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Cocaethylene as a Biomarker in Human Hair of Concomitant Alcohol

and Cocaine Use in a High-Risk Population.

By

Aniket Makarand Natekar

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Pharmacology and Toxicology,

University of Toronto

© Copyright by Aniket Makarand Natekar (2012)

ii

ABSTRACT

COCAETHYLENE AS A BIOMARKER IN HUMAN HAIR OF

CONCOMITANT ALCOHOL AND COCAINE USE

IN A HIGH-RISK POPULATION

Master of Science (2012)

Aniket Makarand Natekar

Department of Pharmacology and Toxicology, University of Toronto

Cocaethylene (CE) is a cocaine metabolite formed during alcohol and cocaine co-

consumption. To our knowledge, no previous studies were conducted assessing CE as a

biomarker indicating chronic excessive alcohol consumption in a suspected high-risk population.

In this study, we hypothesized that hair CE can be an effective marker for alcohol consumption

in a high-risk population. We recorded cocaine, benzoylecgonine, and CE levels in hair samples

from individuals, establishing the predictive value of CE by comparing it to hair levels of the

widely used hair fatty acid ethyl esters (FAEE), direct markers of chronic excessive alcohol

consumption. CE had 14.04% sensitivity and 95.18% specificity in samples separating FAEE

positive/negative results. The positive predictive value was 0.66, showing that the results for

individuals with CE positive results were more than likely to be FAEE positive, but not

conclusively. Thus, CE cannot be used as a definitive marker, indicating chronic excessive

alcohol consumption.

iii

ACKNOWLEDGEMENTS

The past two years of my graduate studies could not have been completed without the

guidance of many individuals, all of whom have contributed to my personal and professional

growth through this amazing time in my life. I would first like to thank Dr. Gideon Koren, for

providing the utmost support, mentorship, and encouragement for my academic pursuits. I feel

truly honoured to have worked in a department where your dedication is marvelled by all, and it

was a gratifying experience to be a part of such a team for two years. I know as time goes on,

many more students will benefit from the same experiences as I have.

I would like to thank Dr. Bhushan Kapur for being a wonderful advisor and for helping

me gain a new perspective on my research. To Dr. Ilan Matok, I would like to extend my

appreciation for all the time you spent helping me understand the statistical tests. I only have the

sincerest appreciation for the time both of you spent with me. I wish you success in all of your

future endeavours.

Ms. Paula Walasek, Mrs. Chitra Rao and Dr. Tatyana Karaskov deserve all the

admiration for their commitment and tireless efforts to ensuring top quality results are produced.

To Mr. Joey Gareri and Dr. Katarina Aleksa, thank you for providing me with opportunities to

counsel and understand laboratory techniques. To the department, I will be forever grateful for

all the pleasant memories.

To all of my friends and family, thank you for standing by me and being there for me. To

Mama, Baba, and Sneha, I am blessed to have all of your unconditional love and support since

the day I arrived into this world. I will never forget it, and will cherish my time with you every

day.

iv

TABLE OF CONTENTS

ABSTRACT..................................................................................................................................................ii

ACKNOWLEDGEMENTS.......................................................................................................................iii

TABLE OF CONENTS..............................................................................................................................iv

LIST OF TABLES.....................................................................................................................................vii

LIST OF FIGURES..................................................................................................................................viii

LIST OF ABBREVIATIONS....................................................................................................................ix

CHAPTER 1. INTRODUCTION.............................................................................................................1

1.1 STATEMENT OF CURRENT KNOWLEDGE.....................................................................................1

1.2 PURPOSE AND STUDY OBJECTIVES...............................................................................................3

1.3 RESEARCH HYPOTHESIS AND RATIONALE.................................................................................4

CHAPTER 2. REVIEW OF THE LITERATURE.................................................................................5

2.1 COCAINE AND ALCOHOL ABUSE ...........................................................................................5

2.1.1 Prevalence and Description..................................................................................................5

2.1.2 Risk Factors of Concomitant Cocaine

and Alcohol Use .................................................................................................................6

2.2 COCAINE.........................................................................................................................................7

2.2.1 Routes of Administration.....................................................................................................7

2.2.2 Receptor Interactions and Effects .......................................................................................8

2.2.3 Cocaine Pharmacokinetics...................................................................................................8

2.3 FATTY ACID ETHYL ESTERS...................................................................................................10

2.4 COCAETHYLENE........................................................................................................................11

2.4.1 Formation and Properties...................................................................................................11

2.4.2 Cocaethylene Toxicity.......................................................................................................14

2.4.3 CE Pharmacokinetics.........................................................................................................15

v

2.5 HAIR TESTING....................................................................................................................................17

2.5.1 Conventional Drug Testing Matrices.................................................................................17

2.5.2 Advantages of Hair Testing...............................................................................................19

2.5.3 Cocaethylene in Hair..........................................................................................................24

CHAPTER 3. MATERIALS AND METHODS....................................................................................25

3.1 SUBJECT RECRUITMENT..........................................................................................................25

3.1.1 Hair Samples......................................................................................................................25

3.1.2 Inclusion and exclusion criteria.........................................................................................25

3.1.3 Sample collection...............................................................................................................25

3.2 COCAINE, BENZOYLECGONINE AND CE PREPARATION.................................................26

3.2.1 Solute extraction................................................................................................................26

3.2.2 Automated solid-phase extraction......................................................................................27

3.2.3 Gas chromatography/mass-spectrometry...........................................................................28

3.3 FATTY ACID ETHYL ESTER SAMPLE ANALYSIS...............................................................29

3.3.1 FAEE extraction.................................................................................................................29

3.3.2 Gas chromatography/mass-spectrometry...........................................................................30

3.4 STATISTICAL ANALYSIS..........................................................................................................30

3.4.1 Data Collection..................................................................................................................30

3.4.2 Statistical Analyses............................................................................................................31

CHAPTER 4. RESULTS.........................................................................................................................32

4.1 DEMOGRAPHICS..............................................................................................................................32

4.2 COMPARISON OF COCAINE, BE AND

CE CONCENTRATIONS WITH FAEE CONCENTRATIONS...............................................................32

4.3 COCAINE ABUSERS WHO ALSO ABUSED

ALCOHOL CHRONICALLY.....................................................................................................................33

4.4 PREDICTING POSITIVE CE RESULTS..........................................................................................34

4.5 SENSITIVITY AND SPECIFICITY OF CE AS

A BIOMARKER OF CHRONIC ALCOHOL ABUSE..............................................................................34

vi

CHAPTER 5. DISCUSSION.....................................................................................................................40

5.1 EFFECTIVENESS OF CE AS A BIOMARKER OF

CHRONIC ALCOHOL CONSUMPTION..................................................................................................40

5.1.1 Explanation of Demographics............................................................................................40

5.1.2 Cocaine and Metabolite Concentrations

Compared With FAEE Concentrations..................................................................................................41

5.1.3 Proportion of Cocaine Use Within

the FAEE Positive/Negative Groups..................................................................................43

5.2 USE OF CE IN GENERAL HAIR TESTING FOR

DRUGS OF ABUSE.........................................................................................................................44

5.2.1 Do cocaine and alcohol use predict positive CE results?..................................................44

5.2.2 Ability of positive FAEE to predict positive CE results....................................................46

5.2.3 Ability of positive CE to predict positive FAEE results....................................................49

5.3 STUDY LIMITATIONS…................................................................................................................51

CHAPTER 6. CONCLUSIONS & FUTURE DIRECTIONS................................................................56

6.1 FUTURE DIRECTIONS...................................................................................................................59

6.1.1 Additional CE incorporation investigations ......................................................................59

6.1.2 Additional CE formation investigations............................................................................60

6.1.3 CE scalp hair elimination rates..........................................................................................61

6.1.4 Possible clinical applications of hair CE testing................................................................62

REFERENCES...........................................................................................................................................64

LIST OF PUBLICATIONS AND ABSTRACTS....................................................................................78

vii

LIST OF TABLES

Table 1. Number of positive cocaine use samples in each range

identified by concentrations in hair................................................................................................33

Table 2. Distribution of results for FAEE positive/negative with CE

positive/negative values with trace CE considered as negative.....................................................35


positive/negative values with trace CE considered as positive......................................................35


positive/negative values for samples positive for cocaine use.......................................................35

Table 5. Distribution of results for FAEE <0.2/≥0.2 ng/mg with CE

positive/negative values with trace CE considered as negative.....................................................36


positive/negative values with trace CE considered as positive......................................................36


positive/negative values for samples positive for cocaine use.......................................................36

Table 8. Sensitivity and specificity of FAEE for CE in different FAEE

Conditions. Trace CE were considered negative...........................................................................37


Conditions. Trace CE were considered positive............................................................................37


Conditions. Only cocaine use samples were considered...............................................................38

viii

LIST OF FIGURES

Figure 1. Cocaine and its various metabolites.................................................................................9

Figure 2. Cocaine metabolism to its metabolites...........................................................................12

Figure 3. Xenobiotics entering hair shaft through blood stream...................................................20

Figure 4. Cocaine and its metabolites are incorporated into adult scalp hair................................22

ix

LIST OF ABBREVIATIONS

CE Cocaethylene

BE Benzoylecgonine

FAEE Fatty Acid Ethyl Esters

CES 1 Human Carboxylesterase-1

CES 2 Human Carboxylesterase-2

EME Ecgonine Methyl Ester

EEE Ecgonine Ethyl Ester

BAC Blood Alcohol Concentration

MDTL Motherisk Drug Testing Laboratory

SPME Solid-Phase Microextraction

GC-MS Gas Chromatography-Mass Sepctrometry

ASPEC Automated Solid-Phase Extraction

PPV Positive Predictive Value

NPV Negative Predictive Value

FASD Fetal Alcohol Spectrum Disorder

1

CHAPTER 1: INTRODUCTION

1.1 Statement of Current Knowledge

In the United States, it is estimated that about 3 million people are regular cocaine users. In

Canada, approximately 238,000 individuals use cocaine. While these statistics are alarming, even

more surprising is that approximately 60-90% of cocaine users also abuse alcohol concomitantly.

It is said that alcohol helps to prolong cocaine-mediated effects, as well as reduce the negative

effects of cocaine use, such as anxiety and uneasiness. While the combination is popular, it has

been shown to be more dangerous than either drug alone. It has been demonstrated that

concomitant alcohol and cocaine use can increase the risk of heart attack and stroke, and also

increase the risk of homicidal thoughts in special populations.

Cocaethylene (CE) is a cocaine metabolite that forms in the presence of ethanol.

Normally, cocaine is hydrolyzed to benzoylecgonine (BE) by human carboxylesterase 1 in the

liver. When ethanol is consumed, about 17% of cocaine is transesterified to CE. Being

pharmacologically active, CE is thought to prolong cocaine-mediated effects, as it acts on the

same receptors and has a longer elimination half-life than cocaine.

Cocaine and BE have been found in scalp hair of cocaine users, and are used as markers

to indicate cocaine use or exposure. Fatty acid ethyl esters (FAEE) are the gold standard in hair

testing to indicate chronic excessive alcohol consumption. CE has been found in scalp hair and

there have been indications for its use as a marker in drug testing to indicate concomitant cocaine

and alcohol use. While CE has been studied in the hair of known cocaine users, the prevalence of

CE in a population whose cocaine and alcohol consumption patterns are unknown is still largely

unstudied. Thus, more knowledge needs to be gathered before CE can be fully used in practice.

2

By understanding the effectiveness of using CE in human hair as a marker to indicate cocaine

and alcohol consumption in a high-risk population, more targeted approaches to drug abuse

treatment and other medical and social strategies can be pursued. With cocaine and alcohol use

still prominent in North America, having the knowledge about CE in hair testing could possibly

aid Children’s Aid societies and legal professionals to make more individualized decisions

regarding the suspected cocaine and alcohol abusers involved. In turn, children living in such at-

risk environments would be protected from harm caused by cocaine and alcohol abusing parents.

3

1.2 Purpose and Study Objectives

The purpose of the study was to determine if CE in scalp hair could be used to indicate

alcohol consumption in a suspected cocaine-user population. The following two objectives were

employed for this investigation:

Objective 1: To determine if positive FAEE values could be used to predict positive CE

values in samples identified positive for cocaine use.

Objective 2: To determine significant relationships between cocaine and metabolite

concentrations in relation to chronic excessive alcohol consumption by testing FAEE

concentrations in scalp hair.

4

1.3 Research Hypothesis and Rationale

The following hypothesis for the present study is below:

Hypothesis 1: A previous study found that CE and FAEE concentrations were related in the scalp

hair of known cocaine users. However, there is no literature on how these relationships are

distributed in a large population for hair testing in a clinical setting. We hypothesized that

positive CE levels could predict positive FAEE levels in people whose cocaine and alcohol use

habits were unknown to us, but were only suspected.

5

CHAPTER 2: REVIEW OF THE LITERATURE

2.1 Cocaine and Alcohol Abuse

2.1.1 Prevalence and Description

Cocaine and alcohol are widely abused in Canada and the United States. The prevalence of

cocaine users in Canada, from 15 years of age and older, appears to have decreased from 1.9% to

0.7% from 2004 to 2010. Also, lifetime alcohol use significantly decreased from 92.8% to

88.9%, while alcohol use within the last 12 months slightly decreased from 79.3% to 77%. The

proportion of heavy frequent drinkers decreased from 7.1% to 4.3%, while the proportion of light

frequent drinkers increased from 27.7% to 32.2%1. In the United States, the most recent statistics

on cocaine and alcohol abuse are on adolescents in grades 9-12. The estimated prevalence of

students from grades 9 to 12 who used any form of cocaine one or more times significantly

decreased from 9.5% to 6.4% between 1999 and 20092, while 2.8% of students surveyed in 2009

admitted to using some form of cocaine within 30 days of the survey3. In the 2009 National

Survey on Drug Use and Health, approximately 30.2 million (12%) people aged 12 or older

admitted to driving under the influence of alcohol4. In North America, an estimated 5.69 million

people were using cocaine in 2009, and approximately 14.25 million to 20.52 million people

worldwide were using cocaine5. This is important because it has been estimated that 50% to 90%

of cocaine users also co-abuse alcohol6-9

. That would mean that approximately 7.125 million to

18.468 million people co-abuse cocaine and alcohol, making it a significant global issue.

6

2.2.1 Risk Factors of Concomitant Cocaine and Alcohol Use

Some reasons for co-abusing cocaine with alcohol is that alcohol prolongs cocaine-mediated

effects, and reduce anxiety, uneasiness, and other psychological adverse effects associated with

the cocaine “crash”10-12

. While the combination of cocaine and alcohol use is very common, it is

also very harmful to the body. In dogs, cocaine and alcohol together were found to decrease the

heart stroke volume by 34% and increase the heart rate by 89%, demonstrating that there is a

synergistic depression of ventricular contraction and relaxation that exceeds the sum of the

depressive effects of cocaine or alcohol alone13

. In humans, the combination of cocaine and

alcohol may increase the risk of cardiologic and neurological complications, such as stroke and

torsades14

. Cocaine and alcohol together demonstrate a trend towards greater-than-additive

effects on heart rate6,10,15-19

. As cocaine alone can cause cardiovascular complications,

concomitant use of cocaine and alcohol may put the user at serious clinical risk for

cardiotoxicity20

. The combination of cocaine and alcohol was found to increase the heart rate of

volunteers by 41 beats per minute compared to placebo21

. Additionally, there seems to be a

cocaine dose-dependent effect on heart rate when in combination with alcohol, as volunteers had

a significantly higher heart rate at a high cocaine dose (1.2 mg/kg) vs. a low cocaine dose (0.3

mg/kg) 22

. Although the volunteers were experienced concomitant cocaine and alcohol users,

four participants felt “nauseous” in almost all of the treatment sessions, and mild headaches were

reported several hours after dosing22

.

In addition to the physical effects of cocaine and alcohol together, there are also psychological

adverse effects. A study conducted on 320 male veterans in treatment for cocaine abuse found

that those who used the combination of cocaine and alcohol rather than cocaine alone were more

likely to attack people physically and more likely to threaten people with a weapon23

. Another

7

study showed that those who used the combination were three times more likely to have

homicidal thoughts or plans than those who drank alcohol only, and five times more likely than

those who used cocaine only24

. In both cases, however, there are other factors that should be

considered before such associations are taken at face-value. For example, individuals in the

military may already have a propensity toward violence, thus a significant relationship between

cocaine and alcohol abuse vs. violence should be expected. The same theory could be applied to

the second study, where the individuals may have already had a tendency toward violent action

prior to drug and alcohol abuse. Additionally, veterans of war may have encountered traumatic

experiences, thus current environmental “triggers” could provoke these individuals to act

violently irrespective of their cocaine and alcohol abuse.

2.2 Cocaine

2.2.1 Routes of Administration

Cocaine is harvested from the coca shrubs Erythroxylum coca and Erythroxylum

novogranatense25

. Once harvested, cocaine is prepared into different forms that are meant for

specific routes of administration. The most common mode of cocaine consumption is through the

intranasal route by sniffing cocaine hydrochloride26

. This route has been demonstrated to take

approximately 14.6±8min to reach peak subjective “high” 27

. “Freebase” cocaine, also known as

“crack” is produced by mixing cocaine with a base, and is typically smoked28

. This has become

the preferred method of cocaine consumption26

since it creates an intense and immediate “high”

within 10-15 sec29

, and the peak subjective “high” was reached within 1.4±0.5 min27

. Cocaine

can also be injected30

, which allows for the highest blood concentration in the shortest amount of

time, and the time to peak subjective effects is 3.1±0.9 min27

. Cocaine can also be swallowed,

usually through the chewing and swallowing of coca leaves31

.

8

2.2.2 Receptor Interactions and Effects

Cocaine mediates its effects for monoamine neurotransmitters in the brain, including serotonin,

dopamine, and norepinephrine32-37

by blocking neurotransmitter re-uptake38

. Short term effects

include increased energy, mental alertness, euphoria, and decreased appetite. Acute cocaine

exposure may induce cardiac arrest, seizures, and/or respiratory failure. Long-term cocaine abuse

can lead to paranoia, auditory hallucinations, irritability, fever, heart attack, gastrointestinal

problems and muscle spasms39-41

.

2.2.3 Cocaine Pharmacokinetics

Once cocaine has entered the blood stream, it is metabolized mostly by the liver, but also by the

heart, stomach, and kidney42,43

. In humans, cocaine is metabolized by a family of

carboxylesterases, particularly carboxylesterase 1 (CES 1) in the liver and carboxylesterase 2

(CES 2) in the intestines43,45,46

. Cocaine is primarily hydrolyzed to benzoylecgonine (BE) by

CES 1 and ecgonine methyl ester (EME) by CES 247

, but is also metabolized to norcocaine48

and

several other metabolites49

. Cocaine’s breakdown into various products is depicted in Figure 1.

9

Figure 1. Cocaine and its various metabolites that are formed in the body (Adapted from

Cone et al., 2003)

The diagram depicts cocaine and its various metabolites that form through enzymatic breakdown

in the human body. Reprinted from Journal of Analytical Toxicology, Vol. 27 (7), Cone et al.

Metabolism of Cocaine. Pg 386-401. Copyright 2003, with permission from Oxford University

Press-Journals.

10

Cocaine’s bioavailability through the nasal insufflation route averages about 80±13% with an

average elimination half-life of 80 min50

. Bioavailability through smoking averages 57±19%

with an average elimination half-life of 56 min. Simulated studies found that only 44% of what

was inhaled was actually cocaine51

, suggesting that most of the un-decomposed cocaine reaches

systemic circulation as its parent form50

. The lower bioavailability is most likely due to

decomposition before inhalation50

. When given intravenously, cocaine has an average

elimination half-life of 78-90 min50,52

. A study found that the elimination half-life of cocaine

through the intranasal and gastrointestinal routes was as high as 5 h53

. While BE and EME have

no bioavailabilities, their elimination half-lives are 7.5 and 3.6 hours respectively48,53

.

Cocaine and its metabolites are excreted in urine and can be detectable for several days

depending on the dose used26,54

. In addition, the same products can also be found in oral fluids55

,

sweat56,57

, and hair58

.

2.3 Fatty Acid Ethyl Esters

Fatty Acid Ethyl Esters (FAEEs) are products of non-oxidative ethanol metabolism by the

conjugation of ethanol to endogenous free fatty acids and fatty acyl-CoA. FAEE can be

spontaneously formed, but is usually catalyzed by microsomal acyl-coA:ethanol O-

aceyltransferase59

. Also, enzymes with FAEE synthetic activity, such as carboxylesterases,

carboxylester lipases, and lipoprotein lipases, in various organs also use ethanol and free fatty

acids as substrates60-63

. For example, FAEE can be found in the liver60

, pancreas62

, plasma64

,

skeletal muscles, and the heart65

. It can be presumed that because alcohol can have effects on

each of the organs mentioned, FAEE could form in the respective organs as well.

11

Although FAEE is produced naturally, they are known to be toxic to the body. FAEE have been

demonstrated to induce apoptosis and necrosis in human peripheral blood mononuclear cells66

.

Additionally, FAEE have been implicated in hepatocellular and pancreatitis-like injury67-69

.

FAEE is normally excreted through the urine70

.

2.4 Cocaethylene

2.4.1 Formation and Properties

Normally, cocaine is hydrolyzed into BE by CES 1. However, when ethanol is consumed at the

same time as cocaine, a portion of cocaine hydrolysis is shifted towards transesterification to

produce cocaethylene (CE) 71,72

. Cocaine metabolism by CES 1 and CES 2 to its respective

metabolites is summarized in Fig 2.

12

Figure 2. Carboxylesterase Metabolism of cocaine and the various metabolites that form

(Adapted from Laizure et al., 2003)

In the presence of ethanol, a portion of the metabolism shits from hydrolysis to

transesterification by CES 1 to produce CE. If enough ethanol remains, then CE can then remain

unmetabolized. CE can be hydrolyzed to BE, or form ecgonine ethyl ester (EEE). Reprinted from

Drug Metabolism and Disposition, Vol. 31, Laizure et al. Carboxylesterase metabolism of

cocaine. Pg. 16-20. Copyright 2003, with permission from American Society for Pharmacology

and Experimental Therapeutics.

13

A study conducted in volunteers given varying doses of IV cocaine with 1 g/kg ethanol found

that plasma CE concentrations reached about 17% of cocaine concentrations, indicating that only

partial transesterification occurred22

. Unlike BE, which is inactive44,45,71

, CE only differs

structurally from cocaine by the substitution of an ethyl instead of a methyl ester47

. As such, CE

has similar pharmacological effects as cocaine at the monoamine transport binding sites in the

brain, and is equipotent in binding to dopamine receptors in the brain as well14,73

. Blocking the

re-uptake of dopamine likely contributes to the increased euphoria73

, thus contributing to the

reinforcement of subjective responses and acute physiological responses of combined alcohol

and cocaine use22,74

. Consequently, the prolonging of cocaine-mediated effects has been

attributed to CE, and this provides a plausible explanation why many cocaine users co-abuse

alcohol75,76

.

It is also important to note that CE can be produced spontaneously in the presence of alcohol for

prolonged periods of time. A study conducted found that cocaine was dissolved in liquor bottles

and smuggled into Canada and was then extracted and converted into its hydrochloride salt.

Analysis of this product found that approximately 20% of the cocaine had undergone

transesterification into CE. Consequently, CE appeared as a major component of the final

cocaine hydrochloride salt product77

. Additionally, the DEA found that the Peruvian method of

processing uses ethanol to purify the cocaine base. Upon analysis of these products, CE was

found in all the samples with an average concentration of 0.12% relative to cocaine, with the

highest CE concentration to be 0.93%. CE reached average concentrations of 0.013% relative to

cocaine hydrochloride, with the highest concentration being 0.084%. Colombian methods

convert cocaine base to cocaine hydrochloride using ethanolic hydrochloride, thus it is expected

to form CE. CE was found to be 2% of cocaine concentrations in Colombian-processed

14

samples78

. As well, CE was found to range from 0.08% to 1.16% of pharmaceutical cocaine

produced for research purposes79

. Thus, the exposure to ethanol for a certain period of time

might be enough to produce CE, and metabolism might not be required. As seen in Fig 2, if CE

is already entered circulation, then the transition from cocaine to CE is bypassed, and only the

second half of the figure would be applicable. With ethanol present, then CE would remain as

itself, and if CE was present in the original cocaine dose, then theoretically the pharmacological

properties of CE could be prolonged.

2.4.2 Cocaethylene Toxicity

In dogs, CE caused hypertension by increased systemic vascular resistance. When CE was

present in high concentrations, it decreased myocardial function, slowed cardiac conduction, and

was arhythmogenic80

. In humans, CE was less potent in elevating heart rate compared to

equivalent cocaine doses. Also, similar differences were found for subjective measures of

“cocaine high”, “rush”, “stimulated”, and “good drug effects”. There was also a significant

increase in systolic blood pressure compared to placebo, but not significant for diastolic blood

pressure81

. Another human study also found that the effect of CE on heart rate was similar to

equivalent doses of cocaine, but may be less potent since higher plasma concentrations were

achieved for CE than cocaine82

. The authors also found that CE is at least as potent as cocaine

with respect to increasing systolic blood pressure82

.

CE has been reported to be more toxic than cocaine in rats83

. CE had an LD50 of 60.7 mg/kg in

females and 63.8 mg/kg in males, while cocaine had an LD50 of 92.4 mg/kg in females and 93.0

mg/kg in males83

. Another study also found CE given through intraperitoneal injection was

significantly more potent than cocaine in causing death (201.5 µmol/kg vs. 237.7 µmol/kg)84

.

Postmortem studies in humans found high CE levels in overdose victims who had recently

15

ingested cocaine and alcohol73,85,86

. One study of post-mortem samples found that CE was not

found when cocaine was absent. As well, even when ethanol and BE were present but CE was

not, cocaine was only in trace amounts, thus CE might have been eliminated85

. In another study,

many had detectable plasma CE concentrations. Cocaine concentrations showed a significant

correlation with CE levels, and CE concentrations were as high 249 µg/L, while cocaine

concentrations reached as high as 441µg/L86

.

2.4.3 CE Pharmacokinetics

In dogs, cocaine and CE had similar clearance values of 0.91±0.22 and 0.79±0.16 L/min and

similar volumes of distribution of 2.6±0.82 and 2.7±0.47 L/kg respectively. In the presence of

ethanol, both cocaine and CE clearance values decreased by approximately 20%, indicating that

both compounds remained in the body for a longer period of time47

. In humans, when cocaine

and CE are given intranasally, CE at low and high doses (0.48 and 0.95 mg/kg) had lower

clearance values than cocaine at a high dose (0.92 mg/kg). With 0.015 and 0.017 L/min/kg for

the low and high CE doses vs. 0.0333 L/min/kg for cocaine, this would explain why the

elimination half life for CE was about 24% longer than cocaine at almost equivalent doses (138

min vs. 111 min respectively) 82

. Also, CE Cmax was found to be much higher than cocaine (251

vs. 144 ng/ml respectively) with a much shorter absorption half-life (13 vs. 17 min respectively)

82. These results suggest that CE is more readily absorbed and remains in the body for a longer

period of time in humans like it did in dogs.

While CE acts on the same receptors as cocaine to prolong cocaine-mediated effects, it is also

metabolized by the same enzymes as cocaine. As shown in Fig 2, if enough cocaine and ethanol

are around, then CE production continues. Also, ethanol can continue to interact with CES 1,

thus preventing CE hydrolysis to BE. Otherwise, it can be metabolized by CES 2 into ecgonine

16

ethyl ester (EEE) or by CES 1 to BE. In dogs, when CE was administered alone, BE achieved

concentrations similar to when cocaine was administered, indicating that BE is a major CE

metabolite47

. A steady dose of ethanol (1 g/kg) with different IV cocaine doses (0.3, 0.6 or 1.2

mg/kg) in volunteers exhibited a dose-dependent increase in CE concentrations. In fact, for every

10 mg of cocaine given, an average of 1.8 mg CE was produced (range 1-4 mg), with

approximately 17±6% of cocaine being converted to CE. Even more important was the fact that

the fractional conversion from cocaine to CE did not differ significantly among cocaine doses22

.

Within post-mortem samples of one study, all seven subjects had different plasma ethanol

concentrations, ranging from 20-240 mg/dl. Plasma cocaine concentrations ranged from 91-4370

mg/ml and plasma CE concentrations ranged from 142-1447 ng/ml85

. There was no discernible

pattern between increasing CE concentrations with ethanol concentrations85

. It has been

postulated that CE formation may not be proportional with low or high ethanol doses22

. This is

further supported by a study conducted in volunteers given cocaine, but no ethanol. The authors

tested the hair of volunteers, and found about 2.33% of the low dose cocaine and 2.22% of the

high dose cocaine was converted to CE87

. It was suspected that soft drinks and other beverages

provided small amounts of alcohol88

. This provides further support to the hypothesis that CE is

not dependent on the amount of ethanol consumed. It is also possible that CE is rapidly

metabolized to BE and EEE hence providing an alternate explanation for the lack of correlation

between alcohol dose and CE production. This theory has not been studied to date, so no

conclusions can be made at this point.

17

2.5 Hair Testing

2.5.1 Conventional Drug Testing Matrices

Cocaine and its metabolites have been detected in urine89,90

, blood91

, and oral fluid92,93

as a part

of substance abuse monitoring. While these drug testing methods are used regularly, they have

disadvantages. For example, cocaine and its metabolites can be detected in urine, but only for a

short time period after use. Intravenous cocaine has been reported to no longer be detectable after

13.5-45 h. BE concentrations (ng/ml) reached single digits after 72.3 h, and EME was almost 0 at

about 60 h26

. When cocaine was smoked, it was no longer detectable after 3.7-37.2 h, while BE

and EME reached single digits after 70 and 69 h respectively. Cocaine taken through the

intranasal route was no longer detectable after 10.5-49.7 h, and BE and EME were almost 0 after

70.8 h for both26

. Although there is a wide range depending on the route of administration,

cocaine will be positive for only 40-120 h in urinanalysis94,95

. Using plasma levels is even more

challenging because of the short elimination half-lives of cocaine than in urine96

. This means that

in order to use plasma levels to monitor cocaine use, sampling has to occur within a short time

frame. As CE has a longer elimination half-life and larger volume of distribution than cocaine74

,

it can be presumed that CE can be detected for longer periods of time than cocaine and its other

metabolites, but not much longer.

Saliva can be used for substance monitoring, and it is less invasive than plasma collection. One

study found that saliva to plasma concentration ratios varied from 0.5-2.9 at different time

points, but had significant correlations of saliva to plasma cocaine levels for three doses of IV

administered cocaine97

. Another study found cocaine to be the predominant analyte found in

saliva, but BE and EME were also found. When volunteers were stimulated to produce saliva by

chewing on candy, cocaine concentrations in saliva decreased. This was likely due to an increase

18

in saliva pH associated with increased saliva flow. Another possible hindrance to using saliva to

screen for cocaine use is that an increase in saliva pH levels impedes the amount of cocaine that

can enter the fluid98

. A study looking at saliva cocaine and metabolite levels in chronic cocaine

users seeking treatment found that unmetabolized cocaine could be found 24 h entering

treatment, and up to 5-10 days during abstinence99

. However, the detection of unmetabolized

cocaine days after the last administration suggests that multiple doses and high cocaine

exposures could lead to accumulation in deep body compartments, and be released slowly back

into circulation and then excretion99

. Having cocaine detectable for up to 5-10 days is beneficial,

but only for a subset of cocaine users. This can affect cocaine use monitoring, especially in

treatment programs99

. CE has also been found in oral fluids, including saliva, and because CE

has similar properties to cocaine, it can be inferred that CE will be found in saliva for similar

periods of time100

.

Another body fluid that can be used for cocaine use monitoring is sweat. Sweat levels can be

monitored through the use of adhesive dermal patches that are placed in areas where sweat is

released101,102

. Drugs can be incorporated into sweat through several different mechanisms,

including passive diffusion from blood into sweat glands, as well as trans-dermal drug passage

across the dermis of the skin103,104

. As such, sweat can be used as a non-invasive method to

detect cocaine use due to the relative ease of sample collection. Additionally, the time frame of

detection of cocaine can range from 24 hours after the last cocaine administration and up to 7

days103,105,106

. CE can also be found in sweat of individuals who used cocaine with good

sensitivity while BE was not found with good sensitivity104

, indicating that CE has similar

properties to cocaine with respect to its incorporation into sweat glands.

19

Normally, testing for alcohol consumption is conducted with the traditional matrices such as

blood, urine, saliva, and expired air for drug licensing and substance abuse monitoring

programs107-110

. However, there are some disadvantages to using these matrices for alcohol

testing. Firstly, ethanol is eliminated rapidly, so multiple samples are required to determine

amount and timing of exposure. Secondly, the body metabolizes about 7 g of alcohol per hour,

equivalent to one drink per hour107

. This equates to a rate of 0.015% of blood alcohol

concentration (BAC) per hour. A person with a BAC of 0.08% (the previous legal limit for

driving in Canada) will not have any measurable alcohol within 5.5 hours of the last drink, and

this applies to breathe testing as well111

. As mentioned previously, measuring ethanol in the

traditional matrices is only beneficial if alcohol is consumed within hours of sampling. FAEE on

the other hand, were found in the serum of volunteers anywhere from 13-24h after ethanol

administration112,113

, and FAEE increased rapidly after ethanol consumption and persisted even

after ethanol was no longer detectable112

. Serum FAEE levels can be used as a marker to indicate

recent, especially binge-type, ethanol consumption113

. Other unique testing methods for FAEE

include human pancreas, liver, heart, brain, and white blood cells114-116

. Additionally, FAEE have

been found in post-mortem liver and adipose tissue117

. FAEE has also been found in meconium,

a matrix that forms during the last two trimesters of pregnancy118

.

2.5.2 Advantages of Hair Testing

While urine, blood, oral fluids, and tissues can be used to detect cocaine and alcohol

consumption, these matrices are useful to detect use within a few hours to a few days of

sampling. Adult scalp hair grows on average 1 cm per month, and can be segmented, which can

reflect both short-term and chronic use and/or exposure119

. This is due in part to the fact that the

hair matrix remains relatively stable for many months120

. Such a phenomenon occurs because

20

hair integrates xenobiotics during its growth, such as drugs and products of ethanol metabolism,

which are present in the blood. Compounds that are trapped within the hair shaft are thus

protected from environmental degradation121

.

Figure 3. Diagram of the simple diffusion model for drug incorporation into hair showing

drugs transferring only from blood to the growing hair cells (Adapted from Henderson,

1993)

A model of how xenobiotics can incorporate into the growing shaft by transferring from the

blood stream surrounding the follicle into the active zone of hair synthesis. Reprinted from from

Forensic Science International, Vol. 63, Henderson. Mechanisms of drug incorporation into hair.

Pg 19-29. Copyright 1993, with permission from Elsevier Limited.

21

With cocaine and its metabolites, hair provides a unique matrix to determine patterns of use over

a long period of time. For example, when compared with urine screening, hair testing had 3.8-

fold higher rates of positivity for cocaine123

. Such a higher rate of positivity is due to the longer

cocaine half-life in hair, which was found to be at a median of 1.5 months for males and 1.5

months for females at an average growth rate of 1 cm/month. BE was found to have a median

half life of 1.46 months for males and 1.48 months for females with the same average growth

rate124

. In fact, it was reported that at least 3 months need to pass before someone who is

abstinent is considered negative for cocaine in the segment proximal to the scalp125

, and this

result was confirmed124

. With such a long period of time before someone will have a negative

result, this indicates that hair is a viable matrix to use for cocaine testing.

An important aspect of hair testing is understanding the possible routes of cocaine and

metabolites incorporation into hair. As stated before, hair incorporates cocaine and its

metabolites from the blood stream that surrounds the follicle121

, but there are also other routes for

cocaine and its metabolites to incorporate into hair.

22

Figure 4. Diagram of a multi-compartment model for drug incorporation into hair from

multiple sources (Adapted from Henderson, 1993)

A more accurate representation of how cocaine and its metabolites are incorporated into adult

scalp hair. External contamination, sweat glands, apocrine and sebaceous glands, as well as skin

also contribute to total cocaine and metabolite incorporation. Reprinted from from Forensic

Science International, Vol. 63, Henderson. Mechanisms of drug incorporation into hair. Pg 19-

29. Copyright 1993, with permission from Elsevier Limited.

23

External contamination, usually through contact with cocaine residues or crack smoke in the

environment, usually can contribute to positive cocaine and BE results since cocaine and its

metabolites remain mostly on the exterior of the hair shaft, but this can be prevented for the most

part through proper decontamination procedures126

. As mentioned previously, cocaine, BE, and

CE can be found in sweat which deposits to the external hair shaft, from which drugs and

metabolites can enter into the hair122

. No clear correlation was found between dose and

concentration in sweat, but the amount of cocaine incorporated into hair appeared to be dose-

dependent127

. Additionally, cocaine and BE found in sebum can be incorporated into the hair

shaft128

. Cocaine was also found within skin as well, but BE was not detected, indicating that

skin may not act as a cocaine resevior, but rather cocaine is distributed rapidly to the skin and

then eliminated128

. Although it is not known how much cocaine is incorporated into hair from

each possibility, hair testing provides a good estimation of cocaine use or exposure within a

period of months depending on the segment length. The International Society for Hair Testing

determined that BE/cocaine ratio must be at least 0.05 or greater to differentiate between use and

external contamination129

.

In our laboratory, the sum of four FAEEs, specifically ethyl myristate, ethyl palmitate, ethyl

oleate and ethyl stearate, are used to determine the amount of alcohol consumption. The four

FAEE have been measured in the hair of alcoholics and used successfully to differentiate them

from social and non-drinkers129,130

. Furthermore, the use of FAEE hair testing has proven to be a

useful tool in substance abuse monitoring to confirm abstinence119,131

. Because the human body

does produce low levels of FAEE from endogenously-circulating ethanol108

, a cutoff was

established by the Society of Hair Testing in 2009. The sum of the four FAEEs must be at least

0.5 ng/mg to distinguish between social drinking and chronic excessive alcohol consumption132

.

24

An FAEE level above 0.5 ng/mg scalp hair to has high sensitivity and specificity (90% for each)

for chronic excessive alcohol consumption129

. Generally, the FAEE level of 0.5 ng/mg

corresponds to chronic excessive alcohol consumption, which is defined by the World Health

Organization as consuming more than 60 g of pure ethanol per day for several months109

. This is

the same definition that is used by the consensus of the Society of Hair Testing as well as by

most laboratories today in hair testing for chronic excessive alcohol consumption. FAEE levels

between 0.2 and 0.5 ng/mg are usually indicative of individuals who consume alcohol socially108

,

defined as one to two standard drinks per day109

. As such, the cutoff of 0.5ng/mg is used

clinically as an indicator to identify a positive result for chronic excessive alcohol

consumption133-135

.

2.5.3 Cocaethylene in Hair

CE, while studied mostly in serum, has also been found in hair of individuals who were known

cocaine users and positive for markers of alcohol consumption136-141

. CE hair concentrations

have been reported to range between 2.5 and 30 ng/mg142,143

, but an acceptable range of detected

CE concentrations has not been established. Recommendations have been made by the Society of

Hair Testing, among other entities, that CE can be used as a marker in hair testing to indicate

simultaneous alcohol and cocaine consumption144

. To our knowledge, there have been no

previous attempts to look at the utilization of CE as a marker of ethanol consumption in a large

high-risk population. Such a biomarker may be practically important, as very few laboratories

worldwide measure FAEE, while numerous laboratories can measure CE.

25

CHAPTER 3: MATERIALS & METHODS

3.1 Subject Recruitment

3.1.1 Hair Samples

Many of the hair samples were from individuals involved with Children’s Aid societies for

suspected drug and/or alcohol abuse in the context of cases concerning child safety. Others were

required to have hair testing for drugs and/or alcohol by the legal system. Individuals consented

to providing a hair sample for testing at the Motherisk Clinic at the Hospital for Sick Children,

Toronto, Canada, on the advice of Children’s Aid societies or legal professionals. If the clients

were unable to visit the clinic, their hair was cut at appropriate sites and mailed to the Motherisk

Laboratory, ensuring that geographic location would not be a limitation for hair testing. The use

of clinical hair testing results available at the Motherisk Laboratory database was approved by

the Research Ethics Committee at the Hospital for Sick Children.

3.1.2 Inclustion and exclusion criteria

Only data from samples which were requested for both cocaine and alcohol testing were used for

this study. All data were collected from adult male and female samples, defined as anyone 16

years or older. Sex, ethnicity and hair colour were not required from social workers or legal

professionals, thus were not recorded. Sex was found by conversing with social workers and

legal professionals, and also based on the name. Samples were included even if they had un-

measurable values for either cocaine and/or alcohol. Additional segments for each person were

given different notations and were counted as separate samples to be included in the study.

26

3.1.3 Sample Collection

Using scissors, a section of hair was lifted from the rear “crown” of the scalp to clear an area for

hair sampling. While firmly holding approximately 75 strands (30mg), hair was cut on the vertex

posterior (“crown”) as close the scalp as possible using disposable gloves. Two separate samples

were required to ensure there was enough hair to test for alcohol and drugs. The root ends were

kept aligned and taped to a blank sheet of paper. The paper was folded and then placed in an

envelope and sealed. The samples were submitted to the Motherisk Drug Testing Laboratory

(MDTL) at the Hospital for Sick Children, Toronto, Canada, along with the Motherisk Chain of

Custody Requisition form (Form MDTL 7030A). The form required the following information:

the sample collector’s statement and signature to identify the collector, certifying that the

collected hair was obtained with informed consent from the donor and photo identification was

provided; statement from the donor certifying it is their hair, was given with their consent, and

sealed in their presence; drugs to be tested; name and address for results recipient and billing

address. For cocaine, cocaethylene, and chronic excessive alcohol consumption using

established methods145,146

. Results from a total of 588 tests were used for the study dating from

September 1, 2010 to May 24, 2011.

3.2 Cocaine, Benzoylecgonine, and CE preparation

3.2.1 Solute Extraction

For cocaine, CE, and BE testing, 10 mg of scalp hair was measured and weighed in a scintillation

vial (Kimble Chase, Vineland, NJ) washed with 1 mL dichloromethane for 1 min. The

dichloromethane was aspirated and the step was repeated. The hair dried for 10 min, and 1 mL

methanol was added. The hair was cut into 1-3 mm sections before the vial was capped and

wrapped with parafilm. After intubation for 18 h at 56°C, the methanol was transferred to clean

27

12x75mm glass test tubes (Fisher Scientific, Ottawa, ON) and dried with 15psi nitrogen on a

35°C pre-heated heating block. The residue was re-suspended in 400µL 1xPBS (pH 7.4). Half

the solution was screened for cocaine using the Cocaine Direct Elisa Kit (Immunalysis, Pomona,

CA). The technique utilizes a competitive binding receptor assay to verify the presence of

cocaine in solution. The method is used qualitatively to identify the presence of cocaine. If

positive for cocaine, results were confirmed by headspace solid-phase microextraction (HS-

SPME) and GC-MS (Mandel Shimadzu GC-MS QP2010 Plus, Guelph, ON) using a method

established in our laboratory145

.

3.2.2 Automated Solid-Phase Extraction (ASPEC)

If the samples were confirmed positive with ELISA, 200 µL of solution was transferred to

13x100mm glass test tubes and 20µL of deuterated standard and 20 µL of 20% methoxiamine

solution were added to each tube. After vortexing and a 1 h incubation, 2mL of 0.1M phosphate

solution were added to the solutions, vortexed, and incubated for 15 min. During the incubation,

100mL of 0.1M HCL and 200mL of elution solution: dichloromethane: isopropanol: ammonium

hydroxide (80:20:2) solutions were prepared for automated solid-phase extraction.

The ASPEC (Gilson, Middleton, WI) was turned on and primed for the sample run. Then

appropriately labelled 12x75mm glass collection tubes were placed in slots corresponding to the

tubes in the collection rack. The test tubes were then placed in their slots and four 700mL

ASPEC reservoir bottles were filled with the following solutions were prepared and placed in the

machine in the following order: (1) methanol; (2) 0.1M phosphate solution; (3) 0.1M HCl; (4)

Elution Solution. The ASPEC program was started and allowed to complete. Once completed,

the solutions in collection tubes were transferred to appropriately labelled solid-phase micro-

extraction (SPME) vials (Sigma-Aldrich, Oakville, ON) and dried with N2 at 35°C. Once the

28

vials were completely dry, a mixture of BSTFA and MSTFA (3:1) was prepared. Once the

heating block cooled, 10µL of derivatizing mixture was added to each vial under N2 and then

placed on the GC-MS rack.

3.2.3 Gas Chromatography/Mass-Spectrometry (GC-MS)

All the GC-MS parameters required to run the samples were completed before the samples were

run. The samples were run using an automated SPME injector and were analyzed using a pre-

programmed method using GCMS Real-Time Analysis (Shimadzu, Laval, QC). Cocaine ions

were identified with m/z ratios of 182, 82, and 303, while CE ions had m/z ratios of 196, 82, and

317. BE ions had m/z ratios of 240, 82, and 361. Cocaine was also identified using the retention

time of 15.602 compared with the standard retention time of 15.583. CE had a retention time of

15.583 and its standard had a retention time of 16.212, while BE was 16.24 and its standard had

a retention time of 16.193. Because BE and CE have similar retention times, if there was a large

BE detected and CE was positive, then the assumption was made that the positive CE could have

been due to the high BE concentration, and thus was considered negative. The lower Limit of

Quantification (LOQ) was 0.4 ng/mg and the Limit of Detection (LOD) was one-third of the

LOQ. Trace CE results were concentrations between the LOD and LOQ and were not

quantifiable. Trace CE values were given a value of 0.13, representing the lowest possible

concentration that can be detected by GC-MS.

29

3.3 Fatty Acid Ethyl Esters Sample Analysis

3.3.1 FAEE Extraction

For alcohol testing, the samples were pre-washed with heptanes (approximately 30 ml) in a 50

mL beaker by stirring with a spatula every 5-10 min for 30 min to remove external

contamination. The solvent was decanted and the hair was allowed to dry for approximately 30

min. Once dry, the samples were cut into 1-3 mm long pieces using scissors and 20 mg were

weighed out for extraction into a 4 ml amber vial and stored in a dry place at room temperature

until testing. When ready for testing, 20 µl IS was added to each vial along with 500 µl DMSO

and 2 ml heptanes. The samples were capped and placed in the shaker at 975 rpm for 15 h

overnight at room temperature. The samples were removed and placed in a cold room (2-8°C) for

60 min until the DMSO became solid, and then the heptane was decanted into 10 mL clear

SPME vials (Sigma-Aldrich, Oakville, ON) in the fume hood. The heptane layer was evaporated

at 40°C under nitrogen flow until the samples were completely dry. Then 1 ml of 0.1M

phosphate buffer, pH 7.6 was added to each sample and they were capped and sent off for the

GC-MS for analysis. Extraction and analysis of four FAEE species (ethyl oleate, ethyl myristate,

ethyl palmitate, ethyl stearate) was conducted by liquid-liquid extraction with a heptane and

dimethyl sulfoxide mixture followed by automated headspace solid-phase microextraction and

GC-MS/EI (Mandel Shimadzu GC-MS QP2010 Plus, Guelph, ON) analysis as previously

reported146

.

30

3.3.2 Gas Chromatography/Mass-Sepctrometry

All the GC-MS parameters required to run the samples were completed before the samples were

run. The samples were run using an automated SPME injector and was analyzed using a pre-

programmed method using GCMS Real-Time Analysis (Mandel, Guelph, ON). Ethyl myristate

had qualifier m/z ratios of 88, 101, and 157 and a quantifying m/z ratio of 256. Ethyl palmitate

had the same qualifier m/z ratios but a quantifying m/z ratio of 284. Ethyl oleate had qualifier

m/z ratios of 88 and 101 and a quantifying m/z ratio of 310. Ethyl stearate had the same qualifier

m/z ratios as ethyl myristate, but a quantifying m/z ratio of 312. Samples were reported as NSQ

if there was not enough hair to run the samples. If the sum of the four did not amount to ≥0.2

ng/mg, then it was reported as negative ≤0.2 ng/mg. If the samples were between 0.2-0.49

ng/mg, then it was reported as negative for chronic excessive alcohol consumption and an

explanation is provided below. However, if the samples ≥0.5 ng/mg, then the results were

reported as positive for FAEE, indicating chronic excessive alcohol consumption. FAEE

between 0.2-0.49 ng/mg can be indicative of low/moderate alcohol consumption, but there are

many confounding factors that prevent the identification of concentrations within this range to be

considered low/moderate alcohol consumption conclusively.

3.4 Statistical Analysis

3.4.1 Data Collection

We identified cases where cocaine, BE, CE, and FAEE were measured, and the concentrations

were collected into an Excel file and organized these based on case sample number. Only

individuals who were tested for both cocaine and alcohol were used in the study. Both positive

and negative results were recorded.

31

3.4.2 Statistical Analysis

Statistical analyses were performed using SPSS Package Software (SPSS, Version 17, Chicago,

Illinois) and GraphPad Prism Software (GraphPad Prism, Version 5, La Jolla, California). The

total population was stratified into two subgroups, one representing individuals who were

chronic alcohol abusers (FAEE ≥0.5 ng/mg of hair), and the other being social/non-drinkers

(FAEE 0.2-0.49 ng/mg of hair). For the purpose of the study, social drinkers were identified with

FAEE concentrations between 0.2-0.49 ng/mg, but this was only used for the study and was not

used when reporting results. Within the alcohol abuse cohort, individuals were stratified into

cocaine users (BE≥5% of cocaine) versus just cocaine-exposed (BE<5% of cocaine). To assess

whether cocaine, BE, and CE concentrations increased with FAEE concentrations indicating

chronic excessive alcohol consumption, we used the Mann-Whitney Test. A Chi-Squared Test

was used to compare proportions of cocaine users who were identified as chronic excessive

alcohol abusers within the study population. Logistic regression was used to identify if being

positive for FAEE and cocaine use could predict a positive CE result. Lastly the sensitivity and

specificity along with positive and negative predictive values of using CE to indicate chronic

excessive alcohol consumption were calculated, using the following formulae:

TP=True Positive TN=True Negative FN=False Negative

FP=False Positive PPV=Positive Predictive Value NPV=Negative Predictive Value

32

CHAPTER 4: RESULTS

4.1 Demographics

A total of 539 individuals were tested during the study time frame, for which a total of 588

samples were tested. Of the 588 samples, 288 were from females and 127 were from males. The

remaining 173 were unknown because the sexes of the individuals were kept confidential by

Children’s Aid societies and legal professionals. The ages ranged from 17-64, with the median

age being 30 years. While many individuals were from Ontario, samples were received from all

across Canada. No information was available on education, socioeconomic background, and

living situation as those were kept confidential.

4.2 Comparison of Cocaine, BE, and CE Concentrations with FAEE Concentrations

Out of 588 samples, a total of 353 non/social drinking and 235 chronic excessive alcohol abuse

samples (≥0.5 ng/mg) were identified based on the accepted cut-offs of hair FAEE. No

comparisons were made between FAEE <0.2 ng/mg and FAEE between 0.2-0.49 ng/mg because

there were too few samples compared to the other groups used for statistical analysis. Overall, 44

samples were found to have quantifiable CE concentrations and 6 had trace CE concentrations.

The number of individuals in each classification of cocaine use (BE≥5% of cocaine

concentration) is listed in Table 1. CE concentrations were found to range from 3.5% to 104% of

cocaine concentrations.

33

Range (ng cocaine/mg of hair) Number of Individuals

Low (0.5-1.23) 74

Medium (1.24-6.72) 90

High (6.73-16.00) 40

Total 204

Table 1. Number of positive cocaine use samples in each range identified by the concentration of

cocaine in hair.

The ranges represent the percentile concentrations of cocaine associated with specific patterns of

use. The low range indicates isolated or sporadic use, the medium range indicates repeated

cocaine use, while the high range indicates frequent cocaine use on more than one occasion.

The mean cocaine levels in the social drinking vs. chronic alcohol abuse population was

significantly different (Mann-Whitney U Test: 273.02 and 326.76, P<0.001) respectively. For

BE, the mean levels in the same groups were also significantly different (Mean Ranks: 277.31

and 320.33, P<0.001). For CE, the mean levels were similar (Mean Ranks: 294.23 and 294.91,

P=0.962). The mean levels for cocaine use in social drinking vs. chronic alcohol abuse

populations were significantly different (Mean Ranks: 280.05 and 315.60, P=0.001).

4.3 Cocaine Abusers who also Abused Alcohol Chronically

Analysis of the social drinking/non-drinking population, consisting of 353 positive samples,

found that 250 were only cocaine-exposed, while 103 were identified as positive cocaine use. Of

the 235 samples in the chronic alcohol abuse population, 136 were identified as only cocaine-

exposed, while 99 were identified as positive cocaine users. Of the 353 social/non-drinking

individuals, 250 were either exposed to cocaine or negative, comprising 70.8% of the subset of

34

the study population. This subset consisted of samples having FAEE concentrations indicating

social drinking, as well as abstinence from alcohol. The remaining 103 were identified as cocaine

use, comprising 29.2% of the same population. Of the 235 chronic alcohol abuse samples

(FAEE≥0.5 ng/mg), 136 were not considered as cocaine use, about 57.9% of the subset. The

remaining 99 FAEE positive samples were also positive for cocaine use, comprising 42.1% of

the subset of the alcohol abuse group. The proportions of cocaine use samples also exposed to

chronic alcohol abuse or social/non-drinking were found to be significantly different from the

proportion of cocaine-exposed or cocaine negative samples (OR 1.767, 95% CI 1.25-2.497,

P=0.002).

4.4 Predicting Positive CE Results

Conducting a logistic regression, cocaine use (BE≥5% of cocaine concentration) was associated

with a positive CE result (OR =15.56, 95% CI 5.95-40.67, P<0.001). A positive FAEE result

(≥0.5 ng/mg) was also found to be associated with a positive CE result (OR =2.44, 95% CI 1.22-

4.87, P=0.012). For the overall cocaine group, which included cocaine users, cocaine-exposed,

and negative cocaine results, no association was found with a positive CE level (OR =1.053,

95% CI 0.98-1.13, P=0.134).

4.5 Sensitivity and Specificity of CE as a Biomarker of Chronic Alcohol Abuse

The values for the CE and FAEE positive and negative results are listed in Tables 2-4. Table 2

included all samples, but considered trace CE results as negative. Table 3 included all the values

for all of the samples tested, while Table 4 was selective to the samples positive for cocaine use.

The values for CE positive and negative with FAEE <0.2 ng/mg and FAEE ≥0.2 ng/mg are listed

in Table 5, Table 6 and Table 7. Table 5 has the same conditions as Table 2, Table 6 the same

conditions for samples as Table 3, and Table 7 the same as Table 4.

35

FAEE Positive (≥0.5

ng/mg)

FAEE Negative (<0.5

ng/mg)

Total

CE Positive 27 15 42

CE Negative 208 338 546

Total 235 353 588

Table 2. Distribution of results for each condition of FAEE positive/negative with CE

positive/negative values for all the tested samples with trace CE considered as negative.


ng/mg)

FAEE Negative (<0.5

ng/mg)

Total



Total 235 353 588


positive/negative values for all the tested samples. Trace CE values were considered as positive.


ng/mg)

FAEE Negative (<0.5

ng/mg)

Total



Total 100 104 204


positive/negative values for samples positive for cocaine use.

36

FAEE ≥0.2 ng/mg FAEE <0.2 ng/mg Total

CE Positive 38 4 42


Total 441 147 588

Table 5. Distribution of results for each condition of FAEE <0.2 ng/mg and ≥0.2 ng/mg with CE

positive/negative values for all the tested samples with trace CE considered as negative.


CE Positive 46 4 50


Total 441 147 588

Table 6. Distribution of results for each condition of FAEE <0.2 ng/mg and ≥0.2 ng/mg with

CE positive/negative values for all the tested samples.


CE Positive 46 4 50


Total 162 42 204

Table 7. Distribution of results for each condition of FAEE <0.2 ng/mg and ≥0.2 ng/mg with CE

positive/negative values for all the samples positive for cocaine use.

The sensitivity and specificity of FAEE to indicate positive/negative CE results, along with the

PPV and NPV of CE to identify positive FAEE results are listed in Tables 8-10. Table 8 included

all the samples and trace CE results were considered negative. Table 9 included all the samples,

where trace values were considered positive. Table 10 was restricted to samples positive for

cocaine use. The distinctions are made between chronic alcohol abuse (FAEE ≥0.5 ng/mg) and

37

social/non-drinking (FAEE <0.5 ng/mg). Also, another comparison is made between non-

drinking (FAEE<0.2 ng/mg) and any amount of alcohol consumption (FAEE ≥0.2 ng/mg).

CE

Value

FAEE

(ng/mg)

Sensitivity

(%)

Specificity

(%)

AUC Sig

(P)

PPV NPV

>0.200 <0.5 or ≥0.5 11.49 95.73 0.5457 0.06012 0.6428 0.6190

>0.065 <0.200 or

≥0.200

4.36 97.28 0.5080 0.7739 0.904 0.2619

Table 8. The sensitivity and specificity of FAEE for CE was assessed in chronic excessive

alcohol abuse population (FAEE >=0.5 ng/mg) vs. a social/non-drinking population (FAEE <0.5

ng/mg). The sensitivity and specificity of FAEE for CE was also assessed in the non-drinking

(<0.2 ng/mg FAEE) versus any amount of alcohol consumption (≥0.2 ng/mg FAEE). The

positive and negative predictive values for each condition were also calculated. Trace CE results

were considered negative.

CE

Value

FAEE

(ng/mg)

Sensitivity

(%)

Specificity

(%)

AUC Sig

(P)

PPV NPV

>0.065 <0.5 or ≥0.5 14.04 95.18 0.5457 0.06012 0.66 0.6245

>0.065 <0.200 or

≥0.200

10.43 97.28 0.5385 0.1618 0.9200 0.2657





positive and negative predictive values for each condition were also calculated. Trace CE values

were considered positive.

38

CE

Value

FAEE

(ng/mg)

Sensitivity

(%)

Specificity

(%)

AUC Sig

(P)

PPV NPV

>0.065 <0.5 or ≥0.5 33.00 83.65 0.582 0.04305 0.66 0.5649

>0.065 <0.200 or

≥0.200

27.95 90.48 0.5009 0.9856 0.918 0.247





positive and negative predictive values for each condition were also calculated. Only positive

cocaine use samples were used.

If trace CE results were considered negative, FAEE≥0.5 ng/mg was found to have 11.49%

sensitivity for positive CE and FAEE<0.5 ng/mg had 95.73% specificity for negative CE for all

the samples. However, when trace CE results were considered positive, the sensitivity of

FAEE≥0.5 ng/mg to detect positive CE increased to 14.04%, while the sensitivity of FAEE<0.5

ng/mg to detect negative CE results decreased very little to 95.18%. When restricted to the

cocaine use samples, the sensitivity increased tremendously to 33.00% and specificity decreased

to 83.65%, but the relationship was found to be significant. The PPV was higher in the condition

where trace CE results were considered positive, thus improving the proportion of positive CE

results that are expected to be FAEE positive. Also, the NPV increased, increasing the proportion

of negative CE results that are expected to be FAEE negative. It did not matter if only cocaine

use samples were considered, as the PPV between all samples and cocaine use samples remained

the same. Unexpectedly, the NPV decreased in the cocaine use condition compared to when all

samples were considered.

39

When comparing FAEE ≥0.2 ng/mg against FAEE <0.2 ng/mg, the results were similar. When

trace CE results were considered positive, the sensitivity of FAEE ≥0.2 ng/mg for positive CE

results went from 4.36% to 10.43% and the specificity of FAEE <0.2 ng/mg for negative CE

results remained the same. The PPV and NPV also increased, although not by a large amount. In

the cocaine use samples, sensitivity increased to 27.95% and specificity decreased to 90.48%.

The PPV decreased compared to all the samples where trace CE results were considered positive,

but were very similar. The same statement can be made for the NPV when comparing the same

conditions. The relationships of sensitivity and specificity were not significant in all three

conditions.

40

CHAPTER 5: DISCUSSION

5.1 Effectiveness of CE as a Biomarker of Chronic Alcohol Consumption

5.1.1 Explanation of Demographics

Of 588 tests during the time period, about 49% of the samples, for which the sexes were known,

were provided by female individuals, the clear majority of the total sampling population. With

173 samples belonging to people of unknown sex, there is a possibility the number would

increase. This seems contrary to what is generally understood about drug use between sexes,

where men usually are more likely to use illegal drugs and consume alcohol than women147,148

.

This could be due to several possible reasons. Motherisk is an organization that is primarily

associated with drug use and safety during pregnancy, which caters primarily to a female

audience. As well, the Motherisk Laboratory conducts adult, child and neonatal hair tests, as well

as meconium tests which cater to a specialized population. Since our laboratory deals mainly

with many children’s aid societies, the need to test pregnant women and women with children is

high. As such, this leads to a majority of hair donors being female.

Additionally, the age range of our population fell within expected ranges as Canadians as young

as 15 have admitted to using cocaine and consuming alcohol1. Equally important is that an upper

age limit has not been established for cocaine and alcohol abuse2-4

. Only individuals 16 years of

age or older were selected for the study due to their ability to give independent consent for hair

testing, and are considered to be adults with respect to hair testing. Another important fact is that

many statistics about patterns of use exist for individuals 15 years of age or older, so it was a

logical decision to select a similar age group.

41

5.1.2 Cocaine and Metabolite Concentrations Compared with FAEE Concentrations

Cocaine and BE concentrations were significantly higher in the FAEE positive (≥0.5 ng/mg)

group than in the FAEE negative (<0.5 ng/mg) group. Cocaine and BE concentrations in the

cocaine use group were also significantly higher in the FAEE positive group than the FAEE

negative group. While there was no obvious dose-response that was detected, the result is still an

important finding regarding cocaine and alcohol use. It was found that cocaine powder users

consumed approximately 108g of ethanol in a drinking episode when not using cocaine, but that

number increased to approximately 147.2g when ethanol was consumed with cocaine.

Furthermore, there was a statistically significant increase in the mean amount of cocaine

consumed during an episode of concomitant cocaine and alcohol use. This increase was in the

amount of cocaine powder consumed during an episode, and for total amount of cocaine

consumed149

. Our results also indicate that there was an increased amount of cocaine detected in

FAEE positive samples. Additionally, there was also an increase in BE concentrations in the

same samples, suggesting the possibility that there was increased cocaine consumption, either in

the amount or total consumption, with ethanol use149

. This is important because our study

assessed cocaine, BE, CE and FAEE concentrations in a high-risk population, without knowing

use patterns conclusively before testing. Our results demonstrate that a high-risk population may

have fewer numbers of positive cocaine and alcohol users, but there are still significant patterns

of cocaine and alcohol co-abuse. For example, as seen in Table 1, majority of the cocaine users

were classified as repeat or frequent users, indicating the possibility of addiction and the

increased likelihood of other risk-taking behaviours. These results are disturbing because the

combined use of cocaine and ethanol can be highly toxic. With long-term concomitant cocaine

and alcohol use being damaging, the combination is potentially lethal.

42

CE concentrations were not found to be significantly higher in FAEE positive samples compared

to FAEE negative samples in the present study. In fact, CE concentrations ranged from 3.5% to

71.44% of cocaine concentrations, suggesting that the rate of CE incorporation into hair does not

reflect serum concentrations17,22

. Another study assessing the concentrations of CE in hair found

CE levels to be between 0%-18.98%, and made the assumption that it relates to heavy alcohol

use150

, as 17±6% was reported in the blood22

. Although this is possible, there are no studies that

have demonstrated the incorporation ratio of cocaine from all the possible sources into hair, thus

this assumption cannot be made. With respect to the CE concentrations found in our study, there

are a few possibilities as to why our results did not match concentrations found in literature.

Firstly, there were approximately 73 CE positive samples that were not tested for FAEE as

alcohol testing was not requested. It is possible that many of the CE positive results would have

also been FAEE positive, thus affecting the significance of CE concentrations in relation to

FAEE positive or negative results. Due to ethical reasons surrounding our testing protocol, these

samples could not be tested for at least another year from the date when the samples were

collected. Secondly, in some cases, CE was classified as inconclusive, which was counted as

negative, but the sample could have potentially been positive for CE. The inconclusive result

could be due to experimental or mechanical error, and many of the samples were not re-tested

due to lack of sufficient hair. Another possible explanation might seem more plausible based on

what has been published in the literature. A theory has been proposed that CE formation is not

affected by the amount of alcohol consumed87

. However, trace amounts of ethanol in soft drinks

can induce CE formation88

; hence it is unknown how much ethanol is actually required for the

transesterification of cocaine to CE to occur. It is important to note that while CE was found in

hair of volunteers who did not consume alcoholic beverages; CE was measured at the pg level87

,

43

while our laboratory measures CE at the ng level, which is 1000-fold higher than in the study.

Thus, any CE formed through the consumption of soft drinks may be inconsequential. Although

the measuring at the pg level may not have clinical significance, there is also the issue of cocaine

spontaneously transesterifying to CE in ethanol bottles. If individuals identified as positive for

CE took the contaminated cocaine, then that could have contributed to such high CE

concentrations in hair.

Another possibility is the order in which alcohol and cocaine were consumed. Most studies were

conducted with alcohol being given before cocaine, but one study where alcohol was given after

cocaine found CE concentrations to range between 4.3%-20.1% of cocaine concentrations151

,

thus lower than when alcohol was given before cocaine. This finding raises the possibility that

the order of administration is important with regards to the formation of CE. If our subjects

consumed alcohol at time points after 80 min, this could lead to a lower concentration of CE

being formed since it was after cocaine’s half-life. Although this theory is plausible, there is no

published literature on the subject, thus no conclusions about the order of administration can be

made from our results.

5.1.3 Proportion of Cocaine Use within the FAEE Positive/Negative Groups

Our results demonstrate that a greater proportion of alcohol abusers did also use cocaine

compared to the proportion of social/non-drinkers that used cocaine, and this was likely to be

concomitant use. When considering it from the perspective of the cocaine use population, the 99

samples represented 49% of the cocaine use samples. While this proportion was closer to 50%, it

was still below the expected range of 50-90%6-9

. This could be due to the fact that the study

population were only suspected of cocaine use, and not actually confirmed cocaine users. In fact,

in the majority of the results, either the individuals were negative for cocaine, or were only

44

exposed to cocaine. It is possible that those individuals were recreational or sporadic cocaine

users, and that their use was not detected due to differences in incorporation, amount of cocaine

consumed, or consumption that occurred prior to the tested time frame. Another fact is that it is

expected that majority of alcohol abusers do not abuse cocaine152

, thus our population may

reflect the accepted statistics regarding alcohol abusers.

Despite the possibilities mentioned above, the results are important to consider on a population

perspective. The sample population represents only a 9 month period from which data were

included only where both cocaine and alcohol testing was requested. There were many more

samples that were positive for cocaine use but not tested for alcohol abuse, so how those values

could affect our proportions is unknown at this time. Even within a suspected population, these

results demonstrate possible high-risk behaviour by many individuals, and how this behaviour

impacts their lifestyle is yet to be determined. While social workers and legal professionals are

aware of cocaine and alcohol abuse trends, these results might suggest the need to emphasize the

benefits of hair testing for both in individuals. With more knowledge of hair testing, we should

see an increase in testing for both cocaine and alcohol.

5.2 Use of CE in General Hair Testing for Drugs of Abuse

5.2.1 Do Cocaine and Alcohol Use Predict Positive CE Results?

We first looked to see if just having a cocaine positive result would predict the ability to achieve

a CE positive result. The group called “cocaine” included cocaine use as well as cocaine

exposure. As majority of the results were only cocaine exposure, it was expected that there

would not be a significant relationship between “cocaine” and CE results. In the context of how a

sample could be considered cocaine exposure, there are a few possibilities. Firstly, there could

have been use, but it was not determined from the hair test. In addition, there may have been use

45

prior to the tested time frame, and the cocaine concentration remaining would be residual

amounts. Also, if an individual touches a contaminated surface, or handles cocaine153

, then the

residues remaining on their hands can be rubbed into hair and enters the hair shaft. As well, if an

individual is in direct, frequent, intimate, physical contact with a cocaine user, then residues from

the user can transfer to the other individual. Lastly, an individual can be positive for cocaine

exposure if they are in an environment where cocaine “crack” is being smoked153

. As expected,

the overall cocaine group positive result does not indicate the likelihood of achieving a positive

CE result. As mentioned previously, CE is formed endogenously in the liver by CES 1 in the

presence of ethanol. If individuals are only exposed to cocaine, meaning that cocaine was not

ingested, then CE should not be detected. Even while CE can be found in the cocaine product

used by individuals, there were not many results that had CE, thus suggesting that CE being part

of the cocaine “formulation” may not have influenced the results.

When only the cocaine use (BE≥5% of cocaine concentrations) results were considered, positive

results were 15.56 times more likely to achieve a positive CE result. This is in line what was

expected since CE is thought to be produced when cocaine has been ingested into the body and

undergone transesterification in the liver. CE was not seen if cocaine was not present, and in all

cases, CE was only present if BE was present in at least trace concentrations. This provides

further support to the expectation that CE was only present when cocaine was consumed, and

that CE was not a part of the original product. Six samples were CE positive but had FAEE

levels below our limit of quantification, equivalent to a teetotaller or someone who drinks on rare

occasions. Those results also provide support to the theory that CE production may not be

dependent on the amount of alcohol consumed, but this has yet to be confirmed. The trace CE

46

results could also be due to the possibility that CE was a part of the original cocaine product that

was used.

FAEE positive results were only 2.44 times more likely to achieve a positive CE result. While

this relationship was significant, FAEE’s ability to predict a positive CE result was much less

than the ability of the cocaine use group to predict positive CE levels. This could be due to a few

possible reasons. The simplest explanation is that many people may consume excess amounts of

alcohol at different times than when they use cocaine. This conclusion was made previously,

among others regarding cocaine and alcohol consumption150

. While this may be true in some

cases, this cannot be assumed for all cases, as cocaine and alcohol use patterns were not reported

by the individuals who provided the samples for testing. Additionally, there are other concerns

that need to be addressed before making such conclusions, and this was discussed in a

commentary154

. Another explanation is that because majority of the samples were FAEE

positive, this indicates that many individuals were chronic excessive alcohol abusers. As already

mentioned, majority of alcohol abusers do not abuse cocaine152

, so it is expected that FAEE

positive results will have a low predictability of CE positive results.

CE positive results where FAEE concentrations were <0.5 ng/mg may have also contributed to

the lower likelihood of predicting positive CE results. This may have occurred due to a few

reasons. For instance, cocaine users may not have consumed alcohol in excess when using

cocaine, and may not have consumed excess amounts of alcohol chronically outside of using

cocaine as well. Also, it might be possible that on the rare occasions when cocaine was

consumed, that alcohol was also abused, and that specific pattern of alcohol consumption would

not be detected. This, however, is only a theory and thus should not be interpreted as a

conclusion. Lastly, the possibility that CE was a part of the final product cannot be ruled out.

47

5.2.2 Ability of Positive FAEE to Predict Positive CE Results

As seen in the results, Tables 2-7 list the number of samples that were positive and negative for

the combination of FAEE and CE concentrations. Table 2 has fewer positive values than Table 3

for the combination of FAEE≥0.5 ng/mg and CE positive results. This is due to the fact that trace

values were considered negative results in Table 2. Our laboratory defines trace concentrations

as between the LOD and the LOQ. Since trace concentrations cannot be quantified, the results

may be interpreted as negative. Tables 8-10 list the sensitivity and specificity of the proportion of

FAEE positive/negative and FAEE <0.2/≥0.2 ng/mg that were positive or negative for CE. The

PPV and NPV in each condition are listed, along with the concentration cutoffs at which the

results apply. In this situation, we demonstrated that 11.49% of FAEE positive (≥0.5 ng/mg )

results tested positive for CE, and 95.73% of FAEE negative (<0.5 ng/mg) results tested negative

for CE. However, because there is no established dose-response relationship between ethanol and

CE, even trace CE concentrations may indicate alcohol and cocaine co-consumption. As such,

we decided to see if there would be any improvement in the sensitivity and specificity if trace

concentrations were considered as CE positive. As expected, sensitivity increased to 14.04% of

FAEE positive samples tested positive for CE. Also, there was very little change in specificity,

with 95.18% of FAEE negative samples testing negative for CE. This demonstrated that

including trace CE results as positive improves the sensitivity, thus increasing the proportion of

FAEE positive results that tested positive for CE. It should be noted that trace CE values should

be used with caution to predict FAEE positive results, as their concentrations could not be

quantified. Another concern is that CE could be incorporated from its presence in the original

cocaine product that was used by individuals. Rather, trace CE concentrations could possibly be

48

used to indicate cocaine use, and possibly some concomitant alcohol consumption, which could

still be dangerous to the cocaine user.

When the results were restricted to only samples positive for cocaine use, the sensitivity

increased to 33.00%, thus further increasing the proportion of FAEE positive results that tested

positive for CE. The specificity decreased to 83.65%, but this was expected as many of the

results with no detectable cocaine, BE, and CE concentrations were excluded. Even with an

approximately 14% decrease, many of the FAEE negative results tested negative for CE. It was

expected that majority of samples positive for cocaine use would also be positive for FAEE, as

majority of cocaine users also abuse alcohol. However, this was not the case, as only 99 samples

were positive for cocaine and FAEE. This could be due to the fact that the samples were

collected from suspected cocaine and alcohol abusers, so our study population is not

representative of a known cocaine use population.

Among the three conditions, sensitivity increased, but never reached a level that would indicate

that majority of FAEE positive results tested positive for CE. This can make it difficult to be

used as a screening tool to confirm co-abuse, because there is a high rate of false negative

results. In other words, many individuals who are suspected of concomitant cocaine and alcohol

use would not be positive for co-abuse. This is in spite of CE being found in cocaine products

before consumption. As discussed in previous sections, the majority of alcohol abusers do not

abuse cocaine152

, thus it is expected that majority of the results would be negative for CE. On the

other hand, the specificity demonstrated the opposite trend, decreasing as the conditions of

samples considered for testing changed. Although there was a decrease, the specificity of

negative FAEE results testing negative for CE remained high. As a screening tool, this is

beneficial because it reduces the chance of false positives that might result if the specificity was

49

low. In a clinical setting, this can be interpreted that if individuals were negative for chronic

excessive alcohol consumption, then it is possible that they would not be positive for CE.

We then wanted to assess the sensitivity and specificity of positive FAEE≥0.2 ng/mg to detect

positive CE results. If trace CE results were considered negative, then FAEE≥0.2 ng/mg were

only positive for CE 4.36% of the time, whereas FAEE<0.2 ng/mg were negative for CE 97.28%

of the time. When trace CE results were considered positive, FAEE≥0.2 ng/mg were positive for

CE 10.43% of the time, while the specificity remained the same. Once again, the increase in

sensitivity demonstrated the benefit of including trace amounts as positive results. Within the

cocaine use samples, the proportion of FAEE≥0.2 ng/mg results that were positive for CE

increased to 27.95%, while the proportion of FAEE<0.2ng/mg that were negative for CE

decreased to 90.48%. Just as in the separation between FAEE≥0.5 ng/mg and FAEE<0.5 ng/mg,

the sensitivity increased. This was expected, as CE should not form if cocaine was not

consumed. Even if the original cocaine product was contaminated with CE, this did not seem to

influence the results; otherwise we would have seen many more CE positive results. Although

the specificity decreased, FAEE<0.2 ng/mg results indicate that CE was not detected in almost

all the cases, decreasing the rate of false positivity tremendously. This is also beneficial as a

screening tool, because if any amount of alcohol was consumed, it was not consumed with

cocaine. Additionally, this indicates the possibility that cocaine contaminated with CE was not

consumed, reducing the potential for health risks in these individuals.

5.2.3 Ability of Positive CE to Predict Positive FAEE Results

The PPV and NPV were calculated to find the proportion of CE positive results that were also

FAEE positive and negative in all three conditions. When trace CE concentrations were

considered negative, 64.28% of positive CE results were also positive for FAEE (≥0.5 ng/mg),

50

and 61.9% of negative CE results were also negative for FAEE (<0.5 ng/mg). As a result,

majority of CE positive results indicated that alcohol was consumed chronically in excessive

amounts. When trace CE concentrations were considered positive, 66% of positive CE results

were also positive for FAEE, and 62.45% of negative CE results were negative for FAEE.

Including the trace concentrations in the samples increases the proportion of FAEE positive

results, as well as the proportion of FAEE negative results. In the cocaine use group, the

proportion of positive CE results that were popular for FAEE remained the same, while the

proportion of negative CE results that tested negative for FAEE decreased to 56.49%. The

decrease in the NPV was due to the reduction of negative values when only selecting for cocaine

use. The results indicate that if positive CE is detected in a particular hair sample for an

individual, then it is more than likely that the same individual could be classified as a chronic

excessive alcohol abuser. From a clinical stand point, the result of 0.66 is not enough to

completely confirm a diagnosis of chronic excessive alcohol consumption. This can be an issue

in alcohol abuse treatment, as other routes of identification are already established. Instead, it is

possible that using CE in combination with other identifiers, such as FAEE, could be of some use

in a clinical setting. If both CE and FAEE (≥0.5 ng/mg) were positive, then this could suggest

that the individual(s) in question are possible chronic excessive alcohol consumers. With NPV

values ranging between 56.49%-62.45%, this indicates that an individual with a negative CE

result would also be expected to be negative for FAEE, but this is not proven conclusively. It

would be recommended to assess the individual’s behaviour and see if there is any cause to

believe that alcohol may be abused by the individual before determining if further testing for

FAEE is required.

51

Although the PPV does not prove definitively that positive CE results were all FAEE positive,

with a proportion of 66%, it is more than reasonable to suspect chronic excessive alcohol

consumption in addition to the cocaine use. Consequently, a positive CE test would suggest that

individual(s) in question are indeed using cocaine, and possibly are using cocaine that is

contaminated with CE. Thus, they should be assessed for additional health risks, as CE is known

to be toxic. Social workers and legal professionals involved with child custody issues could use

the same information provided by the test to their benefit. If CE is positive in hair testing, then

this indicates that parents were possibly consuming alcohol in addition to cocaine use. Equally

important, it could suggest that the parents were using contaminated cocaine. Either way, the

children are at risk for cocaine and/or alcohol exposure. Due to the fact that cocaine is requested

far more frequently than alcohol testing, the use of CE as a marker to indicate possible chronic

excessive alcohol consumption is only a benefit for a population screening of cocaine and

alcohol use. Other than indicating possible co-consumption, positive CE results also indicate the

risky behaviour associated with cocaine use, putting children at even greater danger due to other

factors that are also involved with cocaine use, such as the physical and psychological side

effects.

We also wished to understand the effectiveness of positive CE results to predict any amount of

alcohol consumption, so we calculated the PPV and NPV of positive CE to indicate FAEE≥0.2

ng/mg and FAEE<0.2 ng/mg. When trace CE results were considered negative, the proportion of

positive CE results that tested positive for FAEE≥0.2 ng/mg was 90.4%, and the proportion of

CE negative results that tested negative for FAEE<0.2 ng/mg was 26.19%. When CE trace

results were considered positive, the proportion of positive results that tested positive for

FAEE≥0.2 ng/mg increased to 92% and the proportion of negative CE results that tested negative

52

for FAEE<0.2 ng/mg increased to 26.57%. When the results were restricted to only cocaine use

samples, both the PPV and NPV decreased to 91.8% and 24.7% respectively. The low NPV

values for all three conditions were expected, as only 4 positive CE values had FAEE<0.2

ng/mg. This is important because it suggests that most positive CE results will indicate that

either alcohol was consumed with cocaine at levels that would be considered social drinking, or

cocaine, that was in alcohol for extended periods of time, was used. This is supported by the fact

that the PPV values for all the conditions were at least 90%, implying that alcohol may have

been consumed on more than one occasion, which includes social consumption as well as

chronic excessive alcohol consumption. Further support is also provided to the assumption that

contaminated cocaine was used by the same PPV value. Because it looks at FAEE≥0.2 ng/mg, it

is possible that some alcohol was consumed with cocaine, as almost all of our CE positive results

had quantifiable FAEE levels.

5.3 Study Limitations

There were limitations to the applicability of our results concerning cocaine and alcohol use to

the general population. There is a potential ethnic bias with respect to cocaine incorporation into

hair between Caucasians and non-Caucasians. It was found that nine non-Caucasians

incorporated on average 2.7 times more cocaine into their hair than Caucasians who were given

the same cocaine dose155

. In particular, it is suggested that African-Americans, Asians, and

Hispanics incorporate more cocaine than Caucasians, therefore representing a potential bias in

drug testing using hair155

.It is suspected that darker and thicker hair have higher rates of cocaine

incorporation compared to lighter and thinner hair. Published studies have found greater amounts

of cocaine in pigmented versus non-pigmented hair within animal and human subjects156-160

.

Because a greater proportion of minority groups have darker and thicker hair, this may lead in

53

outcomes that demonstrate a “bias” toward people with dark hair161-163

. However, a study which

looked at other studies that considered ethnicity as a factor found some issues with study design

and some of the conclusions made from results160

. For example, many of the studies, there was a

small sample size which in itself does not allow for anyone to make conclusions based on their

results. Other studies soak the hair in cocaine solutions for an extended period of time, which

does not represent conditions seen in vivo. The authors found that black and brown hair had

significantly higher rates of cocaine positivity than blond, light brown and medium brown hair.

Additionally, grey and red hairs had much less cocaine positivity. Hair and urine analysis for

cocaine were similar, but this could be due the drug prevalence in different ethnic/racial groups,

as found in epidemiological data164,165

. Even though race/ethnicity and hair colour might be a

limitation in the generalization of results, there is conflicting evidence that might prove

otherwise. Thus, the amount of incorporation, or lack thereof, of cocaine and BE could be due to

a person’s ethnicity, but this limitation is taken with caution until the issue of hair colour and

ethnicity affecting hair testing results is resolved.

Another limitation was with regard to the sex of our sampling population. Almost majority of our

study population were female, thus affecting the applicability of our results to the rest of the

population. It is understood that more men use cocaine147

and consume alcohol1 than women,

thus our results might be underreporting the prevalence of cocaine and alcohol abuse in a high-

risk population. Another fact is that cosmetic treatment to hair can affect the amount of cocaine

detected in hair. For example, in the case of bleaching treatments to hair, the melanin content in

hair is reduced165

. As cocaine does interact with melanin molecules156,159

, it is possible this can

lead to reduced cocaine concentrations in hair, and even false negative results. It has been

reported that cocaine and BE concentrations decreased to average concentrations of 10% of

54

starting concentrations166

. However, permed and bleached hair seems to have increased

absorption capabilities for drugs from sweat, sebum, and external sources; this could lead to an

increased rate of false positives166

. As it is assumed that women generally cosmetically treat their

hair compared to men, this could have lead to an over-reporting of cocaine exposure and/or use,

but this has yet to be confirmed.

There is also a potentially important limitation that must be considered with regards to FAEE

concentrations in hair. FAEE have been detected in hair care products, most likely formed during

production or storage from ethanol and lipid traces. These FAEE may contribute to the FAEE

detected in hair, but the concentrations were very low. Permanent wave hair treatment reduced

FAEE concentrations by approximately 12%, hair shading reduced ∑FAEE in hair matrix upto

11%, and ∑FAEE from the hair surface by up to 50%. Ethanol-containing hair spray was found

to cause a false-positive result, where the ∑FAEE concentrations resembled what occurs after

ethanol consumption. In summary, hair lotion and hair spray were found to elevate ∑FAEE

concentrations and lead to false-positive results167

. These results were supported by another

study that found hair care products containing as little as 10% ethanol could potentially elevate

FAEE concentrations beyond the 0.5 ng/mg cutoff, misidentifying a social drinker or teetotaller

as an individual who chronically consumes alcohol in excessive amounts168

. The use of specific

hair care products was not recorded when samples were collected, and no attempts were made to

find out due to the possible impact it could have in many cases, as individuals would buy those

products and pretend to use them. It can be assumed that women tend to use more hair care

products than men, and as majority of our population was female, this could be a confounding

factor that affects the results. Further analysis between sexes is required to assess how this could

influence the results.

55

With respect to CE, there are a few limitations to using CE as a biomarker in hair testing to

predict cocaine and alcohol co-consumption. Firstly, the potential ethnic bias that occurs for

cocaine incorporation into hair may also exist for CE. This is plausible due to its similar

chemical and physical properties47

, meaning that CE could interact with melanin molecules in a

similar manner as cocaine. If so, then higher hair CE concentrations should be seen in ethnicities

that have darker and thicker hair (such as Hispanics, Asians, and African Americans). However,

this requires further study before any conclusions can be made.

Another limitation is the sex bias that occurred within the sample population. Because more

women were tested than men, this could influence the presence of CE within the hair samples.

As women are more likely to undergo cosmetic treatment on their hair, this permed and bleached

hair seem to increase absorption capabilities of drugs from sweat, sebum, and external

sources165

, this could lead to a high rate of false positives of CE. A conflicting study found that

bleaching hair reduced melanin content, thus cocaine and BE decreased to an average of 10% of

their original concentrations165

. Either way, cosmetic treatment affects the rate of cocaine

incorporation, so the same can be expected for CE. In addition to the cosmetic treatment, women

consume less cocaine146

and alcohol1 than men, resulting in a decreased proportion of positive

CE results. Therefore, the results achieved are not equally representative of both sexes in a high-

risk population. Our belief is that Motherisk, as an organization, is associated with mainly

assisting the female population, hence a strong reason for the sex bias. There is also a lack of

awareness about hair drug testing within Canada. If more information was provided, our sample

population would have been more representative of the general high-risk population.

Lastly, the theory that CE is formed exclusively through cocaine and alcohol co-consumption

needs to be explored further before it is accepted. The fact that CE can form from street cocaine

56

manufacturing and smuggling practices77,78

, as well as in pharmaceutical grade research79

, is

concerning if CE is to be used as a biomarker in hair testing. With respect to studying cocaine

production, the issue of CE production through techniques seems to have been solved85

, but it is

still possible that some CE could be produced. In regards to illicit cocaine, it is crucial to note

that there is no published literature about how much of an effect the production of cocaine or the

smuggling techniques have on the concentration of CE found in scalp hair. This must be

considered when interpreting the results.

CHAPTER 6: CONCLUSIONS & FUTURE DIRECTIONS

To our knowledge, our study is the first to investigate using CE as a biomarker of chronic

excessive alcohol consumption in a suspected cocaine-user population. While there was great

variability between CE concentrations compared to cocaine concentrations, important trends

were identified. Our study demonstrated that expected trends in cocaine and alcohol

consumption can be seen in a large sampling population of high-risk individuals. Increased

cocaine and BE concentrations within the alcohol abuse group compared to the social/non-

drinking revealed that cocaine and alcohol co-consumption is still an important issue in Canada,

as it is in the United States6-9

. However, CE concentrations were not higher in the alcohol abuse

samples, probably due to the few CE positive results compared to all of the samples tested. If

more male samples were tested, we might expect to see higher proportions of concomitant users,

but such a hypothesis should be taken cautiously considering the study limitations. Either way,

cocaine use is still prominent in our society despite countless efforts to educate the public about

the dangers of cocaine and alcohol abuse. Clearly, drug prevention efforts are not reaching

everyone, and there are at-risk individuals who need to be identified from an early stage.

57

We also had a greater proportion of positive cocaine use samples in the alcohol abuse group than

the proportion of positive cocaine use samples in the social/non-drinking group. While 42.1% is

not majority of the sampling population, it provided further evidence that most alcohol abusers

do not abuse other drugs. When looking from the cocaine use perspective, approximately 49% of

positive cocaine use samples were also positive for alcohol abuse, supporting the accepted

standard of 50-90% of cocaine users abusing alcohol. The sex bias that we experienced could

have contributed to lower proportions of cocaine and alcohol abusers, which again raises the

question of how the proportions would be influenced with more male subjects.

When we considered if cocaine and FAEE concentrations would be associated with positive CE

results, we were amazed by how similar our results were to our expectations. While these were

common expectations, this is the first study to take into account such associations on a large

scale. When all cocaine samples were considered, including cocaine-exposed and cocaine

negative, there was no association with positive CE results. This was despite the fact that cocaine

can be contaminated with CE before it is used. When only cocaine use samples were considered,

positive samples were more likely to be CE positive as well. Even though positive FAEE results

were also likely to be CE positive, there was not as strong an association as there was with

cocaine use. This was expected because CE is a metabolite of cocaine, and not of alcohol.

Despite that fact, the weaker association between FAEE and CE could be due to the fact

individuals may have been consuming alcohol at different times than when they were using

cocaine. It could also be due to lack of chronic excessive alcohol consumption in our population,

thus more people are unlikely to use cocaine as well. Although CE can be produced

spontaneously in the presence of ethanol, many of our samples still had some amount of alcohol

consumption. Also, with all the CE positive samples, BE was also present at concentrations that

58

were at least 5% of cocaine. Hence, each of the samples was considered positive for cocaine use

as well. For the CE positive samples that had FAEE<0.2 ng/mg, those individuals may have

consumed some alcohol with cocaine, but not in excess amounts chronically. Also, the CE

results could have come from contaminated cocaine. Still, the amount of alcohol required to

produce CE still needs to be studied before any further conclusions can be made regarding dose-

concentration relationships.

Lastly, we calculated the sensitivity and specificity of CE positive values to predict chronic

excessive alcohol consumption, and also social drinking. When trace values were included as

positive results, sensitivity increased, demonstrating that the CE trace levels will positively

impact the effectiveness of its use a biomarker. After isolating for only cocaine use samples, the

sensitivity further increased, demonstrating that cocaine use impacts the presence of CE in hair.

Although the specificity decreased, majority of FAEE negative results were still CE negative as

well. An equally important point was that the PPV increased when trace CE concentrations were

considered positive results. This means that the proportion of CE positive results that were FAEE

positive increased. If an individual has a positive CE result, then it’s more than likely that FAEE

would be positive as well. Thus, the possibility of this occurring due to chance is not a valid

argument against FAEE testing when CE is positive. With no change in the PPV and a slight

decrease in the NPV in cocaine use samples, it can be implied that the PPV value can be applied

to our entire population. However, it must be taken into consideration that the PPV does not

provide a definite association, so it should not be used as the sole marker to indicate chronic

excessive alcohol consumption. Rather, CE could be used as an indicator to test for FAEE in a

positive CE sample. Before this can occur, studies need to be conducted that assess how cocaine

contaminated with CE affects the results attained in a high-risk population.

59

When only considering the sensitivity and specificity of FAEE<0.2 ng/mg vs. FAEE≥0.2 ng/mg,

it was understood that sensitivity would decrease because of the larger number of FAEE≥0.2

ng/mg/CE negative results. However, the sensitivity increased when CE trace concentrations

were considered positive. Similarly, sensitivity increased in samples indicating cocaine use. The

PPV increased when CE trace values were positive, but decreased very little in cocaine use

samples. This showed that a large proportion of CE positive values were also having

concentrations of FAEE≥0.2 ng/mg. While this result does not indicate chronic excessive alcohol

consumption, it does indicate that possibly some alcohol may have been consumed with cocaine

use. Once the issue of contaminated cocaine and CE is resolved, there is a potential application

for the use of CE in substance abuse treatment programs, but this is many years away. Of course,

CE would be used with a multitude of other treatment options, as substance abuse is a very

complex issue.

In conclusion, this study provides critical information on the utility of CE as an indicator to

prompt FAEE testing for possible concomitant cocaine and alcohol abuse. The issue of

contaminated cocaine with CE needs to be taken into consideration when conducting

interpretations of results. Our results demonstrate that cocaine and alcohol co-consumption is an

important public health issue that needs to be discussed more openly in society. Further studies

are required to comprehend the effect of varying amounts of alcohol on CE formation and

incorporation into hair.

6.1 Future Directions

Although our study demonstrated that positive CE levels were more than likely to indicate

chronic excessive alcohol consumption, there are certain factors that prevent us from being able

60

to estimate the amount of cocaine and alcohol co-consumption required to produce various CE

concentrations. Further investigations would be able to examine this issue in greater detail.

6.1.1 Additional CE Incorporation Investigations

Until accurate incorporation rates of CE from blood, sweat, sebum and other routes into hair are

established, no definitive conclusions can be made regarding levels of cocaine and alcohol

consumption. This can be done by conducting similar studies as done previously22,129

using

human volunteers and providing them with cocaine and alcohol. Using radioactively-labelled

cocaine can help determine the cocaine pharmacokinetics in those individuals, and help

determine how much CE was incorporated into each body fluid. Each concentration from every

body fluid must be compared to CE concentrations measured in adult scalp hair. Having this

information will provide estimates of how much CE from each fluid contributed to the

concentrations found in hair.

Also, the issue of ethnicity and hair colour needs to be studied further with respect to CE

incorporation. Conducting the same study as above, but in individuals from different ethnic

backgrounds and having different hair colour and thickness will help to potentially resolve the

issue of ethnicity. It is important to ensure that enough participants are selected to achieve

significant statistical power. As well, there should be equal numbers of men and women in each

group matched for age, socioeconomic background, and education level, as well as other

confounding factors that may influence the results.

6.1.2 Additional CE Formation Investigations

While the theory has been proposed that the amount of alcohol consumed does not affect CE

formation, there is not sufficient support for this theory to be accepted by the scientific

61

community. CE was previously found in hair of volunteers not given alcohol at 2% of cocaine

concentrations, and its levels were attributed to possible trace amounts found in ethanol88

. Even

though this may be a possibility, endogenous levels of ethanol remaining in the body may also

have produced the CE detected. An important note is that the CE concentrations were detected at

the pg level, which is 1000-fold less than our detected concentrations, which was measured in

ng.

A proposed method would be to conduct clinical trials in healthy human cocaine users. The male

and female participants would be from different ethnicities and matched for age, socioeconomic

background, educational level, and other possible confounding factors that would affect the

validity of the study. The participants would be housed in a facility, so their cocaine and alcohol

consumption can be monitored. Each subject would be given different concentrations of cocaine

and ethanol, and the order of both would be different. Cocaine, BE, CE, and ethanol

concentrations would be measured in blood, urine, and other body fluids over periods of time to

track the pharmacokinetics of cocaine and ethanol. Hair concentrations would also be measured

to understand the incorporation of CE from the various sources when different cocaine and

ethanol concentrations were administered. The goals of this proposed study would be to

understand how much CE was formed when different ethanol concentrations were consumed. A

second goal would be to understand if a possible estimate could be made about how much

cocaine and ethanol was consumed based on hair CE concentrations.

Another technique that could be used would be conducting dose-concentration relationship

analyses using enzymatic assays. CES 1 and CES 2 could be exposed to different cocaine and

ethanol concentrations to assess the amount of cocaine hydrolysis that is transferred to

transesterification to form CE. Conducting such a study would be less expensive and less

62

invasive. Additionally, enzymatic assays should be conducted to propose specific concentrations

which can then be tested in animals and humans. On the other hand, in-vitro studies of CE

formation are not representative of what occurs in vivo, where there are many other factors that

could affect cocaine and ethanol metabolism. Thus, the best solution would be to first perform

enzymatic assays, and then move onto clinical trials with human volunteers.

Also, cocaine can be dissolved in different ethanol concentrations for different periods of time to

understand how much ethanol exposure is required for CE to form without enzymatic activity.

Once CE forms in the different solutions, the cocaine/CE compound would be extracted and

provided to human volunteers to assess how much of the CE from the contaminated cocaine

product enters the various compartments, including the scalp hair. This will help to find a

possible answer to the question of how much CE in street cocaine is incorporated in human scalp

hair.

6.1.3 CE Scalp Hair Elimination Rates

Cocaine was found to be detectable in adult scalp hair for a minimum of 3 months until it was no

longer detectable in an individual abstaining from cocaine use or exposure125

. To our knowledge,

there are no published studies about the elimination rate of CE from hair, so there is a possibility

that it stays in hair longer than cocaine. It would be recommended to collect scalp hair samples

of individuals undergoing successful treatment of cocaine and alcohol abuse. Scalp hair samples

would be collected at specific time points during their treatment, and concentrations would be

measured. Samples would also be collected for a period of time after completing treatment to

determine how long CE is detectable in human hair. This would help to prevent false positives in

drug testing; particularly in special cases where individuals claim to be drug and alcohol free for

months, yet concentrations are still detected in their hair. By understanding the rate of hair CE

63

elimination, an estimated period of time between abstinence and hair testing could be provided to

best prevent a false positive result. Such knowledge would not only save time in the laboratory,

but would also save clients large amounts of money.

6.1.4 Possible Clinical Applications of Hair CE Testing

Hair testing is routinely done in many labs for cocaine and alcohol consumption. In many cases,

individuals are suspected of using cocaine and alcohol, and hair testing is useful as a screening

process to indicate several months’ worth of consumption or exposure. Since cocaine is already

identified using GC-MS methods, the addition of CE to standard cocaine testing is not a

complicated process. The methods are similar, with only adding the ability of the analytical

machine to identify CE correctly. In addition to testing parents involved with child custody

cases, the proposal of using hair testing for CE in a medical setting would aid physicians identify

individuals at greater risk for physiological and psychological side effects. By identifying these

risks early on in a person’s life, physicians and medical professionals can implement appropriate

measures to ensure that people attain the correct substance abuse treatment. The complexity of

substance abuse makes it so that CE can be used as a measure alone, but can be a small part of a

large network of treatment and intervention options.

Another possible application of hair CE testing would be considered both medical and social.

Pregnant women represent a special subsection of the general population because their drug and

alcohol use not only affects them, but also their unborn child. Accordingly, hair testing on a

pregnant woman would provide evidence of drug and alcohol use during her pregnancy and also

provide information on what the fetus was exposed in-utero. Since cocaine is requested much

more frequently than alcohol in the Motherisk Laboratory, the presence of CE in scalp hair from

the woman could indicate possible alcohol consumption in addition to cocaine use. In-utero

64

cocaine exposure can affect mother-child interactions170

, spontaneous abortion, preterm births,

placental abruption and congenital anomalies171

. In-utero alcohol exposure is associated with

fetal alcohol spectrum disorder (FASD)172

, which causes several physical and mental

deficiencies173

. As there is still no safe level of alcohol consumption that has been established in

pregnancy173

, any amount of alcohol could pose a danger to the fetus. If CE is detected later in

pregnancy, the appropriate measures could be taken. This will enable medical professionals to

follow up with the baby for FASD and provide necessary assessments and interventions.

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78

LIST OF PUBLICATIONS & ABSTRACTS

PUBLICATIONS

Natekar A, Matok I, Walasek P, Rao C, Clare-Fasullo G, Koren G. Cocaethylene as a biomarker

in human hair of concomitant alcohol and cocaine use in a high-risk population. Ther Drug

Monit: Under Review

Natekar A, Pupco A, Bozzo P, Koren G. Safety of azathioprine use during pregnancy. Can Fam

Physician 2011 Dec;57(12):1401-2.

Natekar A, Koren G. Interpretation of combined hair fatty acid ethyl esters, cocaine and

cocaethylene. Ther Drug Monit. 2011;33(4):560.

ABSTRACTS

Natekar A, Matok I, Walasek P, Rao C, Clare-Fasullo G, Koren G. Cocaethylene as a biomarker

in human hair of concomitant alcohol and cocaine use in a high-risk population. J Popul Ther

Clin Pharmacol 2012: In press.

Natekar A, Aleksa K, Koren G. Cocaethylene as a Marker in Human Hair of Alcohol and

Cocaine Co-Consumption. ACER 2011 Jun:35(Suppl):168A.

Natekar A, Aleksa K, Koren G. Cocaethylene as a biomarker of alcohol and cocaine co-

consumption in human hair. J Popul Ther Clin Pharmacol 2011 May:18(2):e350.



http://www.ncbi.nlm.nih.gov/pubmed?term=%22Natekar%20A%22%5BAuthor%5D


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