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combination of enzyme source, activity units, temperature, and incubation time in sample
preparation by enzyme hydrolysis of codeine-6-glucuronide yields the most accurate results for
David M. Weinberg
Table of Contents
Rationale of Study………………………………………………………………………………....3
Chapter 1……………………………………………………………………………………….6-11
Definitions of Terms………………………………………………………………….....7-9
Assumptions………………………………………………………………..………….9-10
Importance of Study……………………………………………………………………...11
Chapter 2……………………………………………………………………………………...12-18
Chapter 3……………………………………………………………………………………...19-24
Research Methodology…………………………………………………………...…..19-22
Treatment of Data……………………………………………………………………..…22
Data Tables……………………………………………………………………………....23
References…………………………………………………………………………..………..25-28
WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 3
Rationale of Study
The purpose in conducting this study is to optimize the enzyme hydrolysis of codeine-6-
analysis, and hydrolysis reactions are commonly used to convert compounds into more easily
analyzed forms. Codeine-6-glucuronide was chosen for this research because it is known for
being difficult to hydrolyze, even over other drugs in other classes (R. Godwin, personal
communication, October 9, 2018). Clinical laboratories that evaluate the presence of drugs and
drug metabolites in patient samples must ensure that sample preparation methods provide
adequate conversion of glucuronide conjugates to parent drugs, like codeine, in order to obtain
adequate response from instruments to confirm or deny the presence of drugs. This research
serves to optimize the procedures for such preparation. Enzyme source, incubation time, enzyme
activity units, and temperature will be examined because they are influential and cost-inducing
factors in sample preparation (Morris, Chester, Strickland, & McIntire, 2014, pp. 1-2; Taylor,
Flint, Ma, Hill, Clark, & Strathmann, 2017, p. 2). Using beta-glucuronidase as the enzyme for
metabolites, which are pervasive in human metabolism (Hassan, Waheed, Grubb, Klei, Korolev,
& Sly, 2013, p. 1; Liang et al., 2015, pp. 2-3). The researcher undertook this research and the
internship where this research was performed in order to further their knowledge of the
laboratory industry and the field of analytical chemistry, and because it provided the opportunity
Research Concept
Research Design:
The research will be conducted at the researcher’s internship location, Newstar Medical
Laboratories, LLC., over the span of two to three weeks. A review of literature has occurred,
with the researcher studying the differences and respective of advantages of acid/base hydrolysis
and enzyme hydrolysis, as well as the uses and sources of beta-glucuronidase. Once the research
proposal is approved, the researcher will begin preparing experimental samples for data
collection. The researcher will prepare the first samples under standard conditions for Newstar
WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 5
Subsequent samples will see variables manipulated. All samples then will be analyzed in a liquid
discrepancies in results, including finding the percent recovery of codeine from experimental
samples based on expected values. The data will then be analyzed to conclude which set of
Once the research is completed, the researcher will compile his findings, along with any
necessary graphs or tables, the literature review, and his conclusions into a research paper that
will be submitted at the end of the semester. A presentation will also be given on the research
Chapter 1
Statement of Problem:
yields the most accurate results for percent recovery of analytes upon analysis?
Purpose of Study. The purpose of this study is to optimize the sample preparation
procedures for preparing urine samples to be tested for drug metabolites. This
other laboratories.
Foundational Sub-Problems:
Hypothesis. Acid/base hydrolysis introduces acids or bases into water to create solutions
of ions. The conditions that are needed for this can be extreme. Enzymatic hydrolysis
uses enzymes to facilitate this bond-breaking when desired. This method is more
its sources?
Applied Sub-Problems:
temperature, and enzyme activity units in sample preparation afford the most accurate
Hypothesis. An activity of 4,000 units, a time of 2 hours, a temperature of 55°C, and the
E. coli-derived beta-glucuronidase from manufacturer Kura Biotec will result in the most
Independent Variables. The independent variables studied in this research will be beta-
IMCSzyme and Kura Biotec respectively, and one from abalone; time of sample
incubation to allow the reaction to reach completion before sample analysis; and
temperature and activity units of enzyme in samples, which impact reaction rate.
These will include percent recovery based on expected values and coefficient of variation
Definitions of Terms:
substitution reactions. Base hydrolysis involves the hydroxyl (OH-) group of the
base acting as the substitution for something else in a reaction (S. Vander Wielen,
Jenkins, 1997).
(beta-Glucuronidase)”, n. d.).
Enzyme Activity Unit: The amount of an enzyme that catalyzes the conversion of
a specific amount of a substance per minute under optimal conditions for the
(NC-IUB), 1979).
bonds in molecules in the presence of water (Yang, Dai, Ding, & Wyman, 2011).
involving the breaking of bonds in the solute (McNaught, Wilkinson, & Jenkins,
1997).
14, 2018).
a mixture are applied to a solid, or stationary phase, moved along that solid by a
liquid solution, or mobile phase, and separated from each by various interactions
between the individual molecules and the stationary phase (Coskun, 2016).
charge using electromagnetic fields, and detecting them and creating a graph
based on the results (Reusch, 2013). Tandem mass spectrometry uses multiple
Metabolite: “A substance made or used when the body breaks down food, drugs
assure the validity of results and to improve analysis associated with measurement
Acronyms:
GUS: Beta-glucuronidase
Assumptions of Research:
The researcher assumes that the instruments used for analysis are tuned and provide
The researcher assumes that they will have access to enough enzymes/supplies to prepare
The researcher assumes that the equipment used for sample preparation and analysis are
The researcher assumes that the enzymes obtained from manufacturers function as
The researcher assumes that the quality control samples from manufacturers provide
This study does not include any tests on enzymes that are not GUS. Within the confines
of laboratory where the research will be performed, GUS is the only enzyme available for
study.
This study does not perform any tests on variables that are not time of incubation, activity
units, or temperature. Tests on other variables, such as pH, are possible but not within the
This study obtains results for validation of preparation techniques from liquid
obtain the same or similar results, but the study will only be performed using instruments
A limitation of the study is in the “cleanliness” of the enzymes used. Different sources of
GUS yield different levels of “cleanliness” in the enzyme. E. coli sources are generally
considered the cleanest and abalone is less so. However, these factors and their impacts
A limitation of the study is in the time frame the researcher will have to collect data.
Because of time constraints, the researcher will not have time to investigate the effects of
longer incubation times on metabolite recovery results or any long-term cost benefits of
Importance of Study:
This study looks at what enzyme sources and sample preparation conditions yield the
most accurate results for analysis of drug metabolites in urine samples and attempts to optimize
these factors. This study is of immediate relevance and importance in the industry of clinical
laboratories. Laboratories must try to cut costs wherever necessary to provide more cost-
effective services while maintaining quality. Boison, Dowling, Johnson, & Kinar (2016) showed
that recovery of drug metabolites from horse tissue samples is increased by the addition of a
GUS hydrolysis step, so it has been shown to increase yield. However, optimization of that
hydrolysis is required, and any amount of optimization of techniques can give laboratories an
advantage. This is very useful in an expensive but lucrative business like private healthcare.
Thus, the importance of this study stems from its potential to increase accuracy and efficiency of
Chapter 2
Hydrolysis involves the breaking of bonds with water. Two efficient hydrolysis methods
are acid/base hydrolysis and enzymatic hydrolysis. These techniques of hydrolysis are important
in today’s world, both in science and in industry. Hydrolysis is used to create fuel (Greenwood,
Farrell, Zhang, & O'Hara, 2015) and other products and separate useful compounds, including
those to help fight disease, from other larger substances (Peciulyte, Karlstrom, Larsson, &
Olsson, 2015; Liu, Wen, Zhang, Wei, Deng, Xiao, & Zhang, 2017). Generally, hydrolysis
techniques are used because they are more efficient and provide greater products yields than
other methods of substance separation (Greenwood et al., 2015; Boison et al., 2016; Liu et al.,
2017). Much current research is being conducted to improve hydrolysis methods, both to expand
their uses to larger scale operations and to optimize their efficiencies (Greenwood et al., 2015).
This review will cover some of the characteristics of acid/base hydrolysis and enzyme hydrolysis
Acid/base hydrolysis involves using acids or bases to catalyze hydrolysis and break
bonds. Acids and bases are somewhat interchangeable in this manner, as the better option
depends on the substance being hydrolyzed. Often this technique is used in large-scale hydrolysis
of tough biofuels and natural substances, especially plant matter (Greenwood et al., 2015; Liu et
al., 2017; Hilpmann et al., 2016). This type of hydrolysis characteristically requires severe
conditions for reactions to take place efficiently. Due to reaction kinetics, the most influential of
these conditions is temperature. In order for acid/base hydrolysis reactions to occur swiftly, high
temperatures are used, often times near or beyond 100°C (Hilpmann et al., 2016; Greenwood et
al, 2015). Yields often improve at these higher temperatures because of those reaction kinetics
WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 13
(Greenwood et al., 2015). Another main condition affecting acid/base hydrolysis is, of course,
pH. Naturally, these reactions require extremely high or low pHs to proceed (Hilpmann et al.,
2016).
While these reaction conditions can be acceptable in large scale industrial production,
they can often lead to undesired consequences. Hydrolysis at these high temperatures and pHs
can drain resources and money (Greenwood et al., 2015). At temperatures and pHs that are too
high, degradation can occur, leading to the destruction of resources before they can be converted
to useful products. Alternatively, unwanted by-products can be formed in the same way. This can
cause reactions to occur efficiently in only a relatively small window of temperatures and pHs
(Hilpmann et al., 2016). As said previously, these conditions are obtained and accepted in some
catalysts for bond breaking. Uses of enzyme hydrolysis are more exact, including drug analysis
(Boison et al., 2016), and require less severe conditions in order to maximize the activity of the
enzyme and reduce the possibility of degrading the target compounds and enzymes. Increasing
temperature helps increase the reaction rate, as in most cases, but enzymes usually cannot be
subjected to high temperatures and remain effective due to degradation of the enzyme (Liu et al.,
2017; Hilpmann et al., 2016), although there are exceptions (Hilpmann et al., 2016). Another
characteristic of enzyme hydrolysis is that enzymes, by design, are very specific to certain
substances. Enzymes must be able to chemically bind to other substances, but the physical
structure of enzymes used for hydrolysis must also closely match the structures of the substances
that are hydrolyzed. Sometimes this principle is extended to structures surrounding the target
substances. Even within targets for hydrolysis, variations in structure and the attachment site of
WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 14
enzymes can impact how effectively enzyme hydrolysis breaks up substances (Peciulyte et al.,
2017).
One other possible method for hydrolysis is to treat substances with acids or bases prior
to enzyme hydrolysis. This could serve to be more efficient for some substances than either
technique used alone. What can occur in a lot of cases of both hydrolysis techniques being used
is that first treating a substance by acid or base hydrolysis allows enzymes to better access sites
or can prepare certain substances for enzyme hydrolysis that would not have been able to without
pretreatment under the severe conditions of acid/base hydrolysis (Peciulyte et al., 2015). This
As shown, both acid/base and enzymatic techniques of hydrolysis have their respective
benefits and situations where they are used. Acid/base hydrolysis is better for larger scale, more
industrial processes where conditions can be made as severe as possible to speed up reaction
rates. Enzyme hydrolysis is preferable for more precise methods as there is less possibility of
regulated. In the context of the research that will be performed in this study, enzyme hydrolysis
is used because the drug analytes that are studied are not stable under severe reaction conditions
and the instruments of analysis are very finely tuned and thus subject to degradation and damage
under those same conditions. The specific enzyme being used, beta-glucuronidase, will be
Before talking about the enzyme beta-glucuronidase (GUS), one has to talk about the
functions and processes that GUS is related to. In human metabolism, one of the main ways that
humans excrete waste is through urine. In order for waste to be excreted in urine, it has to be
soluble in urine. Not all waste products are soluble in urine, so the body has mechanisms to
change the solubility of certain waste products to make them more easily excretable. One of
glucuronic acid – a type of sugar – substituent to a waste product to make it soluble in urine. One
of the main groups of waste products that are affected by glucuronidation is drugs. Thus, it is
prescribing drugs (Liang et al., 2016; Gagez, Rouguieg-Malki, Sauvage, Marquet, & Picard,
2012).
In the same vein, being able to separate drugs and drug metabolites from their glucuronic
acid conjugates helps to facilitate drug analysis. GUS, the enzyme used for the research proposed
here, has the specific function of cleaving glucuronic acid groups, and sometimes other
substrates, from waste products, including drugs and drug metabolites, carcinogens, and
hormones (Sakurama et al., 2014). Now, other substances are hydrolyzed by GUS: it is used in
the body in regular metabolism to regulate the accumulation of non-harmful substances in the
body (Hassan et al., 2013) and on other substances not related to human metabolism in order to
convert them from a form containing glucuronic acid to another, more useful one without it
(Sakurama et al., 2014). Not every form of GUS – because GUS is actually a class of enzymes –
has the same capabilities. Sakurama et al. (2014) discovered a GUS that has “strict glycon
specificity” (p. 7), meaning that it selectively hydrolyzes only substances with a glucuronic acid
WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 16
group, as opposed to others that have the capability of hydrolyzing other substrates. The use of
GUS that is implemented in the research proposed here is its ability to isolate drugs and drug
This is something that GUS excels at. GUS is used often in clinical laboratories in sample
preparation before drug analysis. The isolation of drugs and drug metabolites allows for clearer
recognition by machines of drug presence in human urine samples. Taylor et al. (2017)
mentioned that “an enzymatic hydrolysis step using ß-Glucuronidase may be implemented to
increase the sensitivity of [benzodiazepines]” (p. 2). Taylor et al.’s study was done with clinical
laboratory testing in mind. In fact, they showed an increase in positive screens for
benzodiazepines of 42.5% with the addition of GUS in sample preparation (Taylor et al., 2017).
Much current research is occurring in optimization of sample preparation using GUS in clinical
laboratories, including the study proposed here. Morris et al. (2014) showed that a specific form
of GUS can give the same amount of hydrolysis “at [room temperature] equivalent to
measurements at 55°C” and “was considerably faster” compared to another form (p. 3). They
also showed that this form was less damaging to instruments over time as many samples were
run through them. These different forms of GUS are another area of ongoing research and
optimization.
GUS is harvested or manufactured from a variety of sources. Humans are one such
source (Hassan, Waheed, Grubb, Klei, Korolev, & Sly, 2013), although human GUS is not used
in clinical settings. Other sources are more easily harvestable, such as abalone (Morris et al.,
2014). Abalone-based GUS was used often in clinical laboratories, but other sources have
become more prominent recently. Bacterial GUS is even more easily acquired than abalone
GUS, so it has become more pervasive. The main source of GUS from bacteria is Escherichia
WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 17
coli and other closely related bacteria; it was thought to be essentially the only bacterial source of
GUS (Krahulec, Szemes, & Krahulcová, 2010). However, Krahulec et al. (2010) discovered
another source of GUS in bacteria. Sakurama et al. (2014) continued this, finding other bacterial
sources, as did other groups, and still more sources of GUS are being sought out. In reality,
human GUS is very similar to bacterial GUS (Hassan et al., 2013), but obviously it is easier to
obtain GUS from bacteria than from humans. The desire for better sources of GUS has begun the
development of synthetic analogues to natural GUS. Manufacturers like IMCSzyme and others
have developed recombinant GUS, and research has begun on characterizing these recombinant
GUS is obviously an important enzyme because of its role in the laboratory. Since
glucuronidation and GUS to be able to monitor metabolism. Since healthcare, including drug
testing in clinical laboratories, is so imperative and expensive, it is important as well to use the
right materials and to optimize resources and processes to provide efficient results and care at
low cost. The research proposed here will use GUS in sample preparation to hydrolyze codeine-
6-glucuronide. GUS source, time of incubation, temperature, and enzyme activity units will be
manipulated to determine which combination of these factors leads to the most efficient and most
A review of the literature has shown a few different sets of conditions for working with
GUS. It is obviously important to ensure the right conditions for both the enzyme and the
substrates that are catalyzed. Sakurama et al. (2014) characterized a recombinant version of the
GUS they discovered and determined that its optimal activity levels occurred at 55°C and with a
pH of 4.5. Morris et al. (2014) relayed that 55°C was also optimal for an abalone GUS to
they also found that a recombinant GUS hydrolyzed almost as efficiently at room temperature
with only five minutes of incubation. Taylor et al. (2017) found that “optimal hydrolysis time
was achieved around 15 [minutes] with 1,600 units of enzyme” (p. 2). The goal of the research
proposed here is to consolidate some of these factors so they are more easily comparable and
sources.
WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 19
Chapter 3
What source of GUS, time of incubation, temperature, and enzyme activity units in
sample preparation afford the most accurate sample analysis by percent recovery of codeine
analytes?
Restatement of Hypothesis:
An activity of 4,000 units, a time of 2 hours, a temperature of 55°C and the E. coli-
derived GUS from manufacturer Kura Biotec will result in the highest percent recovery.
The data that the researcher will need to determine the optimal method of sample
preparation are percent recovery of codeine and percent difference from expected concentrations
of codeine. The researcher will obtain these values by entering experimental samples into an LC-
MS/MS for analysis. Software compatible with the LC-MS/MS will calculate and display results
for percent recovery, from which the researcher will calculate coefficient of variation as well.
Research Methodology:
The researcher will conduct the data collection for this research over the span of two to
three weeks at Newstar Medical Laboratories, LLC. The results and methods of similar studies
were reviewed in the literature review to inform this study’s methodology. Once this proposal is
approved, the researcher will start preparing samples as soon as possible with one of three GUS
sources, two E. coli and one abalone. The two E. coli-based GUS will come from two different
The researcher will prepare the first set of samples with one source of GUS and
manipulate variables at between five and eight data points, depending on the condition variable.
However, the researcher will only manipulate one variable at a time. For example, the researcher
might begin with the IMCSzyme GUS and manipulate temperature. In this case, temperature will
be changed as incubation time and activity units are held constant at 2 hours and 4,000 activity
units, standards for Newstar Medical Laboratories’ practices. The same procedure will follow for
manipulating other condition variables. The researcher will collect results for samples with a
single GUS source, and then work will begin on samples with a different source. This separation
is done to ensure that inherent activity levels of enzymes don’t confound results for preparation
condition manipulations. Incubation of all experimental sample will occur in a Benchmark Incu-
Mixer MP 4-plate. All experimental sample reactions will occur in 100 µL of synthetic urine,
which simulates the actual chemical environment of human urine. The researcher will prepare all
An LC-MS/MS instrument will analyze the experimental samples, after preparation under
the various combinations of factors. This method first separates analytes from each other and
then individually measures their masses so they can be identified. The setup at Newstar Medical
Laboratories contains a Shimadzu 20-Series LC and an AB Sciex API 4500 MS. Analysis results
obtained from these instruments will send to and display on a paired computer with software for
quantitative analysis.
In analyzing each batch of samples to obtain quantitative results, the researcher will
prepare a set of samples alongside experimental samples to prepare the instruments for analysis.
Blanks, samples containing only mobile phase, will go first so that the mobile phase running
through the LC will equilibrate with the stationary phase. Calibrator samples will inject into the
WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 21
instrument next. These will set up a calibration curve on the paired software that will serve as the
reference for assigning samples concentrations based on LC-MS/MS analysis. Quality controls
follow calibrators and have a known concentration of analyte. They are used in clinical practice
to verify that the calibration curve is accurate before any patient samples are run. After running
quality controls, the LC-MS/MS will inject experimental samples for analysis. All experimental
equivalent to a known concentration of codeine. This will serve as the reference value against
which the researcher will compare experimental samples. Each type of sample created will
contain internal standards. These are solutions of analytes of a known concentration that are
slightly modified to distinguish them from target analytes. They serve to reduce the confounding
of response results from LC-MS/MS. By creating a ratio of responses between target analytes
and internal standards, one can protect against data skewing because of common instrument
issues. Because both the targets and internal standards are affected, no change is recorded in the
response ratio.
Once results for the experimental samples are obtained, the researcher will compare them
with the expected concentrations of codeine in samples to determine the percent recovery of
analytes and thus the effectiveness of hydrolysis under experimental conditions. The researcher
will record, tabulate, and illustrate this data graphically, if appropriate. To quantify analysis
results, the researcher will use statistics including percent recovery of codeine for the
acceptable difference in percent recovery from expected is plus or minus 20%. This standard
allows the researcher to assess the viability of experimental conditions for sample analysis for
actual urine samples. The researcher will calculate the coefficient of variation to determine the
WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 22
precision of preparation techniques. He will do this by finding the mean and standard deviation
of percent hydrolysis for each of the combinations of factors. The researcher will then analyze
the results to determine which method for sample preparation yields codeine recovery
Treatment of Data:
Data obtained from LC-MS/MS will transfer to software on the laboratory’s computers
for further analysis and calculations, so the researcher will not have to compute percent recovery
manually. However, the researcher will need to compute the coefficient of variation among
triplicated samples. This will show the precision of the preparation method in hydrolyzing
manipulated variables plotted against percent recovery to show the impact of conditions on
enzyme efficiency. The researcher will determine the optimal method of sample preparation by
determining the closest percent recovery of codeine analytes to the expected value. He will also
factor in the coefficients of variation in weighing whether or not a particular average percent
recovery is reliable.
WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 23
Data Tables:
Time, with a Constant Source, Temperature, and Level of Activity. (Placeholder Data,
The researcher’s qualifications for conducting this research include taking classes such as
AP Chemistry, Advanced Organic Chemistry, and AP Statistics. These provide a background for
the researcher on reaction conditions and mechanisms, chemical analytical methods, and
statistical methods. The researcher also completed courses provided by his mentors on drug
The researcher’s assistants are also his mentors, who both have a B.S. Chemistry and
collectively have almost two decades worth of experience in clinical laboratories. Both have
served as technicians and laboratory management staff, teaching others how to use instruments
WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 24
and prepare samples, and they have conducted research in the field of clinical toxicology before
as well.
Once the researcher’s proposal is accepted, the researcher can begin work on preparing
samples immediately. As soon as each set of samples is finished preparing, the researcher must
insert them into the analytical instruments as soon as possible to ensure the results are accurate to
the desired conditions. This, along with the limited amount of time the researcher has to spend in
the laboratory, might cause the process of preparing all the samples under different conditions to
take a week or more. Once all the data has come back and been tabulated, the researcher can
begin comparing results. Graphical representations and tables will display evidence, but the
process of determining an optimal preparation method will not take more than two or three days.
The researcher will spend subsequent time developing the research paper and presentation. The
researcher will investigate any unexpected or concerning results in a timely manner if they arise
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model for the dilute acid hydrolysis of hemicellulose. Biotechnology for Biofuels, 8(26), 1-
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WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 28
Taylor, L. L., Flint, N. A., Ma, V., Hill, B. M., Clark, C. J., & Strathmann, F. G. (2017). Internal
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