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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
.
Washington, D.G, 2CH02
Foreword
J.'W.GlinUyJv %
Preface
The Editors 4
Negative Ion Mass Spectrometry — a New Tool for the Forensic Toxicologist
H. Brandenherger 48
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
I. Instrumentation
Applications of HPLC to the Analysis of Drugs
B, B. Wheats and R. L. Williams 136
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
V.
Letter From the Administrator
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.
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
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
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-
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.
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
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
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
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-
'
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
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?)-
,
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)
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
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.
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
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)
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-
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).
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 <
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
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
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
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-
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)
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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Lin, S.-N., Stillwell, R. N., and Thenot,
search,’’ in Mas.f SpectromcPy in Drug
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D. I., Dzidic, I., and Stillwell, R. N., in
21. Games, D. E,, Games, M. P., Jackson,
Advances in Biochemical Psychophar-
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macology, Vol. 7 (E. Costa and B. Holm-
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22. Ghisalberti, E. L., “Chemical Ionization 33. Horning, M. G., Brown, L., Nowlin, J.,
Mass Spectrometry in the Identification
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of Drugs and Drug Metabolites,” inMass
Zion, T. E., Clin. Chem., 23 (2), 157-64
Spectrometry in Drug Metabolism (A.
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C. M., Lertratanangkoon, K., Sommer,
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Parfitt,
M., Rodgers, M. S., and Weston, A.,
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Mass Spectrometry and Allied Topics, J. L., Clin. Chem., 22 (11), 1775-88
Washington, DC, May 29-June 3, 1977 (1976).
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(1977). Risby,T. H.,A/ia/. Chem., 49(11), 1501-
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J., S.,
Schulten, H.-R., presented at the
Anal. Chem., 46(14), 2232-34 (1974).
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Washington, DC, May 29~June 3, 1977. C/jem., 48 (4), 726-29 (1976).
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39 .
,
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
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
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-
.
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+,
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
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)
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
attachment torr
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
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
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
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
-
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
57 .
Table IV
intensities at different pressures of the
Anion Ci o f Methaq ualone. Relative anion
reagent gas NaO. —
63.6 “
1 6.6 3.6 26.1 17.7 51.1 100 0.9
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
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
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).
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
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
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
63
, ,
_ ka c
2/?„ s
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
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.
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).
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,
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
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
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
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
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:
r23*i)
JnsoUfltroi
the problem. Clearly, the poorer the
0 i\
signal/noise, the larger the random error L
in the quantitative determination.
‘“C NMR problems is contained in a recent re- flip angle, 400 sec repetition time, de-
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-
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
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
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),
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 .
CIS COMPONENTS
MAY 1978
A
/ \
^
/\ /\ //\ /\ /\
/AQUATIC S
/
/ AMES
TE8T_
\
\
\ /
/0HM-TADS_S^
\
/
/ WATER
DROP
S /
/
A SIHOLE \
/\
/PARTITHJKi
CHy STAl\ /tOWFICjE«1>j
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
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
*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
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.
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 ) ,
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
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).
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-
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,
90 .
by
The Use of Mass
G. W. M.
Spectrometry for Drug A. Milne and H. Fales
N. C. Law
Suburban Hospital,
Bcthesda, MD., 20014
//. DISCUSSION
A. Service Area
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
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
,
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
Ill, RESULTS
A. Case load
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
.
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
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
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
Dalmane more
defies explanation and clearly
data are needed before any commentary on
such trends is justified.
99 ,
Drug 1972 1973 1974 1975 1976 1977 1978 7 Year Total
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
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
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
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
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
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
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
**«A<
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
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
113 .
by
Development of a National
Cornelius G. McWright
Criminalistics Laboratory
FBI Laboratory
Information System Washington, D.C. 20535
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
.
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
'
;
base and increased number of participating
;
laboratories.
Development and Use of Paul B. Ferrara and Michael P. McGee
and
Robert J. McGann
Division of Consolidated Laboratory Services
1 North 14th Street
Richmond, Virginia 23219
tion.
quirements were identified, covering
This includes type and number of Central Processing Unit (CPU) speed,
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.
119 .
CtHtnAl C&HPUfCft ftOJM
TCLeCCMUUHiCWlOHS IIHK .TO STATE DATA
KA^VcTlC ^PflQCESSlHO CtHtEP
rPROCRAMUER S STAtlOH “I
I
»<JOH SPCCO I
J tPUNCH-t20CHAR/5EC |
I LINE PRINTER
H ISO COLUiAHS
• 350 LWES/TAIH
6C/MS
DIGITAL INPUT - INSTRUMENT
SUBSYSTEM (EKI 5 TIN 6 )
• gp PO 3 A •UP TO 6
CHATTNEES CHANNELS
• SCAN RATE •SCAN RATE NUR
• JOPTS/SEC' 6000 PTS/SE( • ' SPECTROMETER •
CHAU HE L [MINI lEYISTINS}
OfNlR
IHVTRUUCHTS
tl J RJrtAPMItS
(C«ST(H6) Cftf
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.
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
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
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
‘
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
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
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
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:
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
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).
133 .
Chromatographic Advances
Session
I. Instrumentation
II. Methodology
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
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
Moderate
'
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
.
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
D. Detectors
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
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-
'
142 .
immunoassay techniques have also been re-
ported (8).
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
7. Cannabis
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
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
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.
4. Electrochemical detection
Conclusion
148 .
,
D. Acknowledgment
We should like to acknowledge the valu-
able contiibutions made by our co-work-
all
REFERENCES
4 . Wheals B B ,
.
. , 7, Cht oma togr. ,107, 402
(1975).
149 .
9.
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).
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
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:
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
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
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-
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)
,
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.
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.
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
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.
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).
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-
matography Detectors” p. 29, Elsevier L. J., and Friedman, L., Biochem., 13,
Scientific Publishing Co., Amsterdam
5060(1974).
and New York, 1977.
16. Buehler, R. J., Flanigan, E., Greene,
5. Kenndler and Schmid, E. R., in “Instru- L. J., and Friedman, L,, J. Amer. Chem.
mentation for High Performance Liquid Soc., 96,3990(1974).
163 .
17. Beuhler, R. J., Flanigan, E., Greene, 31. Kraak, J. C., Poppe, H., and Smedes, F.,
L. J., and Friedman, L., Biocliem. J. Chromatogr., 122, 147 (1976).
20. Schulten, H. R. and Beckey, H. D., A/w/. 34. Sternberg, J. “Advances in Chro-
C., in
C/ie/n.,45,2358 (1973). matography, Vol. 2” (J. C. Giddings and
45. Dark, W. A., McFadden, W. H., and 55. Henion, J. D., presented at the 26th
Bradford, D. L., J. Chroimilogr. Sci., 15, ASMS meeting, paper No. RE-2, St.
Lab,, 9,55(1977).
58. Arpino, P. J. and Krien, P., presented at
48. Baldwin, M. A. and McLafferty, F. W., the 26th ASMS meeting, paper No. RE-6,
Org, Mass Spectrom., 7, 1111 (1973). St. Louis, MO, May 28-June 2, 1978.
50. Arpino, P. J., Dawkins, B. G., and 61 . Hatch, F. and Munson, B,,Amil, Chem.,
McLafferty, F. W.,7. Chromatogr. Sci., 49, 169(1977).
12, 574 (1974),
62. Hatch, F. and Munson, B. Anal. Chem.,,
49,731 (1977).
51. McLafferty, F. W., Knutti, R., Venka-
taraghavan, R., Arpino, P. J,, and Daw- 63. Colin, H. and Guiochon, G., J. Chroma-
kins, B. G.fAnal, Chem., 47, 1503(1975). togr., 141,289(1977).
165 .
64. Colin, H. and Guiochon, G. J. ,
Chroma- 77. Knox, J. H., Laird, G, R.. and Raven,
togr., to be published in “Advances in P. A.,/, Chromatogr., 122, 129(1976).
Chromatography 1978” 78. Jones, P, R. and Yang, S. K., Anal.
65. Colin, H., Ward, N., and Guiochon, G., Chem., 47, 1000 (1975).
Proceedings of the 25th ASMS meeting, Phys. Chem., 46, 456 (1972).
Washington, DC, May 29-June 3 (1977),
80. Tal’Rose, V. L., Grishin, V. D,, Skurat,
p. 189. V. E,, and Tantsyrev, G. D., in “Recent
67. Hunt, D. F., McEwen, C. N., and Har- Developments in Mass Spectrometry”
vey, T. M., A«((/. Chem., 47, 1730 (1975). (K. Ogata and T. Hayakawa, eds.), Uni-
versity Park Press, Baltimore, MD, USA,
68. Melera, A., private communication.
p. 1218(1970).
69. McAdams, M. J., Blackley, C. R., and 81. Karasek, F. W. andDenney, D. W.,A«n-
Vestal, M. L., presented at the 26th
lyt. Letters, 6, 993 (1973).
ASMS meeting, paper No. RE-1, St.
T.ouis, MO, May 28-June2, 1978. 82. Tal’Rose, V. L., Gorodetskii, I. G., Zolo-
toi, N. B., Karpov, G. V., Skurat, V. E.,
70. Udselh, H, R., Orth, R. G., and FuUell,
and Maslennikova, V. Ya., in ‘‘Advances
J. H., presented at the 26th ASMS meet-
in Mass Spectrometry Vol. 7” (N. R.
ing, paper No. RP-4, St. Louis, MO, May Daly, ed.), p. 858, Heyden & Son, Lon-
28-Jfne2, 1978.
don (1978).
71. lakeuchi, 1'., Hirata, Y., and Okumura,
Y., Anal. Chem., 50,659(1978).
166 .
A New Approach to the by
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- . . . ,
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
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.
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
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
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-
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.,
< • •
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
. . .
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
171 .
TABLE II
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
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
currence of ion pair phenomena. This is not search window of 2 hRf-units to see if that sys-
174 .
TABLE til
System: 1 2 3 4 5 6 7 8
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
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
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
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).
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.
A review of methodology that utilizes de- One form of derivatization commonly used i
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) ;
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
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
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
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
(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,
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
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.
pies; though not illustrated in Figure 1, many In summary, the GC derivatization proce-
188 .
Figure 2. Electron Impact Mass Spectra of
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-
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.
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 !
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-
194 .
i
TABLE I
COMPOUND MDQ
Morphine (HFB )2 20 pg
0®-Acetylmorphine (HFB), 100 pg
Codeine (HFB), 80 pg
M0RPHINE(HFB)2
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
derivatives. minutes
As in the case of the desoxymoiphine-A
impurities, these acetylated
normorphine im- tratlng Some Late-Eluting Heroin Impuri-
purities were characterized
primarily from sig- ties Found In an HFB-DerIvatized
nificant ion shifts observed Illicit
in the TMS and Heroin Sample
TMS-dft-mass spectra of 0»,OS-diacetyl-N-
Heroin sample Is subjected to derivatization
TMS-norraorphine. Fragment ions at m/e
326, HFBA as described Jn Reference 72; derIvatUed with
sam-
342, 368 and 385 suggested the ple subjected to GC-ECD analysis under the
Is
presence of ace- follow*
toxyl groups at the C3 and C6 oondtions: Perkin-Elmer 990 QC
equipped with a
positions in the Nl electron capture detector;
tnorphine-type molecule. 6 ft.x4 mm l.d. glass
All of these ions CO umn packed with 3% OV-1 on GCQ
{100-120 M)-
shifted the expected 9 amu column tempGrature-220'>C: injector and
upon deutero-silyla- detector
tion. One of the most significant ions occurred t6mperature-275°C and SOO'C, respectively;
Nj carrier
flow-100 ml/min.
196 .
Figure 12. Electron Impact Maes Spectra
of TMS derivatives of Acetylated Normor- Figure 13. Electron Impact Mass Spectra
phine Impurities Found in illicit Heroin ofHPB Derivatives of Acetylated Normor-
Heroin Impurities O^,0°-dlacetylnormorphlno and O®-
phine Impurities Found In illicit Heroin
acelyl-normorphlne were Isolated from the bulk her- Heroin Impurities O^-acetyl-normorphlne and 0^O«-
oin matrix by liquid-liquid partition chromatography; dlacetylnormorpMne were Isolated from the bulk her-
the Isolated Impurities were derivatized with BSAfor oin matrix by llquEd-llquId partition chromatography;
30 min. at 7SX; the derivatized Impurities were sub- the Isolated impurities were derivatized with HFBA as
jected to QC-MS analysis under conditions described described In Reference 72; the derivatized Impurities
In Figure 0, were sub|ected to QC-MS analysis under conditions
y described In Figure 6.
at iTi/e 100 and represented in part a TMS-sub-
stituted N This ion also shifted 9
function.
amu upon deutero-silylation. Figure 14 illus-
trates some significant TMS- and acetyl-con-
taining ions as well as other ions used in the
MS characterization of the acetylated nor-
morphines. As with the desoxymorphine-A
impurities, details of the isolation and
characterization of the acetylated normorphine
i mpu rities will be published soon.
Though the mass spectra of the HFB deriva-
tives of the acetylated normorphine and other
impurities were obtained, they were not uti-
lized as fully as the mass spectra of the TMS de-
rlyatives for mass spectral elucidation. This is
because most of the intense ions represented
either HFB fragments or losses of HFB frag-
nientsfrom parent ions. Additionally, the lack
of an isotopic analogue of HFBA permitted
Ojhly limited mass spectral evaluation of the re-
sjbltant HFB derivative of the impurity. Fi-
nally, the lesser stability of the HFB
derivatives compaied to their TMS counter-
parts did not allow for their lepetitive mass
eipectral analysis from a single solution.
ACKNOWLEDGMENTS
I would thank Dr. Kenner C. Nice, Medicinal
like to
Chemist ry, National
Instjtutesof Health, Bethesda,
Maryland, far supplying standards of desoxymor-
phIne-A and the acetylated normorphines. I would
also like to extend my appreciation to Mrs. Jean
Nolan for typing the final manuscript.
SUMMARY
Flgure 14. Some Significant TMS- and Ace-
The
application of derivatization techniques
tyl-Containing Ions Used in the Electron
has enjoyed limited use in routine forensic drug
impact Mass Spectral Characterization of
analysis, and this situation is not likely to 0^0^'Diacetyinormorphine and 0®-Acetyl-
phange in the foreseeable future. Derivatiza- normorphine
fion methodology, especially when used in
Parameters describing isolation, derivatl7ation and
coiyunction with GC-FID, GC-ECD, and GC-
GC-MS analysis of acetylated normorphine heroin Im-
MS techniques, has significant potential in fo- purities given In body of paper and In Figure 12.
198 .
rensic drug research. It is especially applica- 13. Lochmuller, C. H. and Souter, R. W. J. ,
ble in the in-depth analysis of illicit samples and Chromatogr., Vol. 113, pp. 283-302
in the characterization of trace drug impurities (1975).
for forensic and intelligence purposes.
14. Cimbura, G. and Kofoed, J., J. Cluoma-
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7. Lawrence, J. F. and Frei, R. 'W., Chemi- 22. Eskes, D., 7. Chromatogr,, Vol. 1 17, pp.
cal Derivatization in Liquid Chromatog- 442-44(1976).
raphy, Vol. 7, Elsevier Scientific
23. See Ref. 1.
Publishing Co., New York, (1976).
24. Clark, C. C.,J.A.O.A.C., Vol. 58, No. 6,
8. Vessman, J., Hartvig, P. and Molander,
pp. 1174-77(1975).
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(1973). Brochmann-Hanssen, E. and Olawu-
25.
9. Hartvig, P. and Vessman, J., Anal. Let- Pharm. Sci., Vol. 58, No. 3,
yioke, T., J.
10. Drozd, J., J. Chromatogr., Vol. 113, pp. 26. Venturella, V. S., Gualario, V. M. and
303-56(1975). Lang, R. E.,7. Pharm. Sci,, Vol. 62, No.
4, pp. 662-68 (1973).
11. Ahuja, S,,J. Pharm. Sci., Vol. 65, No. 2,
pp. 163-82(1976).
27. Hooper, W. D., Dubetz, D. K., Eadie,
12. Nicholson, J. D., Analyst, Parts I and II, M. J. and Tyrer, J. H., J. Chromatogr.,
Vol. 103, pp. 1-10 and pp. 193-222(1978). Vol. 1 10, pp. 206-9(1975).
199 .
28. Street, H. V.,7. Chromatogr,, Vol. 41, 42. Radecka, C. and Nigam, I. C,, J. Pharm.
pp. 358-66(1969). Vol. 55, No. 8, pp. 861 -62 (1966).
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31. Harvey, D, J. and Paton, W. D. M., J. 45. Barron, R. P., Kruegel, A. V., Moore,
J. M. and Kram, T. C.,J.A.O.A.C., Vol.
Chromatogr., Vol. 109, pp. 73-80 (1975).
57.No.5,pp. 1147-58(1974).
32. Turner, C. E., Hadley, K. W., Henry, J.
and Mole, M. L,.,J. Pharm. Sci., Vol. 63, 46. Kram, T. C. and Kruegel, A, V., J. Fo-
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rensic Sci., pp. 40-52
(1977).
33. Harvey, D Chromatogr., Vol. 147,
i.,J.
pp. 291-98(1978). 47. Kram, T. C.,J, Pharm. Sci., Vol. 66, No.
3,p. 443(1977).
34. Nakamura, G. R. and Noguchi, T. T., J.
Forensic Sci. Soc., Vol. 14, pp. 347-53 48. Kram, T, C,, J. Forensic Sci., Vol. 22,
(1974). No. 3, pp. 508-14(1977).
35. Parker, K. D., Wright, J. A., Halpem, 49. Lomonte, J. N. Lowry, W. T, and Stone,
,
39. Joint Committee of the Pharmaceutical 53. Holley, J. H., Hadley, K. W., and
Society and the Analytical Division of the Turner, C. E., J. Pharm. Sci., Vol, 64,
Chemical Society on Pharmaceutical No. 5, pp. 892-94 (1975).
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Osadchuk, M., Ann/. Chem., Vol. 35, pp.
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55. Lee, C. K. and Kim, H. K., Bulletin on
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200 .
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pp. 327-36(1978)
59. and Cooper, D. A., private
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201 .
by
Advances in Chemical
L. Stroinberg & A. C. Maehly
Signature Analyses of
The National Laboratory of
Drugs Forensic Science
Fack
S-581 01 Linkoping, Sweden
202 .
Fig. 1. Chemical FID signatures of hashish samples seized in Norway and Sweden,
respectively.
that such analyses may be carried out without terials proper; and b, impurities in these prod-
exchange of genuine drug samples which often ucts), from products of side reactions (C), and
involve intricate legal problems. Only solvent finally from intermediate co mpounds (D) The .
extracts of the impurities need to be ex- B, C and D compounds which we call key im-
changed These extracts contain drugs only in
. purities can be useful in determining the
trace amounts and are, therefore, of no interest method of synthesis used. This is the subject
from the point of view of drug control. of most of the publications mentioned above
Before going on, it may be useful to consider (1
- 10).
the origin of the impurities and by-products The E, F, G . , . . Z substances, on the
forming a chemical signature. A somewhat other hand, stem from the further fate of the
simplified scheme is rendered in Fig. 2, The raw product obtained at the clandestine labora-
raw product cbfitains the substances of the A, tory. The contribution of these substances
B, C, D group, i.e., impurities from the equip- to the chemical signature can also be of value
ment and the laboratory (A), contributions for the police intelligence work. The F,
from the starting materials (B, the starting ma- G . . Z substances can be subdivided into
. .
203 .
RESULTS
BWtins
cotic drugs has been based on recording simul-
A.Ateriai.9
taneously the signals from an FI- and an
EC-detector, using an effluent splitter. Re-
proiiuctt of 1^1 n iDteni)«d.i ate
products
cently we began using a similar technique but
Bidereactiona product
replaced the ECD by an NP-detector. Obtain-
ing different types of signatures enhances the
possibility of establishing a common origin of
different samples.
We were interested to find out to what extent
impurities in adulterants might contribute to
Owing to its wide-
the total signature pattern.
spread occurrence, we started with the sugats
(Stromberg, L. and Bergqvist, H., to be pub-
i_V“ lished). So far, two kinds of glucose and two
diluent tl
]-[ product fl
I
kinds of sucrose were investigated, using n-
\
heptane for extracting impurities. The signa-
PRODUCT tures of the sugars, especially the sucroses,
were found to be weak, probably due to the
Fig. 2. The origin of the compounds of a high standards of purity requested for food in-
chemical signature. The details are ex- gredients,The glucose samples showed one
plained in the text. or two m^or peaks in their signatures. The
corresponding impurities have been partially
the main adulterants (F, G . . . . Z) and the identified.
impurities in these (f, g . . . .z). The former Next the sucrose and dextrose samples were
are, in general, easily identified. In seizures mixed with samples of seized amphetamine
of central nervous system stimulating amines, sulfate and with amphetamine sulfate synthe-
the following adulterants have been observed sized and twice recrystallized at our labora-
quite frequently in Sweden: sugars, caffeine, tory. As expected, the addition of sucrose did
phenazone, ascorbic acid, ephedrine, nor- not noticeably interfere with the signatures of
ephedrine, pseudonorephedrine, barbituric drugs. The stronger signature of the dextrose
acid derivatives, lidocaine and procaine, as samples, on the other hand, contributed to that
well as mixtures of CNS-stimulating amines. of the specially purified amphetamine. How-
Sugars are the most frequent additives. ever, the signature of most seized ampheta-
Most of these adulterants are themselves mine samples so strong that the sugar
is
sugars, alongside with the more sporadically The dilution of drugs with sugars should, there-
used substances, such as starch or fiour, can be fore, as a rule, not hinder this analytical
regarded as passive diluents, procedure.
Finally, handling and packing can introduce We can now return to the second task of sig-
another type of contaminant (E). nature work, /.e., identifying key impurities in
204 .
.
order to establish the method of synthesis. trometry and the mass spectra were compared
The major part of the work published in the lit- with those published by other workers (cf
erature concerns itself with the key impurities Table I), Peak 1 is impurity nr 8 (6), peak 2 is
in methamphetamine (1, 2, 3, 5, 9, 10), which impurity nr 9(6), peak 3 has not yet been identi-
we have not studied Amphetamine
in detail. fied, peak 4 is impurity nr 6 (4) and peak 5 is im-
workers (8,9). All of this work was concerned al. (3), but to our knowledge not reported in
with the Leuckart method of synthesis {/.e., the amphetamine samples.
reductive amination of phenylacetone by The two remaining larger peaks 6 & 7 have
formamide, or modifications thereof). The identical mass spectra. These spectra are sim-
compounds reported by these authors are ilar to that reported for impurity nr 7. Lomonte
listed in Table I. et (4) published a mass chromatogram
al.
Using our method of analysis, very rich sig- (m/e=91) where a double peak is visible. The
natures were obtained and only a limited num-
,
larger (first eluted) peak was shown by them to
gas chromatogram from a GC/MS experiment stereomeric forms of this tertiary amine
(JEOL D 100 coupled to a Varian gas chromato- The key impurities so far described are those
graph 2700) is shown in Fig, 3, Some of the of Leuckart amphetamine. A
second method
major peaks have been identified by mass spec- of amphetamine synthesis which has been used
205 .
Table I
methyl benzyl a)
1. 134,05 CH,-C0-CH3 (9)
ketone
N-formyl‘ c) (9)
4 163.07 C,eH,3NO
amphetamine
r 1
206 .
Fig. 4. Chemical FiD (left) and ECD (right) signatures of Alles amphetamine.
The reduction probably proceeds step- experiments at this laboratory where the cath-
wise as outlined in Fig. 5Phenylnitropropene
.
ode potential was carefully controlled. Sec-
residues can be identified with the FID/ECD ond, benzyl methyl ketone was found in a
seized reduction mixture of the type concerned
(Fig. 6) probably as a result of the hydrolysis of
0-CIIO + Ctl2-CH3
0 -CII » C-CH3 + lIjO the oxime. The absence of the oxime
may
itself in
be explained by the
KO2 N«2 the seized materials
fact that these reductions were carried out with
207 .
Ee ftw ’Ve «• '«T‘ ^’
Ffg. 6. Reconstructed TIC and computer'processed mass spectrum showing the pres-
ence of benzyl methyl ketone in Alles amphetamine. The first “peak" In th© TIC Is am-
phetamine, the second benzyl methyl ketone,
uncontrolled extreme potentials, leading sufficient di.scrimination power csjiccitilly
directly to amphetamine. The presence of when using a capillary column.
benzyl methyl ketone just mentioned is inter-
esting from another point of view. Evidently, Note added May 17, 1978: Phenylacetoximv
the presence of benzyl methyl ketone residue was found to be present in a seizure of Aiies
in illicitly prepared amphetamine does not nec- amphetamine.
essarily point to the Leuckart method of
synthesis.
Key impurities are obviously of great inter- REFERENCES
est in the forensic analysis of narcotics.
Their
recognition among the numerous gas 1. LeBelle, M,, Sileiku, M., and Romiich,
chroma-
tographic peaks which constitute the M., J. Phar/n. ScL. 62, 862 (1973).
chemical
signature should therefore be an
important 2. Bailey, K., Boulanger,!. G., Legatilt, D.,
question for forensic laboratories
concerned andTailIefer,L.,y./^/imv«,.yc/.. 63, 1575
with the analysis of narcotics. The
“classi-
(1974).
cal” method for recognition of an
organic
compound in a complex mixture is gas chroma- 3. Barron, R. p., Krucgcl, A. V., Moore,
—
tography mass spectrometry. We think, J. M,, and Kram, T. C.,J.
however, that key impurities of drugs Anal. Chem., 57, 1 147 (1974).
may be
recognized also by less sophisticated
(and less 4. Lomonte,
expensive) methods, A gas J. N., Lowry, W, T., and
chromatograph
with a dual detector system
Stone, I, C„ J. Forens. Set., 21, 575
(19) should have a
(1976).
208 .
5. Kram, T. C., personal communication
(1976).
folded in tinfoil. After determining the color Normally three, but at least two, different sys-
210 .
terns of mobilephases are used. For compari- the jury to know whether or not a suspect has
son purposes, a standard heroin solution is been found in possession of a “not small
added to each plate. High performance plates amount” of a drug. Consequently, police la-
show good results in an adequately short time. boratories are often requested to determine the
concentration of heroin in a specific sample.
D. Methods of Detection Various methods have been used in the past,
ranging from UV- and IR-spectrometi'y to titra-
For detection of spots, plates are observed
tion with perchloric acid, but, of course, best
under UV light and sprayed with iodine-plati-
results are gained by. GC analysis using pro-
nate, Dragendorff and van Urk reagents and
caine hydrochloride as internal standard. A
FeCla-iodine solution.
Sigma 10 Integrator (Perkin-Elmer) is a valu-
After completion of this TLC procedure,
able aid.
samples which do not contain heroin are
discarded.
D, Derivative Analysis
211 .
IV. GC-MS ANALYSIS OF HEROIN 2. Anesthetics, i.e., lidocaine, procaine,
cocaine.
For reasons already discussed during this 3. Benzodiazepines, i.e., diazepam.
Symposium, GC-MS is also used for drug anal- 4. Pyrazolone derivatives, i.e., antipyrine,
ysis. Our experience has been with a quadru- aminopyrine.
pole instrument supplied by Finnigan (Model 5. Salicylic acid or phenol derivatives, i.e.,
4000), The highly specific method of identifi- salicylic acid, salicylamide, acetylsali-
cation needs no further explanation. cylic acid.
6. Hypnotics, i.e., barbital, carbromal,
A. Preparation of Sample methaqualone.
7. Alkaloids, i.e., caffeine, quinine, strych-
Preparation of the sample and conditions are
nine, scopolamine.
the same as withGC. Sugars.
8.
9. Inorganic compounds, i.e., CaCOg,
B. Conditions NaHC03,NaC103, KCIO3.
Our application of mass spectroscopy has A change in heroin itself has been noticed.
been mainly El; in only a few cases, Cl also has There is an increasing number of samples con-
been employed with methane, isobutane or am- taining mixtures of free bases and salts of her-
monia as reagent gases. For trace amounts of oin. Another noticeable change in the
heroin, SID has been used successfully.
by-products is the appearance of papaverine
and narcotine.
C. Methods of Analysis
1.
212 .
,
(NH 4 2 C 03
) solution (4 ml) with a flow
rate of 2 ml/min. 2,3-Dimethylnaph-
thalene is applied as internal standaid.
The results are extremely good,and
this method be used in the Berlin
is to
Laboratory, too, employing a Perkin-
ElmerHPLC (Model 3).
2. Use of Data Systems
The high rate of requests for inves-
tigations, as mentioned in the preface,
forces us to apply data techniques, even
in our small laboratory unit. Therefore
the GC-MS system is complemented by
an Incos Data System, including a
library with 23,000 mass spectra. The
IR spectrometers have interfaces in
order to employ an interdata system.
As long as police laboratories remain
responsible for fast and accurate
analyses, they must be supplied with the
necessary technical means. It must be
understood, however, that no computer,
no data system, not even the most
sophisticated one, is able to assume the
responsibility of an expert, and none of
these systems used in forensic chemistry
can see the link between analytical work
and human fates.
by
Street Drugs and the
J. Wells and G. Cimbura
Forensic Toxicologist
Toxicology Section
Centre of Forensic Sciences
Toronto, Canada
A forensic toxicologist is concerned with the drugs. Flame ionization detectois have gen-
analysis of body tissue for drugs and poisons erally been used in these procedures although
and the interpretation of the results obtained. electron capture detectors have found some
It is not surprising therefore that he or she will application The introduction of stable ni-
(2).
come into contact with so called “Street trogen phosphoms (NP) detectors marks a
Drugs”, as some of these drugs are used legiti- m^or advance in GC screening. These detec-
mately in medicine while others such as butyl tors were first used to screen the urine of ath-
nitrite, methylenedioxyamphetamine (MDA), letes competing in the 1976 Montreal Olympics
tetrahydrocannabinol (THC), and phencycli- (3); the urine was extracted with ether and an
dine (PCP) have widespread illicit use. The aliquot injected directly into the GC. We have
analysis of body tissue for many of these non- used a similar approach for screening blood.
medicinal drugs represents a considerable The extraction proceduie is shown in Fig. 1 and
challenge for the toxicologist as adequate as can be seen it is very simple. The choice of
methodology is often not available. The ap- 1 ml of blood and 2 ml of solvent is not critical
proach we have tried to follow with street drugs and was in fact dictated by the volume (2 ml) of
is to incorporate their detection in the general the disposable vials used by the automatic liq-
screening procedures we are presently using uid iryector. Vials with a volume of 100 (x\ are
for the detection of the common prescription available and the use of such vials would enable
and non-prescription drugs. This, of course, is blood volumes of 0.1 ml to be used. The
not always possible and important exceptions
will be dealt with later.
Screening procedures of one type or another
1 ml bl
are in use in most toxicological laboratories; extra etj^vortex
these vary from simple inexpensive thin-layer
chromatography (TLC) procedures to methods
mass spectrometry. The use of gas
utilizing 2 ml toluene, NaOH
chromatography (GC) for screening blood and
urine for basic and neutral drugs is a technique |ac20
in which we have taken a particular interest
We would like to describe our latest work
(1).
in this area as we feel it would be of some inter-
GC
est to the reader.
2 ml vial
GC screening of blood and urine for drugs
has been used by us and others for a number of
years. The differences in the methods used re- 10i|l inj at
volve around the extraction procedures and the
type of GC liquid phases employed. Using
200”C . «
such procedures many drugs are detectable at
0 1 mg/ 1 00 ml a level that is adequate for many
.
240'C
,
overdose situations but inadequate to detect Fig. 1. Extraction procedure for neutral and
therapeutic levels of some of the common basic drugs.
214 .
Rg. 2. Gas chromatographic equipment used in screening blood and urine for basic ana
neutral drugs.
choice of solvent for the extraction is limited in
the sense that halogenated solvents are not rec-
ommended for routine use with NP detectors.
We have found toluene to be adequate for most
of the drugs of current interest.
The GC system is shown in Fig, 2; the small
data system, after suitable calibration, con-
verts retention times into retention indices.
The sample (10 ^1) is injected twice, once at
200'’C and a second time at 240°C. The GC col-
umn system (Fig. 3) is a split glass column
which we have used previously (4); the combi-
nation of OV-1 and Poly A- 103 liquid phases
enables us to screen for a large range of drugs.
Figs. 4-8 illustrate the results; sensitivity is not Rg.3. The split glass column system used
generally a problem as most drugs are detect- NP detectors and packed with 2%
with dual
able below the 0.05 mg/100 ml level. An addi- OV-1 (Back Column) and 3% Poly A-103
tional advantage of the NP detectors is that (Front Column) both on 80/100 mesh Chro-
they do not respond to the lipid material nor- masorb W.
215 .
’I
216 .
Fig. 6. GC Chromatograms obtained at Fig. 7. GC Chromatograms obtained at
200°C from a case biood treated as in Fig. 1 200°C from a case blood treated as In Pig. 1
and containing 1-Caffelne, 2-Amitriptyline and containing 1 -Caffeine, 2-Propoxy-
(0.1 mg/100 ml), 3-Diazepam (0.04 mg/100 phene (0.2 mg/100 ml), 3-Diazepam (0.06
217 .
Fig. 8. GC Chromatograms obtained (at200°C) from a urine sample (1 ml) treated as in Pig.
1 and containing 1-Methyprylon, 2-Diphenhydraniine, 3-Methyprylon metabolite (6--Hy-
droxy), 4-Cafteine, 5-Methaqualone, 6'Acetylated Nordiphenhydramlne, y-Methaqua-
lone metabolites.
niques available to the toxicologist are either zine, can interfere; however, by using a pre-
inadequate or too cumbersome to be of much parative TLC clean-up procedure of the urine
value. LSD (lysergic acid diethylamide), co- extract we were able to increase the specificity
caine, and marijuana are examples of such of the technique (7).
drugs. The use of preparative high pressure liquid
For a number of years we have used a com- chromatography (HPLC) is a distinct improve-
mercially available radioimmunoassay (RIA) ment over TLC and has been used together
method (6) for the analysis of LSD in urine and with RIA in the detection of LSD in urine (8).
blood and found it adequate as far as sensitivity A recent publication describes an elegant
is concerned. A few drugs, e.g., chlorproma- method for the detection of LSD in urine em-
218 .
ploying HPLCwith a fluorescence detector equipped with NP detectors. Using a 2-ft.
(9). The
detection of LSD using a mass spec- glass column (OV-1 and 250°), we have de-
trometer has been described and 50 femto- tected less than 1 ng of LSD injected on col-
grams were repoited to be detectable (10). umn. This level of detectability should be
Our own results using mass spectrometry have adequate for the confirmation of most positive
not been as fruitfuland, in fact, we have ob- LSD cases where uiine levels of 2 ng/ml and
tained greater sensitivity employing a GC greater are the norm.
219 .
Theinteresting point here is that the basic
Benzoylecgonine is the principal metabolite sis.
of cocaine and the substance most likely to be extract is only positive for those urine speci-
found in the urine of cocaine users. We have mens collected up to 2 hours after smoking and
used a GC method, similar to that described by preliminary results indicate that a positive
Wallace et al. (11), for the detection of benzo- basic extract is associated with a reasonably
ylecgonine in which the metabolite is con- high THC blood level. Although much more
verted (by methylation) back to the parent work is required, the positive RIA result of a
cocaine. The method works well, but it is too basic urine extract may be indicative of recent
time-consuming for the routine screening of marijuana smoking. It would be interesting to
urine specimens which are perhaps best see what kind of results we would obtain if the
screened using a RIA method (12). marijuana smoked contained no THC, but, say
The detection of cannabinoids in urine and a high level of cannabinol (CBN) as it is not
blood is a difficult problem for most toxicolo- known if the THC antibody we are presently
gists. In Canada at the present time the au- using is hydroxy metabolites of
sensitive to the
thor’s laboratory is the only forensic CBN or even cannabidiol (CBD).
laboratory which is reporting cannabinoid When MDA and more recently pora-methox-
body fluids. There have been nu-
analysis on yamphetamine (PMA) were first implicated in
merous methods published (13) for the detec- deaths in Ontario (14), we were not aware that
tion of cannabinoids (mostly THC and such compounds were available on the street.
metabolites) in urine and plasma; however, as Although the performance of screening proce-
far as we are aware, no method has been pub- dures is constantly improving, perhaps a more
lished for whole blood. practical step to ensure the detection of street
Our own work in this area* has centered drugs in tissue would be a closer coopei ation at
around the use of a commercially available RIA the local level between those agencies involved
method (6). The antibody was developed for in street drug analysis and those involved in fo-
trates the kind of results we have obtained in tario, forexample, our discovery of the danger
analyzing urine specimens of a smoker who of PMA (14) resulted in massive warnings to
over a period of 20 minutes smoked two the public throughout Ontario and Canada, and
“joints” containing a total of 12 mg of THC. was at least partly responsible for the place-
The intermittent line represents the cannabi- ment of PMA on the list of “Restricted” drugs
noid content (measured as ng/ml THC) of the in Canada.
non-extracted urine. The dotted line repre-
sents the cannabinoid content of an acid ex-
tract of the urine after enzyme hydrolysis and
the solid line represents the cannabinoid con-
tent of a basic extract after enzyme hydroly- ACKNOWLEDGMENTS
'
20 .
REFERENCES
07110.
As recent decades have witnessed a torrent component may be defined by a single chemical
of instrumental developments, analysis of formula, or perhaps as a single optical or posi-
chemical purity and reference standards have tional isomer, or it may be limited to a single
experienced a resultant flood of applications of crystal modification.
instrumental methods. These have become Separation science, using modern instru-
the most important tests in drug purity and ref- mental methods, in particular leaps to mind
erence standard protocols. Each method has a when this general definition is considered.
known scope of applicability, and each has
generated substantial cumulative experience. A. Reference Standard
Reasonable assessments of the impacts of
these methods may be made, therefore, both Most modern instrumental methods give
relative to classical methods and relative to re- quantitative and qualitative data relative to
cently instrumented versions of established concomitant treatment of another sample of
chemical methods. the same component, one which is of known
Our consideration of the impact of instru- composition, that is, a chemical reference stan-
mental methods must be postponed momen- dard. This is in contrast to some older
tarily because definitions are in order. The methods, called “absolute”, in which the
word “purity” is particularly elusive and cul- equipment or reagents are separately standard-
turally loaded. ized or calibrated by some other means, as in
gravimetric or titrimetric methods.
We must define reference standard purity as
/. PURITY CONCEPT known composition with respect to intended
use. Adoption of a chemical reference stan-
This treatment must be limited to chemical dard demands a known purity level which is
purity, although it is recognized that microbio- obtained by meaningful experiments. The ex-
logical contamination is of some forensic sig- perimental results are held up against the al-
nificance. based on experience with over
It is ready known and certain challenges to the
700 different chemical reference .standards standard —the intended uses of the reference
(USP, NF and FCC). Chemical purity was standard, the reasons that itwas called into ex-
best formulated in terms of physical chemistry istence in the first place. Here is the anchor
(1) as the extent and sense in which a substance against aimless drifting on seas of arbitrary nu-
conforms to all known methods of resolving mericism (two, three or four “9’s”) or em-
this or similar substances into more than one piricism. In other words, we can say that, at
component. Unresol vable specimens con- the minimum, a chemical reference standard
form to this definition and are, thereby, called must be suitable for each intended use. Con-
“pure”. We have a specimen of unqualified sider these examples: a chemically pure sample
purity merely because we have failed to discern of one polymorph of a drug is unsuited as a ref-
another component (impurity). To the foren- erence standard if the intended use is to give
sic or medicinal chemist, therefore, purity is positive identification to a different poly-
the extent to which all the atoms in a specimen morph; on the other hand, highest purity is
are accounted for by a single component That . extraneous if the identification is to be made
224 .
by thin-layer chromatography or solution allows decisions as to the “purity” of a speci-
spectroscopy. men, or the scope of suitability of that speci-
Finally, a reference standard is a tool. It is a men as a reference standard for certain stated
means to an end not an
, end in itself. It usually applications. To confuse matters, purity mea-
serves rather limited purposes, and usually is surements give data in different units, i.e,,
consumed in the process. %
mass or mole purity, u. v. -absorptivity acid- ,
225
Information '
Scope^ Overall Impact^
TLC Elemental, Spectral (IR,UV) TLC
HPLC TLC HPLC
GLC Titrimetry;^ Classical func- PSA
PSA tional group methods'* Titrimetry; functional
TItrlmetry;® functional HPLC groups
groups'* Optical Rotation GLC
Optical Rotation PSA Optical Rotation
Thermal Analysis (DSC) GLC Thermal analysis
Spectral (IR, UV) Thermal Analysis Spectral (1R,UV)
Elemental Elemental
in every laboratory, e,g., drying instructions choice for steroids, and is being applied to
should accompany chemical reference stan- most other families of drugs. Each year, the
dards, and results usually are expressed on the impact of HPLC has grown until it is now a
dry basis. mature, routine technique, particularly in
laboratories working with drug purity or ref-
/V. COMMENTS ON erence standards. Rapid development re-
INSTRUMENTAL METHODS sulted from exploitation of existing
capabilities: chromatographic know-how
Gas-liquid and pressurized liquid chroma- about adsorption, partition and ion-ex-
tographies (GLC and HPLC) are instrumented change; and, from gas chromatography, ex-
methods which have obvious potential for re- perience in chromatograph design and data
solving a specimen into components. Al- reduction (not to mention inheriting the ex-
though not usually used in an instrumented pectant, prepared market place).
version, thin-layer chromatography (TLC) also Examples of the specificity achieved by
is an irreplaceable technique. HPLC are the assay of progesterone in the
presence of fixed oils (4) and assay and purity
1. HPLC
evaluation of folic acid (5). These are just
Incorporation of thisnew development two examples of the great impact which
into the existingframework of pharmaceuti- HPLC is having on pharmaceutical analysis.
cal-organic analysis has been nothing less Solid solutions of impurities in the main
than precipitous. The number of published component are a common feature of steroid
accounts forbids selection of a range of ex- chemistry, and often escape accurate assay
amples. It rapidly became the method of by calorimetry or solubility analysis. Ex-
226 .
amples of HPLC resolving such mixtures are tector scope are also of practical signifi-
estradiol dipropionate (2) and testosterone cance, Preparative HPLC presently is too
propionate (acetate as second component). expensive except for limited purposes, but
Our laboratory performs HPLC analyses this already is having impact on reference
on about two-thirds of all candidate refer- standards programs.
ence standards, and has examined all
steroids during the last seven years.
2. GLC
The main pioblem in HPLC has been non- For purity work, low-polaiity phases,
uniformity of packings, but some improve- such as the polysiloxanes, are preferred in
ments in this sophisticated technology have order to minimize thermal degradation.
been made, and the future promises even Where a distillate is analyzed, GLC is an ab-
more useful packings. Limitations on de- solute method; more commonly, it is used to
227 .
compare batches of standards. Sources of
error (2) are injection-port degradation
which gives rise to other distinct peaks, and
non-linear adsorptive losses. The latter is
particularly important in that minor impuri-
ties can be grossly underestimated (see Fig-
ure 2). It is important to analyze different
sample sizes, up to overload, to measure
minor impurities in terms of fractional
areas. This is a common practice because
authentic samples of all possible impurities
‘
needed for ‘spiking’ (i.e. standard addition
’
,
3. TLC (flat-beds)
28 .
SYSTEM COMPOSITION MG/g
solvent and 3 to 10 times the amount of diug 6. Wyatt, D. K., Richardson, W. G., et ai,
which can dissolve, will be enriched in im- J. Pharm. Sci, 65, 680 (1976).
purities relative to the main component by a 7. Mader, W. J., CRC Rev, Anal.
Crit.
precise factor. Another application is to Chem., 1,193(1970).
collect the undissolved, purified crystals
from a solubility analysis. On a large scale,
this is known as swish-purification or iso-
thermal recrystallization, and also is based
on a prior PSA.
229 .
by
Drug Analysis by
Irving Sunshine
Immunoassays
Cuyahoga County Coroner’s
Laboratory
Cleveland, Ohio
ten must have a large molecular weight. The ularly useful when a large volume of antibody is
substances of concern to chemists seldom have desired because there is expectation of great
230 .
—
demand for the antibody. These animals are usually with ammonium sulfate; and (b) ad-
worth their weight in gold.
literally sorption of the labeled antigen on a suitable ad-
Having successfully generated a sensitive sorbent, such as charcoal or silica gel. In (a)
antibody (Ab) how does one use it? Very sim- the precipitate contains the labeled antigen-an-
ply by reacting it with an antigen (A) according
, tibody complex which, when separated from
to the general equation shown below: the supernatant containing the unreacted la-
in this system, one must separate the labeled sive counting equipment. The time requ tred to
antigen-antibody complex from the labeled an- complete one analysis is 60-90 minutes. Re-
tigen, and count either or both of the separated agent costs are high and technician time re-
fractions. The quantitative measurement is quirements are moderate. A
large number of
based on the ratio of the bound label to the free, specimens can be processed simultaneously
the bound to the total, or free to total. The sep- aiid the counting can be done automatically,
aration of these fractions may be achieved in thus reducing the average technician time re-
several different ways, among the most com- quirements for an analysis when a large num-
mon of which are; (a) precipitations of proteins ber are to be done simultaneously, If the
231 .
for a particular assay not high, then men (Figure 1). Measuring this reaction rate
demand is
shelf-life of the reagent may be a cost problem thus measures the amount of drug in the sample
because the material may become useless if not when this rate is compared with those obtained
used in a reasonable time period. by analyzing samples of known concentration
of drug added to drug-free specimens proc-
essed simultaneously with the samples in ques-
in.ENZYME MULTIPLIED tion. No separation of material is required.
IMMUNOASSAY TECHNIQUE (EMIT) The entire procedure can be carried out in one
vessel, and, thanks to modern engineering, the
In an effort to avoid the tedium of the separa- rate can bemeasured in less than one minute.
tion steps required for RIA and the need for a Computations have been simplified by the
“hot” laboratory, another and very ingenious manufacturer who currently markets the most
technique was developed which eliminates frequently usedhomogeneous assay procedure
these problems (Ullman, E. F., et al., J. Biol. (so-named because no separation is required).
Chem., 251, 472, 1976). Called EMIT, it uses Sy va Corporation provides Probit charts which
an enzyme to tag the antigen. In this proce- linearize the standard curve data, but at a
dure, the drug and the drug-labeled antigen price. The middle range permits good pre-
compete for the antibody sites and, as a result cision and accuracy, but this decreases some at
of the equilibrium which is established, some of either the high or low concentration ranges.
the enzyme from the labeled antigen is re- Nonetheless, even in these ranges, the CV's
leased. In the presence of a suitable substrate, obtainable are well within the tolerances ac-
a reaction ensues and its rate is a function of the ceptable for clinical data. The literature is re-
concentration of the drug in the sample speci- plete with data attesting to this.
2.
The manipulative skills required for EMIT ogues. To do so is laborious, difficult, and
analyses are minimal and easily learned. The very expensive, although theoretically possi-
time to analyze one sample is 40-60 seconds in ble. Hence a positive immunoassay indicates
toto. A numerical result ensues, making inter- that a drug in a given family of drugs may be
pretation simple and objective. The reagents present, but does not indicate which one is
are stable for at least six months. The required present. Quantitative data are difficult to
bench space for the entire analysis is about 4 achieve because the specific drug in question
feet. If a qualitative analysis is required pos-— may not be identified by the immunological
itive or negative' —then only three determina- technique. If one knows the drug in question,
tions are required: a blank, a sample containing e.g., clinical evaluation of digoxin or the anti-
the concentration above which a result is con- epileptic drugs, then reasonably precise and
sidered positive, and the test sample. If other accurate data may be achieved. Table I illus-
specimens are to be tested within 1-2 hours trates thisproblem with data for the barbiturate
after the first test sample, it is not essential to assay where the hapten was produced using se-
repeat the first two tests. Reagent costs are cobarbital and all the others are less sensitive,
comparable to those for RIA and total cost is barbital being l/IO that of secobarbital. How-
lower because less technician time is required ever, though this may seem to be a liability in
and no special vials or scintillation fluid are —
some respects, it is an asset in another i.e., in
needed Costs may be significantly reduced, if
. answering the question of whether or not a
many specimens are to be analyzed at one time, given class of drugs is present or absent. This
by using expensive automatic enzyme ana- question is frequently posed in the diagnosis of
lyzers or centrifugal fast analyzers. Their high acute poisoning, and if the immunoassay is per-
cost can be amortized in a reasonable period of formed and it is negative, then you can be sure
time if the volume of analyses is high. that that group of drugs is not present and you
must seek elsewhere for the diagnosis.
Another aspect of immunoassays that must
IV. IMMUNOLOGICAL PROCEDURES
Having described the two principle meth- TABLE 1
odologies now in common use, some general Relative Activity of Five Barbiturates
comments on immunological procedures are in
(100 /zg/liter Secobarbital Standard)
order.
For clinical purposes, when one analyte is in- Secobarbital 100*
volved, this technique compares favorably Pentobarbital 45
with many other procedures However, if one
. Butabarbital 45
must analyze a specimen for more than one Amobarbital is
s^stance, e.g., phenytoin, phenobarbital and Phenobarbital 25
primidone, then three separate analyses must Barbital 10
be performed in contrast to chromatographic ^Secobarbital equIvalentSj /tg/IHer of urine.
procedures which will determine all three sub-
stances in one analysis. (Reprinted from original source; R. Cleeland,
J. Christenson, M. Usetegul-Oomez, J. Neveran,
It is very difficult to produce an antibody that 712-
R. Davis, and £. Qrunberg, Clin, Chem., 22 (6),
is specific for one drug and none of its anal- 72S, 1978)
233 ,
be checked is the specificity of the antibody, this, by ion monitoring. But as yet, few lab-
not only for other drugs related chemically to oratories have this potential. There are a few,
the antigen, but also for its specificity against if any, other procedures that will satisfy this re-
the metabolites of the drug. If these metabo- quirement. Hence one’s ability to certify with
lites are present and the antibody titer for them certainty that a result obtained is precise and
is unknown, some disparate data may ensue. accurate is open to question unless large
Non-specific reactions to most antibodies amounts are present, and these then are detect-
occur from many sources. From the data in able by less sensitive methodology. An im-
Table II, one would hestitate to indicate a sen- munoassay for tetrahydrocannabinol has been
sitivity less than 30 /Ag/l for the '^®I assay and developed and it is to be tested on urines of
less than 50 p,g/l for the assay. Similar, drivers suspected to be “under the influence’’
but not identical data are demonstrable for all to ascertain what role, if any, THC plays in ve-
immunoassays and one must select a realistic hicular accidents. In the test program, a non-
“low cut off’’ value to avoid false positive punitive one, all alleged positive THC results
results. by immunoassay be checked by a sensitive
will
Two other aspects of sensitivity need discus- GC/MS procedure. It will be most interesting
sion. If one obtains a positive immunoassay to learn what degree of correlation will be ob-
result and has to certify that the result is accu- tained in this study. In practical terms, for
rate, then at least a second independent tech- medico-legal purposes, the probability is great
nique is required. To obtain a second be high enough to
that the correlation will not
chemically or physically different analysis allow one to state “without a reasonable
which has the same sensitivity of the immu- doubt’’ that the immunoassay result alone is
TABLE II
V. CONCLUSION
Apparent Concentration of Morphine
in Urines of 100 Normal Individuals With the resurging demand for increased
monitoring of urines for the detection of those
assay assay drugs which may be abused, immunoassay
tiq ME/llter* % % offers a novel approach. If 300 samples are to
be tested, one could pool each of three and ana-
0-9 90 70 lyze the 100 samples thus prepared. As the
10-19 8 17 chart below indicates, even if 10% of the sam-
20-29 2 6 ples were positive, a most unusual situation
30-39 0 5 based on past experience, only 130 analyses
40-49 0 2 need be done instead of 300. This is a signifi-
*Resulls expressed as morphine equivalents cant saving of time, effort, and reagents, and
(ME).
can be done effectively if the sensitivity of
{Reprinted from original source! A. Cleeland, three times the “low cut off’ is adequate.
J. Christenson, M. Usategul-Gomez, J. Heveran,
A. Davis, and £, Grunberg, Clin. Chem., 22 (6), 712- % positive 0 3 5 10
725, 1976) No. of analyses 100 109 115 130
234 .
TABLE III
235 .
Applications: An extensive bibliography of lishment at Aldermaston. They tested the
immunoassays is attached to this article and is eluate from an HPLC column with an RIA
indexed in Table III. Because RIA and EMIT assay, and thus had a very sensitive detector
have not been indicated in Table III, a separate for this chromatographic technique. Again,
Table IV indicates the EMIT applications. further development is needed, but the poten-
A very novel application of RIA was made tial for a new HPLC detector is indicated.
by Moller, et ai which merits attention. They
Other novel approaches include the use of
dropped blood and urine on samples of cotton,
fluorescence as a labelling device rather than
wool, and fired clay and stored these dry or
an enzyme or a radioactive molecule. Appli-
under humid conditions for several days. As
cations to opiates and gentamicin have been
Table V indicates, using RIA the dmg was de-
described (Burd, J. F., et ai, Clin. Chem.,
tectable in the blood stains, but not easily
23 /8 ,
1402 - 1408 1977 ).
,
TABLE IV
EMIT Assays
3. Respiratory drugs
a. Theophylline
4. Anticancer drugs
a. Methotrexate
236 .
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alized as more ingenious investigators apply munol. ImmunopathoL, 1, 53, 1972.
their talents to this area.
2. Adler, F. and Liu, C.-T., Detection
of morphine by hemagglutination-in-
hibition,/. 106, 1684, 1971.
20 ng
fired Olay
++
4* :
5 5.0- 7.6 ng
^
-
9.
Bastiani, R,, Clinical Study #20,
Corp., Palo Alto, Calif., 1976.
C.,
211,1973.
237 .
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10. Battaglia, D. J. and Cianci, M. L., Io- for the determination of digoxin. Bio-
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19. Bohuon, C., Duprey, F., and Boudene,
Med.. 17(9), 847, 1976.
C., Radioimmunoassay of methotrexate
11. Bennett, R., Critical determinants ena- in biological fluids, Clin. Chim, Acta,
mor-
bling an antibody to distinguish 57(3), 263, 1974.
phine from heroin, codeine and
20. Booker, H. E. and Darcey, B. A., Enzy-
dextromethorphan, Immimochemistry,
matic immunoassay vs. gas/liquid chro-
in press.
matography for determination of
and pharmacologic factors on the dispo- 22. Bost, R. D., Sutheimer, C. A., and Sun-
sition of morphine as determined by ra- shine, I., Letters: methaqualone assay
238 .
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1940.
40. Catlin, D. H., Cleeland, R., and
31. Butler,H. A. et al., Eds., Radioimmu-
Grunberg, E., A sensitive rapid radio-
noassay Methods, Churchill Living-
immunoassay for morphine and immu-
stone, Edinburgh, 1971, 635.
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32. Butler, V. P., Jr.,Drug immunoassays, and serum, C/m. Chem., 19,216, 1973.
J. Immunol. Methods, 7(1), 1975.
41. Centeno, E. R. et al. , Int. Arch. Allergy
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262 .
by
Light Microscopy and
Walter C. McCrone
Forensic Drug Analysis
McCrone Research Institute, Inc.
2508 South Michigan Avenue
Chicago, Iliinois 60616
angles, optical properties, melting points, etc.), facial angles, axial ratios and crystal system,
isan excellent microanalytical tool. In the and optical properties such as refractive In-
hands of an experienced microscopist it is rapid dices and dispersion. This has the advantage
and dependable. Even in the absence of that the sample is observed directly without
lengthy training and experience a microscopist dissolving, vaporizing or otherwise modifying
can expect to find polarized light microscopy it. The second is based on microcrystal tests
very useful as a confirmatory technique. If, and these precipitation reactions are based on
for example, GC/MS reports the presence of the known morphology and optics of crystals
seconal it should be a simple matter to compare obtained after dissolving the compound and
crystals in that sample directly with known obtaining a precipitate through use of a specific
crystals of that substance or with data from the reagent. Finally, there are methods based on
literature for that particular compound. heating the sample between slide and coverslip
For any analytical procedure to be useful as a and observing the phase changes which occur
general method for complete unknowns, it is as a result of heating and subsequent cooling.
countered . Unfortunately there are many ex- methods developed by the Koflers. Often the
,
provides the necessary data bank, the light mi- whether the identification is accurate.
might include morphological and optical prop- quantitative measurements that can be made
erties on crystals of the drug itself or it might be on crystals, especially optical properties (2)
the corresponding properties on either precipi- (not to mention crystal lattice parameters de-
tated derivatives of the compound in question termined by x-ray diffraction). Most crystals
263 .,
have three refractive indices measurable to Prqfije angle, Oil A O il in
three or even four decimal places and therefore •100 plane =123^
considerably more accurate than capillary
•
microanalysis.
Formula weight. 296.52
mdrphism was observed during this study. 6.38 1.00 2.82 Very weak
6.09 0.38 2.77 0.09
6.53 0.18 2.70 0.04
5.29 Very weak 2.64 0.04
5.09 0.40 2.60 Very weak
•
264 .
characteristic optic axis interference figure,
Although a crystallographer’s language is
almost entirely incomprehensible even to
other microanalysts, a microscopist familiar
with these techniques would need no more
than five minutes to confirm that a crystallo-
graphic substance he has under the micro-
scope is, or is not, identical with a compound
as described. In the absence of known data,
a known sample will permit an equally
speedy and dependable decision. Unfortu-
nately, this data bank covering crystal mor-
phology and optics is very incomplete;
•
265 .
1 1 1
2a. Cocaine Precipitated with Sodium Carbonate (a). Cocaine Chloroplatinate (p). 130:1
2h. Strychnine. Precipitation with Ammonia (a). Precipitation with Sodium Bicarbonate. 60 :
IV, THERMAL METHODS scope. The identification tables they include
make it any of about eleven
possible to identify
Ludwig and Adelheid Kofler both taught in hundred compounds in just a few minutes.
the Department of Pharmacognosy of the UnL Table I shows a portion of one of their tables
versity of Innsbruck in Austria and this ex- with the data for morphine. Mostoftheii iden-
why their book (5) includes most of the
plains tifications are based on the melting point of the
common drugs They have published a compi-
. compound itself* as well as melting points of
lation of methods for the study of compounds compound with standaid sec-
eutectics of that
and their mixtures using the hot stage micro- ond components. They also usually determine
3f. Morphine, Precipitated by NaHCO^ (1); Precipitated by Na.COg from HCI Solution. (1 ) 90 :
1 ; (2) 60 :
267 .
TABLE I
Kofler Data
the retractive index ot the melt by a reverse im- dures are nest held for confirmatory tests. If,
mersion method using standard glass powders for example, one felt that he had a sample of
of known refractive index. Finally, they note sulfamerazine it would take only a minute or
any evidence of sublimation, polymorphism, two to heat the sample between slide and co-
decomposition and so on. Again, the methods verslip to observe the characteristic sublimate
are straightforward and simple and the data and, finally, to melt the sample and observe
bank is reasonably complete for drugs. crystallization from the melt on cooling; the
It is probably worth mentioning, for com- crystal forms and polarization colors are very
McCrone’s more qualitative
pleteness at least, characteristic of sulfamerazine. If any doubt
procedures (6) which are based on the appear- remained, a few crystals of known sulfamera-
ance of the ciystals as they grow from the zine melted at the edge of the coverslip, so that
melt. The significant parameters include the melt runs under and into contact with the
phase changes, sublimation, decomposition, unknown, would then show (by the absence of
estimated melting point, degree of supercool- other crystals in the zone of mixing) that the
ing, crystal form, crystal growth rate (as a func- two were identical or (by some discontinuity of
tion of temperature), refractive indices relative form or behavior in this zone of mixing) that the
to the melt, extinction angles, estimated bire- two were different. This is an ideal identity
fringence, anomalous polarization colors, in- test, far superior in speed and dependability to
terference figure, optic axial angle, optic sign a mixed melting point,
and dispersion. The limitation of the method
is based on how many compounds one can re-
REFERENCES
Victor R. Matricardi
Utilization in the Crime
Elemental Analysis Unit
Laboratory FBI Laboratory
Washington, D.C. 20535
only elements having atomic number greater ing, requiring great care in the orientation of
than 10 (Ne) can be analyzed. A detailed de- the specimen. Often days of searching can be
scription of the instrument is omitted but the in- spent before completion. Completion does
terested reader is referred to any of several not necessarily mean a positive comparison,
publications on the subject (1,2). but can mean negative or inconclusive results.
The specimen is positioned inside the vac- If the applications mentioned in this section
uum chamber of the SEM-EDX, thereby plac- were all that were possible with the SEM,
270 .
the instrument would be little used in our poisons, some drugs, and whatever other spec-
laboratory. imens enter a crime laboratory.
While the work on fracture comparison was The previous two sections have described
being cairied out, various samples which were two separate instruments: a powerful micro-
not suitable for analysis by other techniques scope (section III) and an analytical instrument
available in our laboratory were brought to the (section IV). However, the greatest informa-
SEM-EDX. The X-ray fluorescence spectra tion can be obtained from the specimen by
of these specimens were obtained and a quali- using these two modes simultaneously. That
tative elemental composition of the specimens is, a particular microscopic item of an aggre-
was given to the examiner who originally con- gate can be analyzed without requiring the item
tributed the samples. The number of speci- to be physically isolated from its matrix. A
mens examined increased with usage and mass sensitivity of 10"'® to 10"'“ grams can be
eventually included specimens which could obtained (2). However, in the analysis of a
have been adequately run by other types of ex- component particle situated in a matrix, the
amination. Two properties of the SEM-EDX contribution of the matrix to the spectrum can-
caused this phenomenon: fast turn-around time not be completely eliminated. Common appli-
(about 10 minutes for a simple qualitative anal- cations of the SEM-EDX are: analysis of
ysis) and non-destructive qualitative analytical individual layers in a multi-layeied paint chip;
capability. Thus, part of the workload in- analysis of each grain in an ore sample to deter-
volved specimens which were being given a mine the distribution of a particular element;
qualitative “once-ovcr” prior to other analy- analysis for small particles of Zn in bomb ex-
ses (for example, neutron activation analysis) plosion debris; and localization of As in a poi-
which would provide additional information soned bread. Another general category of use
(quantitative results or increased sensitivity).
requires the SEM-EDX to provide the exam-
Some of the disadvantages of the EDX in- iner with the two-dimensional distribution of a
clude a relatively high background signal caus- particular element. That is, the instrument
ing a low ambiguous
sensitivity (0,1 to 1%), places a heavy dot concentration wherever it
interpretation of peaks unless secondary peaks finds a previously chosen element. The result-
are considered, and insensitivity to organic ant image is called a “dot pattern” or “elemen-
constituents. tal distribution pattern”.
A voluntary training program was initiated to A typical application was the examination of
introduce the examiners to the use and applica- a knife blade which was suspected to have been
tion of the instrument. The result was an even used to cut a telephone wire during the com-
greater increase in the frequency of examina- mission of a crime. The purpose of the exami-
tions requiring the simple qualitative elemental nation was to identify foreign copper smears or
comparison of items in casework. The speci- the cuttiijg edge. The knife is first examined
mens consist of small chips of paint, metallic under an optical microscope for the presence of
fragments, unknown powders, ores, suspected suspect areas having different light reflecting
271 .
properties. Then these areas are analyzed in VI. SUMMARY
the SEM-EDX for copper and finally the cop-
per is localized in the fine grooves present on The applications of the SEM-EDX in the au-
the sharpened edge. Figure 1 shows the sharp- have been outlined. Most of
thor’s laboratory
ened knife edge on the left and, on the right, the the casework now being handled involves pure
“dot pattern” for copper. The copper is local- analysis or analysis combined with elemental
ized in particular areas which generally con- mapping. The number of work requests are
form with the surface grooves. The dot now in excess of 150 cases per year. The main
pattern technique is most often used to localize reason for the instrument’s use relies on the
surface smears as in bullet grazing or whenever fact that a quick qualitative non-destructive
a softer substance is brought into forced con- analysis can be obtained on a microscopic area
tact with a hatder material. of the specimen. The disadvantages include
v;
'V '
S’
r'i SV'V’ y
MS '-.
:vS. •;
.
I -,!
REFERENCES
273 ,
Indistinguishable From by
Center for
S. Flnkle
Human Toxicology
Promise of Laboratory
University of Utah
Utopia Salt Lake City, Utah 84112
Amongst his many quotable quotes, Winston ing. To select and purchase laboratory instru-
Churchill once stated: “Never overlook an op- mentation as if it were the next family car, on
portunity to remain silent’ ’ This paper, there-
. the basis of dazzling advertising and sex appeal
fore, represents a missed opportunity but the is to invite the retribution and disappointment
challenge to rationally address the problems, of having swapped a Studebaker for an Edsel,
both human and scientific, which accompany (or perhaps a British Airways for an Air France
laboratory transition from distillation appa- Concorde).
ratus, retort-stand and evil smelling organic Beginning in the late forties and progressing
preparation, to the P.C. Board and flashing dig- through the fifties, ultraviolet and infrared
ital lights of the technological scene, cannot be spectrophotometry successfully wooed the fo-
lightly disregarded. This change, without rensic toxicologist to instrumental analysis by
doubt, represents the most dramatic occur- permitting dramatic improvement in sensitivity
rence in forensic toxicology history, and has in- and characterization of drugs and toxic agents
troduced an unequalled turmoil of activity isolated from biological samples. The early
during the past five years or so. It demands as- sixties saw the birth of the gas chromatography
sessment; the aim of this presentation is then, revolution which has continued unabated for
assessment— inference and cautious proposal. well over a decade, until today “optional
Any sufficiently advanced technology is in- extras” so abound that there are extras for
distinguishable from magic. In this one axio- extras and even the illustrious journal, “Sci-
matic statement there is embodied threatening ence”, publishes an annual “Guide to Scien-
danger, promising hope, and a caution. Cer- tific Instruments” which in 1977 stretched to
tainly the technology of television and modern more than 300 fine printed pages.
communication systems is so far beyond the Multiple gas chromatogi aphy systems have
comprehension of a child that it qualifies as become the staple of the toxicology laboratory,
magic. But magic is anathema to science, and (a measure of even
their utility is the fact that
the replacement of occult mysticism, a belief in I
trial lawyers manage pronounce and spell
to
gremlins and “Black-box Reverence” by un- the name correctly), and are now commonly
derstanding and scientific judgment must al- equipped with mechanized sample injection
ways be the first consideration, and is essential devices and pyrolysis units, a variety of detec-
if the introduction of computers and other com- tors and peripheral electronics which minimize
plex instrumentation into the laboratory is not noise and render ever more beautiful peak
to wreak havoc with the principles and objec- shapes. Now, in the seventies, the physicist
tives of our Forensic Science responsibility. and electronic engineer with remarkable imagi-
Alternatively, there is promise of broadened nation have mated the gas and liquid chromato-
laboratory capability; improved analytical sen- graphs to mass spectrometers, and in a stroke
sitivity, creation of a wide uniform data base produced an awesomely powerful analytical
from which to draw reliable inferences, spe- tool-and the need for computer control and in-
cific identification (perhaps the final touch- teractive data reduction systems. Add to this
stone of the analyst), speed and control of automated immunological testing, including ra-
high- volume routine work; but all within the dioimmunoassay, stable isotope techniques,
cautious context of clear-minded understand- and the demand for very high volume drug con-
274 .
trol, traffic alcohol and postmortem screening can be followed in making this decision. Play a
analyses and it is not difficult to appreciate that role of careful, critical observer. There is, in
judiciously applied mechanization is necessary my experience, always a year or so of hysteria
and that computer data-storage, manipulation and hyperbole immediately following the ar-
and calculation provides a solution that cannot rival of the new toy in the scientific nursery.
be ignored. The slump of disillusionment follows the reali-
The “untouched by human hand” analysis zation that it is not a crystal ball, but eventually
has arrived, making possible an unmanned but applied research and development by responsi-
productive laboratory by night and little more ble scientists, and others who bought during
than a maintenance operation by day. It is the of excitement, brings the equip-
first flush
kitchen chemistry begone! Enter Utopia! ment into perspective. Now ask questions:
The drudge is an instrument, and the toxicolo- Can the instrument fulfill a need in the labora-
gist a researcher released from harness to ex- tory which is beyond present capability; or sig-
pound and expertly opinionate from his nificantly improve a definable inadequacy?
experimental data. Only proceed if the answer is YES. Consider
The cry of the young bicycle rider, “Look its forensic applicability; it is essential that the
Father. No hands” is all loo frequently fol- function, purpose and data output can be con-
lowed by “Look Father. No teeth!” How vincingly described and is legally tenable, and
then do we preserve the teeth and face of toxi- that total instrument control (beyond the on/off
cology at a time of bewildering progress and the switch) is in the hands of the scientist. It must
advent of instruments which many aie ill- be capable of built-in quality control and func-
trained to fully appreciate? tion testing, and demonstrably require the op-
The benefits ot saved time, staff manage- erator for all variable or judgment steps.
ment, cost-effectiveness and quality of analyti- Pay a working visit to a laboratory already
cal resultsmake the acquisition of computers, experienced and ask knowledgeable colleagues
and the laboratory instruments they control, to explain differences, often subtle but very
very attractive. But the decision to spend important, between various manufacturer’s
huge sums of precious budget money on equip- equipment. Similarly, visit the manufac-
ment, which once indelibly stamped with a turer’s laboratory for at least a day and analyze
government identification number becomes test samples typical of your particular case
immortal and irreplacable by definition, can in- problems. Observe the analysis, perform
duce white elephant nightmares in a toxicolo- yourself, and do not accept data generated in
gist and send him scurrying to the safety of your absence.
conservatism. It is an act of considerable Ensure that there are adequate training and
courage to buy $100 to $500,000 worth of Uto- education courses available, perhaps spon-
pian hardware, with a percentage of built-in sored by the company or conducted by them.
magic. In the United Slates, at least, there is a endeavor to stay with those manufac-
Finally,
general belief in, “you get what you pay for” turers with aproven record in forensic sci-
and, accordingly, at 100 kilo-bucks and above, ence. These simple precepts can prevent a
the scientist expects infallability, and the doc- hardware store-room full of unfulfilled prom-
tor instant response. The lawyer, of course, ises of yesteryear.
simply wants to know how to beat it! •
In any event be assured that the infallible in-
There arc, howdvei', some safety rules which strument (computer or not) has not arrived and
275 ,
is not likely to bail you out in the forseeable fu- ern technology which is our good fortune can
ture. This symposium about tools; tools of
is exert the widest possible benefit.
the scientific craftsman, and they perform ex- It must be emphasized that there is no single
actly as commanded —
no more and no less. answer to the question of complex instrumen-
There are dangerous implications for the Fo- tation in scientific affairs. For his different
rensic Scientist: without an acute sense of real- purposes, the scientist needs many different
ism it is easy to become trapped in the belief structures —
some simple, some complex,
that the instrument can overcome deficient an- some exclusive and some comprehensive, but
alytical technique and apparently produce irre- the clamor for a final solution continues.
futable data. It must be remembered that the Today we suffer from an almost universal idol-
first rule of GC-MS demands the best chroma- atry of computer technology; it is, therefore,
tography possible as a prerequisite to mass necessary to insist upon the virtue and unique-
spectrometry, and data is simply a means to an ness of the human brain. Men always seem to
end; it must reflect reality. That reflection can need at least two things simultaneously that su-
only be seen and described by a mature, experi- perficially seem to be incompatible. We al-
enced scientist. The Forensic Scientist must ways need both freedom and order. In the
not permit sacrifice of the slightest scientific in- laboratory this paradox should disappear as the
tegrity in justification of more analyses per orderly and purposeful application of instru-
hour or a dazzling printout. mentation provides time for freedom of
The need for quality performance and results thought and a range of experimental testing
is no less important in the small laboratory only dreamed about a generation ago.
than in the large, busy, urban county, or state If a thoughtful being from another world
organization. All too often, justification for were to visit us today, what would be the most
equipment based on case-load and staff con-
is astonishing —
the brilliance of our scientific
siderations alone. Development of central re- achievements or the rising crime rates; the
sources for complex instruments and reference progress of our medicine or the overcrowding
data banks is essential. It is impressive that and cost of our hospitals, the efficiency of our
there are at least six regional forensic science machines or the inefficiency of the system as a
organizations in the USA and Central whole? The implications of technology in the
Research Laboratories in other countries. laboratory can be just as disturbing unless care-
Regular meetings and frank discussion have led fully considered. In human terms, increasing
to the beginnings of real sharing and it is confi- complexity may entail a degree of specializa-
dently anticipated that teletype terminals and tion that destroys work satisfaction and pro-
interconnecting lines will eventually allow the duces fragmentary scientists, too narrow to be
common wealth to be used where it is most wise. However, despite this note of guarded
needed. computer ser-
Similarly, time share reticence, there is reason for optimism. If
real
vices are now available at minimal cost and are the “breakthrough a day keeps the crisis at
well-proven as reference libraries, able to bay” syndrome can be avoided and application
search and identify unknown substances. of the wondrous tools at our disposal is made
Some private research institutes and a few con- with care and controlled within the human
cerned manufacturers offer services and tech- scale to human requirements, the opportunities
nical assistance. encumbent
In short, it is for creative service in support of the natural
upon all of us to work together so that the mod- harmonies of society through forensic science
276 .
have surely never been greater. Our work is
about service, and the enjoyment that comes to
those who know they are involved, at whatever
level, in testing scientific capability— about
grasping for the apparently unobtainable.
Robert Browning the poet wrote: “But a
man’s reach should exceed his grasp; Or
what’s a heaven for?” I trust that there will be
many such moments for all those who have the
courage to enjoy the challenge of employing
modern technological invention in the cause of
forensic science.
Registered Attendees
(by Country)
Hduordo, Anguls G.
Ministerio de Salud
Sail Jose, Costa Rica
280 ,
GRONINGEN, THE SWliZERLAND
NETHERLANDS
Braiuienbeigei, Plans J , Piof Di
DeZeeuw, Rokus A (Dr.) ,
Dept, of Forensic Chemistry
Department of Toxicology
University ofZuiich
State University
Switzciland
Laboi atory for Pharmaceutical
and
Analytical Chemistry
Kouidri, Cherif M.
Antonium Deusmglaan 2
United Nations Niiicotics Laboratoiy
9713 AW Groningen PalaisDen Naliona
The Netherlands
1211 Geneva, Swilzeiland
AUCKLAND. NEW ZEALAND
Nelson, Donald F. APO NEW YORK
Government Analyst
Kulkaini, Shaiadkuniai
Chemistry Division, DSIR
P.O.Box 2224
US Aimy Ciiniinal Invcstigatkm
Lab
Auckland, New Zealand APO New Yoj k 0D757
BELFAST. NORTHERN IRELAND
Morgan, Wm. H D.
Diiector
Noi them licland Foiensic
Science
Laboiatoi y
180 Newtownbreda Road
Belfast, Noi Ihcinliehmd
BT8 4 QR
PRETORIA. SOU'III AFRICA
Neethling, L P. CoI.,Ph D
Direcloi Fuicnsic Science
,
LINKOPING, SWEDEN
Machly, Andicas
Directoi
The National Lab ori ’orcnssic
Science
PACK
Linkoping, Sweden
UNITED KINGDOM
Pearson, E*:iic(Di',)
Home Offlec
CUE
Akicrniuslon, UK
Willinins, Ray I.. (Dr.)
Mctiopolitan Police
Laboratory
London, UK
Registered Attendees
(Alphabetic Listing)
282 .
Blakely, Jocelyn G Byler, Russell Clinton Clemow, Tom
HFD-436 Summit County Coroner’s Office Waters Associates
Food and Drug Administration 31 N. Summit Street 34 Maple Street
200 C Street S W. Akron, Ohio 44308 Milford, Massachusetts 01757
Washington, DC 20204
CanafF,Roger F. Clevenger, Richard Lee
Bobzean, Robert E. Drug Enforcement Administration East Texas Stale University
Houston Police Department Forensic Sciences Division Chemistry Department
61 Riesner St., Room 430 Washington, DC 20537 E.T. Station
Houston Texas 77002
,
Commeice, Texas 75428
Canfield, Dennis Vincent
Boudreau, Morns G. University of Southern Mississippi
Cockciham, Kenneth Ray
New Hampshire State Police Southern Station Box 398
Aciidiana Criminalistics Laboratory
James Hayes Bldg Hazen Drive
,
Hattiesburg, Mississippi 39401
P.O Box 643
Concord, New Hatnpshiie 03301
Caplan, Yale H Newlbeija, Louisiana 70560
Bower, David W. Office of the Chief Medical Examiner
Conaway, Jane M.
WRAMC 1 1 1 Penn Street
Vnginm Bureau of Foiensic Science
Department of Pathology Baltimore, Maryland 21201
40NA Colley Avc.
Ft. Meade, Maryland 20755
Carroll, Robert Baker Norfolk, Viiginia 23507
Bowei Thomas D.
,
ODV. Inc.
284.
1 1
285 .
Kemppainen* Allen E Krikorian, S. Edward Liu, Ray H. Ph.D.
Michigan State Police University of Maryland Ass*t, Professor
Holland Regional Crime Laboratory Dept, of Medicinal Chemistry Box 4348
PO,Boxlll5C 636 W. Lombard Street University of Illinois at Chicago
Holland, Michigan 49423 Baltimore, Maryland 21201 Circle
Dept, of Criminal Justice
Kulkarni, Shaiadkumar
Keppler, Bruce R. Chicago, Illinois 60680
U.S. Army Criminal Investigation
Armed Forces Inst. Pathology (AFIP)
Lab
Division of Toxicology, Rm, 4012 Lorch, Steven K.
Georgia Avenue - WRAMC APO New York 09757
Michigan State Police
Washington, D.C. 20306 Kupferschmidt, Gerald Joseph Forensic Science Laboratory
RCM Police 7 14 S Harrison Road
.
286 ,
Mami, Ismail S, McMahon, Wayne Mullen, Marvin L
Fla. Dept, of Criminal Law Tennessee Crime Laboratory Baltimore Police Dept. Laboratory
Enforcement Chattanooga Branch 601 E Fayette St
Tallahassee Crime Laboratory 3 Milne Avenue Baltimore, Maryland 2 1202
P.O.Box 1489 Chattanooga, Tennessee 37406
Neethling, L. P. Col., Ph.D.
Tallahassee, Florida 32302
Director, Forensic Science
McWnght, Cornelius G. (Dr )
South African Police
Manders, William W. FBI Laboratory
Republic of South Afnca
Armed Forces Inst Pathology Washington, D.C. 20535
Private bag X254
Georgia Avenue - WRAMC Pretoria, South Africa
Washington, D.C. 20306 Meyers, Richard E.
U S Treasury Nelson, Donald F.
Bureau of Alcohol, Tobacco and Government Analyst
Markway, Everett H. Jr.
Chemistry Division, DSIR
,
287 .
Paulig, Gunter, Ph.D., Director Rail a. Fathi Richardson, Drew C.
Der Polizeiprasident in Berlin Forensic Science Bureau FBI
Direktion ITU New Jersey State Police Rm. 3287f
Berlin, Germany North Regional Laboratory Washington, D.C, 20535
RTE46, Little Falls, N.J, 07424
Paulovicks, George E. Richardson, Edwin M,
ArrN:SFC P.J.Bordino
New York State Police Laboratory Wilmington Medical Center
Albany, New York Ramsay, Michael P. 501 W 14th Street
Addiction Research Foundation Wilmington, Del. 19889
Pearson, Eric(DrO 33 Russell St.,Rm.T-607
Home Ofhee-CRE Romano, Anthony, Jr,
Toronto, Canada
Aldermaston, UK Ontario M5S 2S1
Southeast Regional Laboratory
5205 N.W. 84th Avenue
Peindl, Joseph H. Miami. Flonda33166
Detroit Police Dept. Crime Raney, H.T.
Laboratory N .C State Bui of Investigation
. Rosen stein, Jack
1300 Beaubien 421 N. Blount Street South Central Regional Laboratory
Room 647 Raleigh, N.C. 27601 1880 Regal Row
Detroit, Michigan 48226 Dallas, Texas 75235
Reading, Charles N,
Rowe, Walter F,
Penllo. Benjamin Connecticut State Dept, of Health
Geo. Washington University
North Central Regional Lab 3 Tanner St.
Dept, of Forensic Sciences
500 U.S. Customshouse Manchester, Ct. 06040
Geo. Washington University
610 S Canal Street Washington, D.C, 20052
Chicago, Illinois 60607 Reboulet, James E.
Miami Valley Regional Crime Sagei Robert
,
288 .
Smith, Donald J, Stewart, Constance B. Valentour, James Ph.D
Food & Drug Admi n, Toxicologist Chemist Virginia Bureau of Forensic Sciences
200 C Street,^, W. __ Office of the Medical Examiner P.O. Box 999
Washington, D.C, 20204 P.O. Box 427 Richmond, Virginia 23208
Farmington, Conn. 06032
Smith, Edward Van Peteghem, Carlos, H.M, (Dr.)
F.D.A. Stewart, Deborah C. State University of Ghent
Div. of Drug Chemistry Analytical Chemistry Department of Toxicology
HFD420 1155 I6th Street, N.W. Hospitaalstraat 13
Washington, D.C. 20204 Washington, D.C, 20036 B.9000
Ghent, Belgium
Smith, L Gerald Maehly, Andreas, Ph.D.
Tennessee Cnme Laboratory Director
Knoxville Branch Vaney, Robert Dr.
The National Lab of Forensic
1522 Cherokee Trad
Luxembourg Institute of Public
Science
Health
Knoxville, Tennessee 37920 PACK, Linkoping, Sweden
Laboratory of Toxicology and
Snyder, Jack W. Sullivan, Jonn Pharmaceutical Chemistry
Geo. Washington Univ. LEAA
Dept, of Forensic Science Washington, D.C. Vias, Gary
481941st St.,N.W. National Naval Medical Center
Sunshine, Irving Ph.D.
Washington, D.C. 20016 8901 Wisconsin Avenue
Cuyahoga County Coroner’s Lab Clinical Laboratory (Toxicology)
Sobol, Stanley?.
2121 Adelbert Road
Bethesda, Maryland 20014
Cleveland Ohio 44 1 06
Laboratory Director ,
289 .
Wells, John,(Dr,)
CFS Toronto
Centre of Forensic Sciences
25 Grosvenor Street
Toronto, Ontario M7A 2G8 Canada
Wells, William E.
South Carolina Law Enforcement
Div.
P.O.Box 21398
Columbia, South Carolina 29221
Wennmg, Robeit
Laboratoire
De Chimie Toxicologioue
Institute of Hygene el de Saute
Publique
P.O.Box 1102
Luxembourg, Europe
Wong, Alice S.
Toxicology Section
Centre of Forensic Sciences
25 Giosvenor Street
Toronto, Ont. M7A 2G8 Canada
Wuensche, Martin R.
Houston Police Dept. (Crime Lab)
61 Riesner St.
Houston, Texas 77002
290 .