Gas chromatographic-mass spectrometric identification of cocaine and opiate analytes in keratinized matrices of human origin

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Title:
Gas chromatographic-mass spectrometric identification of cocaine and opiate analytes in keratinized matrices of human origin
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xi, 241 leaves : ill. ; 29 cm.
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English
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Ropero-Miller, Jeri Diane, 1967-
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Subjects / Keywords:
Mass Fragmentography -- methods   ( mesh )
Keratin -- chemistry   ( mesh )
Keratin -- drug effects   ( mesh )
Hair -- chemistry   ( mesh )
Hair -- drug effects   ( mesh )
Nails -- chemistry   ( mesh )
Nails -- drug effects   ( mesh )
Substance Abuse Detection -- methods   ( mesh )
Cocaine -- analysis   ( mesh )
Codeine -- analysis   ( mesh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1998.
Bibliography:
Includes bibliographical references (leaves 221-239).
Statement of Responsibility:
by Jeri Diane Ropero-Miller.
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Typescript.
General Note:
Vita.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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ocm51618291
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GAS CHROMATOGRAPHIC-MASS SPECTROMETRIC IDENTIFICATION OF
COCAINE AND OPIATE ANALYTES IN KERATINIZED MATRICES OF HUMAN
ORIGIN














By

JERI DIANE ROPERO-MILLER


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1998














This dissertation is dedicated to the special people in my life:

MY HUSBAND, Jeffrey

MY FATHER, Sydney

MY MOTHER, Kathy

MY SIBLINGS, Kathy and Bill

Each has touched my heart as well as contributed, in some significant way, to my success
in attaining my educational endeavors. For this, I give my endless love and gratitude.












ACKNOWLEDGMENTS


Many individuals supported me in obtaining my lifelong educational dream. Each

was instrumental in guiding me through my long and arduous journey, refusing to let me

give up or accept anything less than the degree I strongly desired.

First, I would like to extend my love and gratitude to my compassionate husband,

Jeffrey, who endured the most difficult times within our relationship. I have often been

told that both individuals build a relationship on compromise, and I can confidently state

that Jeff has done his share of compromising in the best interest of our relationship. It

has been tough, but we persevered! He has truly been my companion, my confidant, my

therapist, and my hero. I eagerly look forward to the rest of our lives TOGETHER!

Next, I would like to thank my loving parents, Sydney Anthony Ropero and

Helen Kathleen Kelly. They have always been by my side offering words of wisdom and

support, displaying parental pride, and giving a "helping hand" if ever I genuinely needed

it. In addition, I can not fail to mention my deceased stepfather, James Dale Kelly, who

began this journey with me but was not able to see me finish. I know he is still

encouraging me in a spirit sense. My stepmother, Patricia Ann Ropero, and Jeff's

mother, Nancy Miller, were also supportive of my education. I was also blessed with a

wonderful sister, Kathleen Parker, and brother, Bill Strickland, who played a part in

making me the person I am today.








Each member of my graduate committee has definitely done their part of seeing

me through my dissertation, for all their efforts I give my sincerest gratitude. Dr. Bruce

Goldberger served as the committee chairperson and my "ever-present" mentor during

my graduate program. Together, I hope we have both grown and learned a great deal

through our mentor-student relationship. He was a very fair advisor, who always allowed

me many opportunities to build my knowledge in the field of forensic toxicology. Dr.

Roger Bertholf could always answer my statistical questions in such a way that made

perfect sense. Many times this was a true task since the research data generally never

followed the "cleaned-up" examples given in most college-level statistics books. Dr.

Catherine Hammett-Stabler nurtured my career as a graduate student and contributed

significantly to all aspects of my graduate work. She offered analytical support,

suggestions for improvement of my public speaking, opportunities to coauthor papers and

student-teach, and overall refinement of skills that would make me successful in my

career. Dr. Ian Tebbett was always helpful in discussing the pharmacological principles

that were important to consider for my research. I was also able to count on his

benevolence for resources such as laboratory supplies and instrumentation. Dr. Donna

Weilbo served on my committee for almost four years, during which time she generously

offered her laboratory facilities for an animal study in which I participated, her

"motivational chats", and helpful advise concerning study design and pharmacology. Dr.

Kathleen Shiverick was added to my committee during the last year of my program. I am

forever indebted to her for agreeing to "step up to bat during the ninth inning" of my

program. I took several courses under Dr. Shiverick and she always bestowed confidence

in my abilities.








My research projects would not have been possible without the contributions of

several collaborators that I would also like to acknowledge. Dr. Ronald Zielke and Ron

Vigarito helped with specimen procurement and manuscript suggestions for the SIDS

study. Drs. William Hamilton and William Maples (deceased) contributed to research

involving postmortem fingernail and toenail analysis. Drs. Edward Cone and Robert

(Ted) Joseph of the Addiction Research Center (NIDA) provided samples, space in their

laboratory during the last part of my research work, and many hours of consultation for

my paired hair and nail study.

Finally, I would like to thank many friends and colleagues from whom I was

fortunate enough to receive endless support during my Ph.D. Program. While the list is

long, I am sure it is not as complete as it needs to be. Although I would like to

personalize the contributions of each of these individuals, I must only list them and hope

that they realize the gratitude I have for their "gifts" to me. Special appreciation is given

to Dr. Ruth Winecker, Dr. Diana Garside, Dr. Chris Chronister, Abraham W/Tsadik, Dr.

Mark Bowman, Rebecca Jufer, Helen Paget-Wilkes, David Darwin, Jonathan Oyler, and

Rose Mills.

Chapter 3 was funded in part by Grant No DA09096 awarded by the National

Institute of Drug Abuse (NIDA). The clinical study and specimen collection were

performed at the Intramural Research Program, NIDA under the direct supervision of

Drs. Edward Cone and Robert Joseph.

Chapter 4 of the dissertation has been published with co-authors Drs. Diana

Garside, Bruce Goldberger, William Hamilton, and William Maples (deceased) in The








Journal ofForensic Sciences 1998; 43(5): 974-979. It was sponsored in part by the

Lucas Grant presented by the American Academy of Forensic Sciences.

Chapter 5 was funded in part by a grant from the University of Florida Division of

Sponsored Research and NIH contract number NO 1 -HD-1-3138. Head hair specimens

were obtained from the Brain and Tissue Bank for Developmental Disorders at the

University of Maryland.














TABLE OF CONTENTS

Dage

ACKNOWLEDGMENTS ............................................... ......................................iii

A B STR A C T ............................ ..................................................................................... x

CHAPTERS

1 INTRODUCTION.... ..................................................................................... 1

Historical Overview...................................................................................... 1
Unconventional Matrices in Drugs of Abuse Testing: Advantages and
D isadvantages ......................................................................................... 6
Anatomy and Physiology of Hair and Nail................................................ 9
H air.......... .... ..................................................................................... 9
N ail ........................ ................................................................... .............. 13
Growth of Keratinized Matrices and Possible Routes of Drug
Incorporation.......................................................................................... 16
Cocaine: A Brief Review ............................................................ .... 19
U se and M isuse .......................................................................................... 19
Neurobiology ....................... ........ .......................................................... .. 22
Pharm acology ......................................................................................... 23
M etabolism .................................................................................................. 24
Adverse Effects and Treatment................................................................ 25
Opiates: A Brief Review ................................................................................ 28
U se and M isuse............................................................... ............................ 28
Chem ical Structure..................................................................................... 30
N eurobiology. ..... ....................................................................................... 30
Pharmacology ....................................... ...................... .. ........ 32
M etabolism ............. .......................................... .......................... ....... 33
Adverse Effects and Treatment ............... .................................................... 33
Scope of Dissertation ............................................................................. ... 35

2 DEVELOPMENT OF A GAS CHROMATOGRAPHIC-MASS
SPECTROMETRIC ASSAY FOR THE MEASUREMENT OF COCAINE
AND OPIATE ANALYTES IN KERATINIZED MATRICES............................... 45

Historical Overview of Methodology for Hair and Nail Analysis .....................45








Developed Assays for the Detection and Measurement of Cocaine and
Opiate Analytes in Keratininized Matrices by GC/MS Analysis ...................49
Collection and Preparation of Specimens.................................................... 49
Decontamination Wash Procedures .......................................................... 51
Isolation of Drug Analytes from Keratinized Matrices................................. 52
Assay Standardization.................................................................... 55
Extraction and Derivatization ................................................................... 56
Instrum entation ......................... ............................................................ 58
Perform ance D ata.............................. ................................................. ...... 60
Summary and Conclusions............................................................................ 62

3 THE DISPOSITION OF COCAINE AND OPIATE ANALYTES IN
KERATINIZED MATRICES OF VOLUNTEERS IN A CONTROLLED
CLINICAL STUDY ............................................. .......... ...... ............... 115

Introduction .............................. .............................................................. 115
Materials and Methods............................................................................... 119
Standards and Reagents................. .................................................... 119
Study D esign............................ ..... .............. ... ................................... 120
Collection of Specimens................................................................. ........... 121
Decontamination Washes. ......................................................................... 122
Enzymatic Digestion ...................... ......... ................................................... 122
Solid-Phase Extraction ............................................................ .............. 123
GC/MS Analysis .................................................................................... 124
Pharmacokinetic Measurements and Statistics............................................. 125
R esu lts.......................................................................................................... 127
Analysis of Washed and Unwashed Specimens ......................................... 128
Analysis of Decontamination Washes ................................................... 130
Comparison of Total Drug Removed from Hair and Nails from the
Same Subject.................................................................................. 132
D discussion ...... ........................................................................................ 133

4 THE IDENTIFICATION OF COCAINE ANALYTES IN POSTMORTEM
FINGERNAILS AND TOENAILS............ ........................................................ 158

Introduction ... ................................................. ........ 158
Materials and Methods............................................................................... 159
Standards and Reagents......................................................................... 159
Subjects.......................................................................... ...................... 160
Solid-Phase Extraction .......................................................................... 160
G C /M S A analysis ............................................ .................................. 161
Statistics..................................................... .................................... .. .... 163
Results.... ...................................................................................... 163
D iscu ssion ........................................................ ....................... 165
C conclusions ............................................................... ................... ........ 169









5 THE UTILIZATION OF HAIR ANALYSIS IN THE IDENTIFICATION OF
DRUG INVOLVEMENT IN SIDS-RELATED DEATHS................................... 175

Introduction ............................ .. ....... ............................................... 175
Sudden Infant Death Syndrome................................................................ 175
Risk Factors and Adverse Effects Associated with Cocaine Use and
Pregnancy............................................ ........................................... 178
Background and Significance of Drug Testing........................................... 181
Hair Analysis as a Measure of Gestational and Prenatal Cocaine
Exposure ...................... ................... ........................................ 183
Materials and Methods..................................................................................... 187
Reagents and Materials................................................................................ 187
Subjects, Specimen Collection, and Data Handling...................................... 188
Specimen Preparation............................................................................... 189
GC/MS Analysis.................................................................................. 190
Statistics................................ ........... .............................................. 19 1
R esults........................................ ...................................................... 192
D discussion ........................................ ................................................... 194
C conclusions ............................. ................................................................ 199

6 SUMMARY AND SIGNIFICANCE OF RESEARCH........................................ 214

LIST OF REFERENCES.................. ................................................................ 221

BIOGRAPHICAL SKETCH................................................................................. 240













Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

GAS CHROMATOGRAPHIC-MASS SPECTROMETRIC IDENTIFICATION OF
COCAINE AND OPIATE ANALYTES IN KERATINIZED MATRICES OF HUMAN
ORIGIN


By

Jeri Diane Ropero-Miller

December, 1998


Chairperson: Bruce A. Goldberger, Ph.D.
Major Department: Pathology, Immunology and Laboratory Medicine

Previous research has demonstrated that analysis of keratinized matrices can be

beneficial in detecting drug exposure. Hair and nails can identify long-term drug use while

blood and urinalysis detect recent drug use.

Gas chromatography-mass spectrometry (GC/MS) was employed to investigate

drug disposition of cocaine and codeine into keratinized matrices and to compare the

sensitivity of hair and nail analysis with conventional techniques.

In project one, paired hair and nail specimens from eight volunteers enrolled in an

inpatient study who were administered cocaine and codeine for a ten-week period were

obtained for drug analysis. A significant dose-response relationship was observed for hair

specimens. The mean peak concentrations after low dosing were half the concentration

observed after high-dose administration.








Generally, no clear relationship was evident between nail drug concentrations and

dose. Decontamination washes removed less than 20% of the total drug present in hair,

but removed most of the drug concentrations (50-100%) in nail.

In project two, postmortem finger and toenail clippings of suspected cocaine users

were obtained and subjected to drug analysis. Cocaine analytes were present in the nails

of 14 out of 18 subjects (82%), whereas only 5 out of 18 (27%) subjects had positive

results by conventional postmortem techniques. This study demonstrated a marked

increase in the detection of cocaine use by nail analysis.

The final project utilized hair analysis to determine if detectability of drug exposure

in SIDS cases could be improved. Head hair samples were obtained from 26 infants.

Positive results were observed in 10 out of 17 SIDS cases (58%) and 1 out of 9 control

cases (11%). Conventional postmortem analysis did not detect cocaine in the infants.

Statistical analysis demonstrated a significant increase in drug detection with the use of

hair analysis.

In conclusion, this dissertation demonstrated that hair and nail analyses are

sensitive techniques for detecting drugs of abuse. In addition, drug incorporation into

keratinized matrices may follow a predictable dose-response profile. However, further

research investigating the mechanism of drug incorporation in keratinized matrices is

needed.














CHAPTER 1
INTRODUCTION




Historical Overview



Since ancient times, man has consumed drugs in an effort to treat ailments, relieve

pain, promote health, or induce pleasure and euphoria. As early as 4000 years ago,

Sumerian and Assyrian/Babylonian cultures recorded opiate use for medicinal purposes

(1). Similarly, cocaine was first used in the sixth century by Incan tribes of Peru and

Bolivia (2-3). In modem times, a large population continues to abuse both licit and illicit

drugs for a plethora of reasons and this has resulted in an epidemic in many global regions.

In fact, the 1996 National Household Survey on Drug Abuse conducted by the Substance

Abuse and Mental Health Services Administration reported that the number of current

illicit drug users in the United States was approximately 13 million (4).

Drug abuse impacts society through decreased job productivity and earnings and

increased crime, drug-related fatalities, health costs, prevention costs, and social welfare

recipients. The Lewin Group for the National Institute of Drug Abuse and the National

Institute on Alcohol Abuse and Alcoholism estimated that the total economic cost of

alcohol and drug abuse in the United States was over 245 billion dollars in 1992. Of this








cost which are primarily absorbed by the government, almost 40% was attributed to drug

abuse. These cost estimates increased nearly 50% from the 1985 data (4).

Rapidly increasing drug abuse among Americans has forced the Government to

address the issue by creating agencies and new laws to regulate drugs in the United States.

Agencies such as the Food and Drug Administration (1862), the National Institute of Drug

Abuse (1974), and the Drug Enforcement Agency (1989) have emerged to address

specific aspects of drug purity and control regulation. Likewise, many important

legislative milestones concerning drugs have been established. In 1848, Congress passed

the Drug Importation Act requiring U.S. Customs to prevent the passage of adulterated

drugs from overseas into the States (5). In 1906, the Pure Food and Drug Act gave the

Federal government more authority over the production, distribution, and marketing of

food and drugs. This bill also deemed the U.S. Pharmacopeia "the legal standard of

official preparations," requiring label disclosure of ingredients not found in this document

(3). The Harrison Narcotic Act of 1914 imposed severe restriction on the sale and

distribution of opiates and cocaine due to their adversities and addictive nature (1). New

safety provisions were the focus of the 1938 Federal Food, Drug, and Cosmetic (FDC)

Act. The FDC Act required manufacturers to demonstrate that newly marketed drugs

were safe, including establishing safe tolerance doses. In 1965, the Drug Abuse Control

Amendments were enacted to combat the growing abuse of depressants, stimulants, and

hallucinogens (5).

The Schedule of Controlled Substances Act of 1972 placed many drugs on a

schedule categorizing drugs based on their potential for abuse and their medicinal

purposes. For example, certain narcotics, hallucinogens, and cannabinoids were classified








as Schedule I, restricting their legal use because of their high abuse potential and limited

medicinal purposes. To minimize drug abuse in the workplace, the Drug-Free Workplace

Act was enacted in 1988 by the National Institute of Drug Abuse (NIDA) requiring

implementation of programs and policies for Federal employees, contractors, and grantees.

Shortly afterwards, businesses within the private sector adopted similar workplace drug

testing policies and procedures for pre-employment screens, random testing to promote

safety, and potential drug-related accident investigations (6).

Drug testing has become a multi-billion dollar industry, whether its primary

purpose is to investigate illegal behavior, to promote a drug-free workplace, to assist in

clinical diagnosis and treatment, or to investigate accident and death scenes. In the past,

many biological specimens have been investigated for their ability to demonstrate drug use

by an individual. Biological tissues and fluids utilized for drugs of abuse testing include

urine (7), blood (8-11), saliva (11-13), sweat (14-15), hair (16-18), nails (19-22), vitreous

humor (7), cerebral spinal fluid (23-24), bile (7), meconium (25-28), amniotic fluid (29-

30), breast milk (31-32), semen (33-34), and organs such as kidney, brain, and liver (7).

In most instances, the conventional matrices chosen for drug testing are blood and/or

urine. Use of these specimens provides sufficient quantities of material for testing,

adequate concentration of the drug, and confirmation of recent drug use.

Although blood and urine are the most prevalent matrices for drug testing, there

are several disadvantages associated with them. Both of these matrices require

processing, and in some cases preservation and refrigeration to prevent degradation of the

endogenous and exogenous chemical constituents. The collection of blood and urine can

be invasive both physically and socially. Many individuals experience embarrassment








when a urine specimen is requested from them. The presence of a drug in blood or urine

reflects use within a period determined by both metabolism and elimination. Drug in

blood occurs within minutes to hours while drug in urine occurs within hours to days.

Negative test results can occur depending on the elimination rate. For example, many

cocaine and opiate abusers escape detection by conventional urine analysis since these

analytes are eliminated rapidly, usually within 72 hours of use. All samples collected after

elimination of drug is complete will have negative test results although drug was ingested.

Conversely, false positive test results may result with the incorrect identification of an

analyte due to interference. For example, oxaprozin (Daypro6), a structurally unrelated

nonsteroidal anti-inflammatory drug, produces false positive results with a number of

benzodiazepine immunoassays (35). Due to these problems associated with blood and

urinalysis, alternative matrices have been investigated as a complement to conventional

matrices.

Hair and nails are two unconventional matrices recently investigated as

complimentary specimens to blood and urine. As I will show, the complementary traits of

these keratinized matrices make them a reasonable choice to increase detection of drug

exposure. Numerous studies have shown that results of hair tests provide valuable

information regarding relatively recent and past drug use (18, 36-41). Despite some

controversy such as environmental contamination and color bias, results of hair tests have

been utilized in clinical, forensic, and epidemiological studies, historical research, and have

even been presented as evidence in civil, criminal, and military courts of law (42-44).

Hair analysis has entered the legal system with two specific purposes as

testimonial evidence and as an acceptable specimen for workplace drug testing. The








admissibility of hair analysis in American courts has made a gradual progression. At first,

admissibility of hair analysis was based on the previously established Frye standard (1923)

requiring that scientific principles be sufficiently established with general acceptance by the

scientific community. In 1975, the Federal Rules of Evidence were adopted, allowing

expert witnesses to give testimony "in the form of an opinion or otherwise" if it assisted

"the trier of fact to understand the evidence or to determine a fact in issue" (45).

However, these rules were not utilized until 1993 after a Supreme Court ruling in the

Daubert v. Merrell Dow case. This new standard implemented a more liberal outlook

allowing the judge to determine whether or not hair analysis evidence would lend

understanding to the case, hence, superseding the "general acceptance" approach for

admissibility (42).

Although hair analysis remains controversial, it has been deemed an "acceptable

matrix" for drug testing in some States and agencies. The State of Florida Drug-Free

Workplace Act was amended in 1996 to include hair as an acceptable specimen for drug

detection. The U.S. Armed Forces have used hair testing since the late 1980s to

investigate drug use and to serve as evidence in court-martial cases. The Federal

Government is also considering hair as an alternative matrix to blood and urine for drug-

free workplace testing.

Nail analysis for drugs of abuse has also been investigated since the early 1980s

but research has been limited. The scientific community is just beginning to realize the

potential of nail analysis. Due to its unfamiliarity, nail analysis has not entered the legal

system like hair analysis. No matter the degree of general acceptability of these matrices,








their advantages and disadvantages should be carefully evaluated for each specified

application in the forensic and clinical fields.


Unconventional Matrices in Drugs of Abuse Testing: Advantages and Disadvantages


Keratinized matrices offer drug testing laboratories many advantages which

complement blood and urinalysis. One of the most complementary characteristics of

keratinized matrices is their reflection of long-term drug use. Depending on the amount of

growth available for collection, hair and/or nail analysis can potentially represent drug

exposure from several months to years. Once incorporated into the final keratinized

structure, the metabolic activities occurring within the body no longer influence

internalized drug. Therefore, the hair or nail acts as a record of the metabolic events

occurring at the time of its formation (46). Hair has also served as a "chronological ruler"

using segmental analysis in which measured hair length was correlated with average hair

growth to estimate the timing of drug exposure (47-49). The use of hair analysis to define

the timing of drug exposure has proved beneficial in prenatal drug exposure, rehabilitation

abstinence compliance, and therapeutic drug monitoring (23, 44, 50-51). Hair analysis has

also been used to determine the pattern of drug use, differentiating between

frequent/infrequent drug use (43, 52). Finally, temporary abstinence from drug use will

not lead to a negative test result as seen with conventional matrices. Hence, several

aspects of timing for drug exposure can be determined with hair analysis.

In addition to timing drug exposure events, hair analysis offers other advantages.

Hair analysis can sometimes distinguish which form of the drug was actually ingested. For

instance, the differentiation of opiate use as illicit heroin, prescribed morphine or codeine,








or consumed poppy seeds is possible in some cases. If codeine and morphine are

detected, determination of licit or illicit drug use or poppy seed consumption can not be

confirmed (53-54). However, if heroin or its major metabolite, 6-acetylmorphine, are

detected, then illicit drug use is confirmed. Since heroin and 6-acetylmorphine are only

detected in the blood and urine for minutes and hours after ingestion, respectively, finding

these analytes within these specimens is rare. Nevertheless, these opiate analytes can be

detected for a much longer period in hair and this increases the ability to detect illicit

opiate use (55).

Another advantage of keratinized matrices is their chemical composition, which

makes them very stable and less susceptible to environmental conditions. In postmortem

cases, hair and nail are sometimes the only matrices present at the time of the body's

discovery. In addition, the drug itself is more stable under normal conditions (i.e., no

chemical treatment or physical stress) once it is incorporated into the interior regions of

hair and nail. Drug analytes within the hair and nails are protected from further enzymatic

degradation unlike blood and urine. The stability of drugs in hair has led to successful

detection of drugs in the hair of Peruvian and Egyptian mummies dating 200-1500 AD and

1070 BC-395 AD, respectively (56-57). Stability also makes adulteration or manipulation

of drug content in hair and nails more difficult to achieve (23). Therefore, the more stable

nature ofkeratinized matrices provides many complementary advantages to conventional

matrices.

Keratinized matrices also have advantages in specimen collection and analysis.

The collection of hair and nails is relatively noninvasive and specimens are easy to obtain.








The ability to detect drugs in a small quantity of hair and nails is also beneficial. As little

as 10 to 25 mg of keratinized tissue is required to detect many drugs.

Conversely, the inherent nature of hair and nails also leads to disadvantages of

these matrices for drug testing. First, the mechanism of drug incorporation into hair and

nails remains unknown. This, of course, complicates the interpretation of data for these

matrices. Secondly, the lag period of drug incorporation into hair and nails makes them

unacceptable for assessing current clinical impairment or very recent use (19, 48, 52).

Several reports have estimated that it takes 3-7 days for a drug to enter these matrices and

proceed to a point where collection of an appropriate specimen and identification of the

drug are possible (58-60). Another potential problem of hair and nails is their inherent

exposure to the environment and this subjects them to potential contamination leading to

false positive test results. Decontamination washes are not always successful at removing

the entire parent drug concentration. Researchers have demonstrated that 10 to 75% of

the drug dose remained after standard wash procedures with organic solvents depending

on the type of contamination (vapor or aqueous solution) and the type of drug

investigated (cocaine, heroin, or 6-acetylmorphine) (48, 61). Some investigators have

proposed "wash kinetics" to determine the contribution of environmental contamination

while others do not believe these calculations are proven methods (48, 62). Finally, the

chemical treatment of the hair including bleaching and shampooing can influence drug

concentrations (48, 63-64).

Another controversy for keratinized matrices is a demonstrated color bias. Studies

suggest that lighter-colored hair incorporates drug to a much lesser degree than darker-

colored hair (62, 65). A possible mechanism for this color bias is the different








concentrations of pigment protein such as melanin present in hair (see following hair

anatomy section). Since darker hair is more prevalent in specific ethnic groups compared

to other groups, this color bias has been very detrimental for hair analysis (66-67). Since

our judicial system is founded on equal treatment of all individuals, the legal system

cannot risk any bias towards certain ethnic groups in accepted drug testing methodology.

Specimen preparation and laboratory handling of keratinized matrices also suffer

from some disadvantages. Laboratory procedures to prepare hair and nails for analysis are

often more tedious, time consuming, and costly than blood and urinalysis. These matrices

usually require additional decontamination and solubilization/isolation steps that are not

necessary for other matrices. Furthermore, technological standardization and quality

assurance have not been established in this relatively new field of science (37, 52).



Anatomy and Physiology of Hair and Nail


Hair

A chemical analysis of hair shows the composition to consist of 65-95% proteins,

1-9% lipids, and 15-35% water. Hydration influences the percentage of each component

(68).

The matrix proteins found in hair are referred to collectively as keratin. Hair and

nail consist of hard keratins as opposed to the soft keratins found in skin. Keratins are

fibrous in nature, insoluble in common protein solvents such as trypsin, and rich in sulfur

content. These keratinized proteins are composed of several strands of highly oriented

polypeptide chains wound into an a-helical structure known as microfibrils which combine








to form macrofibrils of the hair shaft (51, 46, 69). The amino acids cysteine, lysine,

histidine, glutamic acid, and aspartic acid form the framework of the matrix proteins.

Cysteine accounts for 11-18% of the keratin matrix (68, 70-71).

Strong covalent bonds are formed from cross-links between sulfylhydryl groups

within the amino acid make-up (69). Electrostatic, hydrophobic, and ionic interactions

can occur between small molecules and keratin's functional groups such as carboxyl,

amine, hydroxyl, and sulfylhydryl substituents. These interactions lead to the

incorporation of exogenous components such as drug and metals into the hair matrix (65,

72-73). Trace elements and heavy metals partitioned into the hair matrix vary between

0.25% to 0.95%. The proteinacious bonds may be broken with such chemical treatments

as permanent wave, bleaching, and enzymatic or acidic digest used in the laboratory. In

addition, temperature extremes can denature matrix proteins (51).

The hair lipids include free and ecosinic fatty acids, phospholipids, cholesterol, and

sulfates (73). The phospholipids may be chemically linked to keratin by their free fatty

acid side chain (46). Alcohol and ester groups of these lipids may all contribute to both

specific and nonspecific binding of drug to hair (65).

The anatomical structure of the hair consists primarily of the follicle, hair shaft,

and surrounding glands. Figure 1-1 depicts the anatomy of the hair. The hair shaft, with

an average diameter of 0.1 mm, is imbedded 3 to 4 mm below the surface of the epidermal

epithelium of the skin. The hair shaft is a long cylindrical structure that protrudes from the

hair follicle (46). The shaft is made up of tightly compacted cells found in three distinct

regions outer cuticle, cortex, and inner medulla. The elongated, overlapping cells of the

cuticle protect the hair from the environment and anchor the shaft to the follicle. The








cuticle is not always present as it can be removed by chemical or physical stress to the

shaft leaving only the inner regions (74). The shaft is composed of long, keratinized cells

(100 mun) which form fibers through cross-linking bonds (51). The majority of the inner

shaft is composed of bundles ofmacrofibrils that make up the cortex region. The inner

most region is a hollow central area referred to as the medulla.

The hair follicle is a sac-like organ that houses the hair shaft, sebaceous gland, and

in some regions the apocrine gland. There are approximately 100,000 follicles covering the

human scalp (75). Apocrine glands secrete sweat in axillary, pubic, and perianal regions

of the body. Apocrine glands secrete a specialized milky sweat that is rich in lipids (46).

Eccrine glands are sweat glands within the epidermis, in close proximity to the hair follicle.

Sweat of the eccrine glands is composed of a mixture of water, inorganic salts, amino

acids, and waste products such as urea, uric acid, and lactic acid. Sebaceous glands

secrete oily sebum composed of fats, cholesterol, proteins, and inorganic salts that keep

the skin soft and pliable. Both secretions of the sebaceous and apocrine glands are

released from ducts opening into the follicle. These secretions coat the skin and hair and

act as a physical barrier to water, bacteria, and fungus.

Two capillary plexuses supply blood to the hair bulb (subcutaneous plexus) and to

the epidermal area just beneath the stratum coreum surface (papillary plexus). Finally, a

network of nerves surrounds the follicle at the level of the sebaceous gland (76).

Functionally, the follicle has three distinguishable growth zones. The zone of hair

synthesis occurs at the base of the follicle where mitotically active matrix cells form the

hair shaft. These cells change morphologically by elongating and increasing in size and

volume. The cells move up the follicle to the keratogenous zone where the structural








protein, keratin, is synthesized. The final zone of permanent hair occurs after the cells

lose their nucleus and dehydrate forming a condensed, cornified structure.

Hair color is attributed to pigment granules. The most predominant pigment is

melanin, a copolymer composed of a protein matrix foundation with repeating 5,6-quinone

units linked to form a polymer (77-78). Melanin contains many free carboxyl, phenolic

and/or quinnonoid groups. Scatchard analysis of in vitro binding assays between melanin

and several organic cations (i.e., chlorpromazine, chloroquine, and paraquat) indicate that

more than one melanin binding site must exist for all investigated drugs. The binding of

drugs to melanin is thought to occur through cation-exchange activity of the ionic drug

with the carboxyl groups and phenolic groups of the melanin pigment granules. The

protein moiety of the pigment granules may also bind to substances (79-80).

Melanin is synthesized in small organelles, melanosomes, located within specialized

cells, melanocytes, of the hair bulb and other tissues such as skin. The melanosomes are

subsequently transferred to keratinocytes within the medulla and cortex regions of the

shaft. The average number of melanocytes on the scalp ranges from 1000-1200

melanocytes/mm2. Melanin pigment granules originate from various substrates including

tyrosine, L-dopa, dopamine, and catechol. Some melanin-synthesizing pathways occur in

the presence of enzyme. For example, tyrosinase catalyzes the conversion of tyrosine to

melanin. There are various melanin types; each vary in size, structure, and physiochemical

properties (81).

Two types of melanin pigment human hair eumelanin and pheomelanin. The

types of melanin deposited, and the size, structure, distribution and density of the granules

deposited within the cortex determine hair color (75). In a general, eumelanin produces








brown or black shades, pheomelanin produces blond, ginger and red shades, and lack of

melanin leads to graying hair. In most hair, both melanin types exist and eumelanins are

the most prevalent. The melanin content among ethnic groups is known to vary. For

example, the melanin content of Chinese black hair (3%), European brown hair (1.2%),

Irish red hair (0.3%), and Scandinavian blond hair (0.07%) varies as much as 40% (51).

Melanin content has become an issue for hair analysis because some researchers

believe drugs vary in affinity for the different types of melanin. Researchers have

proposed that drug binds to the functional chemical groups of melanin (48, 65, 69, 82-83).

In vitro studies by Joseph et al. (65) demonstrated that more drug was detected in black or

dark brown hair when compared to blond hair. These investigators suggested that the

melanin fraction was the primary binding site, whereas the lipid fraction played a minor

role. Animal studies with coexisting dark and light hair on individual rats showed that

drug content was highest in black hair followed by brown then white (67, 84). Likewise,

Reid et al. (86) demonstrated that hair analyzed from graying individuals had 1.3 to 6

times higher concentrations of cocaine analytes in pigmented hair when compared to

nonpigmented (i.e., white) hair of the same individual. Nonetheless, the extent to which

drug incorporation into hair is influenced by melanin content or the mechanism by which

this relationship exist remains uncertain.



Nail

The chemical composition of the nail is very similar to the hair. Modified horny

cells of the epidermis form the nail tissue. Nail is composed of a specialized keratin

referred to as onychin. Like proteins of hair, the nail matrix cannot be dissolved by weak








acids/bases or pepsin digest, but stronger corrosives or enzymes can solubilize the nail.

Essentially, growth occurs by the transformation of living cells of the nail matrix into

layers of hardened, dead cells, forming a nail plate. Primates are the only mammals

possessing true nails rather than claws. The tactile surface of nails is detached from the

digit and this greatly increases sensitivity for exploration, scratching, grooming, and

manipulation (87).

The basic structure of the perionychium, or nail and surrounding tissues, includes

the nail root, cell matrix, lunula, nail bed, nail plate, eponychium, cuticle, and the distal

free edge. Figure 1-2 illustrates the anatomy of a fingernail. The nail root constitutes the

most proximal end of the germinal cell matrix located at the base of the nail plate just

beneath the cuticle. The germinal matrix is the cell-producing component that contains

lymph, blood vessels, and other nourishment required for nail formation. The lunula is the

white, crescent-shaped nail area where the matrix connects with the nail bed. The lighter

color persists either because the cells of the proximal nail plate are not entirely cornified

and still contain keratohyalin granules, and/or due to variation in the nail plate's adherence

to the nail bed (87). The cuticle is the skin that overlaps the nail plate at its base and the

eponychium is the point at which the nail enters the skin just beneath the cuticle. The nail

fold guides the direction of the nail growth and it is divided into a dorsal roof and a ventral

floor. A nail bed of highly vascularized and innervated tissue is located directly beneath

the hardened nail plate. Two arterial arches run parallel to the lunula and free edge just

above the distal phalanx. In addition, capillaries are abundant at the ventral floor, the

major site of nail production (72, 87). The nail plate extends from the nail root area,








where it is anchored to the nail bed, to the distal free edge where it becomes detached

from the soft tissue underneath it.

Nail growth occurs at the dorsal roof and the ventral floor of the nail fold as well

as the nail bed located at the free margin of the nail. Utilizing silver-staining techniques

with scanning electron micrographs, Zook et al. (87) demonstrated that the mature nail is

composed of three layers formed by three separate processes. Nail layers and their

respective formation process are as follows: 1) the dorsal nail arises from the proximal

dorsal roof and ventral floor of the nail fold due to swelling of the cells, disappearance of

nucleuses, and cell collapse; 2) the intermediate nail grows from the ventral floor and

lateral walls of the distal nail fold to the lunula by parakeratosis (a gradual process where

the epidermal cells broaden and flatten but keep the nuclei); and 3) the ventral nail layer

originates from the distal lunula to the free edge of the nail. Though not always present,

the ventral nail layer compensates for dorsal nail wear and leads to a thicker nail plate

(72).

Nails also contain melanin housed in melanocytes, although the average

melanocyte concentration is 5 to 10 times less than the amount present in hair of the scalp.

In 1971, Hashimoto (88) was the first to demonstrate the presence ofmelanocytes in the

normal nail matrix using histologically prepared tissue and high voltage electron

microscopy. This study revealed that the development ofmelanosomes within

melanocytes ranged from fully melanized in black subjects, to well developed in Japanese

subjects, to poorly developed in Caucasian subjects. Melanosomes were rarely transferred

to keratinocytes in Caucasian and the frequency of mature, dense melanosomes transferred

to keratinocytes increased even more for Japanese and black nails (88). Another study








using L-dopa staining and immunochemistry with specific monoclonal antibodies to

tyrosinase and proteins of the melanosomal vesicle, showed the proximal nail matrix

contained unmelanized, immature melanocytes, whereas the distal nail matrix contained

dormant and functionally differentiated and active melanocytes. The average count in the

unmelanized compartment of the proximal epithelial sheets was 21784/mm2, the distal

matrix was 13234/mm2, and the nail bed was 4525/mm2. The active, melanized count

was 10317/mm2 for the distal matrix, relatively few in the proximal matrix, and none in

the nail bed (89). Utilizing similar techniques, Higashi et al. (90) determined melanocyte

count within the nail matrix to range between 208 to 576/mm2. These investigators also

determined that the maximum melanocyte count occurred in the distal portion of the nail

matrix and the lower two to four layers of the matrix cells (90-91). Hence, depending on

the region of the nail, melanocytes will have more or less melanin present. So if

melanocytes having functional melanin do not occur until the distal portion of the nail,

drug incorporation into nail may not be as strongly influenced by melanin content.



Growth of Keratinized Matrices and Possible Routes of Drug Incorporation


The growth of keratinized matrices is important to consider when evaluating drug

exposure. How these matrices grow and the factors influencing their growth strongly

affect the degree of drug incorporation. For example, drug content in plucked hair is

different from shed hair because shed hair undergoes a resting stage before falling out

(23).








Hair grows at an average rate of 0.35 mm/day, or about 1 cm/month and nail

grows at an average rate one-third that of hair (48, 71, 92). On average, nail produced at

the proximal nail fold takes between 70 to 160 days to reach the distal free margin (87). It

takes approximately 10 to 15 days for newly incorporated matrix cells of the hair to move

from the root to the surface of the scalp.

Growth rates for keratinized matrices are influenced by many biological and

environmental factors. Factors influencing intra-individual growth rate of hair and nail

include age, gender, ethnicity, heredity, climate, health, injury, and physical stress. In

addition, location on the body influences growth (92-93). Average hair growth rates at

different anatomical locations are as follows: scalp- 1.0 cm/month, axillary- 0.9 cm/month,

facial 0.8 cm/month, pubic- 0.9 cm/month, and chest 1.3 cm/month (75, 94). Growth rate

for the scalp displays regional variations ranging from the fastest growth occurring at the

vertex (1.3 cm/month) to the temporal (0.87 cm/month) (75). Fingernails grow

approximately four times faster than toenails. In addition, growth rate among digits varies

slightly with longer digits having the fastest growth rate (72).

Hair undergoes a cyclic cycle; periods of growth are punctuated with periods of

rest. The cycle includes a growth or anagen stage followed by resting (telogen) and

degradation (catagen) stages lasting 2-5 years, 100-150 days, and 35 days, respectively.

During anagen stage, matrix cells of the follicle undergo active mitotic division.

Transition from the anagen phase to the catagen phase leads to rapid degeneration of the

lower follicle matrix cells and subsequent shrinking of the follicle to only a small amount

of undifferentiated cells. These undifferentiated cells form the new follicle. The portion of

the follicle surrounding the hair base is lost and the hair develops a proximal swelling and








dies. Telogen hair is no longer secure and may fall out or is pushed out by the newly

developing hair of the next anagen phase. The length of each cycle and the ratio of

growth to rest vary depending on the body region, age, and gender (75). Chest hair has an

equal or longer rest period lasting for two or more weeks, whereas the growth-to-rest

ratio of scalp hair is 9:1. Scalp hair growth can last for years.

Data regarding the incorporation of drugs and drug metabolites into hair and nail is

sparse and primarily hypothetical. However, based upon results from recent studies,

several processes of internal and external incorporation of drug analyte in keratinized

matrices have been proposed including

a) transport of the analyte directly into the hair follicle and hair cells through the

blood supply to the hair follicle and nail bed;

b) diffusion of drug into secretions that can bath the hair follicles, maturing hair

fibers, and nail perionchyium;

c) exposure of the hair fibers and nail surface to the external environment including

drug residues, contaminated surfaces, or vaporized drug (48); and

d) exposure of the hair fibers and nail surface to outward transdermal diffusion of

drug from the skin (95-96).

Many factors influence drug incorporation into keratinized matrices. First, the

functional groups of the hair and nail matrix components enhance drug binding to

intracellular components of hair cells. Acidic, basic, and peptide bonds will cause certain

drugs to bind while other drugs will not. Because of this phenomenon, melanin and

keratin content influences drug incorporation. Second, membrane permeability of

keratinized matrices favors the incorporation of basic drugs such as cocaine and codeine








(high pKa) over neutral or acidic drugs. This is in agreement with the normal

physiological ionization gradient between the follicular environment (pH 7.4) and the hair

matrix (pI 4.0) allowing the unionized drug to cross the membranes (48, 51). Third,

lipophillic drugs partition into hair and nail more readily than polar drugs. Previously

published reports have demonstrated that cocaine is found in hair at a greater

concentration than its metabolites, benzoylecgonine and ecgonine methyl ester (41, 97).

This is contrary to cocaine analyte concentrations for urine, a more polar matrix. Finally,

the metabolic profile of the drug determines the rate at which each analyte can potentially

incorporate into these matrices (48).



Cocaine: A Brief Review


Use and Misuse

Cocaine is a naturally occurring alkaloid found in the leaves ofErythroxylon coca,

a shrub indigenous to the warm climates of South America, Indonesia, and the West Indies

(2). Radiolabeled carbon dating of tribal artifacts has documented the first known use of

cocaine, by pre-Inca inhabitants, to be nearly 2000 years ago. The native tribes of Bolivia,

Peru, and other South American countries used cocaine in religious rituals to treat

ailments such as mountain sickness and fatigue (98). When the Spanish invaded and

conquered these territories, they introduced cocaine to the European culture upon their

return.

In 1857, Albert Neimann isolated cocaine from the coca leaf and found it to be the

active alkaloid (99). For almost 20 years, anecdotal investigations from chemists and








physicians were reported within the literature but cocaine remained unnoticed by most. In

the early 1870s, Charles Fauvel began experimenting with a tincture of alcohol and

cocaine to relieve the painful sensations existing with surgical procedures of the throat and

vocal chords. He introduced this concept to his cousin, Angelo Mariana, who brilliantly

created a wine containing coca leaves. This wine, Vin Mariani, was successfully marketed

to relieve many ailments including fatigue, insomnia, and despondency (3). In the mid-

1880s, two well-known physicians, Sigmund Freud and Karl Koller published manuscripts

promoting cocaine's uses to treat morphine and alcohol addiction and as a local anesthetic

in eye surgeries, respectively.

By the late 1890s as cocaine became a household product contained in the

tinctures, wine, and syrup-based drinks (Coca-Cola), America's addiction to cocaine

flourished. Adverse effects and addiction of cocaine and narcotics led to the enactment of

the first American Drug laws of the early 1900s. These laws are the origin of drug

regulation that continues today. In the mid-1980s, a highly addictive form of cocaine

known as "crack" became available and it continues to dominate the Nation's illicit drug

problem.

In 1996, the estimated number of current cocaine users reported by SAMHSA's

National Household Survey on Drug Abuse was 1.75 million, which was slightly higher

than the previous year (1.45 million). Although this statistic is lower than the peak

incidence of 5.7 million reported in 1985, this survey estimated that over 650,000

Americans had tried cocaine for their first time in 1995 (4).

Cocaine is central nervous system (CNS) stimulant and sympathomimetic agent

categorized as a Schedule II drug. Clinically, cocaine is used as a local anesthetic (4-10%








solution) and vasoconstrictor, especially for surgery of the nose, throat or cornea. Less

common combination therapy includes tetracaine, epinephrine, and cocaine (TAC) used to

treat scalp and facial lacerations in children and "Bromptom's mixtures" prepared with

cocaine and an analgesic-phenthiazine solution used to treat oncology patients in

European countries (2).

Unfortunately, cocaine has become a popular drug of abuse due to its highly

desirable euphoric effects, availability, and low cost. The routes of administration vary

depending on the form of cocaine ingested. The hydrochloride salt is administered

intranasally (IN), orally (PO), or intravenously (IV), while free-base and cocaine base

("crack") are smoked (SM). Coca paste is also smoked by applying it to the end of a

tobacco or marijuana cigarette (98, 100-102).

The 85-90% purity of crack and free-base cocaine create a greater risk to the user.

In addition, crack cocaine is readily absorbed, whereas absorption ofinsufflated cocaine

hydrochloride is limited by its vasoconstrictive effects on the nasal mucosa (2). The

normal illicit dose ranges between 10-120 mg by intranasal "snorting" or smoking (103).

On the street, cocaine is often mixed with inert powders or other drugs to increase the

amount for sale (lactose, talc, and sucrose) or to heighten the response (amphetamine,

heroin, PCP) (98). Moreover, many cocaine users are drugs polydrug users, which can

lead to a greater toxicity. For example, 30-60% of all cocaine users coingest alcohol; this

can lead to more toxic metabolites and greater CNS effects (104).

Cocaine's maximum safe adult intranasal dose ranges from 80-200 mg (1-3 mg/kg)

and an adult fatal dose can be as high as 1.2 g. Solutions of the hydrochloride salt should

not exceed 10%. The average therapeutic blood level reaches 0.2 mg/L one hour post-








administration (98). The toxic dose of cocaine averages greater than 200 mg resulting in

detected blood levels approximating 4.6 mg/L for cocaine and 7.9 mg/L for

benzoylecgonine, the major metabolite (103). A recently published report by Logan et al.,

(105) cautions that postmortem blood concentrations of cocaine analytes may not

accurately reflect the perimortem concentrations due to "site-dependent differences and

time-dependent changes believed to result from competing processes of tissue release and

chemical and enzymatic degradation." Hence, the presence of any postmortem

concentration of cocaine could reflect toxicity and as such the investigators should

evaluate the history, autopsy, and death scene of suspected cocaine-related deaths more

thoroughly before assigning the cause of death.



Neurobiology

Pharmacologically, cocaine has three mechanisms of action on the nervous system.

Cocaine acts on noradrenergic nerve terminals by inhibiting reuptake of catecholamines,

such as dopamine and norepinephrine, following release from the presynaptic cells.

Subsequently, these neurotransmitters pool at the postsynaptic receptors contributing to

cocaine's reinforcing and addictive effects (102). The increase in neurotransmitters also

leads to cocaine's sympathomimetic actions resulting in vasoconstriction, hyperthermia,

hypertension, respiratory irregularities, and heart rate elevation (100, 106-107). Cocaine

also influences the brain's "stimulation-reward centers" consisting ofdopaminergic,

serotonergic, and opioid systems, playing an important role in psychotropically driven

effects associated with feeding, drinking, male sexual motivation, and self-stimulation.

Because cocaine can initiate this endogenous positive reward system, it encourages repeat








administration leading to high dependence rates (107). The quality and intensity of

cocaine's psychotropic effects is dependent on the route of administration and the rate of

rise of plasma levels (i.e., greater in the ascending portion of the plasma level vs. time

curve plot) (106). Finally, cocaine's local anesthetic properties arise from its ability to

block Na+ channel conductance thereby increasing the threshold required to generate an

action potential (102).

Cocaethylene is a known CNS-active metabolite. In comparison to the parent

drug, cocaine, CE binds with equal affinity to the dopamineric receptor increasing the

euphoric effects, but is 40 times less potent at the serotonin receptor. The serotonin

receptor is believed to lessen the dysphoric effect associated with chronic COC use. CE

persists longer and demonstrates a greater toxicity than its parent drug (108).



Pharmacology

The pharmacokinetic properties of cocaine originate from the body's ability to

effectively metabolize and excrete this drug. The basic pharmacokinetic properties of

cocaine in man are listed in Table 1-1. Cocaine, like many other basic drugs, binds to

serum proteins such as albumin and a-l-acid glycoprotein (106). The onset of action for

cocaine ranges from seconds (IV, SM) to minutes (IN, PO) (2). Cocaine's adsorption

kinetics are route dependent with peak plasma levels achieved between 5 minutes (IV,

SM) and 60 minutes (PO). Cocaine has a relatively short half-life of less than 1.5 hours.

The cocaine effects of a given plasma level differ depending on whether the plasma

concentration is rising or falling. For example, fewer psychological effects are manifested








during the downward slope of the dose-response curve (98). Biotransformation of

cocaine produces predominantly inactivated, polar metabolites excreted in the urine.



Metabolism

Cocaine's basic chemical structure includes a lipophilic benzoyl ring, an ionizable

nitrogen-containing base (methylecgonine), and its ester substituents. The four primary

metabolic pathways of cocaine include N-demethylation by P450 phase I monoamine

oxidases, hydrolysis by liver esterases and plasma cholinesterases, spontaneous or

chemical hydrolysis, and conjugation by P450 phase II enzymes. Other types of

metabolism include hepatic transesterfication and hydroxylation, resulting in rapid

hydrolysis of both ester linkages to inactivate cocaine (103).

Both chemical and enzymatic hydrolysis convert cocaine to its major metabolites,

benzoylecgonine (BE) and ecgonine methyl ethyl (EME), respectively. Both metabolites

can be further hydrolyzed to form ecgonine (ECG). Benzoylecgonine can be hydroxylated

to form m-hydroxybenzoylecgonine (MOHBE) or N-demethylated to form

norbenzoylecgonine (NBE). The N-demethylation of cocaine results in norcocaine

(NCOC) which can further hydrolyze to form NBE. Liver carboxyesterases form a

transesterfication product, cocaethylene (CE), with congestion of cocaine and ethanol.

Subsequent hydrolysis and N-demethylation of cocaethylene forms ecgonine ethyl ester

(EEE) or BE, and norcocaethylene (NCE), respectively. Both CE and NCOC have been

associated with hepatotoxicity (103-104, 109). Finally, crack cocaine forms a pyrolysis

product, anhydroecgonine methyl ester (AEME). A schematic of cocaine metabolism is

depicted in Figure 1-3.








Cocaine elimination normally follows first-order kinetics, but with saturation zero-

order kinetics may occur. Cocaine's rapid and complete metabolic disposition leads to

low levels of unchanged drug in body fluids. As little as 1-9% (pH dependent) of

unchanged drug can be readily detected in urine 4-6 h post-administration. The majority of

a cocaine dose is excreted as urinary, polar metabolites. Cocaine's major metabolites can

be readily detected in the urine 24-72 h after administration. The combined data of

twenty-four hour urine excretion studies show the percentage of each cocaine analyte to

be as follows: 1-5% COC, 29-45% BE, 32-49% EME, 0.7% CE, 2.6-6.2% NCOC, 0%

ECG, and trace amounts of other demethylated and hydroxylated products (98, 103, 106).



Adverse Effects and Treatment

Cocaine overdose can occur at relatively low doses resulting in a rapid death due

to cardiovascular collapse, respiratory depression and arrest, dysrhythmias, and seizures.

The pathophysiology of acute toxicity originates from cardiovascular and

neuropsychiatric complications associated with cocaine ingestion. The symptoms of acute

toxicity include:



vasoconstriction myocardial infarction

cerebrovascular accident tachycardia

hyperthermia intense paranoia

sudden collapse bizarre and violent behavior

hypertension stroke and grand-mal seizures

pulmonary dysfunction coma








rhabdomylosis atrial and ventricular fibrillation



One aspect of cocaine's lethality is hyperthermia. Psychomotor hyperactivity,

stimulation of hepatic calorigenic activity, and possibly stimulation of the hypothalamic

thermoregulatory centers contribute to hyperthermia (98). Animal studies have

demonstrated that major causes of death are linked to psychomotor agitation and

hyperthermia (2). Cardiotoxicity associated with acute cocainism include myocardial

ischemia and infarction, supraventricular and sinus tachycardia, systemic arterial

hypertension, platelet aggregation, and in-situ thrombus formation. Both the left and right

coronary arteries are subject to vasoconstriction leading to myocardial infarcts. These

infarcts are commonly associated with delayed, atypical chest pain and can occur in

subjects having no prior cardiac dysfunction (2). Pulmonary complications begin with

repetitive short, deep breaths, progress to edema and depression, and ultimately to

collapse (Cheyne-Stoke breathing). The vasoconstrictive nature of cocaine can also affect

the vasculature of the skeletal muscle bed, resulting in severe rhabdomylosis. Individuals

that conceal cocaine in body cavities, also known as body "packers" and "stuffers" may

suffer acute gastrointestinal ischemia, colitis, and perforations.

Cocaine induces other adverse effects that are not acutely life threatening such as

mydriasis, nausea and vomiting, aberrant psychological behavior, insomnia, and anorexia.

Again, most of these symptoms are attributed to cocaine's affect on the central nervous

system.

Chronic exposure signs and symptoms include rhinitis, distorted perception,

shortness of breath, increased and labored respiration, accelerated athersclorosis,








tachycardia, cold sweats skin cellulitis/abscess, chronic cough and bronchitis,

hypersensitive bronchial mucosa ("crack" lung), weight abnormalities, violent protective

behavior, nasal septal perforation, psychiatric complications, and profound mood swings

(2, 98, 110). Continual vasoconstrictive episodes and/or recurrent or diffuse ischemia can

result in dilated cardiomyopathy. In rare cases, the aorta can actually dissect and rupture

secondary to the mechanical stress of chronic hypertension and tachycardia. With

continued IV use of cocaine, individuals are predisposed to bacterial endocarditis and deep

vein thrombosis (98). Other behavioral effects mediated by stimulation of the "reward

system" include self-medicating to control of euphoric response, anorexia, hyperactivity,

and profound sexual excitement.

Cocaine toxicity requires initial treatment of the most life-threatening symptoms to

stabilize the subject. Sedative-hypnotics like diazepam are administered to control the

neurological complications (agitation, anxiety, seizures), sinus and supraventricular

tachycardia, hypertension and other sympathomimetic symptoms. Calcium channel

blockers verapamill, nifedipine), oxygen, and cooling relieve hyperthermia and cardiotoxic

effects (2). In addition, sodium carbonate is given to correct respiratory acidosis. If

treatment occurs at the early excitatory phase during hypertensive effects, sodium

nitroprusside or nitroglycerin drip may be administered. In addition, propanolol may

reverse cardiovascular stimulation. During the late depressive phase of hypotension,

administration of dopamine or norephedrine is more appropriate (98). Other treatment of

cocaine toxicity mainly involves treatment of specific symptoms. Activated charcoal

efficiently absorbs cocaine taken orally or in the cases ofgastrointestinal drug smugglers

(111). Psychotic reactions are treated with haloperidol or other antipsychotics. For








comatose or lethargic patients, naloxone should be considered in case of simultaneous

ingestion of heroin (98).



Opiates: A Brief Review


Use and Misuse

"Opiates" are naturally occurring analgesic alkaloids derived from the opium

poppy, Papaver sominferum. Opium, derived from the Greek name for juice, is

comprised of more than 25 distinct alkaloids, including morphine and codeine obtained

from the milky exudate of the unripened, opium poppy seed (112). Morphine, the

principle alkaloid of opium, was named after the Greek god of dreams, Morpheus, who

was often depicted with poppy flowers (1, 113). "Opioids" are natural and semisynthetic

alkaloid derivatives prepared from opium, in addition to synthetic surrogates whose

actions mimic those of morphine. Therefore, opioids include opiates, synthetic opioids,

and opiopeptins endogenouss neuropeptides such as endorphins and enkephalins).

Opium alkaloids have been utilized as analgesics since the early 1800's following

the chemical isolation of pharmacologically active morphine (1803), codeine (1832), and

papaverine (1848). These discoveries along with the invention of the hypodermic needle

led to a predominant use of pure alkaloids rather than crude opium preparations. By

World War I, opiate addiction became a problem prompting the clinical field to introduce

opioids, like meperidine and methadone, which possess morphine-like analgesia but with

less addiction potential. Then in the 1950s, opioid antagonists like nalorphine and

naloxone were developed to reverse heroin and morphine toxicity (112-113).








Opiate analgesics are effective and common medications for treatment of mild to

severe pain. Strong opiate analgesics are commonly referred to as narcotics, which is

derived from the Greek word for stupor (113). Based on subjective patient reports,

opioids are effective analgesics because they have the ability to change pain perception by

raising the pain threshold. The Controlled Substance Abuse Act categorizes most opiate

analgesics as Schedule II. They are also used to treat congestive heart failure (pulmonary

edema) and anxiety. Opioids are commercially available as elixirs, pills, powders,

suppositories, and solutions (114-116). Some commercial preparations are manufactured

in combination with acetaminophen (Phenergan, Tylenol with codeine) or aspirin

(Empirin). Common doses, given every four to six hours, are 15-60 mg (PO) for

codeine, 2.5-10 mg (IM, SC, PO) for morphine, and 5-10 mg (PO, IM, SC) for heroin

(112, 114).

Both the licit and illicit forms of opioids are often abused due to their desirable

central nervous system (CNS) effects, especially euphoria. Heroin, first synthesized from

morphine by Wright in 1874, is an illicit Schedule I drug that has no medicinal applications

in the United States. Many opioids are highly addictive leading to physical and

psychological dependence (116). Reports by the Drug Abuse Warning Network (DAWN)

show a general trend towards an increased use of opioids during the period of 1992 to

1994 (117). Moreover, SAMHSA's 1996 National Household Survey on Drug Abuse

reported an increasing trend in new heroin users from 1992 to 1995, with estimated "past

month" heroin users increasing from 68 to 216 thousand during this time period (4).








Chemical Structure

Phenanthrenes (4,5-epoxymorphinans), the most well known and characterized

opioids, follow the basic morphine chemical structure illustrated in Figure 1-4. Drugs in

this group possess various modifications including methylation or acetylation of the C3

and C6 hydroxy groups, oxidation of the C6 hydroxy group to a ketone functionality,

saturation of the C7 C8 double bond, and hydroxylation at the C14 position. These

subtle molecular differences among opioids can change their pharmacological action (e.g.,

convert an agonist to an antagonist), receptor affinity, metabolic resistance to first-pass

hepatic metabolism, and lipid solubility (103, 112, 114).



Neurobiology

The neurological mechanism of action for opioids is mediated through specific

receptors located at various sites in the CNS and other peripheral organs. Opioids exert

their pharmacological effects by mimicking endogenous neuropeptides (114). Opioid

receptors are also located on several immune response cell types such as neutrophils,

lymphocytes, and monocytes which are commonly associated with endogenous opioid

peptides produced during a state of stress (116). Primary receptors include:

1) p. (mu) receptors, responsible for euphoria, supraspinal analgesia,

respiratory depression, miosis, reduced gastrointestinal motility, and

physical tolerance and dependence

2) K (kappa) receptors, mediate spinal analgesia, sedation, sleep, miosis,

physical dependence, and limited respiratory depression








3) 8 (delta) receptors, mediate dysphoria, delusions, hallucinations,

respiratory stimulation, and vasomotor stimulation

4) a (sigma), purported to have effects similar to the 8 receptor.

These CNS receptors are part of the limbic system. The limbic system is involved

in the arousal of emotion of man including and painful sensations and its negative

emotional component and in some cases euphoria. Opioids bind to CNS receptors on

terminal nerve endings and block the release of neurotransmitters involved in the

transmission of pain stimuli (114).

On a cellular level, opioid receptors exert their effects through changes in Ca2 and

K flux associated with the cyclic AMP (cAMP) system of the nervous system (114). On

a molecular level, opioid receptors are distinctly different. Through receptor binding and

molecular cloning studies, it has been determined that these distinct opioid receptors are

encoded by different genes expressed in discrete neuronal pathways or cell types. In

addition, each opioid has differing selectivities for each receptor, which determine its

pharmacological effect (113). For instance, heroin is a strong [i agonist while codeine is a

weak gi and K agonist, resulting in heroin's analgesic potency being 10 times that of

codeine. Based upon their pharmacologic activity on the receptors, opioids are classified

as full agonist, mixed agonist/antagonist, or full antagonist. Antagonist can preferentially

displace agonist, which is why naloxone is so effective at reversing toxicity of opioid

agonist. The ability of an opioid to bind to its receptor and the relative binding affinity are

influenced by molecular structure and stereospecificity. For example, the levororatory

isomer of morphine produces analgesia while the detrorotartory isomer oflevorphanol

(dextromethorphan) acts as an antitussive agent (116).








Prolonged use ofopioids leads to tolerance. Tolerance begins with the first dose,

but is usually clinically insignificant until about 2-3 weeks of chronic use. To minimize

tolerance, opioids should be administered in small doses given at frequent intervals.

Cross-tolerance among opioids is a prevailing characteristic (116).

Physical and psychological dependence makes opioid withdrawal and

detoxification extremely painful and difficult. Typically, withdrawal signs from strong

agonists appear within 6-8 hours after the last administration and peak at 36-72 hours.

Psychological dependence produces strong craving that can lead the individual to pleas,

demands, and manipulative behavior.



Pharmacology

Opioids share common pathways for their pharmacokinetic parameters including

adsorption, distribution, metabolism, and elimination.

Since opioids are well absorbed by most tissues, numerous routes of administration

are effective. Depending on the application, opioids can be ingested orally or injected

intramuscular, intravenous, or subcutaneous. Some cautionary heroin users choose nasal

insufflation (snorting) or inhalation of drug vapors ("chasing the dragon") for safety

reasons. For instance, intravenous heroin use has a greater risk of overdose, transfer of

diseases such as hepatitis and autoimmune deficiency syndrome (AIDS), and injection-site

injuries. For analgesic and anesthetic purposes, alternate routes of administration include

transdermal patches, epidural, and intrathecal injections (118-120).

The distribution of opioids depends upon specific drug properties, both chemical

and physiological. The extent of first-pass metabolism influences opioid bioavailability








and pharmacological effects. Opioids can be as much as 95% bound by plasma proteins.

Opioids concentrate in the tissues of highly perfused organs such as the lungs, brain,

kidney, liver, and spleen. Opioids further accumulate in skeletal muscle and lipid

reservoirs and cross-placental barriers to varying degrees (103).



Metabolism

Opioids are chiefly metabolized in the liver forming pharmacologically active and

inactive metabolites. Some active metabolites have analgesic effects that are stronger than

the parent drug itself. Metabolic pathways include reduction, oxidation, N- and 0-

dealkylation, hydroxylation, and conjugation. Both parent drug and metabolites are

excreted through enterohepatic or renal circulation. The majority of opioids and their

metabolites are excreted in the urine, with only a small amount of glucuronide conjugates

eliminated in the feces (103). Figure 1-4 depicts the chemical structure and metabolism of

common opioids.



Adverse Effects and Treatment

Individuals using opioids often experience adverse effects in addition to the desired

therapeutic effects (Table 1-2). Common complaints include nausea, vomiting,

constipation, and mood swings. The most life-threatening adversity, commonly observed

following an overdose, is respiratory depression. Other risks associated with opioid

toxicity include coma, hypothermia, seizures, and hypotension.

Treatment of opioid overdose consists mainly of supportive and antidotal measures


such as:








1. Monitoring cardiovascular and respiratory status

2. Ventilation to reestablish respiratory exchange

3. Oxygen and anticonvulsants to combat seizure disorders

4. Intravenous fluids and vasopressors to regain normal blood pressure

5. Naloxone to counteract CNS and respiratory effects (115-116)

Generally, hemodialysis and forced diuresis are ineffective in treating opioid

overdose patients.

Opioid abusers normally require a treatment program for successful detoxification.

Hallmarks of withdrawal reflecting the physical dependence include irritability, insomnia,

anorexia, violent yawning and sneezing, gastrointestinal abnormalities, elevated heart rate,

profuse sweating, and piloerection. Strong pains in the bones and muscles, and

uncontrollable muscle spasms are consistent with withdrawal (98). The culmination of

these symptoms is widely referred to as the abstinence or withdrawal syndrome. In most

cases, these symptoms follow a characteristic chronology as shown in Table 1-3. The

pharmacological activity of the opioid determines the severity of the withdrawal

symptoms.

If the withdrawal symptoms are severe or prolonged, a methadone management

protocol is followed to alleviate undesirable effects. Methadone is a slower acting opioid

with a lower abuse liability that is frequently used in substitution pharmacotherapy.

Patients are stabilized for 2-3 days before the methadone dosage is gradually decreased.

Buprenorphine and propoxyphene have also been investigated as alternatives in the

treatment of opioid dependence. Other treatments to alleviate withdrawal symptoms








include fluids and electrolytes, antispasmodics (propantheline), sedative-hypnotics

(phenobarbital), and anti-adrenergics (clonidine) (116).



Scope of Dissertation


This dissertation embodies three projects, detailed below, that investigated the

utility of hair and nail analysis for the identification of drug exposure. The current

techniques for detection of drug exposure are flawed in many respects. Self-reports are

notorious for underestimating drug use due to feared repercussions by the individual.

Reported toxicological data derived from the analysis of conventional matrices, blood and

urine, have many deficiencies which limit the sensitivity of the analytical methods.

Therefore, the first objective of this dissertation was to develop and evaluate a more

sensitive analytical technique using keratinized matrices to improve the detectability of

drug exposure.

In the past two decades, hair analysis has undergone a metamorphosis from an

unrefined research tool to a highly sensitive forensic technique. Despite the extensive

research that has already been conducted, many questions and controversies remain. In

addition, the potential utility of another keratinized matrix, nail, for identifying drug

exposure is for the most part unknown.

The second objective of this dissertation was to investigate unknown aspects of

hair and nail analysis that will contribute to a greater understanding of drug incorporation

and detection of drugs in hair and nails. Examples of unanswered questions include: 1)

Can hair and nail analyses improve the detection of drugs in comparison to conventional








matrices? 2) Is the current methodology utilized for hair appropriate for nails? 3) Do

keratinized matrices demonstrate a dose-response relationship? 4) Is there a relationship

between dose, time profile and concentration of drug in hair and nail? To accomplish this,

highly sensitive and specific gas chromatography-mass spectrometry was employed to

obtain analytical results for all three projects.

Chapter 1 is an introduction presenting a historical overview of the applications of

drug testing, the laws addressing drug testing, and the matrices utilized in drug testing. It

continues with a description of hair and nail anatomy, physiology, growth, possible routes

of drug incorporation, and factors influencing drug incorporation. The advantages,

disadvantages, and controversies associated with hair and nail analysis are also discussed.

This introductory chapter also reviews the pharmacology, metabolism, and adverse effects

of cocaine and opiates since these were the drug classes investigated during the

dissertation.

The methodology employed for analysis of specimens within this disseration are

discussed in Chapter 2. It begins with a historical overview of analytical techniques used

in the past and the present for identifying drugs in keratinized matrices. It focuses on each

step of the analysis process including specimen collection and handling, decontamination

wash procedures, isolation of analytes from keratinized matrices, and instrumental

analysis. Performance data including accuracy, precision, recovery, and linearity are also

presented.

Chapter 3 discusses the analysis of specimens collected and analyzed from an 11-

week controlled clinical study in which eight black, male subjects were administered low

and high doses of cocaine and codeine. Scalp hair from the posterior vertex region and








fingernail scrapings were collected weekly. The analytical results served to answer

pharmacological questions about drugs in hair and nails. It also compared paired results

of hair and nail to determine the potential utility of nails since much less is known about

drug incorporation into this matrix. Lastly, it discusses the decontamination wash

procedures and quantifies the amount of drug removed during each wash step.

Chapter 4 discusses a forensic application of nail analysis. The analytical results of

conventional postmortem results using blood, urine, vitreous humor, and other tissue,

were compared with the results of hair analysis. Nine cocaine analytes were investigated.

Results of the cocaine analytes present in nails were compared to the subject's history and

the significance of certain unique analytes was discussed. The advantages, disadvantages,

and potential applications of nail analysis were further presented.

Chapter 5 discusses a clinical application of hair analysis. Epidemiological studies

have linked drug use, particularly cocaine, to some cases of sudden infant death syndrome

(SIDS). Since the mechanism of SIDS is unknown and the risk factors are multifactorial

and in many cases confounding, it is very important to identify all risk factors associated

with an infant's death. This project utilized a more sensitive toxicological technique, hair

analysis, to identify the presence or absence of drug exposure in 26 deceased infants.

Chapter 6 summarizes the conclusions ascertained by these three projects and

discusses the significance of this research.





































Figure 1-1. The Anatomy of a Hair Follicle












Nail Bed Germinal Matrix
Free Edge Nail Plate I Lunula


Figure 1-2. The Anatomy of a Fingernail








Table 1-1. Phamacokinetics of Cocaine in Humans (2, 103-104, 106)



Plasma Half-life (h): 0.7-1.5 (COC)
5-8 (BE)
3.5-8 (EME)
2.5 (CE)


Volume of distribution (L/kg):

Plasma protein binding (%):

Bioavailability (%):


Body clearance (L/min):

Peak plasma level (ng/mL):



Time of peak plasma level (min):


1.6-2.7

96

20-60
90-100


IN
PO
IV and SM

IN
PO
IV and SM


100-500
50-90
500-1000

15-60
50-90
5


(IN and PO)
(IV and SM)


(1.5-2.0 mg/kg dose)
(2.0 mg/kg dose)
(32-50 mg dose)

(1.5-2.0 mg/kg dose)
(2.0 mg/kg dose)
(32-50 mg dose)


pKa:


Abbreviations: cocaine (COC), benzoylecgonine (BE), ecgonine methyl ester (EME),
cocaethylene (CE), intranasal (IN), oral (PO), smoked (SM), and intravenous (IV).









COC
CH3 CH3 AEME
'N
SCOOCH4 COOCH3



NCOC H y transeterification
H Izyme \ /C CE
N hydrolysis N
C OOCH T(cholinesterases) COOC2H5
"-O P3 \chemical P 4
OCOCH N/CH3 EME hydrolysis
N-OCOC Hs N \ ; OCOC6Hs
ri 0 [C O O C H 3 \ IH

,COH /CH3 BE /H NC
ECG / HCOOH COOCH2C
NCH3 z \
/COOH N-COCC6Hs \ OCOCsHs


OH hydroxylation
hydrolysis on CH3 EEI
V N
ENB MOHBE L. ,Cooc2H
NH NBE N-denethylation CHMOHBE CO
N/CH3 "
4COOH N COOH H


SOCOCOC6H40H





Figure 1-3. The Metabolic Pathway of Cocaine

Abbreviations: pyrolysis (A), cocaine (COC), anhydroecgonine methyl ester (AEME),
benzoylecgonine (BE), cocaethylene (CE), ecgonine (ECG), ecgonine ethyl ester (EEE),
ecgonine methyl ester (EEE), meta-hydroxybenzoylecgonine (MOHBE),
norbenzoylecgonine (NBE), norcocaethylene (NCE), and norcocaine (NCOC).









CH3
I
N





CH3COO 0 OCOCH3

HEROIN





CH3






CH3O 0 OH


CODEINE


CH3
I





HO O COCOCH3

6-ACETYLMORPHINE





CH3
N





HO 0 OH


MORPHINE


Figure 1-4. Chemical Structure and Metabolic Pathway of Common Opioids (112)








Table 1-2. Therapeutic and Adverse Effects of Opioids (121)


Central Nervous System Effects:


Euphoria
Analgesia
Sedation
Mental clouding and mood swings
Pulmonary
Respiratory depression
Decreased responsiveness


Gastrointestinal


Other


Peripheral Effects:


Cardiac


Gastrointestinal


Genitourinary


Neuroendocrine


Nausea
Vomiting

Dizziness
Cough suppression
Miosis
Truncal rigidity
Flushing and warming of the skin
Sweating and itching



Bradycardia
Orthostatic hypotension when system stressed
Stroke

Constipation
Decreased motility
Increased tone
Decreased gastric secretions
Biliary tract constriction of smooth muscle

Decreased renal plasma flow
Increased urethral and bladder tone
Prolongation of labor
Menstrual abnormalities
Sexual dysfunction

Increased antidiuretic hormone (ADH) release


Nervous








Table 1-3. Chronology of Opioid Abstinence Syndrome in Humans (121)


8-12 Hours


Lacrimation
Yawning
Rhinorrhea
Perspiration


12-14 Hours


48-72 Hours
(peak of syndrome)


Irritability
Piloerection ("Goose flesh")
Restless sleep
Weakness
Mydriasis
Tremor
Anorexia
Muscle twitching

Increased irritability
Increased heart rate
Insomnia
Hypertension
Marked anorexia
Hot and cold flashes
Sneezing
Alternating sweating/flushing
Nausea and vomiting
Piloerection
Hyperthermia
Rapid or deep breathing
Abdominal cramps
Aching muscles


Syndrome duration:


7-10 days















CHAPTER 2
DEVELOPMENT OF A GAS CHROMATOGRAPHIC-MASS SPECTROMETRIC
ASSAY FOR THE MEASUREMENT OF COCAINE AND OPIATE ANALYTES IN
KERATINIZED MATRICES




Historical Overview of Methodology for Hair and Nail Analysis


The analysis of keratinized matrices to detect drugs of abuse is a relatively new

field of study. The uniqueness of hair and nail requires specialized pretreatment steps to

adequately measure drug incorporated into these matrices. In much the same way that

plasma proteins are precipitated from blood to reduce interference prior to measuring an

analyte of interest, keratinized matrices require pretreatment steps to prepare them for

analyte extraction. The chemical composition, structure, and exposure to the

environment introduce new issues to the toxicologist interested in analysis of hair and

nail.

The analysis of drug analytes in hair is a multi-step process involving the following:

(1) decontamination of the surface of the hair fibers through washing; (2) sample

preparation to facilitate ease of handling and release of drug analytes; (3) incubation or

digestion to release drug analytes; (4) extraction and purification of drug analytes; and (5)

analysis by an immunochemical or chromatographic technique. Conventional analysis of

urine and blood usually only requires the last two steps. However, research has








demonstrated that the additional three pretreatment steps are necessary for the successful

analysis ofkeratinized matrices (48, 62, 122).

First, keratinized matrices require decontamination procedures to remove

unwanted interferents (i.e., lipids, oils, cosmetics) and exogenous analytes (e.g., drug)

coating the surface from environmental exposure. A survey of the literature shows that

researchers have investigated organic solvents, phosphate buffers, water, soaps, and

various combinations of these for decontamination wash procedures. Table 2-1

summarizes representative decontamination wash procedures presented in the literature.

The extent to which keratinized matrices can be decontaminated from exogenous

analytes depends on factors governing penetrance of the drug into the hair matrix such as

route of drug administration, chemical treatment, and the water solubility of the drug.

Drug entry into keratinized matrices is facilitated by water and the pH of the environment,

both of which make decontamination procedures less effective (122).

Regardless of the procedure employed, the efficacy of decontamination washes has

remained controversial. Some researchers are convinced that in most cases externally

bound drug can be removed through multi-step decontamination processes (62, 123-124).

If extensive washing does not remove residual drug, some investigators further propose

that passive exposure and active ingestion are distinguishable through "wash kinetic

criteria." Wash kinetics compares the drug concentrations of the wash fractions and the

extracted hair specimens (123). Others oppose complete efficacy of decontamination

procedures, demonstrating that residual drug may remain after extensive washing

procedures (18, 125-127).








Another required pretreatment step requires special preparation of hair and nails to

optimize solubilization of these matrices during extraction or digest procedures.

Researchers have found that by cutting hair and nails into small segments or pulverizing

them into a fine powder, reagents can more readily enter the protein matrix, thereby

releasing the analyte into solution. Moreover, measuring the segments of hair during this

pretreatment step allows chronological analysis to estimate the timing of drug exposure.

Hair and nails are synthesized from hardened, structural proteins such as keratins

that form a stable framework utilizing a variety of chemical bonds. Many of these bonds

are strong and cannot be easily broken (e.g. covalent and electrostatic). Consequently,

special reagents or environmental conditions must be employed to denature keratinized

proteins. While some proteins can be denatured with reagents such as mineral acids,

trypsin, or increased temperature (>370C), complete solubilization of keratinized proteins

requires harsher conditions.

Normally, to completely denature more resistant proteins, prolonged boiling or

exposure to thiol-containing compounds and detergents (i.e., sodium dodecyl sulfate) or

hydrogen bond-breaking reagents (i.e., urea or guanidine hydrochloride) are required

(128). However, for drug testing of hair and nail, these treatments need to successfully

solubilize the protein matrix to effectively remove the drug without destroying the drug

itself. For example, extreme temperature or acidic environment should not be permitted in

pretreatment steps for cocaine analysis because this would lead to degradation of the

parent drug to its metabolite benzoylecgonine. In addition, chemical bonds between

functional groups of drug analytes and other pigmentation proteins like melanin must be

broken. Treatment with a strong acid or base depends on whether the drug is basic,








acidic, or neutral. Basic drugs are more readily extracted with strong bases while

acidic/neutral drugs are normally extracted with strong acids.

It is not enough to subject keratinized matrices to simple liquid-liquid extraction

(LLE) or solid-phase extraction (SPE) utilized for conventional liquefied matrices (i.e.,

blood and urine) to efficiently isolating the analyte. Rather, these matrices require

degradation procedures prior to common extraction methods. The three primary

degradation techniques employed by researchers include enzymatic digestion, caustic

digestion, or solvent extraction. A myriad of these techniques exists in the literature and

Table 2-1 summarizes a representative assay utilizing all of these. Many digest and extract

solutions also contain chemicals like dithiothreitol and detergents that can break disulfide

bonds of the protein matrix. These procedures are generally carried out under heated

conditions and/or for prolonged periods to facilitate the degradation process. Once these

degradation processes are completed, further extraction is necessary to improve analyte

isolation and recovery.

There are two methods used in the field of forensic toxicology to isolate drugs

from biological matrices. The principle of LLE is based upon the partitioning coefficient

of a drug. The coefficient is determined by the degree of affinity an isolate has for a

chosen organic solvent and partitioning strength of the analyte between aqueous and

organic phases. Given these criteria, an acidic drug is more soluble in an organic solvent

than an aqueous solvent at an acidic pH and a basic drug is more soluble in an organic

solvent than an aqueous solvent at a basic pH. A combination of extraction-back

extraction-reextraction steps is employed to move the drug analyte from organic to

aqueous to organic to obtain cleaner extracts using LLE (28).








Conversely, SPE is a sorbent phase bonded to a solid support such as silica

contained within a column. There are many types of commercially available phases

including hydrophobic (C18 octadecyl), hydrophilic (cyanopropyl), anion-exchange

(quaternary amine), cation-exchange (benzene sulfonic acid), and copolymeric

(hydrophobic and cation-exchange). SPE employs both physical and chemical properties

to reproducibly interact with drug analytes to remove them from the liquid (i.e., serum,

urine, amniotic fluid) or homogenate (i.e., liver, meconium) matrix in which they are

contained. Multiple washings with various organic solvents can remove unwanted

interferents prior to elution of the drug analyte from the SPE column (28).

Previously published methodology, describing the analysis of drugs in keratinized

matrices, have utilized predominantly immunochemical or chromatographic technique. A

description of each is outside the scope of this dissertation, however, Table 2-1

summarizes a cross-section of analytical techniques reported in the literature.



Developed Assays for the Detection and Measurement of Cocaine and Opiate Analytes in
Keratinized Matrices by GC/MS Analysis


Collection and Preparation of Specimens

For this dissertation, several processes were employed to prepare the keratinized

matrices. In all cases, hair was collected from the crown area of the scalp, or posterior

vertex region. This region was chosen because it is purported to have the most follicles in

active (anagen) growth at a given time. Growing at an approximate rate of 1.0 cm/month,

this region also has a faster growing rate than other regions of the scalp (75). In addition,

hair collection methods include cutting the hair as close to the scalp as possible or








plucking the hair from the scalp to include the root portion as well as the shaft. In all

projects of this dissertation, hair was collected as close to the scalp as possible, knowing

that the most recent drug exposure would not be detected.

In Chapter 3, hair was collected by the staff of the Intramural Research Program

(IRP), NIDA. Grooming clippers were employed to remove the first collection of scalp

hair from subjects, collecting different regions of the scalp (temporal, frontal, nape,

posterior vertex, and anterior vertex) separately. For the study reported herein, only hair

from the posterior vertex region was analyzed. The remaining stubble was removed and

discarded with shaving cream and a straight edge razor. Hair from initial collection was

stored in ZiplockT plastic bags at room temperature until hair could be finely cut with

scissors and transferred to separate glass vessels for storage at -300C. For the remainder

of the study, scalp hair (approximately 2-3 mm in length) was collected as close to the

scalp as possible using a cleaned electrical shaver (Norelco). Again, remaining stubble

was removed and discarded using a straight edge razor. Since collected hair specimens

only represented one week of growth, the hair was in small sections that could be weighed

and used for analysis without further manipulation.

Infant hair analyzed in Chapter 5, was cut as close to the scalp as possible using

cleaned scissors. Specimens were weighed into polyethylene vessels and 5 to 6 glass

beads (0.5 mm) were introduced into the vessel to promote mechanical disruption of the

hair. These specimens were pulverized into a fine powder by a Mini-beadbeater-8TM Cell

Disrupter (Biospec Products, Bartlesville, OK) set at 80% power for 5 minutes. The hair

powder was transferred to a disposable glass culture tube and the residual hair was

removed from the polyethylene vessel using 8 to 10 methanolic rinses, which were








included in the subsequent overnight methanolic incubation step. Glass beads were

removed after the overnight methanolic incubation period to prevent loss ofanalyte

present in hair that coated the surface of the beads. It was believed that the glass beads

were inert in the methanolic solution and would not interference with noise to the assay.

Nail was collected from the digits of the hand and foot for the postmortem analysis

study detailed in Chapter 4. Clean nail clippers were utilized to remove as much of the

distal nail portion as possible. The nail was then finely cut into small pieces for analysis.

Fingernail specimens were also collected from subjects enrolled in the controlled

clinical study (Chapter 3). The ventral surface of the nail was scraped with a sterile

scalpel blade. Each digit was scraped 50-100 times and all scrapings were combined for

analysis. These scrapings could be used for analysis without further manipulation. Nail

scrapings were transferred to separate glass vessels for storage at -300C until time of

analysis.



Decontamination Wash Procedures

Two types of decontamination wash procedures were utilized in this dissertation.

Both procedures have been previously employed by several research groups (18, 60, 123,

127, 129).

The first decontamination procedure (Chapter 3) used a multi-step process

combining hydrating and non-hydrating solvents. First, a 15-min isopropanol wash (3.0

mL) was utilized to remove loosely bound lipids, soaps, and drug analyte present on the

keratinized surface. Next, three successive 30-min washes in 3.0 mL of a 0.1 M

phosphate buffer (pH 6.0) solution (1.36 g potassium phosphate monobasic in 1.0 L








deionized water) were employed to hydrate the keratinized specimen and remove analyte

from its porous surface. All four steps were performed at room temperature and agitated

by stirring or placing in an oscillating water bath. These wash fractions were collected for

further analysis to determine the percentage of drug removed in each fraction.

Alternatively, Chapters 4 and 5 utilized a single-step decontamination wash

procedure that was much less involved and less time-consuming. The prepared hair or nail

specimens were transferred into a disposable culture tube and 3.0 mL of methanol was

added to each. The specimen was then vortexed for 15 to 20 seconds and the methanol

was immediately decanted and retained for further analysis. Specimens were not allowed

to remain in the methanol longer than the time required for vortexing in order to minimize

analyte extraction from the interior regions of the keratinized matrices.



Isolation of Drug Analytes from Keratinized Matrices

For the isolation of drug analytes from keratinized matrices, two different

procedures were performed for this dissertation. Comparison studies of these two

isolation procedures gave insight into the advantages and disadvantages of each.

A comparison study of enzymatic digestion and methanolic incubation

demonstrated that both performed similarly and each had advantages over the other

procedure. Replicate analyses (n=10) of an authentic drug-positive hair specimen were

prepared using both isolation techniques. Specimens were otherwise subjected to the

same sample preparation procedures and GC/MS analysis. Specimens were analyzed

during the same run to minimize differences due to instrumental analysis. This comparison

study demonstrated that the enzymatic digestion was able to isolate more drug analyte, but








it also resulted in more interference, and hence, a greater signal-to-noise ratio was

observed. Mean concentration data for cocaine, cocaethylene, benzoylecgonine, and

morphine are summarized in Table 2-2.

A comparison was also performed to determine which enzyme resulted in the best

isolation of analyte. Two enzymes were chosen based on recommendations from the

NIDA scientific group from which we obtained specimens. The enzymes, Tritirachium

album and Subtilisin A were employed to isolate drug from the hair of a known drug

abuser. Both Tritirachium album (proteinase K- Type XI) and Subtilisin A (protease -

Type VIII) were purchased from Sigma Chemical Company, St. Louis. In addition to the

respective enzyme, the digest solution also contained dithiothreitol (DTT-60mg/10 mL)

and sodium dodecyl sulfate (SDS-20 mg/10 mL) to assist in breaking down the protein

matrix of the hair. Mean concentration data for cocaine, cocaethylene, benzoylecgonine,

and morphine are summarized in Table 2-2. Results were comparable for the two

enzymes, however, Tritirachium album was ultimately chosen because it demonstrated

greater recovery of minor analytes. In addition, these studies also demonstrated that SDS

contributed background signal and did not improve recovery of analytes from the matrix.

Therefore, SDS was not included in the digest solution during later specimen preparation.

A brief description of the enzymatic digest procedure used to degrade the protein

matrix of hair and nail specimens collected from subject enrolled in the inpatient study

(Chapter 3) follows. The enzymatic digest solution was prepared fresh daily by combining

60 mg of DTT, 0.5 mg of Protease XI (Tritirachium album), and 10 mL of0.05M Tris

buffer (preset pH 7.4).








Weighed samples were placed into 4.0 mL fitted filter (RFV02F4P-United

Chemical Technologies; Bristol, PA) and 1.0 mL of digest solution was added to each.

Trideuterated internal standards for major analytes (ds-benzoylecgonine, d3-cocaine, d3-

ecgonine methyl ester, d3-codeine, and d3-morphine) were added at a concentration of 100

ng/mg and minor analyte internal standards (d3-cocaethylene, d3-6-acetylmorphine) were

added at concentration of 50 ng/mg.

Micro stir-bars were added to samples, flitted filters were capped and placed

overnight (-16 hr) into a heated water bath (400C) placed on top of stirring plates set at

80% of maximum speed. After completion of the digestion, the digestate was eluted from

the flitted filters and collected into conical-shaped disposable culture tubes. The filters

were rinsed with 2 x 2 mL volumes of 100 mM phosphate buffer (pH 6.0) which was

collected into the same tube as the digestate. The filtered digestate was centrifuged at

4000 rpm for 10 minutes prior to SPE.

Specimens for studies presented in Chapters 4 and 5 employed an organic solvent

incubation to isolate drug analytes from the keratinized matrices. Briefly, weighed and

decontaminated specimens were placed in disposable culture tubes and 3.0 mL of

methanol was added to each tube. Specimens were capped, vortexed, and placed

overnight (-16 hr) into a heating manifold (400C). Specimens were vortexed additional

times during the incubation period. After the overnight methanolic incubation, samples

were centrifuged at 3000 rpm for 5 to 10 min. The drug-containing supernatant was

decanted from the hair pellet and evaporated at 400C under a stream of nitrogen.

Residues were reconstituted in 0.025 M phosphate buffer in preparation for SPE.








Assay Standardization

Calibration curves were prepared using working standards prepared in water or

acetonitrile and stored at -300C until needed. Calibrators and controls were prepared

using drug-free hair or nail obtained from laboratory personnel. A minimum of six

calibration points was included to construct a curve spanning a concentration range of

0.10-10.0 ng/mg. An unextracted standard was included in each run to assist with

instrument setup and to help troubleshoot any potential problems with specimen

preparation (i.e., to help determine if poor signal response was due to poor extraction of

the specimen or instrumental problems). Negative controls containing drug-free hair or

nail, with and without addition of internal standards, were included in each batch to assist

in data interpretation to evaluate background noise and interference due to the matrix or

reagents added during specimen preparation. Positive controls were included throughout

the run (beginning, middle, and end) to measure curve stability. Positive controls were

prepared fresh daily with standard materials prepared separately from calibrator materials

and if possible, from different sources. Both high and low concentrations (0.5 ng/mg and

5.0 ng/mg), with respect to the calibration curve, were analyzed. Within-run and between-

run variability for the positive controls are discussed later in the performance data section

of this chapter.

Also included in each batch were hydrolysis controls for cocaine and 6-

acetylmorphine ranging in concentration from 5 and 40 ng/mg depending on the project

and the matrix being evaluated. Hydrolysis controls measured the spontaneous hydrolysis

occurring during preparation and extraction methods. Table 2-3 summarizes the

hydrolysis control data obtained for each project. Generally, hydrolysis was less than 5%








for cocaine and less than 10% for 6-acetylmorphine. Mean cocaine and 6-acetylmorphine

concentrations were within 20% of the target concentration. Analytes detected due to

hydrolysis included ecgonine methyl ester (EME), benzoylecgonine (BE), and morphine

(MOR). Mean concentration ranges for detected analytes were as follows: EME- none

detected (ND) to 1.0 ng/mg; BE- ND to 3.2 ng/mg; and MOR- 0.42 to 1.6 ng/mg.



Extraction and Derivatization

For this dissertation, SPE technology was chosen based on previous experience

and the potential advantages offered. Since multiple analytes were being simultaneously

analyzed, a copolymeric phase using hydrophobic and cation-exchange properties was

chosen. Several commercially available SPE columns were compared prior to the

selection of United Chemical Technologies CleanScreen (ZSDAU020, 200mg/10 mL)

column.

The same SPE procedure was employed during all projects of this dissertation. A

step-by-step description of the procedure is presented. After completion of the

appropriate isolation techniques (enzymatic extraction or methanolic reflux), specimens

were reconstituted in phosphate buffer (0.01 M or 0.025 M; pH 6.0) prior to SPE using a

vacuum manifold system. The extraction columns were conditioned with elution solvent (1

mL), methanol (3 mL), deionized water (3 mL), and phosphate buffer (pH 6.0, 3 mL).

Specimens were added to SPE cartridges followed by deionized water (2 mL), 100 pM

acetate buffer (pH 4.0, 2 mL), methanol (3 mL), and acetonitrile (1 mL). SPE columns

were aspirated to dryness under a full vacuum. The analytes were collected into clean

tubes inserted into the vacuum manifold system by addition of 4.0 mL of elution solvent








consisting of methylene chloride-isopropanol-ammonium hydroxide (80:20:2 v/v/v).

During conditioning, sample introduction, and elution steps, the pressure was adjusted to

maintain a flow rate of approximately 1-2 mL/min (20 mm Hg). Columns were not

permitted to dry between addition of the phosphate buffer and sample introduction.

Samples were evaporated under a nitrogen purge using a heated water bath set at

40C (Zymark TurboVap). Specimen tubes for Chapter 4 and 5 study received

derivatizing agent, BSTFA [N,O bis(trimethylsilyl)trifluoroacetamide] with 1% TMCS

(trimethylchlorosilane) at a volume of 40 pL, whereas specimens for Chapter 3 received

20 IL of BSTFA and 1% TMCS and 20 pL acetonitrile. Specimens were vortexed before

derivatization at 650C for 30 minutes. Derivatization was performed either directly in the

autosampler vials (Chapter 3) or in a capped, disposable culture tube that was parafilmed

around the seal. The later specimens were centrifuged for 5 min to allow adequate

collection of the small volume. These specimens were then transferred to autosampler

vials for analysis. In all cases, 1 iL of derivatized specimen was injected for GC/MS

analysis.

The addition of a silyl derivative improves sensitivity in a number of ways. First,

the trimethylsilyl (TMS) derivative increases molecular weight (MW) of the analyte of

interest. Higher MW compounds demonstrate greater sensitivity and specificity due to

fewer interferent signals associated with fragmentation products of other chemical

products. Silyl derivatization also prevents interaction of the analyte functional groups

with the GC column and this reduces analyte time on column and peak tailing due to

column interactions.








Instrumentation

GC/MS analyses were performed using one of two Hewlett-Packard GC/MS mass

selective detectors (Hewlett-Packard Company, Little Falls, DE).

Specimens in Chapter 4 and 5 were analyzed by a Hewlett-Packard (HP) 5890A

Series II gas chromatograph and HP 7673 autoinjector interfaced with a HP 5972A series

mass selective detector (MSD). The gas chromatograph was equipped with a cross-linked

95% dimethyl, 5% diphenylpolysiloxane capillary column (HP-5; 30 m x 0.10 mm i.d. x

0.10 pn film thickness). Automated injections were made in the splitless mode using a 2-

mm i.d. silanized borosilicate liner in the injection port.

Similarly, a newer HP model was used during the Chapter 3 study. Analyses were

performed with a HP 6890 Series II gas chromatograph and automatic liquid sampler

interfaced with a Hewlett-Packard 5973 MSD. The gas chromatograph was equipped

with a HP-1 crosslinked 1% diphenyl, 99% dimethylpolysiloxane fused-silica capillary

column (12 m x 0.200 mm i.d. x 0.25 pm film thickness. Automated injections (1 .L)

were made in the splitless mode, and a 4-mm i.d. silanized borosilicate liner with a glass

wool plug was utilized.

Similar GC/MS parameters were employed for all techniques. The injection port

and transfer line temperatures were maintained at temperatures of 250C and 2900C,

respectively. The oven temperature program was set at an initial temperature of 900C

(hold time- 0.5 min) and programmed to ramp at two intervals: 22.50C/min up to 2250C

and 17.50C/min to a final temperature of3200C (hold time- 4.0 min). The helium carrier

gas was set at a constant flow rate of 1.0 mL/min. The septum purge flow was set at 2 to








3 mL/min. The MSD was operated in the selected ion monitoring mode at a dwell time of

20 ms.

A quantitating ion and at least two confirming ions were monitored for each

analyte. Quantification of analytes was based upon the ratios of the integrated target ion

areas to the corresponding deuterated internal standard analogs for each analyte. Those

analytes which did not have commercially available internal standards were compared to

the trideuterated analogue most similar in chemical structure. For example, norcocaine

was quantitated based upon the ratio of its integrated ion peak-area to the integrated ion

peak-area oftrideuterated cocaine. The quantitating and confirming ions differed among

projects due to observation of interference for some ions. For instance, the 182 m/z was

normally utilized to quantitate cocaine concentrations but specimens digested with the

enzyme demonstrated a significant interferent for this ion. Consequently, the molecular

ion, 303 m/z, was employed for Chapter 3. Table 2-4 summarizes all ions monitored for

each analyte during this dissertation but does not differentiate between quantitating and

confirming ions. Details of chosen ions are included in the respective chapters.

Analytes were identified based upon comparison of retention time and ion ratio

with the corresponding values of calibrators assayed in the same run. Ion ratios were

calculated by dividing the ion peak-area of the confirming ion by the ion peak-area of the

quantitative ion. Quantification of analytes was based upon the ratios of the integrated ion

peak-areas to the corresponding trideuterated standard analogues. Acceptance criteria

were that ratios had to be within 30% of the ratio value of a calibrator of similar

concentration.








Figures 2-1-A through 2-1-M illustrate analyte abundances for monitored ions of a

25 ng/mg calibrator. Each figure depicts a representative selected ion chromatogram of

the quantitation ion and a selective ion monitoring (SIM) spectrum showing the relative

abundance for quantitating and confirming ions. In addition, Figures 2-2-A through 2-2-F

illustrate representative selected ion chromatograms for the following specimens:

unextracted 250 ng calibrator, extracted 250 ng cocaine and opiate calibrator in hair

matrix, extracted negative control in hair matrix, and extracted posterior vertex hair and

fingernail specimens from Subject M. The selected ion chromatograms depict signal due

to SIM quantitating ions only and were normalized to the highest peak abundance.



Performance Data

Validation studies of new methodologies are necessary in order to verify

performance, assess potential interference, and compare hair analysis to established

methods. Standard analytical procedures were employed to evaluate analytical parameters

including recovery, precision, accuracy, linearity, and sensitivity.

The limits of detection (LOD) and quantitation (LOQ) were determined by

replicate analysis of a series of standards decreasing in concentration. LOD and LOQ

studies were performed during a 2 to 3 day period in separate batches in order to obtain

more representative values. The LOD was defined as the concentration corresponding to

a signal-to-noise ratio greater than or equal to 3.0. The LOQ was generally defined as the

lowest standard that did not deviate from the target concentration by more than 20%. At

very low concentrations (0.1 ng/mg) a less stringent criteria of 30% was utilized. In

addition, ion ratios and 20% coefficient of variance (%CV) for the replicates were also








evaluated in determining the limit of quantitation. Tables 2-5, 2-7, and 2-10 summarize the

LODs and LOQs for all studies

The linearity of the standard curve was measured by two separate methods during

the investigation. The initial assessment of linearity was determined by replicate analysis

(n =5) of standards prepared at a concentration of 10, 20, and 30 ng/mg. The calibration

curve was considered linear if the mean concentration values did not deviate 20% of the

target concentration. All analytes were linear to at least to 10 ng/mg, which was the

designated highest point of the calibration curve. Table 2-5 includes the limit of linearities

demonstrated by this initial evaluation. Although some analytes demonstrated linearity

above 10 ng/mg, results were reported as greater than 10 ng/mg for consistency.

Assessment of linearity during routine analysis was determined by established criteria that

acceptable calibration points of the curve were within 20% of their expected value.

Along with visual observation of the standard curves, correlation coefficient (r2) greater

than or equal to 0.990 was considered acceptable.

Intra- and inter-assay variability were determined by analysis of controls prepared

at low (0.5 ng/mg) and high (5.0, 7.5, and 10 ng/mg) concentrations. Seven to ten

replicates were performed for both matrices and by both assay procedures. Within-run

precision was determined by analyzing seven replicates. For between-run precision, data

from positive controls included in each run were combined to calculate %CVs for each

analyte. Tables 2-6, 2-8, and 2-11 summarize the precision studies for both assays and

both matrices (hair and nail). Generally, the %CVs were acceptable at both the low and

high concentrations. Analytes that demonstrated lower precision data included AEME,

EEE, NBE, and NCOD.









Summary and Conclusions


Overall, the presented assays utilized for this dissertation performed favorably and

reproducibly for the identification of cocaine and opiate analytes in hair and nails. These

assays followed multiple-step procedures for specimen preparations that are generally

accepted by forensic toxicologists for analyzing keratinized matrices. Specifically,

specimens were decontaminated, subjected to an isolation procedure to remove analytes

from the protein matrix, further purified by SPE, derivatized to improve specificity and

sensitivity, and analyzed by GC/MS. Method validation studies for both assays presented

in this dissertation, demonstrated acceptable efficiency, precision, and sensitivity for the

analysis of keratinized matrices.








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Table 2-2. A Comparison Study of Enzymatic Digest versus Overnight Methanolic
Incubation (n=3)

Analyte Isolation COC CE BE MOR
Technique ng/mg* ng/mg* ng/mg* ng/mg*

Enzymatic Digest using 63 8.1 0.62 0.05 10 0.78 1.40 0.16
Tritirachium Album
[16 h @40C]
Enzymatic Digest using 47 + 1.4 0.56 0.08 12 0.17 1.96 0.01
Subtilisin A
[16 h@40C]
Methanol Incubation 29 1.9 0.24 0.01 5.6 0.24 0.36 0.06
[16 h @ 40C]__

Abbreviations: cocaine (COC), benzoylecgonine (BE), cocaethylene (CE), and morphine
(MOR).

*Mean concentration results for replicate analyses of hair collected from a known drug
user.








Table 2-3. Mean Concentration Data for Cocaine and 6-Acetlylmorphine Hydrolysis
Controls (5 ng/mg) included in each Analytical Batch

Hydrolysis COC EME BE 6-AM MOR
Control (ng/mg) (ng/mg) (ng/mg) (ng/mg) (ng/mg)
Concentration
(ng/mg)
40 ng/mg (n=3) 38.4 1.6 1.0 1.3 0.16 0.28 NA NA
20 ng/mg (n=18) 17.9 1.5 0.62 0.24 3.2 0.70 22.6 3.5 1.6 0.81
10 ng/mg 8.6 1.2 ND ND 9.6 0.13 0.49 0.85
(n=3)
5 ng/mg 5.9 1.7 NDto<0.20 0.49 0.01 5.0 0.13 0.42 0.40
(n=76)

Abbreviations: cocaine (COC), benzoylecgonine (BE), ecgonine methyl ester (EME), 6-
acetylmorphine (6-AM), morphine (MOR), not analyzed at this concentration (NA), and
not detected (ND).








Table 2-4. Cocaine and Opiate Ions Monitored during GC/MS Analysis

Analyte Ions (m/z)

Anhydroecgonine methyl ester 152, 166, 181

d3-Ecgonine methyl ester 85, 99, 274

Ecgonine methyl ester 82, 96,271

Ecgonine ethyl ester 83, 96, 240, 285

d3-Cocaine 85, 185, 275, 306

Cocaine 82,182,272,303

d3-Cocaethylene 85,199, 275, 320

Cocaethylene 82, 196, 272, 317

d3-Benzoylecgonine 143, 243, 259, 364

Benzoylecgonine 140,240, 256, 361

Norcocaine 140,240, 346

Norbenzoylecgonine 140, 298, 404

m-Hydroxybenzoylecgonine 82,210, 240,449

d3-Codeine 181,237, 374

Codeine 178,196, 234, 371

Norcodeine 254, 292, 429

d3-Morphine 236, 417, 432

Morphine 236, 414, 429

Normorphine 254, 308,487

d3-6-Acetlymorphine 290, 343,402

6-Acetlymorphine 287, 340, 399





68


Figures 2-1-A through 2-1-M. 25 ng/mg Calibrator: Selected Ion Chromatogram of
Quantitation Ion and Selective Ion Monitoring (SIM) Spectrum for Analytes Investigated






























Figure 2-1-A. 25 ng/mg Cocaine Calibrator: Selected Ion Chromatogram of Quantitation
Ion and Selective Ion Monitoring (SIM) Spectrum for Analytes Investigated Illustrating
the Relative Abundance for Quantitating and Confirming ions









25 ng/g Calibrator:


30

20


10


Cocaine (m/z 303)


6.0 6.2 6.4 6.6
Minutes


Cocaine SIM Spectrum
182


182


303
1I


300


m/z


8I I I l2 I I I I I 2I 2 0
80 100 120 140 160 180 200 220 240 260 280






























Figure 2-1-B. 25 ng/mg Benzoylecgonine Calibrator: Selected Ion Chromatogram of
Quantitation Ion and Selective Ion Monitoring (SIM) Spectrum for Analytes Investigated
Illustrating the Relative Abundance for Quantitating and Confirming ions










25 ng/g Calibrator:


Benzoylecgonine (m/z 240)


6.3 6.5 6.7 6.9
Minutes

Benzoylecgonine SIM Spectrum


140
1


140 160 180 200 220 240


240


361


I I
260 280


I I I I
300 320 340 360


m/z


90
80
70
60
50
40
30
20
10


I361






























Figure 2-1-C. 25 ng/mg Anhydroecgonine Methyl Ester Calibrator: Selected Ion
Chromatogram of Quantitation Ion and Selective Ion Monitoring (SIM) Spectrum for
Analytes Investigated Illustrating the Relative Abundance for Quantitating and
Confirming ions










25 ng/g Calibrator:
Anhydroecgonine Methyl Ester (m/z 152)


3.3 3.5 3.7 3.9
Minutes

Anhydroecgonine Methyl Ester SIM Spectrum
1152


181


166


m/z


10


150 160 170 180 190































Figure 2-1-D. 25 ng/mg Cocaethylene Calibrator: Selected Ion Chromatogram of
Quantitation Ion and Selective Ion Monitoring (SIM) Spectrum for Analytes Investigated
Illustrating the Relative Abundance for Quantitating and Confirming ions










Cocaethylene (m/z 196)


6.2 6.4 6.6 6.8
Minutes


Cocaethylene SIM Spectrum
1196


272


317


m/z


70
60
50
40
30
20
10


190 200 210 220 20 240 250 260 270 280 290 300 310 320I I I
190 200 210 220 230 240 250 260 270 280 290 300 310 320


25 ng/g Calibrator:






























Figure 2-1-E. 25 ng/mg Ecgonine Methyl Ester Calibrator: Selected Ion Chromatogram
of Quantitation Ion and Selective Ion Monitoring (SIM) Spectrum for Analytes
Investigated Illustrating the Relative Abundance for Quantitating and Confirming ions










25 ng/g Calibrator:
Ecgonine Methyl Ester (m/z 96)


4.0 4.2 4.4 4.6
Minutes

Ecgonine Methyl Ester SIM Spectrum
182


96


1 271

80 100 120 140 160 180 200 220 240 260
80 100 120 140 160 180 200 220 240 260


m/z


70
60
50
40
30
20
10






























Figure 2-1-F. 25 ng/mg Ecgonine Ethyl Ester Calibrator: Selected Ion Chromatogram of
Quantitation Ion and Selective Ion Monitoring (SIM) Spectrum for Analytes Investigated
Illustrating the Relative Abundance for Quantitating and Confirming ions










25 ng/g Calibrator:
Ecgonine Ethyl Ester (m/z 96)


4.2


4.4 4.6 4.8


Minutes

Ecgonine Ethyl Ester SIM Spectrum
182


240


m/z


30-


20-


10-


80 100 120 140 160 180 200 220 240 260 280
80 100 120 140 160 180 200 220 240 260 280


285
I

































Figure 2-1-G. 25 ng/mg Norcocaine Calibrator: Selected Ion Chromatogram of
Quantitation Ion and Selective Ion Monitoring (SIM) Spectrum for Analytes Investigated
Illustrating the Relative Abundance for Quantitating and Confirming ions










25 ng/g Calibrator:


Norcocaine (m/z 140)


Norcocail


6.4 6.6 6.8 7.0
Minutes

Norcocaine SIM Spectrum
1240


140


1I 1I I 1 0 I 22
140 160 180 200 220


240 260 I I I I
240 260 280 300 320 340


m/z


30

20


10


346


360
360






























Figure 2-1-H. 25 ng/mg Norbenzoylecgonine Calibrator: Selected Ion Chromatogram of
Quantitation Ion and Selective Ion Monitoring (SIM) Spectrum for Analytes Investigated
Illustrating the Relative Abundance for Quantitating and Confirming ions










25 ng/g Calibrator:
Norbenzoylecgonine (m/z 298)


5.9 6.6 7.4 8.1
Minutes

Norbenzoylecgonine SIM Spectrum


140


298


404


140 160 180 200 220 240 260 280 300 320 340 360 380 400


m/z






























Figure 2-1-I. 25 ng/mg Codeine Calibrator: Selected Ion Chromatogram of Quantitation
Ion and Selective Ion Monitoring (SIM) Spectrum for Analytes Investigated Illustrating
the Relative Abundance for Quantitating and Confirming ions










25 ng/g Calibrator:


Codeine (m/z 371)


6.8 7.0 7.2 7.4


Minutes

Codeine SIM Spectrum

1178


196


371


234


m/z


70
60
50
40
30
20
10


I I I 2 I I I I I I I 3
180 200 220 240 260 280 300 320 340 360 380































Figure 2-1-J. 25 ng/mg 6-Acetylmorphine Calibrator: Selected Ion Chromatogram of
Quantitation Ion and Selective Ion Monitoring (SIM) Spectrum for Analytes Investigated
Illustrating the Relative Abundance for Quantitating and Confirming ions










25 ng/g Calibrator:
6-Acetylmorphine (m/z 399)


7.2 7.4 7.6 7.8
Minutes

6-Acetylmorphine SIM Spectrum


287


340


m/z


30

20

10


399


2 0 2 0I 30I I 0 3 I I I I
280 290 300 310 320 330 340 350 360 370 380 390 400 410






























Figure 2-1-K. 25 ng/mg Morphine Calibrator: Selected Ion Chromatogram of
Quantitation Ion and Selective Ion Monitoring (SIM) Spectrum for Analytes Investigated
Illustrating the Relative Abundance for Quantitating and Confirming ions