Running Head: WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 1

Between 3 enzymes, 2 E. coli-based and 1 abalone-based beta-glucuronidases, which

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

percent recovery of analytes upon analysis?

David M. Weinberg

Center for Advanced Studies at Wheeler High School

 WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 2

Table of Contents

Rationale of Study………………………………………………………………………………....3

Research Concept and Design……….……………………………………………………....….4-5

Chapter 1……………………………………………………………………………………….6-11

Statement of the Problem………………………………………………………………….6

Subproblems and Hypotheses…………………………………………………………...6-7

Definitions of Terms………………………………………………………………….....7-9

Assumptions………………………………………………………………..………….9-10

Delimitations and Limitations…………………………………………………..……10-11

Importance of Study……………………………………………………………………...11

Chapter 2……………………………………………………………………………………...12-18

Foundational Sub-problem 1: Review of Literature…………………...……………..12-14

Foundational Sub-problem 2: Review of Literature…………….………………...….15-17

Applied Sub-problem 1: Review of Literature……………....…………………………..18

Chapter 3……………………………………………………………………………………...19-24

Restatement of Applied Sub-problem 1 and Hypothesis………………………………...19

Data Needed and Means of Obtaining Data……………………………………………..19

Research Methodology…………………………………………………………...…..19-22

Treatment of Data……………………………………………………………………..…22

Data Tables……………………………………………………………………………....23

Qualification of Researcher and Assistants………………………………….……….23-24

Outline of Proposed Study……………………………………………………………….24

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-

glucuronide by beta-glucuronidase. All analytical laboratories must prepare samples before

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

analysis is also important because it is designed to hydrolyze glucuronide-containing drug

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

to apply such knowledge.

 WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 4

Research Concept

Research Concept Map:

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

Medical Laboratories’ practices. These will be elaborated on in Chapter 3 of this proposal.

Subsequent samples will see variables manipulated. All samples then will be analyzed in a liquid

chromatography-mass spectrometry instrument. Statistics will be performed to measure any

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

conditions for sample preparation yield the most accurate results.

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

that was conducted.

 WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 6

Chapter 1

Statement of Problem:

Overarching Question. Between 3 enzymes, 2 E. coli-based and 1 abalone-based beta-

glucuronidases, which 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 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

optimization would be implemented at Newstar Medical Laboratories, LLC. and possibly

other laboratories.

Foundational Sub-Problems:

Foundational sub-problem 1. What are the differences between acid/base hydrolysis

and enzyme hydrolysis, and what are their respective advantages?

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

selective but requires more strict control of conditions.

Foundational sub-problem 2. What does beta-glucuronidase do and what are some of

its sources?

Hypothesis. Beta-glucuronidase separates drug metabolites from sugars in solution. It

can be harvested from abalone or E. coli, or it can be synthetically manufactured.

 WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 7

Applied Sub-Problems:

Applied sub-problem 1. What source of beta-glucuronidase, time of incubation,

temperature, and enzyme activity units in sample preparation afford the most accurate

sample analysis by percent recovery of codeine analytes?

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

accurate analysis results.

Independent Variables. The independent variables studied in this research will be beta-

glucuronidase source, including two enzymes from E. coli, from manufacturers

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.

Dependent Variables. The dependent variables studied in this research will be

measurements of sample analysis accuracy, as compared to expected concentrations.

These will include percent recovery based on expected values and coefficient of variation

among triplicated samples.

Definitions of Terms and List of Acronyms:

Definitions of Terms:

 Acid/Base Hydrolysis: Acid hydrolysis uses protons from acids as catalysts in

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,

personal communication, October 11, 2018).

WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 8

 Aliquot: A known amount of a substance, generally a liquid, that is usually a

fraction of a whole (McNaught, Wilkinson, & Jenkins, 1997).

 Analyte: A part of a substance that is being analyzed (McNaught, Wilkinson, &

Jenkins, 1997).

 Beta-glucuronidase: Beta-glucuronidase is an enzyme that is used in the

hydrolysis of glucuronides from bodily fluids prior to analysis (“β-Glucuronidase

(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

enzyme (Nomenclature Committee of the International Union of Biochemistry

(NC-IUB), 1979).

 Enzyme Hydrolysis: A reaction in which an enzyme facilitates the breaking of

bonds in molecules in the presence of water (Yang, Dai, Ding, & Wyman, 2011).

 Hydrolysis: Solvolysis by water, solvolysis being the reaction with a solvent

involving the breaking of bonds in the solute (McNaught, Wilkinson, & Jenkins,

1997).

 Incubation: The catalyzation of a reaction by controlling environmental

parameters around the reaction (R. Godwin, personal communication, September

14, 2018).

 Internal Standard: “A compound added to a sample in known concentration to

facilitate the qualitative identification and/or quantitative determination of the

sample components,” (McNaught, Wilkinson, & Jenkins, 1997).

 WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 9

 Liquid Chromatography: Liquid chromatography is a method where molecules in

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).

 Mass Spectrometry: Mass spectrometry is a method of measuring the

characteristics of molecules by ionizing a sample, separating them by mass and

charge using electromagnetic fields, and detecting them and creating a graph

based on the results (Reusch, 2013). Tandem mass spectrometry uses multiple

mass separators to obtain a higher resolution of ions (“Tandem Mass

Spectrometry (MS/MS)”, 2015).

 Metabolite: “A substance made or used when the body breaks down food, drugs

or chemicals, or its own tissue,” (“Metabolite”, n. d.).

 Quality Control: Quality control is an aspect of laboratory management taken to

assure the validity of results and to improve analysis associated with measurement

in the lab (Howanitz & Howanitz, 1983).

Acronyms:
 GUS: Beta-glucuronidase

 LC-MS/MS: Liquid chromatography-tandem mass spectrometry

Assumptions of Research:

 The researcher assumes that the instruments used for analysis are tuned and provide

accurate results for percent recovery.

 The researcher assumes that they will have access to enough enzymes/supplies to prepare

enough samples for the study.

WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 10

 The researcher assumes that the equipment used for sample preparation and analysis are

available for use when the researcher needs them.

 The researcher assumes that the enzymes obtained from manufacturers function as

expected and as advertised by the manufacturers.

 The researcher assumes that the quality control samples from manufacturers provide

accurate calibration for experimental samples.

Delimitations and Limitations of Research:

 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

scope of this research.

 This study obtains results for validation of preparation techniques from liquid

chromatography-mass spectrometry. The mass spectrometer is specifically of the triple-

quadrupole variety. Other techniques, such as gas chromatography, could be used to

obtain the same or similar results, but the study will only be performed using instruments

available in the lab where the research will be performed.

 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

on sample hydrolysis are outside of the researcher’s control.

WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 11

 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

one enzyme over another.

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

reporting and to drive down care costs.

WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 12

Chapter 2

Foundational Sub-problem 1 - Review of Literature:

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

and discuss their relative advantages and disadvantages.

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

operations, but the production process is obviously very intense.

Enzyme hydrolysis, contrastingly, is a more precise technique. It employs enzymes as

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

method of preparing substances for enzyme hydrolysis by a pretreatment technique extends

beyond acid/base hydrolysis to gelatinization and liquefaction (Liu et al., 2017).

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

degradation or unwanted by-products, even though conditions must be more stringently

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

looked at in the next review.

WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 15

Foundational Sub-problem 2 - Review of Literature:

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

these is a process called glucuronidation (Liang et al., 2016). Glucuronidation attaches a

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

very important to understand glucuronidation as a part of drug metabolism when developing or

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

metabolites from their glucuronic acid conjugates.

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

enzymes and evaluating their usefulness (Morris et al., 2014).

GUS is obviously an important enzyme because of its role in the laboratory. Since

glucuronidation is such a ubiquitous mechanism in metabolism, it is important to understand

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

accurate results for drug concentration in samples.

WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 18

Applied Sub-problem - Review of Literature:

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

hydrolyze benzodiazepine glucuronides at an incubation time of forty-five minutes. However,

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

also analyze differences in efficiency of codeine-6-glucuronide hydrolysis between enzyme

sources.
WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 19

Chapter 3

Restatement of Applied sub-problem 1:

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.

Data Needed and Means of Obtaining Data:

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

manufacturers, IMCSzyme and Kura Biotec.

WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 20

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

experimental samples in triplicate.

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

samples will contain, because of prior preparation, an amount of codeine-6-glucuronide

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

experimental samples based on expected recovery concentrations. The industry standard of an

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

percentages closest to the expected values.

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

codeine-6-glucuronide. The researcher will create graphical representations of the various

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:

Table 1. Hydrolysis Results in Percent Recovery Based on Trials of Varied Incubation

Time, with a Constant Source, Temperature, and Level of Activity. (Placeholder Data,

Other Tables Will Be Similar and See Other Variables Manipulated)

Trials Enzyme Incubation Activity Temperature Mean Coefficient of

Source Time Units of Percent Variation (%)
Enzyme Recovery

Trial 1 Abalone 15 mins 4,000 units 55°C 70% 13%

Trial 2 - 30 mins - - 77% 11%

Trial 3 - 45 mins - - 81% 17%

Trial 4 - 1 hr - - 87% 12%

Trial 5 - 1.5 hrs - - 90% 10%

Trial 6 - 2 hrs - - 99% 9%

Trial 7 - 2.5 hrs - - 106% 12%

Trial 8 - 3 hrs - - 115% 8%

Qualification of Researcher and Assistants:

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

metabolism and clinical toxicology testing methods.

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.

Outline of Proposed Study:

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

over the course of the research.

 WHICH PREPARATION YIELDS MOST ACCURATE RESULTS 25

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