography
termined by inter-laboratory experiments Systems, GC=Gas Chromatography Re-
depends uponZ,).
(this tention index, Dl= Discrepancy index.)

131 .
IV. USE OF THE HOCRE COMPUTER terials for identification purposes;
BY OPERATIONAL FORENSIC (iv) the running of a selection of statistical
SCIENCE LABORATORIES and data processing activities;

The information held on the computer at (v) a general puipose computing facility in
HOCRE is used by staff at the UK forensic sci- support of research and development
Queries from these labora- in forensic science.
ence laboratories .

tories are dealt with by telephone or telex. The new mini-computer will have on-line ter-
The officer in charge of the computer accepts minal facilities, 128K of store, a disc storage fa-
the inquiry, carries out the necessary work and cility (SOM bytes), and a similar range of
then passes the information back to the in- The con-
peripherals to the existing machine.
quirer. At the moment the Enquiry Centre of figuration of the present HOCRE computer is
the Information Division at HOCRE handles shown in Figure 5 while Figure 6 shows the
over 200 queries each month configuration of the proposed machine.
On-line facilities have been considered but
have been rejected because of the age and limi-
tations of the present computer. Experimen-
tal acoustic coupler-computer links have been
successful for some of the UK forensic science
laboratories but depend upon the quality of the
telephone links.

V. FUTURE DEVELOPMENT
Because of the successful way computer
usage has developed at HOCRE, plans are well
Figure 5. Present Home Office Central Re-
advanced to replace the HP 2100A with an-
search Establishment computer configura-
other computer in 1979. The existing HP
tion. (CPU = Central Processing Unit.)
210OA probably be “dedicated” to an or-
will
ganic mass spectrometer already in service.
The new computer will be required to support
on-line terminals in the UK forensic science
laboratories for:

(i) an interactive literature retrieval sys-


tem;

(ii) an interactive retrieval project for the


collection of data from cases examined
in forensic science laboratories;

(iii) an interactive information retrieval Figure 6. Proposed Home Office Central


project for the collection of analytical Research Establishment computer config-
and reference data on various ma- u ration. (CPU - Central Processing Unit.

132 .
VL CONCLUSION 7. Owen, G. W., and Brown, C., Home Of-
fice Central Reseat ch Establishment, Re-
A computer-based system foi the storage poitNo 253, (Restricted Circulation)
and retrieval of forensic science literature in- HOCRE, Baughurst Rd., Aldermaston,
cluding drugs has been described. The Betkshire, England, 1977.
HOCRE computei system also involves a co-
8. Ardrey, R. E., and Brown, C., Home Of-
operative scheme of information exchange for
fice Central Research Establishment, Re-
dmgs and toxic materials encountered in foren-
sic science. Analytical and reference data are
No 194A, (Restricted Circulation),
pot t

stored and “unknown” drugs can be identified


HOCRE, Baughurst Rd., Aldermaston,
Berkshire, England, 1976.
by use of a discrepancy index. Data on other
evidence types of interest to forensic scientists 9. Ardrey, R. E., and Brown, C.,7. Foren-
are also coded and stored. sic Sci. Soc.,
17,63(1977).
The system is to be developed in the UK into
10. Moffat, A. C., Smalldon, K. W., and
an interactive on-line system involving opera-
Brown, C,,J, Chromatogr., 90, 1 (1974).
tional forensic science laboratories. It is

hoped that the on-line system will be opera- 11. Owen, P., and Moffat, A. C., Eighth In-
tional in late 1979 or early in 1980. ternational Meeting of the Association of
Fotensic Sciences, Wichita, May 1978,
Int. Mic. J. of Leg. Med., (to be
published).
REFERENCES
12. Moffat, A. C., Home Office Central Re-
search Establishment, Collection of Ana-
1. Curry, A. S., and Kazyak, L., Sixth In-
ternational Meeting of the Association of
lytical Data for Drugs, HOCRE,
Baughurst Rd., Aldermaston, Berkshire,
Forensic Sciences, Edinburgh, Septem-
England, 1976.
ber 1972, Int. Mic. J. of Leg. Med,, 8
(1973). 13. Moffat, A. C., J. Chromatogr., 110, 341
(1975).
2. Kazyak, L,, J. Forensic Sci., 19, 147
(1974).

3. Bryan, J. L,, Icove, D. V., Fiie Journal,


20(1977).

4. Gardner, G. Y., Carnahan Conference on


Crime Countermeasures, p 149, College
of Engineering, University of Kentucky,
May 1977.

5. Blaisdell, B. E., Anal. Chein., 49, 180


(1977).

6. Heller, S. IL., Science, 195, 253 (1977).

133 .
Chromatographic Advances
Session

I. Instrumentation

II. Methodology

III. Drug Analysis


by
Applications of to HPLC
Brian B. Wheals & Ray L. Williams
the Analysis of Drugs
Metropolitan Police Forensic
Science Laboratory
109 Lambetli Road,
London SEl 7LP
England

/. INTRODUCTION matography (TLC) and gas chromatography


(GC) were the principle methods of analysis for
During the 1970’s there was an amazingly diugs. The method of operation of both these
rapid development in high performance liquid techniques had an important bearing on the de-
chromatography (HPLC). This arose partly sign of HPLC instruments since, in general, it

from the needs of the pharmaceutical industry was the manufacturers of TLC and GC equip-
for new analytical techniques and partly as the ment who extended theii activity to include
result of the efforts of instrument manufac- HPLC.
turers, mainly in the United States, to satisfy A comparison of the three methods is shown
these demands and also create new products in Table II.
for their factories. The
three final points are the ones which
Although we commenced work with HPLC have most influenced the development of
in 1971, it soon became clear that the factors
1)
HPLC in our laboratory and need to be elabo-
noted above had resulted in equipment which rated. The solvent used in HPLC plays a very
was not particularly well suited for use in foren- important part in a separation (unlike the car-
sic science laboratories. For instance, in rier gas in GC) and slight compositional
Table I are listed some of the major differences changes are often accompanied by large
in analytical requirements between these lab- changes in the elution volumes of particular
oratories and those of the pharmaceutical compounds. Instrumentally it is inconvenient
industry. and time consuming to change solvents, and to
Prior to the advent of HPLC, thin layer chro- avoid this problem it is necessary either to use

Table I

Analytical requirements of pharmaceutical and forensic laboratories

PHARMACEUTICAL INDUSTRY FORENSIC LABORATORY

High throughput of samples of the 1) Widely differing range of sample


same type type, and numbers
2} The components in a sample are 2) Wide ranging differences In com-
usually known and the levels position, particularly with illicit prep-
should be within well defined limits arations

3) Analysis carried out by a specialist 3) Analysis to be carried out by indi-


group vidual reporting officer or their
assistants

4) Quantitative analysis of primary 4) Most emphasis on qualitative anal-


Importance ysis,aithough quantitation is Impor-
tant in toxicology cases and for
comparative drugs analysis

136 .
each instrument to carry out one particular continuous deactivation of the column, making
analysis, or to select different analyses which it difflcult to ensure long-term reproducibility
can be achieved with the same column and sol- of elution volume. Emphasis has therefore
vent system. The high cost of commercial in- concentrated on developing column separa-
struments would make this approach very which the eluting solvent is itself highly
tions in
expensive. polar.Thus, our strategy for HPLC (1) can be
The sequential nature of HPLC is another as- summarized as follows: to develop low cost
pect of the technique which has had a marked systems, for specific analyses based on the use
influence on the way in which we us.e it. For of polar eluting solvent, avoiding complica-
many years TLC has provided the most conve- tions such as gradient elution. Furthermore,
nient method of screening for drugs in a foren- the HPLC would be used for those applications
sic laboratory, and it seems logical to use where existing separation methods, particu-
analogous conditions to achieve separations by larly GC, could not be employed to supplement
HPLC. In practice, however, most TLC the information obtained from the initial
methods use a silica adsorbent and a non-polar screening of samples by TLC. Before con-
solvent and these are piobably the worst type sidering some examples of such analyses it is
of conditions for ensuring reproducible analy- appropriate to detail some areas where con-
ses with a sequential method such as HPLC. siderable savings in the cost of equipment can
Itdoes not matter in TLC whether polar com- be achieved while still maintaining adequate
ponents (e.g., water) introduced with the sam- performance.
ple deactivate the adsorbent, for the plate is

used once only and many samples can be run //. LOW COST EQUIPMENT
simultaneously. With HPLC, however, the
introduction of such a sample would lead to a For routine analysis based on an isocratic

Table II

Comparison of TLC, GC and HPLC as analytical methods


TLC GC HPLC

1) Applicable to volatile 1) Applicable only to 1) As for TLC


and involatile com- volatile compounds
pounds
2) Difficult to quantitate 2) Readily quantitated 2) Readily quantitated

3) Versatile 3) One instrument read- 3) Solvent specificity


ily used for a variety makes Instruments
of analyses less flexible

4) Simultaneous analy- 4) Sequential analysis 4) Sequential analysis


sis

Moderate
'

5) Low cost 5) Moderate cost 5) to high cost

137 .
4)

(i.e., the same solvent system throughout the The way in which substituting a liquid

analysis) elution the equipment for an HPLC mobile phase for a gaseous one i educes
system can be greatly simplified and can con- the flexibility of an instrument was not
sist of: appreciated. A single GC can be used
for many different analyses, wheieas
Cotnmeicuil cost Our cost
HPLC equipment is best used in a dedi-
a) pump up to £2500 £250-300 cated mode.
b) injector up to £500 £15-25
Cumulatively these “mistakes” led to the
c) column up to £150 £15-20
commercial development of equipment of high cost, oc-
d) detector up to £2000
cupying much space and having a performance
detectors
that was often much better than required. This
mainly used
point becomes more apparent when the com-
Thus, savings of close £3000 per system
to ponents of the HPLC are considered in detail.
can be achieved. It should however be em-
phasized that this is on equipment for routine
A. Pumps
analysis. advantageous for method de-
It is

velopment to use the more expensive pumps, The key features of most of the pumps spe-
although we would still use these in conjunc- cifically developed for HPLC are:
tion with low cost injectors and columns.
1) A virtually pulse free solvent delivery.
On the other hand, commercial instruments
have a number of disadvantages compared 2) A wide range of pressure capacity —^typ-
with laboiatory assembled units which arise ically from 0-6000 psi.
because of the influence of OC design con-
3) Variable and constant delivery fate from
cepts. These can be summarized thus:
O-lOml/min.
1) The design of liquid chromatographs
4) Low internal dead volume, useful when
with all the equipment housed in a single
changing solvents or carrying out gradi-
box rather like a gas chromatograph.
ent elution.
This tends to be inconvenient (particu-
larly if solvent leaks occur) and adds to In practice for analysis using 14” o.d. col-
expense. umns packed with microparticulate packing
material, much of a pump’s performance is su-
2) The success of temperature program-
perfluous, for most chromatographers use a
ming in GC led to the mistaken assump-
flow rate of 1 -2 ml/min with a pressure require-
tion that gradient elution could be
ment of about 500-2000 psi. Hence, pumps
equally effective in HPLC. In fact, gra-
with a performance specification well below
dient elution imposes considetable de-
that of a commercial HPLC pump can be of
sign and operating difficulties in many
value. Pulse free flow is important because
instances.
many of the detectors used in HPLC are sensi-
3) The role of temperature was overempha- tive to solvent pulsing, but it is important to ap-
sized in the early liquid chiomato- preciate that pulsing must be absent from the
graphs. There is no need for elaborate solvent stream as it emerges from the column
thermostatting of columns. not necessarily from the pump . It is compara-

138 .
lively simple to remove much pump induced lication /. Liquid Chromat.)
pulsing by incorporating a Bourdon tube gauge The method of injection is ci ucial for highest
into the solvent line between pump and col- performance. Thus, injection directly onto a
umn. Moreover, the packed bed of the column metal frit at column as in many
the top of the
also provides a smoothing effect. It has been commercial instruments (Figure 2A) gives low
our experience that the flow from single piston efficiencies because of stagnant solvent on ei-
reciprocating pumps can be smoothed suffi- ther side of the capillary inlet, Similarly, injec-
ciently to cause the minimum of disturbance to tion onto a metal mesh on top of the stationary
many commercially available HPLC detec- phase with or without a metal holding collar
tors. Fortunately for the “do-it-yourself’ (Figures 2B and 2C) is unsatisfactory since the
chromatographer such pumps are readily avail- injected solution back diffuses into the solvent
able at low cost as they have industrial uses and above the column.
have been employed for many years for fluid We have found that the best method is to
metering. Although these pumps often have a pack the column with a layer of 80-100 mesh
large internal dead volume this does not detract glass beads and inject the sample into the
from their value for routine analysis where sol- center of these using a hypodermic syringe
vent of the same composition may be pumped (Figure 2D). The sample, which can be up
for weeks or years at a time. 10-15 p,l, is thereby confined to a small volume
well away from the walls of the column and effi-
B. Injectors ciencies are greatly improved Figure 3 shows
.

chromatograms obtained with a 25 cm column


The introduction of a sample to the top of the packed with 5 fxm silica modified with
chromatographic column is one of the key
areas in HPLC, for a badly designed injector
can lead to appreciable loss in the separating
power of a column. Some excellently engi-
neered iiyectors have been developed to allow
a syringe injection to be made with no drop in
pressure in the column. Ironically, despite
their high cost such injectors rarely give the
best performance since appreciable band
spreading occurs as the sample leaves the in-
jector and enters the column. Again, the “do-
it-yourself’ chromatographer Is in luck for the
low compressibility of liquids can be used to
good effect if stop-flow injection is made.
Thus, by stopping the flow in the column, al-
lowing the pressure to drop to atmospheric and
making a direct syringe injection to the top of
the packing material bed, followed by resump-
i

tion of pumping, highest column efficiency can Figure 1 HPLC injectors. A, commercial
.

be obtained with a simple TEE joint, as shown valve injector; B, simple Vto" tee for stop-
in Figure 1 .
(B B Wheals, submitted for pub-
. .
flow Injection.

139 .
groups on the surface. The use of glass beads
doubles the plate value of the column,

C. Columns
Much mystique has been built around the
columns used in HPLC since special slurry
techniques are necessary for their packing.
Without going into details it is true to say that
with a pneumatic amplifier pump costing less
than the price of a single commercial column it

is pack high quality columns. The


possible to
ability to do this opens up the whole field of
HPLC for the chromatographer to then begin
to experiment to produce packing materials ap-
propriate to his special analytical problems.
We have never used a commercially packed
Figure 2. Modes of Injectfon. A, onto frit at column in our laboratory but have instead
top of column; B, onto mesh resting on col- taken a small particle size silica, e.g., Partisil 6
umn packing; C, onto mesh held by metal ^m (Reeve Angel, Maidstone, England) and
collar; D, into glass beads at top of column. graded this by sedimentation to improve the
particle size range (2) In this way efficiencies
.

of up to 18 ,000 plates have been obtained with a


25 cm column.
It is also possible to modify the silica quite

easily by treatment with reagents such as octa-


decyl trichlorosilane so that reverse phase
chromatography can be carried out (see 3). A
particularly interesting development in this re-
spect Is the use of vinyl trichlorosilane to intro-
duce reactive —CH—CHa groups onto the
silica. These can then be used as the sites for
addition of other vinyl monomers, such as
acrylonitrile or diethylaminoethyl acrylate (4)

D. Detectors

There are a variety of these based on differ-

ent principles of operation, e.g., refractive


index, moving-wire, etc., some of which are
Figure 3. Column performance resulting shown Table
in III.

from different injection modes. Figures In- Because of its simplicity and reliability we
dicate plate numbers for same column with have based our HPLC machines on ultra-violet
Injections indicated. (UV) absorption using an inexpensive variable

140 .
Table III

Detectors for Liquid Chromatography

DETECTOR TYPE DETECTION LIMIT (g)

UV SELECTIVE 1q_8
FLUORIMETRIC SELECTIVE 10-"
MOVING WIRE/FID UNIVERSAL io-»
REFRACTIVE INDEX UNIVERSAL 10-=

wavelength UV spectrometer, e.g., Cecil In- fluorescent ergot alkaloids and related com-
struments C2I2. This limits analyses to those pounds on a column containing 5 /^m silica
compounds which have UV absorption but the eluted with an ammoniacal methanol/water
restriction is not as narrow as would be ex- solvent (Figure 4). The separation mecha-
pected since many drugs have such chromo- nism is not clearly understood but seems to
phores. involve siloxane groups on the silica surface
The alternative means of detection which we rather than silanol groups . The same type of
selected was fluorescence. For this either a conditions are used here for many basic
commercial spectrofluorimeter (Perk’in-Elmer drugs and it has the advantage of being a

MPF 2A) with a laboratory constructed capil- chromatographic system that is stable and
lary cell (5) or a single home made filter-based the silica is obviously not in a form which
fluorimetrlc detector (3) is used. The and UV leads to poisoning by the addition of water,
fluorescent detectors can also be coupled in se- which is the most common contaminant for
ries to monitor the effluent from the column. columns based on adsorption with a non-
aqueous eluent.
III. APPLICATIONS In the case of LSD the fluorescent detector
gives a high level of specificity to the analysis
The analysis of drugs and their metabolites as well as the sensitivity to cope with the low
by HPLC has recently been reviewed by level of doses associated with LSD (i.e.,
Wheals and Jane (6) What follows hereafter is
.
100-150 /rg per microdot). Only aportion of
an indication of the uses to which the technique the whole tablet is required and is merely
has been put in our laboratory. crushed with the eluting solvent.
LSD can also be analyzed when present in
A. Drugs Arising From Illicit Possession
blood, urine (7) and viscera (J, M. Wiles,
Cases
J.Hughes,!. Christie & M. White, to be pub-
Most of the applications have been of this lished J. Chromatogr.y, the extraction pro-
kind, one of these being in the screening of ly- cedures used convert some of the LSD to
sergic acid diethylamide (LSD) microdots for iso-LSD. Hence, there is a second peak in
the active ingredient. the chromatogram to reinforce an identifica-
tion made on the retention volume of the
1. Lysergic acid diethylamide.
LSD peak. Similar HPLC procedures in-

LSD can be readily separated from other cluding monitoring the column effluent by

141 ,
Figure 4. Separation of ergot alkaloids. A. Figure 5. Separation of amphetamine type
Synthetic mixture. B. Extract from LSD mi> drugs. Column 25 cm x4.9 mm I.D. packed
crodot. Column 15 cm x4.9 mm
I.D. packed with Partlsit 5 (7 fim) Solvent methanol: 2N
with Partisil 5 (7 ^m) Solvent, methanol: ammonium hydroxide: IN ammonium ni-
0.2% ammonium carbonate (3:2) Plow 1 ml trate (27:2 :1 ) Flow 1 ml min~ at 1 500 psi. De-
'

min~‘ at 1600 psi. Fluorimetric detector. tector UV at 254 nm.


1. Lysergic acid 1. Benzphetamine
2. Lysergamide 2. Phendimetrazine
3. LSD 3. Phenmetrazine
4. Lysergol 4. Amphetamine
5. iso-LSD 5. N-methylephedrine
6. Ephedrine
7. Methylamphetamlne
8. Methphentermine

142 .
immunoassay techniques have also been re-
ported (8).

2. Amphetamine type drugs


Figure 5 shows that the separation of many
amphetamine type drugs can be accom-
plished rapidly by isocratic elution on a silica
column (1, 2).

3. General basic drugs

One of the advantages of a UV detector is


that monitoring the column effluent at differ-
ent wavelengths enables a wider range of
drugs to be analyzed than with other detec-
tors. This is illustrated in Figure 6 for exam-
ple, wherein ephedrine, amphetamine, and
methylamphetamine are detected by their
absorption at 254 nm whereas papaverine,
thebaine and dihydrocodeine are monitored
better at 278 nm. The analyses are carried
out with a 25 cm silica column. Further ap- 5.
plications of such general analyses are listed Figure 6. Separation of basic drugs, show-
in references 1 and 2. ing effect of d Iff erent detector wavelengths
A, 278; B,254 nm. Other conditions as In

4. Opiates Figure 5.
1. Benzphetamlne
The opiate drugs are readily separated on a 2. Papaverine
25 cm silica column, Figure 7, and this sys- 3. Protopine
tem has also been used for the quantitative 4. Thebaine
analysis of the so-called “Chinese” heroin 5. Amphetamine
preparations which were in circulation in 6. Codeine
London a year or so ago (1). The latter, in 7. Morphine
addition to heroin and monoacetylmorphine 8. Ephedrine
or monacetylcodeine, contained caffeine 9. Methylamphetamine
and strychnine. The procedure involved 10. Dihydrocodelne
HPLC analysis of the original material fol-
lowed by a second analysis after the sample
Psllocin and psilocybin
had been hydrolyzed with alkali to give mor-
phine and codeine since the monoacetyl de- These substances are the active principles
rivatives of these were not separated in the in psilocybes mushrooms which grow quite
initial chromatogram. readily in the U.K. Unfortunately, the En-

143 .
ammonia. The eluted compounds are moni-
tored at 254 nm.

6. Diconal

This material, which contains cyclizine


and dipipanone, is becoming increasingly
prominent in cases submitted to our labora-
tory. The components are not resolved
under the normal conditions for the separa-
tion of basic drugs on silica and elute almost
at the solvent front. By treating the silica
with (3-mercaptopropyl)-trimethoxysilane it

is possible to attach mercaptopropyl groups


and carry out elution with
to the silica the
same solvent system as that used for the
basic drugs (B. B. Wheals, to be published,
J. Chromatogr , ). An excellent separation is
obtained, Figures.

7. Cannabis

Surface modification of the silica with oc-


tadecyl-trichlorosilane is necessary in order

Figure 7. Separation of opium alkaloids on to achieve the best separations of the canna-
binoid constituents. Figure 9, most of which
silica: use in opium comparison. Column
25 cmx4.9 mm I.D., packed with Partisil 5. have been identified by comparison of their
Chromatographic conditions as in Figure chiomatographic behavior with authentic
5. Samples 1 and 2 are different opium ex-
specimens or by measuring the mass spectra
tracts: (a) papaverine, (b) narcotine, (c) of the eluted peaks (9).

thebaine, (d) codeine, (e) morphine HPLC is especially useful for the detec-
tion of the thermally labile acidic cannabi-
noids and provides a convenient method for
glish law present not clear as to whether
is at
the comparison of samples in order to estab-
or not their possession is iilega! but neverthe-
lish common origin (10), particularly if detec-
less samples have been submitted for analy-
tion is carried out at two wavelengths (Figure
sis by HPLC. A methanolic extract from
10). Quantitation can be carried out quite
the mushrooms is redissolved in methanol /
satisfactorily as is shown (1 1) by the data in
chloroform onto the col-
(9:1) for injection
Table IV.
umn. Aready separation can then be
achieved with 25 cm column packed with 5 Because of the reliability of quantitation it

jam Partisil-5 silica using methanol, water, is also possible to carry out studies on the
IN ammonium nitiate (240: 50: 10) eluent riseand fall in concentration of various com-
which has been adjusted to pH 9.7 with 0.88 ponents as the sample ages (Figure 1 1).

144 .
Figure 9.Separation of cannablnoid con-
stituents. Column 25 cmx4.9 mm I.D.
packed with 5 /xm silica modified with C,g
groups on the surface. Solvent methanol:
0.02N sulphuric acid 86 :14. UV detector at
A,220; B, 254; C,280 nm
1. Cannabidiol CBD
2. Cannabidlolicacid CBDA
3. Cannabinol CBN
4. A^tetrahydrocannabinol A^'THC
Figure 8. Separation of diconai constitu- 5. Cannabichromene CBCh
ents. Column 25 cm x4.9 mm i.D., packed 6. Cannabinoilcacid CBNA
with 5 fim silica modified with mercapto- 7. A^-tetrahydrocannabinolic acid
propyl groups. Other conditions as in Fig- A»THCA
ure 5. 8. Cannabichromenic acid CBChA

145 ,
1.

Figure 10. Comparison of different Paki- Figure 11. Aging of cannabis extracts.
stani cannabis resins. Conditions as for Conditions as for Figure 9.
Figure 9, but with UV detector at A, 220; 1. Cannabidioi CBD
B,254,nm 2. CannabidioiicacidCBDA
3. Cannablnol CBN and cannablgerolic
B. Toxicoiogical Applications acid CBQA
4. A'*tetrahydrocannablnol A'^THC
Naturally fluorescing materials
5. Cannablnolic acid CBNA
HPLC is less easy to use for toxicological 6. AMetrahydrocannabinolic acid
analysis because of the relatively low sensi- AOTHCA
tivity of the UV detectors, which in general 7. Dibutylphthalate (internal standard)
will respond only to concentrations corre-
sponding to massive overdoses of drugs, the case with LSD (7). Warfarin is another
On the other hand, fluorescence is inherently such substance.
more sensitive and it is possible to exploit
2. Chemical modification of the drug
this property (see Table III). Occasionally
the substance is naturally fluorescent as is In the absence of fluorescence or where it

146 .
Tabfe IV

Quantitative HPLC of Cannabis

CANNABINOID PERCENTAGE IN PERCENTAGE STD


RESIN DEVIATION
Tetrahydrocannabinol 3.9 2.1
Cannabidiol 0.7 1.2
Cannabinol 0.4 6.8
Tetrahydrocannablnolic acid 4.8 1.9
Cannabidiolic acid 1.5 2.0
Cannablnolic acid 0.4 1.7
23 Analyses

is only weak, as is the case with morphine, it


is necessary to resort to chemical treatments
to produce more strongly fluorescing de-
rivatives. For example, moiphine can be
oxidized with alkaline ferricyanide to
pseudomorphine, which is strongly fluores-
cent. This reaction has been applied to the
estimation of morphine in urine, but since
the process is very dependent on the co-ex-
tractives present, it is necessary to add dihy-
dromorphine as an internal standard <13).
Figure 12 illustrates this and levels of mor-
phine as low as O.OI /ug/ml can be detected
quantitatively in urine.

3. Derivatization

The use of fluorigenic reagents to attack Figure 12. Chromatograms of blank urine
functional groups in a drug and thereby at- and morphlnlzed urine (2 /xg/ml). A, 2 /al
tach a fluorescent label to the substance is blank urine extract; B, 2 fi\ blank urine ex-
not a new one. The methods are not yet of tract plu82ferricyanide; C, 2 fxi morphln-
ix\

routine application even though reagents ized urine extract; D, 2 ^il morphlnlzed
such as fluorescamine, NBD chloride (4- urine extract plus 2 {A ferricyanide re-
chloro-7-nitrobenzofurazan), dansyl chlo- agent. Column 25 cm x4.6 mm
I.D. packed

ride (5-dimethylamino- 1-naphthaIenesul- with Partisil 5 (7 /zm). Solvent methanol:


phonyl chloride) and o-diphthalaldehyde are 2N ammonium hydroxide: IN ammonium
available. We have had some success nitrate: 30:20:10. Fluorescent detector
with EDTN (2,4-dichloro-6-(4-ethoxy-l- A«xc 320 nm Xcmis^se nm.

147.
naphthyi)-s-triazine) as a labelling reagent
and it is possible to prepare variants of this
by reacting cyanuric chloride with other aro-
matic moieties.

An example of the use of dansyl chloride is


given in Figure 13, wherein is shown the
chromatograms obtained by injection onto
thecolumn of 1 .25 ng and 10 ng quantities of
norephedrine, ephedrine, amphetamine and
methylamphetamlne after dansylation (14).

4. Electrochemical detection

Quite recently we have been examining


the possibility of electrochemical detectors
based on glassy carbon electrodes (15). A
prototype cell has been constructed and
shows considerable promise for detection of
morphine in blood or urine. Figure 14
shows the peak produced by iryection of a
sample of urine extract containing 1 ng mor-
phine onto the column. The sensitivity is al-
ready fairly good and can be improved
considerably as only a small proportion of
the morphine is being oxidized at the elec-
trode’s surface.

Conclusion

Although there is a wide range of commer-


cial equipment available, HPLC is still at a
stage where there is ample scope for the
chemist to exercise his talents. Apparatus
Figure 13. HPLC of dansyl derivatives.
to suit particular analytical problems can be
Coiumn 25 cm x4.9 nm I.D. packed with Par-
put together quite easily and inexpensively.
tisil5 surface modified with Cis groups.
It can provide reliable results for many
Solvent methanol; water;85:15. Fluores-
cent detector Xcx* 339 nm; Xem* 420 nm. months before the columns need repacking.
a norephedrine It is also possible by the exercise of skill
b ephedrine and chemical ingenuity to bring the analyti-
c amphetamine cal problem well within the scope of this
d methylamphetamlne powerful technique.

148 .
,

D. Acknowledgment
We should like to acknowledge the valu-
able contiibutions made by our co-work-
all

ers, especially I. Jane, R. N. Smith and M.


Whitehouse.

REFERENCES

1. Wheals, B. B., J. CJiromatogr., 122, 85


(1976).

2. Ja.n&, 1., J. Chromatogr,, 111,227(1975).

3. Wheals, B. B., Vaughan, C. G., and


Whitehouse, M. J.,y. Chromalogr., 106,
109(1975).

4 . Wheals B B ,
.
. , 7, Cht oma togr. ,107, 402
(1975).

5. Vaughan, C. G., Wheals, B. B., and


Whitehouse, M. J., J. Chwmatogr. 78,
203(1973).

6. Wheals, B. B., and Jane, I., The Analyst,


102.625(1977).

7. Christie, J., White, M. W., and Wiles,


J. M.,/. Chromatogr., 120,496(1976).

8. Ratcliffe, W. A., Fletcher, S. M., Moffat,


A. C., Ratcliffe, J., Harland, W. A., and
Levitt, T. A., Clin. Chem., 23, 169(1977).

Figure 14. Electrochemical detection of


morphine. Column 25 cmx4.9 mm I.D.,
packed with 5 /.tm silica. Solvent metha-
nol: 2M ammonium hydroxide: 1M ammo-
nium nitrate: 27:2:1. Flow rate 1 ml min-‘.
Detection, oxidation at glassy carbon elec-
trode (+0.6 V versus Ag/AgCI reference) Ag
auxilliary electrode.

149 .
9.

Smith, R. N., J. Chromatogr., 115, 101


(1975).

10. Wheals, B. B., and Smith, R. N.,J. Chro-


ma togr., 105,396(1975).

11. Smith, R. N., and Vaughan, C. G., J.


Chromatogr. 129,347(1976).

12. Smith, R. N, and Vaughan, C. G,, J,


Phurm. Pharmac., 29, 286 (1977).

13. Jane, I., and Taylor, J. F., J. Chroina-


togr., 109,37(1975).

14. Wheals, B. B., in “High Pressure Liquid


Chromatography in Clinical Chemistry”
(P. F. Dixon, C. H. Gray, C. K. Lim, and
M. S. Stoll, Eds.) p.21 1 , Academic Press,
London, 1976.

15. Lankelma, J., and Poppe, H.,7. Chroma-


togr., 125,375,(1976).

ISO.
by
On-line Liquid
Patrick Arpino
Chromatography Mass J.

Ecole Polytechnique
Spectrometry: the Laboratoire de Chimie
Monitoring of HPLC Analytique Physique
Route de Saclay
Effluents by a Quadrupole 91128 < Palaiseau France
Mass Spectrometer and a
Direct Liquid Inlet
Interface (DLI).

SUMMARY work is beginning to appear now; users of com-


mercially available machines are showing in-

On-line coupling of HPLC to a mass spec- and new methods are being suggested.
terest,
trometer is a promising technique as a detec- Today one may write that LC/MS is alive and
tion system for HPLC. It provides good opens to a vast field of promising develop-
sensitivity, a wide range of applications, and is ments, Despite its relative youth, many re-

also an identification method capable of ana- view articles on LC/MS have already been
lyzing nanogram amounts of pure substances published (1-5).
eluted from the column. The advantages of an instrument which
The method for the direct introduction of liq- would combine a separation method such as
uid solution from the HPLC column into the high pressure liquid chromatography (HPLC)
source block of a mass spectrometer through a to an identification method such as MS are ob-
“Direct Liquid Inlet” interface is discussed. vious to any analytical chemist. However,
The technique does not extend to all of the dif- such a combined technique has been regarded
ferent aspects of modern liquid chromato- for many years as utopia, because the two tech-
graphic methods; however, it is well adapted to niques appear fundamentally non-compati-
reverse phase chromatography on chemically ble. Mass spectrometry requires a high
bonded stationary phases or on carbon vacuum and ionization of molecular species in
adsorbents. the gas phase, whereas liquid chromatography
The potential and the future of the technique is intended to analyze those substances which
are presented with respect to recent develop- lack sufficient volatility to be analyzed by gas
ments of MS techniques which make many chromatography (GC),
nonvolatile substances amenable to mass spec- Thus, even today, some authors consider
trometric analysis.
that off-line techniques (6-8), such as collection
of liquid fractions eluted from the LC column,
7. INTRODUCTION evaporation of the solvent, and transfer of the
solute onto the solid probe of a mass spectrom-
Liquid chromatography/mass spectrometry eter are the only realistic LC/MS techniques.
(LC/MS) is a recent technique; after a few pre- An evaluation of off-line LC/MS was reported
liminary attempts, it effectively began in the by Hubert? fl/. (8) It is true that this procedure
years 1973-74 with the publication of the re- may be simplified and partly automated.
sults obtained by E. C. Horning, P. P. -W. Often by dipping the tip of the MS probe into
Scott, and F. W. McLafferty (see Table I). the collected fraction, enough solute is trans-
During the following two years only a few ferred, and the complete evaporation of the sol-
papers appeared in the literature. As LC/MS vent may take place during the introduction of
is a costly and difficult research topic, a con- the probe through the MS vacuum locks.
siderable amount of work was achieved by only Off-line LC/MS coupling may use for the MS
a very small number of research teams. Their analysis new techniques recently developed

151 .
for the analysis of non-volatile molecules. LC/MS avoids ail of these problems. In addi-
These methods often require the desorption of tion, we believe that MS might be the only de-
the solute from a surface introduced into the tectorwhich offers a broad field of application
MS source block and include field desorption to organicmolecules, good sensitivity (10~‘*^g),
(FD) (9), lasei assisted FD (10), election im- and a wide dynamic range (lOHo 10“) suitable
pact/desorption (EI/D), (11, 12) and the veiy for modern HPLC. Therefore, the rest of this

simple method of chemical ionization/desorp- text will deal exclusively with on-line LC/MS
tion (CI/D) (13) in which no high electrical field techniques.
or sharp needles on the emitter tip are required
to obtain I esults which previously could be ob- II. A DIFFICULT PROBLEM
tained only by conventional FD. Other
methods, such as direct chemical ionization The detection system in HPLC is of such
(14), rapid evaporation from inert surfaces paramount importance that it has even been
(15-17), laser enhanced vaporization (10, 18, suggested that a cheap mass spectrometer,
19), pyiolysis FD/MS (20), plasma desorption with a low mass resolution, could be used. We
induced by Californium-252 fission fragments feel that, with the technology now at hand, this
(21-25), and electrohydrodynamic ionization is unlikely to happen. Even a simple quadru-
mass spectrometry (26-28) have been sug- pole mass spectrometer with a vei-y low mass
gested for off-line LC/MS methods. resolution, used only as a low mass filter
On the other hand, the manual collection of eliminating the solvent ions from the solute
fractions from LC systems is long and tedious. ions, or as a total ion integrator for the solute
It may be automated, but the following step, ions, will always be a relatively expensive de-
the introduction of the solute alone into the tector. This is because mass spectrometry re-
mass spectrometer, ismore difficult to achieve quires precisely machined parts, high vacuum,
(see, for instance, the work of Lovins el al., and sophisticated electronic controls. An
29-30). The collection of fractions is impossi- LC/MS interface should be an accessory as
ble to achieve in the case of fast eluting peaks, part of a general multipurpose mass spectrome-
such as those now encountered in modern high ter combining different inlet systems for solid,

performance liquid chromatogiaphy (HPLC): gas, and liquid samples, and for GC and LC

columns with plate number as high as 50,000 introduction.

may be produced (31), but extracolumn effects It is a simple matter to define what the ideal
are of such dramatic impoi tance (32-34) that the interface should be:

analyst is often unable to take full advantage of


1 . The interface must not pose limitations
the se high performances Off-line coupling re-
.
on the operating conditions of the LC; it
quires that a greater amount of solute be in-
must tolerate different solvent systems
jected, and it requires also that a conventional
under different flow conditions.
HPLC detector be used to monitor the fraction 2. The interface also must not restrict the
being collected to reduce the number of frac- operating conditions of the mass spec-
tions further examined by MS. The detector in trometer. Electron impact ionization
HPLC systems is a very critical part of the in- (El) and Cl, under controlled pressure
strument, and most of the detectors used suffer and selected nature of the reagent gas,
from some limitations. (4) Clearly, on-line must be available.

152 .
3 . The interface, when regarded as a post- Table I

column dead volume, should have a time List of the systems proposed
constant less than one second so that
for LC/MS
fast eluting peaks are not broadened (34)
during their transfer through the A) Systems with complete elimination
interface. of the solvent before entering the
4 . The lower sensitivity limit should be less mass spectrometer:
than nanogram, and the dynamic range
1

should be better than 10^


—modified solid probe
Lovins 29,30
5. The interface should carry into the mass
spectrometer a high percentage of the
— moving wire
Scott 42,43
solute eluted from the LC column.
6. The transfer of the solute through the in-
— moving belt
McFadden 44-47
terface should be quantitative, inde-
pendent of the nature of the solute and
— membrane
Jones 78
the solvent system used during the LC
separation.
—collimated molec-
ularbeam
7. The solute should not be degraded Takeuchi SO
(chemically altered or pyrolyzed) during Vestail 69
its passage through the interface. Futrell 70

Consideration of the results obtained so far B) Systems which let the solution en-
show that none of the described interfaces is ter the mass spectrometer:
perfect; at least one,and often many, of these — DLI interface and El
had to be sacrificed. The
idealistic definitions Tal’Rose 79,80,82
methods are listed in Table 1. — DLI Interface and Cl
It is not the purpose of this paper to review McLafferty 49-54
and evaluate the different methods now offered Arpino 58, 66
for LC/MS . The reader is referred to the origi- Henion 55, 56
nal papers, with a specific emphasis on the Meiera 57
atmospheric pressure ionization source of — API source and
Horning c/ al., (35-41) the moving wire of Scott plasma chromato-
(4, 42, 43) and the moving belt of McFadden. graph
(44-47) Karasek 81
The LC/MS interface, referred to here as the — API source and
interface with “Direct Liquid Introduction” quadrupole MS
(DLI), was originally developed in 1973 in the Horning 35-41
laboratory of Prof. F. W. McLafferty at Cor-
nell University. Preliminary work (48) was on introduced through the solid probe inlet of a
dilute solutions of peptides in hexane or liquid mass spectrometer model AEl MS 902 modi-
ammonia, kept in small glass vials with one end Cl work. Cl spectra resulting from sol-
fied for

partially sealed until a small aperture restricted vent/solute quadrupole mass spectrometer
the flow of liquid out of the vial. It was then were used.

153 ;
HI. THE DU INTERFACE back desorption
ing liquid nitiogen; there is not
of the molecule. Thus, the pumping speed of
A. Principle the cryotrap is controlled by the surface of the
trap, and by the conductance of the pathway
The principle of an inlerface based on DLI is
between the source block and the trap (59, 60).
simple. The major portion of the solution
The pumping speed is even faster when the par-
eluted from the HPLC column, which is com-
tial piessure of the solvent vapor in the vicinity
patible with the vacuum pumping of the MS, is
of the surface is close to lO"-' t, as viscous flow
sucked into the MS. The amount of liquid in-
takes place (60). Such is the case in our proto-
troduced is controlled by the liquid pressure
type, which includes a large hemicylinder,
drop across the DLI interface; it is convenient
chilledby liquid nitrogen, with an inner surface
to place a restriction in the interface to lower its
of 326 cm^ directly in line of sight with the
flow permeability. The piessure conditions in
source block, and within 2 cm distance from
the mass spectrometer depend on the amount
of solution introduced, on the pumping equip-
the source block. Up to 100 /xl/min. of acetoni-
ment, and on the vacuum conductances of the
trile may be
introduced over periods of many
hours (5 to 10), without lowering the vacuum
different elements of the MS.
pumping speed of the cryotrap. However,
A quadrupole MS requires operating
under such high flow rates, the pressure be-
pressures of 10"^ t in the source envelope, and
10”® tween the ion exit slit and the entrance aperture
t in For Cl conditions in the
the analyzer.
of the quadrupole rods is too high. This causes
source block, the vacuum conductance of the
a mass discrimination for ions greater than
source block is adjusted so that inside source
m/e=300; thus, we routinely introduce 30 to 50
pressures from 0.2 to 1 torr are obtained during
LC/MS operations. Most commercial quadni- /il/min. of solution of polar solvents. With this

pole MS designed for GC/MS work include fast


liquid flow rate, we observe a source envelope
vacuum pumps and a differentially pumped pressure of 4x 10~* t and an analyzer pressure
source. They may accept the direct intioduc-
of4xl0~® t, as read without correction on hot
tion of about to 10 /il/min. of solution
cathode type high vacuum gauges.
1 from a
HPLC column, and good LC/MS data, using The cryotrap may be regenerated within 30
only oil diffusion pumps for evacuating the sol- min. after isolation of the oil diffusion pumps,
vent, have been previously presented. (49, 55, introduction of dry nitrogen in the vacuum en-
56) The use of very large diffusion pumps, with velope of the MS up to atmospheric pressure,
opening size larger than 20 cm, is not recom- warming up of the cold surface, and pumping
mended as they are expensive, bulky, and have down to vacuum by the roughing pumps. This
a high level of oil vapor back diffusion. The al- procedure is repeated two or three times.
ternativemethod to increase the vacuum Other procedures which allow the solvent
pumping speed is cryogenic pumping (50, 57). vapors to go through the oil diffusion pump,
even when they are turned off, are not
B. Thecryopump recommended.
Most solvent molecules, from solvent sys-
tems used for HPLC, have a C. The source block
condensation
coefficient equal to one when they are ad- In the source b)ock, enough thermal energy
sorbed on a surface at the tempeiature of boil- is supplied to the liquid solution for its com-

154 ,
plete vaporization. In our instrument, as in ion of the solute appears at mye=M-l, (M
most of the other instruments of the same type, being the molecular weight of the solute).
(55-57) the thermal energy is taken from the Chloroform, when void of traces of the metha-
heated soui ce block In one case a laser beam
. , nol used as a stabilizer, yields a mixture of hy-
has been used (54) to supply this energy. As dride abstraction and protonation. All polar
the solution expands in the source block, a solvents (tetrahydrofuran, acetonitrile, metha-
pressure of about 0.5 1 is reached, this pressure nol, water) give chemical ionization with pro-
being practically equal to the partial pressure of tonation, so that the quasimolecular ion of the
the solvent raolecujes. Ionization of the sol- solute appears at m/e=M+l. It is one of the
vent molecules to produce a plasma of primary most attractive features of a DLI interface to
reactant solvent ions is induced by the inter- match well the requirement of reverse phase
action of the solvent molecules with an elec- HPLC, the most widely used and the most
tron beam of 100 eV energy. The electron promising of the HPLC techniques (63 -65).
beam is obtained from a conventional heated LC/MS operations, using chemically bonded
rhenium filament. A plate with a pinhole was silica (58) or graphitized thermal carbon black
placed between the filament and the source (66) as the stationary phase, have been run in
block to focus the electron beam, and to hide our laboratory. Melera has shown (57) that a
the filament from the solvent vapors which exit buffered aqueous solution of volatile inorganic
from the source block. This protects the fila- salts , ammonium acetate (up to 0. M in
such as 1

ment which does not burn when oxygen con- water), may be continuously introduced in a
taining solvents, such as water or methanol, cryopumped quadrupole.
are used; its average life time is one month. The quadrupole mass spectrometer may be
operated mode (67), and de-
in the negative ion
D. The mass spectrum tailed studies of the behavior of the commonly
used solvent systems under such an operating
The mass spectrum results from chemical
mode should be performed, but preliminary re-
ionization between the plasma of the solvent
sults (57) appear very promising.
ions in the mass range from m/e=I00 to the
higher mass limit of the mass spectrometer.
Usually the MSrecords the ions in the solute E. The liquid/gas interface
mass range, thus discarding solvent contribu-
Anqther critical part of the instrumentation
tion to the mass spectra. On the other hand,
in a DLI system is the restriction in the inter-
the monitoring of one of the reactant ions from
face which limits the amount of liquid intro-
the solvent may give a useful LC chromato-
duced into the mass spectrometer. In the
gram (61, 62).
original model (49 -5 1 53) a long (30 cm) thick
,
,

Different solvents have already been tested wall glass capillary tube (6.35 mm ODxO.075
in view of their use in a DLI system: pentane, mm ID) was used. Excess glass at the end in-

hexane, tetrahydrofuran, chloroform, acetoni- troduced in the source block was removed by
trile, methanol, and water. All of these sol- dissolution in HF, and a pinhole of about 0.010
vents perform well under DLI conditions mm was obtained by glassblowing. The same
(49-5 1). Pure alkane solvents, such as n-pen- technique has been used by others (55, 56).
tane or n-hexane, yield Cl reaction with hy- The same permeability is more easily prepared
dride abstraction, so that the quasimolecular by inserting a thin metallic wire inside the capil-

155 .
lary tube, starting at the end normally placed lyzer. Vestal (69) and Futrell (70) have re-
inside the MS source block (58). Adjustment of corded on their instruments either pure El
the permeability to the required value is easily spectra, or Cl spectra from nonvolatile mole-
obtained, and the tube is rapidly cleaned if it cules, such as nucleosides. However, their
becomes plugged. Howevei, any DLI inter- prototypes which include many differentially
face which makes use of such a long and nar- pumped zones and a laser (69), or a sonicating
row capillary tube, either with a pinpoint horn (70) to break the liquid into well homog-
restriction or with a regularly decreasing enized droplets, are far more complex in-

pressure drop as with the wire model, suffers struments than the modified quadrupole MS
from a very severe limitation when solutions of equipped with DLI interfaces (56-58).
a nonvolatile solute are percolated through the Finally, the main theoretical advantages of a

interface (58, 68). Because of the viscosity of DLI interface have not been obtained yet. As-
suming that the conclusions from a theoretical
the polar solvent, the capillary forces opposing
study done by Giddings et al. (72) are valid, we
the flow of liquid through the tube, and the
may expect that the breakage of the solution
range of pressure of liquid one may develop
during a rapid expansion leaves the solute mol-
across the interface (0-100 bars), the speed of
ecule in the gas phase during a very short (10”“
the solution through the tube is about 10
sec.) period of time, befor*e solute molecules
cm/sec. This is too low to insure the transport
recombine to form a solid crystal. Thus, one
of the liquid into the vacuum of the mass spec-
should try to ionize the solute molecules in the
trometer without vaporization of the solvent
gas phase during this brief period of time. The
inside the capillary tube, as the liquid reaches
demonstration of possible isolation of ma-
the end of the capillary tube under vacuum.
croions in the gas phase was realized by Dole
Thus, nonvolatile solutes and high molecular
litai, (73) but none of the described LC/MS sys-
weight impurities, present in either the solvent
tems have been able to provide such an effect.
cr resulting from degradation of the chemical
bonding of the HPLC column, accumulate in-
F. The chromatographic system
side the glass tube and plug it. Heating the end
of the glass tube, either by thermal convection The HPLC instrument should be considered
from the source block, or by a laser beam, does as a full part of the interface, and be optimized
not bring any improvement, as it simply moves to provide the best LC/MS operations. Most
he liquid/gas transition zone deeper within the analytical HPLCcolumns utilize small parti-

:apillary tube. Such a problem is avoided and column inside diameters


cles (5 to 10 /u,M)

vhen a water-cooled diaphragm is used instead of 2 to 6 mm. The output flow rate should be
rf a long capillary tube (57) or when the physical 0.5 to 2 ml/min, which is 10 to 40 times greater

parameters at the end of the capillary tube are than tolerated by a cryopumped quadrupole
such that a supersonic molecular beam of solu- MS On the other hand
. microcolumns (74 -76)
,

tion molecules, expanding in a jet, is produced 1 mm have


with internal tube diameter of 0.5 to
(69-71). In a freely expanding jet a skimmer an output flow rate of 5 to 50 /xVmin, which
may be installed to discard the outer layers, matches well the requirements of a DLI inter-
A'hich contain solvent molecules, and to iiyect face. ‘Different authors, including ourselves,
only the central part of the beam, which con- have used such microcolumns in conjunction
tains the solute molecules, into the mass ana- with LC/MS systems (55, 58 , 71). Unfortu-

156 .
nately, their chromatographic performances
are poor, compared to the separating power of
larger diameter columns. We have tried to ad-
just the injection parameters m a 4 mm column,
using a divided flow injection mode (32). Then
only the central part of the column was trans-
ferred through the DLI inteiface. A consider-
able enrichment was observed for non-retained
solute, such that 10% of outlet solution in the
central zone contained 60% of the injected so-
lute. Unfortunately, no enrichment was ob-
served for retained peaks with K' > I, which
sets a severe limitation on the practical advan-
tages of the procedure.
Research work on improved microcolumns,
and on on-line solvent concentrators may re-
sult from efforts to adjust the HPLC system to
the DLI interface for LC/MS.

Photo 1 Photo 3

157 .
Figure 3. LC/MS detection of 2 to 5 micro-
grams of a triazine derivative injected onto
a HPLC column. Solvent: acetonitrile at 1
ml/mln. and an Inlet pressure of 30 bars;
Figure 2. Schematic drawing of the MS column: IS cm longx4 mm id filled with 5 fi
source block with the DLI interface being C,8 bonded flow rate of solution in-
silica;
connected; (1) movable metallic probe troduced into the MS: 30 /tl/min; electron
shaft; (2) insulated tip; (3) end of the capil-' energy: 100 eV. A' total ion Integrator
iary glass tube; (4) MS source block; (5) synchronized with the MS scan was used
vents; (6) electron beam for primary ioniza- for tracing the chromatogram; Integrated
tion of solution vapors; (7) Ion exit slit. mass range 800-1200 amu In 1.5 sec.

158 .
Figure 5. LC/MS analysis of 400 ng of a
phenanthrene/chrysene mixture analyzed
on a micro LC column directly coupled to
the MS. Top trace; reconstructed LC chro-
matogram from ions in the mass range 130
-500 amu. Bottom trace; mass chromato-
gram of m/e 179 (MH*^ of phenanthrene).
Column: 43 cm longxi mm id filled with 5fi
Figure 4. Dual recording of the signal ob- C,8 bonded silica. Solvent; acetonitrile at
tained from a conventional UV detector, 30 and an Inlet pressure of 40 bars.
/ul/min.
and from the LC/MS during the analysis of 1 Plate number for the phenanthrene peak:
fig of porphine. HPLC and MS conditions 1 1 00. The data were processed by a Rlber-

are the same as in Figure 3; Integrated mag 400 data system; MS scan: 130-500
mass range 130-500 amu in 3 sec. amu In 1.5 sec.

IV. RESULTS the cryotrap: it is a hollow hemi-cylinder which


surrounds the Cl source block of the mass
The advantages of a DLI interface may ap- spectrometer. Details of the DLI interface ap-
pear more clearly after the presentation of pear in Photo No. 3. The injector head of the
some of the results obtained on the instniment HPLC is directly mounted on the column; the
assembled at the Ecole Polytechnique, which LKB detector monitors the major part of the
is shown in Photo No 1
, .The HPLC pump is a eluent left after it exits the interface. Figure 1

gas pressurized coil, seen on the left end; the is a schematic representation of the interface
quadrupole is in the center, and the electronic and of its holder; Figure 2 represents the cut
controls are on the right. Photo No. 2 shows section of the Cl source block with the inter-

159 .
*

N
Ml
— —p~r— —
I I
T I
^
r ^r I —r~T— — —r-i—
i i
Flgura 7. Single ion recording of m/e 203
30 20 10 0 (MH+) during the introduction of 10 pg of
fluoranthene through the DLI interface Into
Figure 6. LC/MS analysis of 1 ^l of lemon the MS. No HPLC column was used. Dead
essential oil. Column: 14 cm longx4 mm id volume between the injection point and the
filled with 5fi C,g bonded silica. Solvent: MS source block: 7 /u.1. Flow rate of aceto*
acetonitrile at 0.5 ml/min. and an inlet nitrile: 33 fd/min. Retention time of the
pressure of 20 bars. Integrated mass solute: 12.6 sec. Volume of the solution
range 100-700 In 3 sec. Other MS condi- injected: 1 fii.
tions are the same as In Figure 3.

troduction of the total of the output solution


face In running position. into the mass spectrometer. Figure 5 shows an
The LC/MS analysis of a high molecular example of such a coupling for the analysis of a
weight substance is presented in Figure 3 . In- polyaromatic hydrocarbon mixture (phen-
creasing amounts of a triazine derivative (from anthrene and chrysene); only 1100 plates were
2 to 5 ^g) were injected on the HPLC column; measured for the phenanthrene peak, which is
the signal the integrated ion currents of ions
is selectively displayed on the computer gen-
in the mass range m/e 800-1200. Figure 4 erated ion chromatogram for mass 179
shows the dual parallel recording of the signal (M-fH+).
seen by the MS and by the UV detector, after Better results are obtained with a conven-
iiyection of 1 //g ofporphine. tional large bore HPLC column, as shown in
It is possible to connect directly a micro LC Figure 6. It is the total ion chromatogram for
:olumn to the mass spectrometer, with the in- ions in the mass range m/e 120-500, after injec-

50 .
Figures. Mass spectra of fluoranthene, chrysene, and coronene after Injection of 100 ng
of each sample. LC conditions are the same as in Figure 7. Data were acquired by a
Ribermag 400 data system. Mass range 150-500 amu In 1.5 sec.

tion of 1 fA of lemon oil on a C-18 reverse phase ferent research groups who use similar

HPLC column. machines. Polyaromatic hydrocarbons, with


The sensitivity of the method is illustrated in increasing number of aromatic rings, or the se-
Figure 10 pg of fluoranthene is introduced
7: ries of the oligomers of polystyrene polymer
into the Cl source block, while recording the could serve as valuable test probes. As an ex-
output signal of the quasimolecular ion ample, we havedemonstrated that benzene,
(M+H' =203). The dynamic range of detec- naphthalene, phenanthrene, and chrysene are
tionwas shown to be 10® for this analysis. transferred without significant peak broad-
Further work is needed to obtain the higher ening. However, coronene and pentacene
limit of volatility a solute should present to be are partly retained due to adsorption of the
analyzed by LC/MS with a DLI interface. Se- solute on the glass tube of our DLI interface

lected test substances should be run by the dif- (see Figures 8 and 9) , This arises from the fact

161 .
Figure 9. Computer reconstructed mass chromatograms for the quasimolecular Ions of
the aromatic hyorocaroons during the analysis in Figure 8. Fluoranthene and chrysene
peaks are eluted from the DU interface without peak broadening,
whereas coronene Is
strongly adsorbed on the glass of the interface.

that the solvent evaporates inside the glass


CONCLUSION
tube before the solution reaches the Cl source
block. We are now working on a rather differ- The direct introduction of liquid solution in-
ent system, compared to the model shown in side the Cl source block of a mass spectrome-
Figures 1 and 2, which keeps the DLI concept
ter appears to be a simple and reliable method
for a more efficient transfer of the liquid solu-
to achieve on-line LC/MS coupling. The sen-
tion into the Cl source block.
of the detection aud the range of solutes
sitivity

amenable to the device make the mass spec-


trometer the most sensitive and universal
HPLC detector although it is a costly and com-
,

162 .
plex detector. Among the results, which we Chromatography” (J.F.K. Huber, ed,),
assume now to be well-established, there is the Elsevier Scientific Publishing Co., Am-
absence of problems posed by the routine in- sterdam and New York, 1978.
vacuum of
troduction of liquid solvents into the
the MS. No
long term degradation effects 6. Elbert, S., Gruhn, B., Wipfelder, E., and
have been observed, and routine LC/MS oper- Heusinger, H., Anal. Chem., 48, 1270
ations are possible on a general multi-pui-pose (1976).
instrument. As most of the problems around
the DLI interface have been solved, we 7. Majors, R, E., and Wilson, B.,

may now concentrate our efforts on the inter- Greewood, H., and Snedden, W., Bio-
face, itself, to And the design with optimal chemical Society Transactions, p. 867
performance. (1975).

8. Huber, J. F. K., Urk-Schoen, A. M. van,


ACKNOWLEDGMENTS and Sieswerda, Z.,Anal. Chem. 264, 257
I am F. W. McLafferty (Cornell Univer-
giatefiil to Prof, •
(1973).
sity, USA), and to Piof. G. Giuochon (Ecote Polyteclt-
nique, France) for helpful advice during the LCJMS work. 9. Schulten, H. R. and Beckey, H. D., J.
The help from Drs. G. Devant and P. Krien (Ribermag Co.,
France, and Fiber Data System, USA) made possible the
Chromatogr., 83,315 (1973).
consiriictlon of a prototype in the author's laboratory.
This research work was made possible by the financial sup-
10. Schulten, H. R., Lehman, W. D., and
port of the French D.G.R.S.T. (contracts 75.7.1040 and Haaks, C., presented at the 26th ASMS
77.7.1606). meeting, paper No. TA-9, St. Louis, May
28 -June 2, 1978.

REFERENCES 11. Holland, J. F., Soltmann, B., and Swee-


ley, C. C., Biomed. Mass Spectrom., 3,

1. Bollet, C.,Analusis, 5, 157 (1977). 340(1976).

2. Aipino, P. J., La Recherche, 6, 769 12. Soltmann, B., Sweeley, C. C., and Hol-
(1975). land, J. F., Ana/. Chem., 49, 1164(1977).

3. Dawkins, B. G. and McLafferty, F. W., 13. Hunt, D. F., Shabanowitz, J., Botz,
in “GLC and HPLC Analysis of Drugs. F. K., andBrent, D. A.,Ah£j/. Chem,, 49,
Vol. 1” (K. Tsuji and W. Morozowich, 1160(1977).
eds.), Marcel Dekker, New York, N.Y.
1977.
14. Baldwin, M. A. and McLafferty, F. W.,
Org. Mass Spectrom., 7, 1353 (1973).
4. Scott, R. P. W., in “Journal of Chroma-
15. Beuhler, R. J., Flanigan, E., Greene,
tography Library Vol. II. Liquid Chro-
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166 .
A New Approach to the by

Optimization of Rokus A. de Zeeuw, Paul Schepers,


Jan E. Greving and Jan-Piet Franke
Chromatographic Systems Department of Toxicology
and the Use of a Generally Laboratory for Pharmaceutical

Accessible Data Bank in and Analytical Chemistry


State University
Systematic Toxicological Deusinglaan 2, 9713 AW
Groningen
ii^alysis
The Netherlands

/. INTRODUCTION Despite their crucial importance for STA,


these aspects do not seem
have receivea to

Systematic toxicological analysis, Le. the much attention. This may be due to the large
undirected search for a potentially harmful amount of work that is necessary for this type
substance whose presence is unsuspected, rep- of research and/or the lack of suitable criteria
resents a most difficult analytical problem. for adequate evaluation and optimization. In
This applies to both forensic and clinical toxi- recent years, mqjor contributions came from
cology, regardless whether the sample a bio-
is three groups; Moffat et al. (1-6) classified PC-,
logical fluid or tissue, a drug formulation, or TLC- and GLC-systems on the basis of their
some stuff traded in the streets To a large ex-
.
discriminating power (DP); Massart el al. (7-
due to the almost infinite number of
tent this is 10) did the same for TLC-systems, using the

drugs that have to be taken into account, either concept of information content (IC), and
as single substances or as mixtures. Muller e/ n/. (1 1) evaluated TLC-systems using
separation quotients (qg), in combination with
During recent years, chromatographic tech- color reactions on the plate (12).
niques, such as thin-layer chromatography
All three approaches are system-directed, in
(TLC), gas-liquid chromatography (GLC) and
high-peiformance liquid chromatography that they provide information on the efficacy of
single systems and combinations of systems.
(HPLC), have gained general acceptance as
basic tools for toxicological analysis. At first
Though quite helpful in selecting proper sys-
tems, they give little or no information on indi-
sight, when looking into the literature one may
vidual substances, for example if substances a
get the impression that a large number of sys-
and h can be separated in system z or if sub-
tems is already available. Yet, most of these
stance c can be unequivocally identified in the
systems were developed for a limited number
presence of substances d, e,f, g etc.
of substance classes and/or individual compo- . . . ,

nents. However, in order to be suitable for


Therefore,we have developed a new approach
which IS both system- and substance-
systematic toxicological analysis (STA), it is
directed. Utilizing the concept of identifica-
necessary that chromatographic systems be
tion power
(IP), systems can be evaluated and
evaluated with a large number of substances,
substances can be identified by means of their
representing a variety of classes. Moreover,
chromatographic retention behavior, pro-
as obviously no single separation system will
vided adequate consideration is given to repro-
be adequate, combinations of systems have to
ducibility and spot or peak size.
be tested. The latter may comprise two or
more systems based on the same technique This paper reports the first results obtained
(e.g. two TLC-systems), or based on different with this concept. It has been applied to a se-
techniques (TLC and GLC; GLC and HPLC). lection of ninety basic drugs which were chro-
Evaluation of separation systems combined matographed in eight TLC-systems, Four of
with other analytical methods such as spectro- them were “normal” systems in which the
photometry, mass spectrometry, and color drugs migrated as bases; the other four repre-
reactions may also be envisaged. sented ion pair adsorption systems in which the

167 .
drugs migrated as ion paiis. The latter was tems would be of advantage for STA.
done to get an insight into the efficacy in STA of In order to be able to properly study the ef
the newly developed ion pair adsorption sys- feet of the ion pair formation on the chromato

tems (13) as compared to that of the noimal graphic retention we tried to select our systems
systems. in such a way that, preferably, the ion paii
TLC-system differed in only one aspect (the

presence of the counter ion) from a corre-


II. THIN LAYER CHROMATOGRAPHY sponding, normal system. A second factor in
selecting our systems was that we wanted to

A. Choice of TLC-Systcms achieve optimum reproducibility for reasons


that will be explained later. As we had ob-
It is well known a dmg
that identification of
served that the presence of basic components
by using a combination of chromatographic
such as NaOH, KOH, ammonia, and diethyla-
systems can best be achieved if the Rf-valiies
mine usually lowered reproducibility, without
for compounds in one system are independent
being necessary to let basic drugs migrate as
of their values in another system, i,e., the Rf-
bases (13), we tried to circumvent the use of
values should have low inter-system correla-
these basifying components, which could be
tion (2, 14). This may be achieved by selecting
realized in three of the normal systems. This
widely different developing solvents, or by ap-
resulted in the selection of the eight solvent
plying a different physico-chemical separation
systems listed in Table I. All further chroma-
principle. An example of the latter can be
tographic conditions were as described
found in the TLC of barbiturates. When using
previously (13).
a neutral solvent, e.g. chloroform-ether
(75+25), barbiturates migrate as undissoci-
B. Substances
ated acids, but with an alkaline solvent such
as chloroform-isopropanol-conc. ammonia Ninety basic drugs were investigated. They
(45+45+10) the substances migrate as anions allbelonged to the selection of 100 substances
(15). Unfortunately, this principle cannot be evaluated by Moffat et al. (2, 4,5), the remain-
applied to basic diugs. With alkaline or neu- ing 10 substances not being available at the

tral solvents they migrate as undissociated time. The bases were each dissolved in metha-
bases, but an acidic solvent nol to give solutions containing
about 1 mg/ml,
if is used, the re-
sulting piotonated base BH+ usually becomes of which 2-5 were spotted. After develop-
/xl

too polar to migrate on silica gel. Yet, we re- ment, drugs were localized under UV light of
cently introduced simple TLC-systems in 254 nm and by spraying with acidified iodopla-
which basic drugs are converted to ion pairs ac- tinate spray.

cording to the equations


C. Reproducibility of the TLC-Systcms
B+H+;=:^BH+
BH++X-^=iBHX- In order to
be able to apply the concept of IP,
it is necessary to know for each system the
in which X" represents a suitable, negatively
standard deviation of its Rf- values. This was
charged counter ion, usually bromide or perch- assessed by measuring the Rf-values of 10 sub-
lorate (13). As the basic dnigs now migrate as stances in six repetitive experiments on six dif-
ion pairs we were interested to see if such sys- ferent days. The 10 compounds were selected

168 .
so that they were evenly spread across the midity, temperature, quality of sorbent and sol-
plate in each system: benzocaine, bromodi- vent) andyor to allow inter-laboratory
phenhydr amine, dimethoxanate, mepyramine comparisons, we corrected Rf-values accord-
nikethamide, nortriptyline, pethidine, pipama- ing to Galanos and Kapoulas (16), using de-
zine, promazine, and strychnine. It could be fined substances as reference compounds to
shown that the variance in Rf-values was es- convert observed Rf-values to corrected Rf-
sentially constant across the plate.. values (Rf'^-values). It has been shown that
this approach results in a remarkable increase
D. Correction of Rf-values in inter-laboratory reproducibility with both
single- and multicomponent solvent systems
In order to correct for systematic changes (17, 18). The original method uses two refer-
(e.g.. geometry of the chamber, relative hu- ence substances t and u for which corrected

TABLE I

Normal TLC-systems and ion pair adsorption TLC-systems for basic


drugs as used in the study*

Normal systems Ion pair adsorption systems

System 1 System 2
Solvent: Methanol Solvent: Methanol, 0.1 M In NaBr
Unsaturated chamber Unsaturated chamber

System 3 System 4
Solvent: Methanoi-Butanol Solvent: Methanol-Butanol (60+40), 0.1
(60-1-40) M in NaBr
Unsaturated chamber Unsaturated chamber

System 5 System 6 **
Solvent: Chloroform-Methanol Solvent: Chloroform-Methanol (90+10),
(90-1-10) saturated with NaBr
Saturated chamber Saturated chamber

System 7 System 6**


Solvent: Ethyl acetate-Cyclo- Solvent: Ethyl acetate-Cyclohexane-
hexane-Methanol-conc. NH 4 OH Methanol (70+15+15), saturated with
(70+15-1-10-I-5) NaBr
Saturated chamber Saturated chamber
* were Silica gel 60 F2S4 (E. Merck, Darmstadt, G.F.R.)
Alt plates
** These solvent systems do not allow dissolution of 0.1 M NaBrso tha t plates had to be Impregnated with a
0.1 M solution ofNaBrln methanol prior to development.

169 .
values have already been established. The a set of parameters obtained in more than one
substances t and u are then run together with system or with more than one technique.
the substance to be determined, p, and the fol- In this work we have evaluated 8 TLC-sys
lowing corrections are applied: terns. In order to be able to adequately handk
the large amount of data, we developed a com-
(Rf%=a puter program TOXIP (19), written in Pascal,
{Rn-{R.n,
. and made use of the CYBER 74-18 computer at
Rf-Rfu the University Computer Center in Gronin-

b^{Rr),-ciRf, gen. Pascal has the advantages over Fortran


thatit allows more compact statements, that
It should be observed that this method works
more efficient use is made of memory capacity,
best if Rf,</?/p</?/„ and if the differences be-
and that it is faster. In the following sections
iweenRf„Rf,, andi?/„ are small. Therefore, in
the mathematical-statistical approach to IP is
our investigation where we are dealing with a
given for these TLC-investigations, but the
large number of substances, we have increased
principles are equally well applicable to other
the number of reference compounds to 3 and
systems and techniques.
have also used two fixed reference points,
namely the starting point S with Rf- value 0 and
the position of the solvent front F after de- B, Mathematical Approach
velopments with Rf-value 1 . The correction Given are N different TLC-systems e
y, J
method then works as follows:
{!,... , N} and A/ different substances /,/
Q<Rf,,<Rf,: use references S and ( {I, .,M). For each system^, a number of
. .

Rfi<Rfp<Rfu' use references t and « Rf-observations were carried out for each sub-
Rfu"^fp<Rfr use references u and v stance i in order to calculate Rf'^-values and
Rfv<Rfp<l'. use references v and F mean RF-values.
For reasons of simplicity we arbitrarily se- If we are to identify
an unknown component
lected codeine (t), nikethamide (u), and benzo- belonging to the population M, we can analyze
caine as refeience substances for all
(v) this substance in k of the systems and we N
systems. Their respective RF-values were de- want to know how good that set of k systems
termined by averaging Rf-values of 9 repetitive Oil ijJ will be for this purpose, i.e.,
< • •

measurements, which system or set will have the highest IP.


If RF-values have been determined for
M substances in N systems and if
111. THE CONCEPT OF
(xTo),, represent the outcomes of the
• •

IDENTIFICATION POWER n independent determinations, the mean Re-


value a,j of substance i in system will be
A. j
General
1 ”
The IP of an analytical methodology is the
number of substances that can be unequivo-
cally identified out ofalarge population of com-
pounds. The methodology may be based on a
This will result in a total of MxN mean
Rf®-values, representing the matrix
single parameter obtained in one system, or on

170 .
C« Definitions in which n is the number of obsei vations and
the excentricity factoi foi a probability a. Foi
be clear that in order to distinguish be-
It will
10 substances and 6 has
obseivations ii
tween two substances p and q in a system 7,
10(6-1)=50 degrees of fieedom and can be
there has to be a minimum distance between p
found in tables for t-distributions. For a prob-
and q, the discriminating distance This
ability of 95%, // = 1.64; for a probability of
leads to the following definitions:
99%, «=2,33. The factor 2 has been intro-
Definition 1 Substances p and q are discern-
.
duced to take into account the sizes and shapes
ible in system j if and only if: of the spots as well as the fact that sizes and
shapes may change with the amount of sub-
stance piesent.
(Forp,<?e{l, . . . ,M}j 6{), . . . ,N)) Thus, |a„j-‘^^j|>^/jguaiantees that the obser-
vation of a certain Rf^- value is correctly appro-
Definition 2. Substances p and q are discern-
ible in a set of systems and
if
priated to substance p and not to </
if a choice
has to be made between these two.
onlyif p and q are discernible in at least one
system^ of this set.
E. Computer Calculations
Definitions, Substance p can be identified in
setOt, if and only if p is discern-
In these investigations, for W systems,

ible
. . .

from all other substances pi^q in this set.


M substances and NxM Rf^-values
.AT.
^nd for N discriminating dis-
Definition 4. The identification power (IP) of tances {dj}j=i calculations were made for:
set {Ji Jii} is the number of sub-
a. The IP’s of the single systems
stances p (pejl, . . . ,M}) that can be iden-
tified in that set. From Definitions 1 and 2, it
b, The IP’s of combinations O'l, , . j’j^}
p and q are discernible in set
.

follows that
of A' systems among the N given systems
{Ji . • • • . a} if and only if there exists at
k being 2, 3, or 4.
least one system j • •
./J) in which
These calculations weie canied out for 95%
and 99% probability, lespectively, for com-
D. The Discriminating Distance dj parison purposes. It v/ill be obvious that in

is dependent on
piactice one should work with 99% probability
The discriminating distance
the standard deviation of the system j and a sta-
tistical factor:
IV. RESULTS AND DISCUSSION
d}=Ci(Ts

each system was Excellent ion pair adsorption chromatog-


The standard deviation for
found as described in ILC., by using 10 sub- raphy could be obtained with biomide as
The counter ions, provided that the counter ion
stances in 6 repetitive experiments, fac-
concentration applied to the systems was in the
tor Cj is defined as;
order of 0, 1 M, With the more polar systems
=2 'b” 1-4, this can be easily achieved by dissolving
Cj
the corresponding amount of NaBr or KBr in

171 .
TABLE II

Rf‘^*values x 100 for the 90 basic compounds in 8 TLC-systems.

Substance System: 1 2 3 4 5 6 7 8

ACETOPHENAZINE 33 41 18 32 13 17 34 9
AMETAZOLE 5 41 4 42 0 0 8 11
AMETHOCAINE 39 46 30 39 25 17 66 22
AMITRIPTYLINE 23 50 17 51 21 31 73 31
AMPHETAMINE 11 71 8 75 5 5 43 45
ANTAZOLINE 7 67 4 66 4 14 47 23
ATROPINE 6 27 4 28 2 12 22 9
BENZOCAINE 82 83 87 87 56 58 71 95
BROMODIPHENHYDRAMINE 54 17 48 18 25 73 24
BUPHENINE 30 84 29 83 11 24 57 72
BUTACAINE 42 76 43 76 27 39 76 65
BUTETHAMINE 45 61 36 55 34 34 77 35
CAFFEINE 63 65 53 55 49 52 46 59
CARBETAPENTANE 18 58 12 49 12 33 12 23
CARBINOXAMINE 11 22 6 16 6 11 56 7
CHLORCYCLIZINE 35 50 28 52 33 35 71 48
CHLORDIAZEPOXIDE 78 79 75 77 43 47 43 80
CHLORPHENIRAMINE 10 23 5 21 5 18 58 10
CHLORPROMAZINE 21 45 15 45 18 32 72 39
CINCHONINE 19 55 13 61 13 41 45 52
CLEMIZOLE 69 67 73 73 56 52 76 9
COCAINE 29 43 22 30 29 16 76 14
CODEINE 20 26 13 22 16 18 31 . 10
CYCLIZINE 35 50 28 52 31 40 72 42
CYCLOPENTAMINE 6 67 4 68 12 1 39 28
DESIPRAMINE 7 69 6 71 10 34 44 54
DIAMORPHINE 26 29 17 33 23 33 52 23
diAzepam 81 81 84 85 66 67 70 95
DIMETHOXANATE 21 43 14 38 15 20 71 23
DIPHENHYDRAMINE 25 52 17 50 21 28 72 30
DIPHENYLPYRALINE 18 44 12 48 18 38 68 36
EPHEDRINE 9 63 6 64 0 0 31 27
ETHOHEPTAZINE 12 41 8 41 11 29 59 21
ETHOPROPAZINE 24 60 17 55 16 37 79 43
FLUPHENAZINE 42 57 27 49 17 23 48 26
GUANETHIDINE 0 22 1 30 0 0 0 4
HYDROXYZINE 54 71 50 65 37 26 55
HYOSCINE 29
47 48 32 33, 23
IMIPRAMINE 12 45 17
18 *»« 13 47 17
IPRONIAZINE 34 73 35
70 67 66 69 28
ISOCARBOXAZID 28 29 53
81 84 87 86
ISOTHIPENDYL 63 64 68 95
20 40 14 35 15
LEVALLORPHAN 27 71 19
35 74 32 73 15
LIGNOCAINE 34 72 58
68 73 69 69 58
LYSERGIDE 37 74 39
59 63 52 59 35 -30
MECLOZINE- 55 37
79 78 87 88 67
MEPIVACAINE 66 82 95
58 67 52 60 34 20 63 21
Substance System: 1 2 3 4 5 6 7 8
MEPYRAMINE 15 40 9 33 8 20 68 15
METHADONE 14 62 10 60 11 31 78 37
METHAPYRILENE 12 32 7 24 9 19 52 10
METHAQUALONE 79 75 84 84 68 70 71 94
METHOTRIMEPRAZINE 28 53 20 49 25 39 77 38
METHYLAMPHETAMINE 9 63 6 63 9 22 44 32
MORPHINE 16 24 11 23 5 7 11 9
NAPHAZOLiNE 4 54 3 52 3 2 32 13
NIALAMIDE 71 69 62 64 18 16 2 25
NICOTINE 36 33 24 22 22 11 61 6
NICOTINYLALCOHOL 70 70 70 69 25 24 32 SO
NIKETHAMIDE 70 66 65 67 49 49 52 56
NITRAZEPAM 83 78 85 86 48 44 51 93
NORTRIPTYLINE 9 68 5 71 7 30 56 37
ORPHENADRINE 23 51 18 49 18 27 73 29
PAPAVERINE 72 72 75 74 64 58 62 72
PERPHENAZINE 36 48 23 40 17 21 42 20
PETHIDINE 27 42 19 40 21 32 80 28
PHENELZINE 29 S3 18 82 9 0 72 96
PHENINDAMINE 38 55 30 49 29 33 74 35
PHENIRAMINE 14 34 10 26 9 20 60 11
PHENMETRAZINE 25 45 18 45 23 37 70 45
PHENYLPROPANOLAMINE 12 74 8 75 5 9 25 46
PHENIPAMIDOL 78 82 85 86 43 34 66 82
PIPAMAZINE 41 62 29 52 9 10 37 18
PIPERIDOLATE 53 62 47 52 47 32 78 31
PIPEROCAINE 21 60 18 56 20 38 74 30
PRAMOXINE 62 69 58 60 56 47 73 60
PROCAINE 31 52 22 42 12 14 64 17
PROCYCLIDINE 16 70 13 68 17 39 75 52
PROMAZINE 14 37 8 35 13 37 66 28
PROMETHAZINE 26 45 18 44 24 32 69 40
PROPIOMAZiNE 36 64 27 52 32 38 72 50
PROTHIPENDYL 12 41 7 29 8 24 66. 18
PYRROLBUTAMINE 22 69 18 66 24 45 74 52
QUININE 25 63 16 65 17 41 40 63
STRYCHNINE 7 10 4 11 10 40 33 14
THENYLDIAMINE 19 45 12 36 10 17 68 15
THIORIDAZINE 18 58 13 55 22 44 72 44
THONZYLAMINE 22 40 14 31 15 22 68 18
TRANYLCYPROMINE 34 69 26 67 18 17 59 45
TRIPELENNAMINE 16 41 9 34 7 18 69 15
YOHIMBINE 63 72 57 70 26 15 57 25

173 .
entirely the case with systems 7-8. When
the developing solvent. The less
polar sys-
and replaced the aim
tems 5 and 6 cannot dissolve 0. 1 halide salt M introduced halide salts
pair system,
nia by water to obtain an ion
and therefore the plates need to be impregnated and an une
water caused solvent demixing
by dipping in a 0.1 Af solution of the halide
salt
distribution of bromide. Therefore, watet 1

in methanol prior to development.


Moreover,
to be omitted) which resulted
in the conrpi
the solvent must be saturated with the halide
tion of system 8. Migration differences <

prevent a wash-out of bromide during


salt to
tained with system 8 as compared with syst
development. The systems 7 and 8, though ca-
7 may thus be due to ion pair phenomena and
pable of dissolving 0.1 A/ halide salt in the sol-
the absence of ammonia and water.
vent, give rise to a,/3 and y-fronts during
an uneven distri-
in Table gives the Rf'^-values for the 90 ba
II
development (20) resulting
bution of counter ion across the plates. Im- substances investigated. In Tabic III the stt
pregnation of the sorbent and saturation of the dard deviations for the system.s are listed, i

gether with the resulting discriminnti


solvent, as with systems 5 and 6, circumvent
distances and identification powers for t\
the latter problem.
When applying systems 5-8 in unsaturated confidence limits.
chambers it was occasionally observed that the Decimal values for dj, which can be e
solvent fronts tended to sag somewhat, which pressed in millimeters or hRf-units, we
rounded off to the lowest For exar
full unit.
was accompanied by an uneven migration of
the substances as described by Geiss and San- pie, if ai/jOf3.8 was calculated, the value 3 wi

droni (21). As this affected reproducibility, it introduced In the computer program. Tofulfi
was decided to use saturated chambers for sys- \apj-aJ>(lj, a value of 4 will then be taken int
tems 5-8. The tanks were lined with filter account as a search window for th

paper and filled with solvent 1 hour prior to the IP-calculations.


introduction of the plate. Systems 1 -4 could It is interesting to note the large difference
be us^d in unsalurated chambers with adequate in reproducibility between the systems (a fac
reproducibility. tor of 3-4 going from system 1 to system 7)
The application of the substances to the plate The simplest system, plain methanol, has thi
was done as bases, dissolved in methanol. It best reproducibility and the general rule seem;
should be observed that they may equally well to be that the reproducibility decreases witi
be applied as salt in methanol or as halide ion the number of components in the system, be it i
pair in methanol, regardless of the system, as second or a third solvent component or tlie ad
this does not affect Rf-values. Of course, the dition of bromide. The deleterious effects or
diameter of the starting spot must be kept as the reproducibility of ammonia as basifying
small as possible which may be byfacilitated agent is reflectedby the standard deviation of
selecting a relatively non-polar dissolution me- system 7, being by far the largest of all systems
dium. In our case we selected methanol for investigated.
simplicity and uniformity reasons. As can be expected, the large differences in
Systems 1 -2, 3-4, and 5-6 represent combi- reproducibility have a severe impact on the c/j-
nations differing only in the presence or ab- factors for the various systems and, conse-
sence of halide salts. Thus, differences in quently, on their IP’s. For example, with a tlj
migration behavior may be attributed to the oc- of 2 for system 1 the computer will apply a
,

currence of ion pair phenomena. This is not search window of 2 hRf-units to see if that sys-

174 .
TABLE til

Standard deviations (o-), discriminating distances (df) and identification


power (IP) for 8 TLC systems.

System: 1 2 3 4 5 6 7 8

0.61 1.40 0.75 0.98 0.89 1.61 2.0 1.38

For a^.05
dj 2 5 2 3 3 6 7 5
IP 1 1 4 2 0 0 0 0

For a=0.01
dj 2 6 3 4 4 7 9 6
IP 1 1 2 2 0 0 0 0

As ihe developing distances were always 10 cm, o^values and dj values can be read In millimeters or In
hfthunlh

TABLE IV

IP’s of optimum combinations of TLC-systems for two confidence limits.

For a=0.01 For a=0.05

Best Two 1+4 IP=35 1+4 |p=40


Best Three 1+4+8 IP=56 3+4+7 IP=6p
Best Four 1 +2+7+8 IP =65 3+4+5+8 IP=72

tern can identify a substance p from the 89 re- Tables II and III it can be seen that with a search
maining ones. Any substance within a range window of 2, Butethamide (hRf=36), Buta-
of 2 hRf-units from substance p will be consid- caine (hRf=43), Piperidolate (hRf=47), and
ered tocoincide. Yet, for system?, with ar/jof Nialamide (hRf=62) can be identified with a
9, a search window of 9 hRf-units will be ap- probability of 95%.Yet, if we would like to
plied in two directions, thus covering 18% of identify with 99% probability, a search window
the total separation distance. of 3 must be applied which means that Piperi-
Clearly, with a large selection of substances dolate can no longer be distinguished from Hy-
such as in the present investigation, the IP- droxizine (hRf=50) and Nialamide no longer
values for single systems are of limited value. from Nikethamide (hRf=65).
For 90 substances and a total separation dis- The value of the IP-concept becomes fully

tance of 10 cm, these IP-values will always be visible in the evaluation ofcombinations of
very close to zero as only a very limited num- TLC-systems. Table IV gives the IP’s of the
ber of substances will have a chance to migrate best combinations of any 2, 3, or 4 systems,
to areas in which no other substances are pres- whereas in Table V IP’s of some less suitable
ent. An example is given by system 3. From combinations have been given, including the

175 .
TABLE V
IP’s of some less suitable combinations of TLC-systems, for confidence
limit <x=0.01

Normal Ion Pair Mixed


Combinations Combinations Combinations
1+7 IP=26 4+8 IP=12 1+8 IP =28
1+5 IP =16 4+6 IP=11 3+4 IP =20
5+7* IP= 7 2+6* IP= 4 5+6* IP= 2
1+3+7 IP =35 4+6+8 IP=21 1+2+7 IP =53
3+5+7* IP =29 2+4+6* IP=14 5+6+7* IP=10
1 +3+5+7 IP =47 2 +4 +6 +8 IP =28 2 +6 +7 +8* IP=27
* Indicates worst combination In that category.

worst combination in each category. are compared with regard to their correlation
As can be seen, there are striking differences coefficients, identification power and therZ/s of
in IP-values, emphasizing the pitfalls of ran- the system used.
domly combining systems without proper cri- The systems H-3, which are highly corre-
teria. Even with two "established” systems, lated as expected, have an IP comparable
still

such as 5 and 7, only seven compounds can be to that of the systems 7-h8 with a correlation
on the basis of the Rf-values,
reliably identified coefficient of 0.283, while systems 4+7, with

which can be attributed to a large extent to the the lowest correlation coefficient of 0. 146 have
low reproducibility of the systems involved. an IP of only 14 It is evident that for identifi-
.

Yet, tjie combination of system 5 with the more cation purposes correlation coefficients are of
reproducible system 1 increases the IP-value to limited importance if the systems have bad re-

16. Furthermore, it should be noted that the producibility and, accordingly, large df
combinations with the highest IP-values al- values. On the other hand ,
it will be clear that,
ways include one ion pair system.
at least besides having good reproducibility, the indi-
Thus, the ion pair adsorption systems can be vidual TLC-systems must provide a good
considered to be a very useful and effective al- spread of the substances across the plate.
ternative for STA and even better results may From it may be concluded
the above results
be expected by further optimizing the ion pair that the concept of IP provides a very useful
systems with regard to their reproducibility and relevant tool to evaluate and to optimally
and separation selectivity. This has not been combine chromatographic systems. Not only
included in the present investigation, in which does it show how optimum combinations can
we simply added bromide to a suitable normal be achieved, it also indicates that the gain in in-
system. Yet, the resulting systems may not formation decreases again when using combi-
necessarily represent the best basis for ion pair nations of 3 or 4 systems. The results further
adsorption systems. indicate how dangerous it is to identify a sub-
The impact of the reproducibility on the IP’s stance on the basis of two or three retention
of combined systems is demonstrated in Table data from randomly selected chromatographic
VI in which a number of relevant combinations systems, a procedure which is not uncommon

176 .
in toxicology. With our best two systems, technique to discriminate between the listed
only 35 out of 90 substances can be identified drugs. If that technique is not available or can-
correctly and should be realized that this
it not be applied, the second best choice may be
number will decrease if a larger population of asked, etc. Obviously, the computer will be
basic drugs is taken into account. most useful in the more complicated and/or un-
Although this report deals with TLC-sys- common intoxications, but it should be empha-
sized thatit can also assist in the seemingly
tems only, the IP-concept has almost universal
easy cases with the more common drugs by
applicability as other separation systems such
checking if there are other possible drugs
as GLC, HPLC, and PC can be included as
which fit a particular analytical behavior, and
well. Moreover, the computer program also
allows incorporation of other physico-chemi-
by providing suggestions how to discriminate
against these other possibilities^
cal parameters, such as UV and IR absorption
itic cuacepi of iP together with the TO^IP
data, mass spectrometric data including molec-
program thus seems to offer a number of impor-
ular weight, melting and boiling points, color
tant advantages for STA. Yet it will be clear
reactions, solubility and extraction behavior,
that a lot of work still remains to be done.
etc., provided that for each system or tech-
With regard to the evaluation and optimization,
nique adequate reproducibility data are
a great many systems and techniques will have
available.
to be tested with relevant selections of drugs,
The presenf approach is not only suitable to Then, after having selected the most suitable
evaluate and optimize systems. As it is sub- systems and combinations, data collections are
stance-directed, can also be used for the iden-
it to be made for these systems with as large a
tification of unknown components. For this number of substances as possible. The latter
purpose one measures some relevant parame- should not only include drugs and other rele-
ters as they become available in STA, such as vant poisons, but also metabolites, endoge-
TLC- and GLC-behavior, UV and mass spec- nous compounds, exogenous interferences
trometry. These data are fed into the com- such as plasticizers and antioxidants, etC/ As
puter together with the reproducibility factor it would be unrealistic as well as undesirable to

for each parameter and the computer then sorts try and carry out this work in a single labora-
out the drugs that fulfill these data. If more tory, it is hoped that it can be undertaken as a
than one drug be possible to
is listed, it will also joint effort between various cooperating
ask the computer about the next most suitable institutions.

TABLE VI

Correlation coefficients (r) and IP’s of combinations of two TLC-systems, in


relation to their d/s (a=0.01)

Combination IP dj
1+3 0.985 10 2 and 3
1+8 0.613 28 2 and 6
1+4 0.582 35 2 and 4
7+8 0.283 9 9 and 6
4+7 0.146 14 4 and 9

177 .
V. SUMMARY REFERENCES

A new approach, based on the concept of 1. Moffat, A. C., Smalldon, K. W., and
Identification Power, is described to evaluate Brown, C.,J. Chwmaiogr, 90, 1 (1974),
and to optimally combine chromatographic
2. Moffat, A. C. and Smalldon, K. W., J.
systems with regard to their applicability in
Chromatogr. 90, 9 (1974).
systematic toxicological analysis Contrary to .

earlier aporoaches, which are only system- 3. Moffat, A. C., Stead, A. H., and Small-
directed, the present IP-approach is both don, K. W.,y. Chromatogr. 90, 19(1974).
system- and substance-directed, allowing
4. Moffat, A. C. and Clare, B., J, Pharm.
substance identification by means of chromato-
Pharmacol. 26, 665 (1974).
graphic retention data. Special considerations
had be
to given to system repro- 5. Moffat, A. C., J. Chromatogr. 110, 341
ducibility and spot or peak size, whereas data (1975).
handling and data retrieval were achieved by a
6. Moffat, A. C., J. Chromatogr. 113, 69
special computer program TOXIP, written in
(1975).
Pascal.
The approach was tested in a study compris- 7. Massart, D. L., J. Chromatogr. 79, 157
ing 90 basic drugs which were chromato- (1973).
,
graphed in four classical thin layer
8. Massart, D. L. and Smits, R,, Anal.
chromatographic systems and in four ion pair
adsorption TLC-systems. Combinations of a
Chem. 46, 283 (1974).

normal system with an ion pair system proved 9. Massart, D. L. and De Clercq, 'H.,,Anal.
to be best suitable for systematic toxicological Chem. 46, 1988(1974).
analysis. Other systems based on paper-,
gas-, and high-performance liquid
10. De Clercq, H. and Massart, D. L., J.
chromatog-
raphy can be evaluated and optimized in the
Chromatogr. 115, 1 (1975).
same way. 11. Mailer, R. K., Mdckel, W., Wallenborn,
A special featuie of the present
approach is H., WeihermUller, A., Weihermailer, C.,
that other physicochemical parameters such as and Lauermann, I., in “Beitrage zur
UV- and IR-absorption data, mass spectral Gerichtlichen Medizin” (W. Holczabek,
properties including molecular weight, color Ed.), Vol. 34, p. 265. Verlag Franz Deu-
reactions, etc., can also be included, thus pro- ticke^ Vienna, 1976.
viding a data bank with suitable flexibility for
the identification of unknown substances. 12. Lauermann, I, and MOller, R. K., Pro-
ceedings, Europ. Meeting TIAFT, 1977
VI. ACKNOWLEDGMENTS Leipzig, DDR, in press.

We are indebted to Dr. A. C. Moffat, Home Office Cen- 13. De Zeeuw, R. A., Van Mansvelt,
tralResearch Establishment, Aldermaston, U.K., for pro-
viding a large number of reference compounds and for F. J. W., and Greving, J, E.,J, Chroma-
stimulating discussions. Thanks are also due to Professor togr. 148,255(1978).
‘•V. Schaafsma and H. Akkerboom, Mathematical Insti-
tute, State University, Groningen, The Netherlands, for 14. Connors, K. A., Anal. Chem. 46, 53
their help and advice.
(1974).

178 .
15. De Zeeuw, R. A. and Feitsma, M. T,,
Pharm. Weekhlad 101, 957 (1965).

16. Galanos, D. S. and Kapoulas, V. M.,7.


Chromatogr. 13,128(1964).

17. Dhont, J. H., Vinkenborg, C., Compaan,


H., Ritter, F. J., Labadie, R. P., Verweij,
A., and de Zeeiiw, R. A., 7. Chromatogr.
47,376(1970).

18. Dhont, J. H., Vinkenborg, C., Compaan,


Labadie, R. P., Verweij,
H., Ritter, F. J.,
A., and deZeeuw, R. A., 7. Chromatogr.
71,283(1972).

19. Schepers, P., to whom requests for fur-


ther details should be directed.

20. De Zeeuw, R. A. ,
in “Progress in Separa-
tionand Purification” (E. S. Peiry and
C. J. van Oss, Eds.), Vol. 3, p. 40. Aca-
demic Press, New York, N.Y., 1970.

21. Geiss, F. and Sandroni, S.,7. Chroma-


togr. 33,201 (1968).

Note aJdetl In the final vei.tion of the manuscript;


We leccntly discoveied that some erroneous IP- values
woie given at the oral presentation of this paper in Wash-
ington, D.C., for which we apologize. The IP-values in
this maiiusci ipt ni e correct to the best of our knowledge.
by
The Application of
James M. Moore,
Derivatization Techniques
Drug Enforcement Administration
in Forensic Drug Analysis Special Testing and Research Laboratory
7704 Old Springiiouse Road
McLean, Virginia 22101

A review of methodology that utilizes de- One form of derivatization commonly used i

rivatization techniques in the analysis of illicit in thepresumptive testing of drugs and in th


drugs and related substances is presented. In- determination of their enantiomeric compos
cluded in the review is a discussion of the inter- tion using microcrystallographic analysis. (1

relationship of the derivatization technique In general, theste procedures involve reactio


with determinative steps such as crystal- of the drug with heavy metal salts and observ
lography, chromatography, infiared and mass ing characteristic crystal formation using m
spectrometry. The various applications of croscopic techniques. These procedure
these techniques include (a) the use of deriva- eiyoy the advantages of using small sampl
tizing reagents in the determination of the sizes as well as speed of analysis. Derivatiza
stereochemical composition of illicit drugs, (b) tion techniques are also used to introduc
the utilization of derivatization reagents and fluorophores into drug stimctures to allow fo
their isotopic analogues, in the mass spectral their detection at trace levels using spectro
characterization of trace quantities of synthetic fluorometric determinative steps. (2-5) De
by-products of illicit drugs, (c) the use of de- been used to render drug,
rivatization has also
rivatizing reagents in the gas chromatographic and other substances suitable for analysis b;
quantitation of illicit drugs and their synthetic thin-layer chromatography (TLC) (6) and higi
by-products, and (d) miscellaneous applica- performance liquid chromatography (HPLC
tions of derivatization techniques in forensic (7).
drug analysis. The forensic significance of the
Undoubtedly, derivatization procedure
above applications is discussed in general
have been most widely associated with thi
terms.
analysis of drugs by gas chromatograph]
The analysis of illicit drugs and related sub- (GC). These chemical derivatization proce
stances invariably involves a qualitative, and dures include techniques using gas chromatog
often, a quantitative determination. In most raphy-flame ionization detection (GC-FID)
instances, these analyses may be accomplished gas chromatography-electron capture detec
without altering the elemental composition of tion (GC-ECD) and gas chromatography-mass
the drug. In some cases, however, the modifi- spectrometry (GC-MS). In these chemica
cation of a drug’s structure is desirable, and derivatization procedures, functional groups
sometimes essential, to allow for its full charac- are introduced into the drug molecule in ordei
terization. For this purpose, the term “deriva- to: (a) render those drugs and other substances,
tization” most often used in conjunction with
is
which possess poor chromatographic behav-
techniques employed in the structural modifi-
ior, less polar and more volatile to allow foi
cation of drugs and other substances. Ideally,
their GC quantitation and GC-MS identifica-
derivatization procedures should require a tion; (b) enhance the electrophilic character ol
minimum number of manipulations and should the drug to allow for its detection and quantita-
proceed in rapid one-step reactions to comple-
tion at ppb levels using GC-ECD; (c) effect the
tion. The derivatized drug species can then be GC resolution and quantitation of certain drug
characterized by a number of determinative enantiomers; and (d) allow for the more facile
steps, including crystallography,
chromatog- mass spectral characterization of the drug spe-
raphy, infrared and mass spectrometry (MS).
cies and related impurities, especially in
con-

180 .
junction with the use of deuterated analogues mass spectrometry.
of the derivatizing reagent. Derivatization techniques are commonly
In chemical derivatization procedures using employed in legitimate pharmaceutical analy-
GC-FID, GC-ECD and GC-MS as determina- ses. This is due to the abundance of drugs not
tive steps, the substance to be derivatized amenable to direct GC or GC-MS analysis.
usually contains an active hydrogen atom. The analysis of drugs in clinical and toxicologi-
These active protons are found in functional cal situations routinely use derivatization pro-
groups such as RNHj,, RiRjNH, RCOOH, cedures. In these analyses, trace quantities of
ROH, RCONH 2 , RSH and R=NH. The ex- drugs and their metabolites in biological ma-
change of these active protons with the appro- terials are often derivatized to allow for their
priate substituent can be accomplished by detection and quantitation using sensitive de-
procedures such as esterification, silylation, terminative steps such as spectrofluorometry,
acylation, alkylation, perhalogenation, etc. GC-ECD, GC-MS and mass fragmentography.
Though some drugs do not contain func-
that The application of derivatization techniques
tional groups with active protons —
such as ter- has erjoyed limited use in routine forensic dmg
tiary amines —
can be chemically derivatized, analysis. This is because most controlled drug
these procedures are not widely used because substances are amenable to direct quantitation
they involve multiple steps, are often time-con- by GC-FID and identification using GC-MS
suming and sometimes result in less-than-de- without prior derivatization; alteinately, those
sirable yields of the derivative. (8, 9) drugs not suitable for analysis by GC-MS or
A detailed discussion ot chemical derivatiza- GC-FID may be unequivocally identified using
tion procedures used in conjunction with GC infrared spectrometry (IR) and quantitated by
and GC-MS analyses is not necessary in the ultraviolet spectrometry (UV), HPLC, etc.
present paper, as they are adequately reviewed Additionally, in most cases the sample size
in the literature. Drozd (10) describes the de- is usually sufficient (mg quantities) to allow
rivatization of a wide variety of substances for the leady identification of the drug using IR
using a multiplicity of derivatizing reagents. and GC-MS techniques and quantitation by
Included in the article are over 600 references. UV, or, more often, by GC-FID; this is unlike
Ahuja’s (II) excellent review of the subject toxicological examinations in which analyses
deals primarily with pharmaceutical prepara- frequently requiie derivatization .of ng-/u,g
tions and includes over 200 references. A re- quantities of the drug and/or its metabolite to
cent review by Nicholson (12) describes render it suitable for identification and quanti-
derivative formation in the GC quantitation of tation using sensitive GC-ECD and mas^ frag-
pharmaceuticals. This comprehensive review mentographic procedures. Finally, the time
includes over 450 references. Lochmuller and limitations imposed upon the forensic' drug
Souter (13) review the GC analysis of enan- chemist discourage the use of the more time
tiomers using derivatization procedures. Cim- consuming derivatization procedures,
bura and Kofoed (14) describe derivatization In the present paper we review derivatiza-
techniques used in forensic toxicology. tion procedures that have applicability in rou-
Pierce’s text on the subject of derivatization tine forensic drug analysis, as well as report on
deals primarily with silylation techniques. recent studies that have used derivatization
(15) McCloskey, et al. (16) report the use of techniques in forensic drug research analysis.
deuterium-labeled trimethylsilyl derivatives in Though most illicit drugs can be successfully

181 .
zures for common source determination by de- tion was essential in order to detect and subse
tecting trace impurities in these samples. In quently quantitate these and other impurilie
this study, thesamples were subjected to anal- by GC-FID at levels as low as 0.2% (based oi

ysis by GC-FID and GC-ECD. Stromberg (52) 50 mg heroin and2 cc dilution); (b) detect ant
has also conducted this type of comparison quantitate impurities in the 10"^-10"'^% rangt
analysis on hashish samples. Holley, et al. (based on 10 mg heroin and 2 to 50 cc dilution)
(53) examined marihuana samples in geo- this necessitated the introduction of perhalo
graphic origin studies. Davis and co-workers genated groups into the drug impurity to allow
(54) utilized GC and paper chromatography in for subsequent GC-ECD analysis; (c) allow foi
determining the origin of cannabis. Lee and the GC separation of numerous trace impuri-
Kim (55) have investigated geographical differ- ties that were not resolvable in underivatized
ences of Korean opium based on the alkaloidal samples; and (d) facilitate in the mass spectral
content. Van der Slooten and van der Helm characterization of new trace impurities in il-
(56) have analyzed illicit heroin samples in- licit drugs; the utilization of the deuteiated ana-
depth for common source determination using logues of certain derivatizing reagents is
GC-FID. Clark and Miller have reported
(57) invaluable in these characterizations.
the forensic characterization of dyes in brown The selection of the appropriate deriva-
heroin samples as an aid in drug comparison tizing reagent in the characterization of trace
studies. In this study, the dyes were charac- drug impurities is critical. Though many and
terized using LC techniques. Miller (58) has varied derivatizing reagents are available, we
also studied the GC analysis of trifluoroacetyl have utilized a select few in our studies. These
derivatives of sugar diluents in illicit heroin include N,0-bis-(trlmethylsilyl)-acetamide
preparations for sample
comparison pur- (BSA), deiiterated analogue N,0-bis-(tri-
its
poses. More recently, Clark and Cooper (59) methyl-do silyl)-acetamide (BSA-d„), hepta-
have reported a GC derivatization proce-
fluorobutyric anhydride (HFBA), and acetic
dure for the characterization of sugar diluents
anhydride (AcjO) and its deuterated analogue,
in illicit drug samples. In their procedure, acetic anhydride-dg (ACgO-dg).
the sugars are chromatographed as TMS BSA was selected as one derivatization re-
derivatives.
agent of choice because:
In the procedures described above, none uti-
lized derivatization techniques in the charac- (a) Its well-recognized TMS-donating
terization of trace drug impurities associated properties often allowed for complete
with clandestine drug manufacture. In our derivatization of the drug impurity in
laboratory, we have been concerned
primarily usually less than 1 hour at temperatures
with the characterization of manufacturing im- of TO^C and below; in most instances
purities in illicit cocaine and heroin. In these the use of catalysts was not necessary;
studies, the development of derivatization
(b) TMS derivatives of the impurities stud-
methodology was essential in order to: (a)
ied invariably exhibited good chroma-
allow for the GC and GC-MS analysis of certain
tographic behavior on columns of
drug impurities that normally exhibit poor
widely varying polarity; this was essen-
chromatographic behavior (e.g., ecgonine and
tial in the resolution of overlapping
benzoylecgonine in cocaine and morphine and
peaks and in providing reproducible
0«-acetylmorphine in heroin); this derivatiza-
and accurate quantitative results using

184 .
Parker, et al. (35) have reported on the derivati- salt, namely gold chloride, and the resultant
zation of morphine, codeine, O^-acetylmor- characteristic crystals examined microscopi-
phine and 0®-acetylmorphine followed by GC cally. This procedure allows for the differen-
analysis. In this procedure, TMS deiivatives tiation of d- and /-cocaine.
were formed using BSA. Grooms (36) sub- As previously mentioned, GC-FID, GC-
jects heroin samples to derivatization with ECD and GC-MS derivatization techniques
BSA for the GC-FID analysis of morphine and have limited applicability in routine forensic
0*-acetylmorphine. Rasmussen (37) quanti- drug analysis. Their greatest potential is un-
tates morphine by means of GC with on-col- questionably in the area of forensic drug re-
umn silylation. Brugaard and Rasmussen (38) search analysis. This research Involves the
determine morphine and codeine by GC after in-depth analysis of illicit drug samples and in-

on-column acylation. More recently, method- cludes identification and quantitation of active
ology has been reported for the GC-FlD quan- drug components, characterization of trace
titation of morphine and codeine in opium as quantities of their manufacturing by-pro4,ucts
TMS derivatives. (39) and characterization of m^or and minor dilu-
The analysis of hallucinogenic drugs can ents as well as other adulterants . The in-depth
usually be accomplished readily without sub- characterization of illicit drug samples is of im-
jecting these drugs to derivatization. The di- portance for forensic and intelligence purposes
rect GC analysis of LSD is difficult, however, in that it may allow forensic chemists and law

unless subjected first to derivatization. Sperl- enforcement officials to: (1) compare various
ing (40, 41) describes the GC and TLC analyses drug seizures for common source determina-
of LSD as the TMS derivative after reaction tion in drug conspiracy and related cases, as
with BSA. Radecka and Nigam (42) subject well as determine drug distribution routes; (b)
LSD to hydrogenation followed by GC-FID determine, in some cases, the precursor chemi-
analysis. cals used in the manufacture of the illicit drug;

The routine forensic analysis of cocaine may this is of importance in thesubsequent moni-
be accomplished without subjecting the sample toring of the distribution of these chemicals (c) ;

to derivatization. Nonetheless, Hammer, differentiate between illicit drugs manufac-

et al. (43) describe an improved GC characteri- tured clandestinely and those drugs produced
zation of illicit cocaine by reacting it on-col- legitimately but diverted from legitimate chan-
umn with trimethylanilinium hydroxide. nels forillicit use; and (d) ascertain in general

Recent controversy has arisen concerning the terms the geogra phica l origin of the drug.
forensic chemist’s ability to differentiate co- A number of studies have been reported on
the in-depth analysis of illicit drug samples,
caine from its diastereoisomers as well as dis-
tinguish d- from /-cocaine. Though cocaine Barron, et al. (45) and Kram (46-48) have char-
may be differentiated from its diastereoisomers acterized a number of impurities associated

by IR, the determination of its enantiomeric with the clandestine manufacture of illicit
composition poses another problem. Unfortu- methamphetamine. Lomonte, et al. (49) have
nately, cocaine is not amenable to enantio- studied manufacturing by-products in illicit

meric analysis by derivatization and GC amphetamine samples. Bailey and co-^ork-


analysis. To resolve this isomer problem ers (50) have investigated impurities associated

Allen and Cooper (44) describe a procedure in with illicit methamphetamine. Stromberg (5 1
which cocaine is reacted with a heavy metal has compared various illicit amphetamine sei-

183 .
characterized without prior derivatization, The direct GC analysis of barbituric acid de-
there are some that require derivatization to rivatives sometimes difficult owing to their
is

allow for their more facile analysis. These poor chromatographic behavior. Brochmann-
drugs include phenethylamines, barbiturates, Hanssen and Olawuyioke (25) report on the GC
cannabis components, opium and coca alka- analysis of barbiturates by flash-heater meth-
loids and hallucinogens. ylation using trimethylanilinium hydroxide.
The analysis of amphetamine and metham- Venturella, et al. (26) describe the use of di-
phetamine samples using various derivatiza- methylformamide dimethylacetal in the de-
tion procedures has been reported. Since the rivatization of barbiturates for GC analysis,
biological activity of these compounds is de- Hooper, et al. (27) assay phenobarbital by GC
pendent upon their enantiomeric composition, using on-column butylation. Street (28) de-
it isof forensic importance to be able to differ- scribes the characterization of barbiturates and
entiate between their optical isomers. Wells other drugs by the GC analysis of their trimeth-
(17, 18) describes a GC-FID procedure for the ylsilyl (TMS) derivatives. Heagy (29) de-
resolution of amphetamine enantiomers. In scribes a rather novel infrared method for
this procedure, a derivative is formed with N- distinguishing optical isomers of amphetamine.
trifluoroacetyl-{l)-prolyl chloride which allows Though the major cannabinoid components
for the GC resolution of d- and /-amphetamine in marihuana and hashish samples can be chro-
and subsequent quantitation of these enan matographed directly, improved methodology
tiomers. Beckett and Testa (19) have studied has been reported for their GC analysis as a va-
similar derivatives of phenylisopropylamines. riety of derivatives. Knaus, et al, (30) have
Ment and Marion (20) differentiate the optical characterized cannabinoids in hashish by ana-
isomers of amphetamine by thermal analysis of lyzing their t-butyldimethylsilyl, trlmethylsil-
their benzoyl derivatives. Choulis (21) re- ylacetate and diethylphosphate derivatives.
solves d- and /-amphetamine isomers on alu- Determinative steps used included HPLC, GC
mina, cellulose and silica gel thin-layers in the and GC-MS. Harvey and Paton (3 1) report the
presence of optically active mandelic and tar- use of trimethylsilyl and other homologous de-
taric acids. Eskes (1) describes the differentia- rivatives in the analysis of certain cannabinoids
tion of amphetamine and methamphetamine by GC-MS. Turner, et al., (32) describe the
isomers by derivatization with N-trifluoroace- routine analysis of Cannabis sativa L. by the
tyl-l-prolyl chloride followed by TLC analy- GC determination of its components as tri-
sis. Classically, the determination of the methylsilyl derivatives. Harvey (33) reports
enantiomeric composition of amphetamine and the GC and GC-MS characterization of canna-
methamphetamine has been done by their reac- binoids as substituted silyl derivatives.

tion with heavy metal salts and observing the In the forensic analysis of opium constitu-
characteristic crystal formation by means of ents and related substances, the characteriza-
high-power microscopy. (,/:») Clark (24) re- tion of morphine and similar drugs is enhanced
ports on an improved method for the GC-FID by derivatization. Nakamura and Noguchi
analysis of amphetamine. In this procedure, (34) describe methodology for the GC-FID de-
amphetamine is reacted with cyclohexanone to termination of morphine in opium by analysis
yield a Schiff-base derivative that exhibited of its di-TMS derivative after reaction with
good GC behavior. N, 0-bis-(trimethyIsilyl)-acetamide (BSA).

182 .
GC-FID; additionally, drug impurities (g) The TMS derivatives of some of the
that would not otherwise chromato- drug impurities we have studied were
graph were detected using GC-FlD and amenable to GLC fraction collection
eventually characterized; techniques and subsequent solvent
treatment with a minimum amount of
(c) No “clean-up” was necessary prioi lo degradation of the derivative; this was
derivatization in most drug samples important when attempting to isolate
studied; this was an important factor
trace drug quantities from bulk drug
when attempting to minimize sample matrices; and
analysis time; additionally; direct de-
rivatization of highly adulterated sam- (h) The formation of TMS derivatives of

ples obviated the use of liquid-liquid drug impurities with BSA has proven
partitionschemes to isolate the drug invaluable in their mass spectral char-

matrix from the diluents; this was im- acterization; e.g., TMS derivatives of

portantwhen attempting to minimize morphine-related alkaloids often yield


drug decomposition and to allow for characteristic fragmentation patterns;

more reproducible and accurate quanti- BSA-do


additionally, the availability of

tative results necessary in drug conspir- in pure form has provided considerable
acy cases; structural information which is usually
only obtained through high resolution
(d) No adverse effects were noted when mass spectrometry (HRMS); these fac-
BSA solutions were introduced directly tors are especially critical when charac-
into GC-FID and GC-MS (El and Cl) terizing impurities at ultratrace levels,
systems over a prolonged time period where supporting infrared and nuclear
(note: BSA solutions do degrade the magnetic resonance spectroscopic data
performance of GC-nitrogen-phos- are often not available.
phorous (GC-NPD) and GC-ECD
systems); Though not as versatile as BSA, we have
found HFBA invaluable as a derivatization re-
(e) The TMS derivatives we have studied agent in selected cases. Some of the charac-
were stable for several days; addition-
teristics of HFBA, as well as HFB derivatives
ally, samples with relatively high mois-
of drug impurities, are given below:
ture content were usually derivatized in
a quantitative manner under mild reac- (a) The most obvious advantage of 'HFBA
tion conditions; is its ability to readily introduce HFB
group(s) into the drug impurity, which
(f) The relatively low volatility of BSA al- allows for its detection and subsequent
lowed for reaction conditions using (jX quantitation at levels as low as 10”%
volumes of BSA at elevated tempera- (based on 10 mg of heroin and final dilu-
tures without significant loss of the re- tion of 2 cc) using GC-ECD;
agent; this was an important factor
when attempting to minimize dilution (b) Derivatization of certain heroin impuri-
effects in the analysis of ultratrace ties with HFBA allows for their gas

quantities of drug impurities; chromatographic resolution not attain-

185 .
;

able as TMS derivatives (e.g., the chro- (g) were not as stable as the correspondin{
matographic resolution of 0®- and TMS derivatives, in that hydrolysii
0“-acetyl-morphines); was noted after 1 -2 days;
(c) The HFB derivatives studied generally Unlike their corresponding TMS deriv
exhibited acceptable gas chromato- atives, it AVas found that in most cases
graphic beliavior; however, on col- GC the mass spectra of HFB derivatives oi
umns that were not well-conditioned, heroin-related impurities yielded lim-
we have observed some interaction of ited structural information; in general,
the HFB-derivatized drug impurity the mass spectra of the HFB deriva-
and column substrate; these interac- tives were not as abundant in struc-
tions appeared as pre- and post-peak turally significant fragment ions as theii

inflections; TMS counterparts.


(d) Most of the heroin impurities we have
Acetic anhydride and its deuterated ana-
investigated were derivatized rapidly
logue have been used primarily in the charac-
and to completion by HFBA in an ace-
terization of acetylated impurities in illicit
tonitrile medium at room temperature;
heroin. Its use has been limited primarily in
(e) The HFB esters and amides studied did the mass spectral analysis of these impurities
not hydrolyze readily in weakly basic and is often useful in supporting structural as-
aqueous media; this allowed for the signments made on the corresponding TMS de-
rapid one -step extraction of the HFB rivatives, It has not been employed as often as
derivative from a sodium bicarbonate- BSA or HFB A because, in general:
water-acetonitrile solution into an ap-
(a) Reaction rates were not as rapid nor
propriate organic solvent (usually pe-
yields as favorable under mild reaction
troleum ether); this eliminated the
conditions
necessity of removing the HFBA sol-
vent via evaporation procedures; the (b) Acetic anhydride has a greater degrada-
obvious advantages were in the reduc- tive action on GC column substrates
tion of analysis time as well as eliminat- when compared to BSA;
ing undesirable side-effects associated
with evaporation techniques (such as
(c) The resultant acetyl derivatives did not
exhibit significant sensitivity towards
the application of heat, which may
'result in undesirable side-products);
BCD detection; and
unlike HFB dei ivatives, other perhalo- (d) The chromatographic behavior of ace-
genated derivatives, such as trifluoro- tyl derivatives of the impurities studied
acety! esters, apparently hydrolyze was not as good as the corresponding
when using the extraction procedures TMS derivatives.
mentioned above;
As mentioned above, most of our work uti-

(f) The HFB derivatives of the heroin im- lizing derivatl;2atlon techniques has been in the
were stable in petro-
purities studied characterization of impurities in illicit cocaine
leum ether during the course of a and heroin. Cocaine is a widely abused stimu-
normal working day; however, they lant. It also has legitimate medicinal value as a

186 .
topical anesthetic. The majority of illicit co- FID derivatization methodology which allows
caine is believed produced from the South for the comparative analysis of illicit cocaine
American coca plant by extraction of the alka- samples. Figure 1 illustiates the derivatiza-
loid from the coca leaf, followed by a series of tion of impurities in cocaine samples with BSA
purification steps. On the other hand, pharma- and subsequent GC-FID analysis. For com-
ceutical cocaine is produced by the double es- parison purposes, a GC-FID derivatization
terification of ecgonine. The total synthesis of profile of pharmaceutical cocaine is included in
cocaine can also be achieved as described by Figure 1. After an analysis of the derivatiza-
Willstatter. (60) Due to differences in manu- tion method and chromatogiams in Figure 1,

facture, cocaine contains some impurities


illicit the following observations can be made; (a) it is

not associated with pharmaceutical cocaine. possible to make a rapid, yet in-depth, com-
Moore has identified the presence of cis-
(61) parison of illicit cocaine seizuies for common
and /ran^-cinnamoylcocaine in over 50% of il- source determinations; Figure 1 clearly dem-
licitcocaine samples examined. The cinna- onstrates that samples A, B and C were not
moylcocaines are natural components of the derived from a common batch source; (b) the
coca leaf and are found in illicit cocaine due to sample containing the cinnamoylcocaines was
their co-extraction with cocaine from the leaf. probably produced clandestinely from the coca
In all samples examined to date, when cinna- plant; this is of forensic significance when at-
moylcocaine was detected, it was present as tempting to differentiate between samples of
both cis- and trani-isomers in roughly equal naturally-occurring cocaine and samples con-
quantities. Most illicit cocaine samples also taining cocaine produced synthetically; (c)

contain varying quantities of benzoylecgonine though sample B does not contain detectable
and ecgonine, both being acid hydrolysis prod- quantities of cinnamoylcocaines, the relatively
ucts of cocaine. While a number of papere high methylecgonine content would suggest
have been published on the analysis of ecgo- that the cocaine was naturally-occurring rather
nine and benzoylecgonine in biofluids (62) than synthetically produced; (d) it is possible to
using derivatization techniques, little work has sample that has degraded to an
relate a cocaine
been reported on their analysis in illicit cocaine undegraded sample from the original source by
samples until recently. In our studies, we doing a total alkaloid analysis; this type of anal-
have reported methodology for the detection of ysis is usually supported by additional com-
benzoylecgonine and ecgonine in illicit cocaine parative data; (e) in many cases, illicit cocaine
by the GC-FID analysis of their TMS deriva- samples can be distinguished from high-grade
tives. (63) Majlat and Bayer (64) separated pharmaceutical cocaine; due to the manufac-
benzoylecgonine and ecgonine in pharmaceuti- turing process used, pharmaceutical cocaine
cal cocaine by paper chromatography. An- will not contain detectable quantities of meth-
other Impurity present in virtually all illicit ylecgonine or cinnamoylcocaines commonly
cocaine is methylecgonine. (65) It is believed found in illicit cocaine samples; additionally,
formed primarily as a result of the potassium the quantities of ecgonine and benzoylecgo-
permanganate oxidation of the cinnamoylco- nine in pharmaceutical cocaine would expect-
caines during the cocaine manufacturing proc- edly be significantly lower than in its illicit

ess. Methylecgonine can also be detected by counterpart.


GC-FID as a TMS derivative. The minimum quantities of coca alkaloids
In our laboratory, we have developed GC- and related impurities that can be accurately

187 .
Figure 1. GC-FID Chromatograms of Uncut Cocaine Samples Subjected to Derivatization
with BSA
sample composition! <A) llllcll cocaine containing; cocaine-86%, benzoyiecgonine-S%, tnethylecgonine~3%,
ecgonlne-2%, c;»-cInnanioylcocalne-2%, lrans-cinnamoylcocaine-2%; (B) Illicit cocaine containing: cocalne-
95%, benzoylecgonlne-3%, melhyiecgonine— 1%, ecgonlne-1%; (C) pharmaceutical cocaine containing: co-
caine-100%.
Derlvellzatlon procedure: To a tube containing about 50 mg of cocaine la added 0.5 ml at BSA and 0.5 ml of
CHCI3
(containing 1 mg/ml of octadecane and tetracosane Internal standards); the tube Is healed at 7S°Cfor about 15
min.; about 2 /Jot the solution Is injectectinto theGC under conditions given below.
GC parameters: Perkin-Elmer 3920 GC equipped with FID detection; 6 ft. x4 mm i.d. glass column packed with
3% OV-1 on GCQ (100-120 M); column Is temperature programmed with an Initial temperature of 140“C at an
Initial hold of 8 min. and a program rate of 4‘’C/min. with a final
temperature of 270°C; Injector and detector tem-
perature -275 °C; air and H2flow to FID— 500 and 50 ml/min., respectively; carrier. Ilow~60 ml/mln; amplifier
sen8illvlty-64x5 chart spoed-10 mln.fln.

quantitated by this derivatization procedure samples contain another cocaine hydrolysis


lie in the 0.2-1.0% range using GC-FID product,namely benzoic acid; using the de-
(based on 50 mg cocaine and
hnal dilution of 2 rivatizationprocedure described in Figure 1*
cc); this level of sensitivity adequate for the
is benzoic acid elutes as a TMS derivative in the)
comparison of many uncut illicit cocaine sam- solvent front.
|

pies; though not illustrated in Figure 1, many In summary, the GC derivatization proce-

188 .
Figure 2. Electron Impact Mass Spectra of

Some TMS Derivatives of Impurities Found


in illicit Cocaine Samples

Mass spectra acquired on a FInnIgan 4000 quadru-


pole mass spectrometer Interfaced with a Model 9610
GC and Model 6110 data system; QC column used was
glass, 6 ft.x2 mm l,d„ packed with 3% OV-1 on GCQ
(100-120 M); column temperature was programmed
from about UOXto 270‘’C; Injeotor, separator, trans-
fer line and Ionizer temperatures were all maintained
at about 270°C; helium carrier flow rate-20 ml/mln; El
spectra were acquired under the following conditions:
emission current-0.3S mA, electron multlpller-1000
V, electron energy-70 eV end amplifier sensltlvlty-
10'“A/V.

dure described above for cocaine was found to mation of cocaine-related impurities. The!
be rapid, accurate and of adequate sensitivity obvious limitation of applying the above
for most samples examined. Preliminary re-t method to highly adulterated cocaine sampled
suits indicate that common cocaine diluents do lies in the relatively insensitive GC-FID deter-j
not interfere with the analysis. These diluent^ minative step.
include benzocaine, procaine, Udocaine, caf- It should be noted that in the comparative
feine, lactose, and dextrose. It should be analysis of illicit drugs such as cocaine, a thor-
noted that a diluent sometimes seen in illicit co- ough knowledge of the various manufacturing
caine, namely boric acid, does inhibit TMS for- processes is important. Additionally, a statis-

189 .
tically sound data base is desirable when at- 0®-acetylmorphine (based on 50 mg heroin and
tempting comparative analysis on illicit drug of 2 cc). Highly adulterated her-
final dilution
samples. Finally, the comparative analysis oin samples can also be compared with one an-
should be complemented with other intelli- other using a modified GC derivatization
gence data, procedure. Figure 4 represents a GC-FID pro-
In the forensic comparison of samples such file of a derivatized heroin sample adulterated
as illicit cocaine, it is desirable to obtain ade- with procaine, mannitol, lactose, and dextrose.
quate identification of the TMS derivatives o? Derivatization techniques have also played
the impurities being quantitated (as well as| an integral role in the characterization of im-
non-derivatized impurities, such as the cinna-^ purities heretofore not detected in illicit her-
moylcocaines) . To accomplish this , the BS A-
CHCI3 solution of the cocaine samples may bej
GC-MS analysis. Figure 2
subjected to direct
mass spectra of the derivatized
illustrates the
cocaine impurities utilized for forensic com-
parison purposes. Though not illustrated in
Figure 2, cis- and tranj-cinnamoylcocaine^
yield virtually identical El mass spectra.
Derivatization techniques have also been
used extensively in the characterization of il-

licit heroin impurities. Heroin


produced il- is

licitly by the acetylation of moiTphine, which is

obtained from the opium poppy, Papaver som-


niferum L. Most all illicit heroin samples
contain well-recognized impurities. These in-
clude heroin hydrolysis products, namely 0®-
acetylmorphine and morphine, as well as
codeine alkaloid and its acetylated product,
namely 0®-acetylcodeine. Recently, Sobol
and Sperling (66) have reported methodology
which allows for the comparison of uncut illicit
heroin samples for forensic purposes. In this
procedure, the heroin samples are subjected to Figure 3. GC-FID Chromatogram of Uncut
derivatization with BSA in order to form TMS Illicit Heroin Sample Subjected to Deri^atl-

derivatives of morphine, codeine, and 0®-ace- zatlon with BSA


tylmorphine. The derivatized samples are Uncut heroin sample subjected to derivatization
Illicit
subjected to GC-FID analysis. Figure 3 illus- with BSA(see Ref. 66) and then analyzed by GC-FID
trates a GC-FID chromatogram of a typical under the following conditions: Perkin-Elmer 900 GC
uncut illicit heroin sample. Though acetylco-
equipped with FID; 6 ft. x4 mmglass column packed
with 3% OV-25 on GCQ (100-120 M); column tempera-
deine is not derivatized, it is included in the ture-26$°C; Injector and detector temperature-
comparison analysis . As with the cocaine pro- 27S°C; air and H: flow to FID-50Q and 50 ml/mln., re-
spectively; Ng carrier flow-60 ml/mln. (Note; after
files, the practical quantitative limitations fall
elution of acetylcodeine, column temperature In-
in the 0.2-1 .0% range for acetylcodeine and creased to effect rapid elution of heroin.)

190 .
Figure 4. GC-FID Chromatogram of Adul’
terated Illicit Heroin Sample Subjected to
Derivatizatlon with BSA
Adulterated Illicit heroin sample subjected to derivatl*
zallon with BSA and then analyzed by QC-FID under
the following conditions: Hewlett-Packard 5S40A QC
equipped with FID; 6 ft.x4 mm
l.d. glass column

packed with 3% OV-1 on GCQ (100-120 M); column Figure 5. GC*FID (jhromatogram of Uncut
temperature-230'’C; Injector and detector tempera- Illicit Heroin Sample Subjected to Derlvatl-
ture-27S°C; air and flow to FID-500 and SO ml/mln., zation with BSA and Chromatographed on
respectively; carrier flow-60 ml/mln.
OV.225
Uncut, Illicit heroin sample subjected to derivatizatlon
oin. Klein (67) has identified the presence of with BSA and then analyzed by GC-FID on OV-225
triacetylnorheroin in illicit heroin samples. under the following conditions; Perkin-Elmer 900 GC
This characterization was made possible, in equipped with FID; 6 ft.x2 mm l.d. glass column
packed with 3% OV-225 on GCQ (80-100 M); column
part, by mass spectral analysis of the acety- tomporature-240X; Injector and detector tempera-
lated and deutero-acetylated derivatives of turo-275°C; air and H 2 flow to FID-500 and 50 ml/mln.,
morphine N-oxide, a substance believed pres- respectively; Nj carrier flow-60 ml/mln.

ent in crude morphine base.


During extension of the original work by GC fraction collection of the minor impuritiet
Sobol and Sperling, additional unidentified represented in the chromatogram was perhaps
peaks were noted in the GC-FID chromato- the most expedient means of isolating these im-
grams of some BSA-derivatized illicit heroin purities for mass spectral characterization.
samples using columns of very high polarity. The heroin sample was subjected to derivatiza-
Figure 5 illustrates such a chromatogram on tion in BSA and BSA-dp and chromatographed
OV-225, Since these new impurities were on OV-225. The GC effluent of peaks 1 3 and ,

present in only trace quantities in a rather com- 6 (Figure 5) were condensed in a melting point
plex matrix and to minimize problems asso- tube at room temperature. The condensates
ciated with hydrolysis, it was determined that were washed from the tubes with ethyl ether.

191 .
O^.O^'-OlACETYLOESOXYMORPHfWE-A
’**1 *1 '*< «*J '»«
Figure 6. Electron Impact Mass Spectra of
1 !

Some New Heroin Impurities Isolated From


>
Heroin by GC Fraction Collection
i( X I
Illicit

Uncut, Illicit heroin sample subjected to derivatizatlon


with BSA and chromatographed on OV-22S; minor <3C
peaks (see Fig. 5) were isolated by QC fraction collec-
tion and subsequently subjected to GC-MS analysis
under the following conditions Finnigan 4000 quadru-
:

pole mass spectrometer Interfaced with a Model 0610


GC and Model 6110 data system; GC column used was
glass, 6 ftx2 mm i.d., packed with 3% OV-1 on GCQ
(100-120 M): column temperature-230°C; Injector,
separator, transfer line and Ionizer temperature-
270°C; helium carrier flow rate-20 ml/min.; El spectra
were acquired under the following conditions: emis-
sion current-0.35 mA, electron mullipller-1600 V,
electron energy~70 eV and amplifier sensitIvity-IO'®
A/V. (Note; monoacetylated desoxymorphIne-A Is
probably a mixture of 0'^- and 0 ‘-acetyl isomers.)

the ether evaporated and the residues reconsti- mass spectra of peak (subsequently charac-
1
tuted in microliter volumes of BSA and BS A-dQ terized as desoxymorphine-A). This analysis
for mass spectral analysis. The El mass spec- was enhanced by observing amu shifts of sig-
tra of the isolated impurities are given in Figure nificant TMS-containing ions after deutero-
6. These impurities are desoxymorphine-A, silylation. Certain ions that did not shift upon
monoacetyl-desoxymorphine-A and diacetyl- deutero-silylation were also used in making
desoxymorphine-A. The identification of structural assignments.
these impurities was made possible initially by The molecular ion in the di-TMS derivative
a detailed analysis of silyl and deutero-silyl of desoxymorphine-A occurred at m/e 413 and

192 .
Figure 7. Some SignificantTMS- and Acetyl-Contalningjons as well as Other Ions Used
In the Electron Impact Mass Spectral Characterization of Desoxymorphine-A and its
Acetyl ated Analo gues
Parameters describing Isolation and electron I mpact mass spectral analysis of desoxymorphlne-related heroin
Impurities are given In body of paper and In Figures 6 and 6.

shifted an expected 18 amu upon deutero-sil- significant TMS- and acetyl-containing ions as
ylation. The next most important ion in the well as other ions used in the MS characteriza-
spectrum appeared at m/e 308 and represented tion of desoxymorphine-A and its acetylated
the loss of the stable phenethyl radical from the analogues A paper describing in detail the GC
.

molecular ion. This fragment ion also shifted isolation and MS characterization of these des-
an expected 18 amu upon deutero-silylation. oxymorphine-A impurities will be published in
This loss of 105 amu’s is prominent in the mass the near future.
spectra of desoxymorphine and desoxyco- As demonstrated above, the utilization of
deine-related alkaloids and its presence was deutero-derivatization techniques is essential
important in making the proper structural as- in the MS characterization of impurities using
signment for the impurity. After MS charac- low-to-medium-resolution mass spectrom-
terization of desoxymorphine-A as its di-TMS etry. This requirement is amplified in the
derivative, the presence of mono- and di- absence of supporting spectroscopic data
acetyldesoxymorphine-A was confirmed by provided by IR and NMR. It is also useful
the analysis of their silyl-deutero-silyl and ace- to correlate low- to medium-resolution mass
mass spectra (note; the
tyl-deutero-acetyl spectral data on derivatized impurities with
monoacetyldesoxymorphine-A isolated from high-resolution data obtained previously on
illicit heroin is probably a mixture of 0®- and structurally-related non-derivatized com-
0'‘-acetyl isomers). Figure 7 illustrates some pounds. A number of studies have reported

193 .
the HRMS analysis of morphine and related
compounds. (68-71)
Much of the GC-derivatization work de-
scribed above was done with FID detection.
In trace drug studies, FID detection suffers
from an inherent lack of sensitivity. In order
to characterize impurities at levels as low as
10-®% (based on 10 mg heroin and final dilution
of 2 cc), we have conducted studies utilizing
derivatization of the impurities with HFBA
and detection using the sensitive GC-ECD de-
terminative step. Moore (72) has recently
reported methodology for the GC-ECD
quantitation of morphine, codeine and 0®-ace-
tylmorphine in illicit heroin as HFB deriva-
tives. Figure 8 illustrates a typical sample
chromatogram of uncut illicit heroin subjected
to HFB derivatization and GC-ECD analysis.
Using this procedure, we have been able to
quantitate morphine in some heroin samples at
levels as low as 10"®-10”'‘% (based on 10 mg
heroin and final dilution of 2 cc) while using a
minimum of manipulative steps. The excel-
lent sensitivity enjoyed by this procedure is
demonstrated by the i-esults given in Table I
which illustrate the minimum detectable quan-
tities of morphine, codeine and 0®-acetylmor-

phine as HFB derivatives.


During utilization of the above-mentioned
GC-ECD procedure, a number of additional
peaks of unknown composition appeared in the
chromatograms of illicit heroin samples. One
such peak has been recently identified by
Moore and Klein (73) as 0*-acetyImorphine.
Figure 9 illustrates a GC-ECD chromatogram

Figure 8. GC-ECD Chromatogram of Illicit


Heroin Sample Subjected to Derivatization
with HFBA for Morphine and 0®-Acetyl-
morphine Quantitation
See Reference 72 for sample analysis and chromato-
graphic parameters.

194 .
i

TABLE I

Minimum Detectabie Quantities


(MDQ) of HFB Derivatives of Morphine,
Codeine and 0'’-Acetyimorphlne
Subjected to GC>ECD Anaiysis*

COMPOUND MDQ
Morphine (HFB )2 20 pg
0®-Acetylmorphine (HFB), 100 pg
Codeine (HFB), 80 pg
M0RPHINE(HFB)2

*Derlvatlzatlon and GC-ECD parameters given In


Reference 72.

of an HFB-derivatized brown heroin sample


containing a significant quantity of 0®-acetyl-
morphine. The identification Of this impurity
was accomplished by the mass spectral analy-
sis of its HFB deiivative. It should be noted
that themass spectrum of 0®-acetylmorphine
(HFB), is significantly different from the mass
spectrum of its isomer, 0®-acetylmorphine
(HFB),. This is illustrated in Figure 10. The
presence of 0“-acetylmorphine in heroin sam-
ples is due primarily to the acid hydrolysis of
heroin, while the presence of 0®-acetylmor-
phine is due mostly to the incomplete acetyla-
tion of morphine.
In addition to 0®-acetylmorphine, other late-
eluting peakswere noted in the GC-ECD
chromatograms of HFB-derivatized hetoin
samples. Two of these impurities have been
recently identified as 0®,0®-diacetylnormor-
phine and O'acetylnormorphine. Figure 11 il-

Figure 9. GC-ECD Chromatogram of HFB-


Derivatlzed Illicit Brown Heroin Sample
T I I I I I I r Containing Significant Quantity of 0®-Ace-
0 2 4 6 8 10 12 14 tylmorphine

MINUTES See Reference 73


graphic parameters.
for sample analysis and chromato-

195 ,
CHpC~0.

CH,-C
I.

'
J '
I
I
T '
M M 1

nVo

mXhlne'
Spec, o, MFB Dert.a.l, es of Q.- and pl^
See Reference 73 for electron Impact mesa spectral
parameters.
1 0^0®- ON HFB MORPHINE

0®-MFB-COO£il,’E
lustrates the GC elution of these compounds as 2 0^-HFC-0®.AC6TVL MORPHINE
HFB derivatives in an illicit heroin sample 2 O^-ACE TV L-0®-HFB -MORPHINE
using BCD detection. Due to their rather low 4 O^H-Dt-HFe-O®-ACeTYl.N0nM0RPHlNE
levels in illicit heroin (10-2^10-®%)
GC fraction 5 HEHOIK

colleption of these peaks for e O^.O^*OIACBTYi-N-HF0-NOnMOHPK(NI


subsequent mass
spectral characterization was
not practical.
They were therefore isolated from the
bulk her-
oin matrix using liquid-liquid
partition chroma-
tography. The isolated impurities were
subjected to derivatization with
BSA, BSA