Printer Friendly

Drugs in hair. Part I. Metabolisms of major drug classes.

TABLE OF CONTENTS

INTRODUCTION
I. HAIR DEVELOPMENT AND STRUCTURE
  A. Development and Structure
  B. Glands Adjacent to Human Hair Follicles
    1. Eccrine Sweat Glands
    2. Apocrine Glands
    3. Apoeccrine Sweat Glands
    4. Sebaceous Glands
II. METABOLISM AND PHARMACOKINETICS
  A. Drug/Drug Metabolite Distribution
  B. Basic Metabolism
    1. Cannabinoids
    2. Cocaine
    3. Amphetamines
    4. Opiates and Opioids
    5. Phencyclidine
    6. Benzodiazepines
    7. Ethyl Alcohol
III. INCORPORATION OF XENOBIOTICS INTO HAIR
REFERENCES
ABOUT THE AUTHOR


INTRODUCTION

One of the fundamental requirements in clinical and forensic toxicology is to demonstrate the presence or absence of a xenobiotic such as a drug. The mere presence of a controlled substance like benzoylecgonine (BZE) in urine may be sufficient to precipitate discharge from a clinical treatment program or an adverse civil action such as violation of probation. Conversely, the absence of a drug in a patient's random urine may be sufficient grounds to discharge a patient from a clinical rehabilitation program. In addition to their presence in blood/blood products, urine and oral fluid, many drugs and their metabolites are also incorporated in hair; thus they can be detected reliably [171,178]. Thus, hair is a useful matrix for the demonstration of the presence or absence of drugs and/ or their metabolites.

Even though the receipt of a final report may be read as absolute, the presence or absence of a substance in toxicology should be linked to a cutoff or limit of detection (LOD) for the report to be forensically or clinically meaningful. An example of hair testing cutoffs taken from the 2004 Proposed Mandatory Guidelines [214] is given below in Table 1.

Presence or absence are also toxicologic terms that need to be considered within established timeframes. The whole blood taken from a motor vehicle driver by EMS, 10 minutes post-accident, has an extremely high probability of indicating that the driver was intoxicated by ethyl alcohol if the laboratory result on the whole blood was 0.18 mg/dL [126]. In an entirely different set of circumstances, the finding of cocaine metabolite in an employee's urine using a cutoff of 150 ng/mL [213] cannot be rationalized with a statement such as "I tried some coke in college 5 years ago." For blood and blood products, oral fluid, and urine, other numerous short-term xenobiotic level-timeframe permutations exist. For a timeframe within which an individual may have used a drug, the hair provides a unique "long look-back" or extended window of detection as described below.

Levels of drugs including ethanol in blood and blood products usually have the shortest window of detection of all of the common matrices. Oral fluid, which has a window of detection similar to that found for drugs and drug metabolites in blood, also shows great promise in linking drug use to a window that defines a reasonable time of last use. Like blood and blood products, oral fluid has the advantage of targeting parent drugs, although metabolites certainly can be seen routinely. For an even longer window of detection than blood/blood products and oral fluid, urine, which has a "look-back" on the order of days to weeks, is commonly employed by toxicologists as an analytical matrix. As a waste product, urine many times contains larger amounts of metabolite than parent drug for some drugs/drug classes. Thus, urine may use the presence of a metabolite to indicate the use of the parent drug (e.g., the presence of BZE indicates the use of cocaine). Hair drug testing, which currently depends primarily on the incorporation of parent drugs into hair rather than metabolites, provides the longest window of detection in drug testing. However, this may be weeks to months and, possibly, years after drug use, unless the hair is cut or shaved off completely. A summary of the useful detection windows for each common clinical or forensic toxicologic matrix is given in Figure 1 [43]. Timeframes for deposition of metabolites in hair would be expected to follow a pattern similar to that seen for their parent drugs.

One major drawback to the use of hair as a toxicologic matrix has been and continues to be external contamination by parent drug. As a general question, "Did the drug X found in a hair specimen originate from incorporation of the substance after the use of drug X, from external contamination such as smoke containing drug X, or a combination of both?" In a recent proposal for interpretations of results from different types of specimens, the US Department of Justice stated that "An examiner may not report or state an opinion that a drug or poison finding in hair is proof of ingestion of the drug or poison unless a metabolite that is unique is also identified and/ or validated wash procedures have been performed that can differentiate between exposure and ingestion." [215] Thus, a metabolite that demonstrates the actual use of a substance in question can be of major importance in hair testing. A metabolite that demonstrates actual use cannot be a non-metabolic degradation product or a contaminant that may be found as a product used for the manufacture and processing of the substance in question. As an example, BZE is usually the major metabolite of cocaine, but it can also be produced by the in vitro spontaneous hydrolysis of cocaine in an aqueous solution [14]. Thus, the presence of parent cocaine and BZE on a hair sample might be viewed just as environmental exposure to cocaine with the subsequent partial hydrolysis to BZE or environmental contamination of the hair sample by partially hydrolyzed cocaine. Needless to say, a metabolite that demonstrates the actual use of a parent drug cannot by itself be a separately marketed drug, e.g., oxymorphone (Numorphan[R], Opana[R]), which is an active metabolite of oxycodone. Thus, oxymorphone cannot be employed to demonstrate that oxycodone was used by the hair donor. From the presence of only oxymorphone and oxycodone, one could conclude that there might have been only a simple environmental exposure to both drugs as parent substances.

Although some level of hair washing probably is necessary to remove external contamination, it is the purpose of this review to demonstrate that the analysis of hair for drug metabolites to indicate the use of a parent drug is a distinct possibility for most, if not all drugs of abuse.

For this review, the author prepared all chemical structures using PerkinElmer ChemDraw Prime 15.0 (Perkin-Elmer: Waltham, MA) and referred to the following two general references for medical terminology, chemical names, and structures: (a) Borland's Illustrated Medical Dictionary [5] and (b) The Merck Index: An Encyclopedia ofChemicals, Drugs, and Biologicals [162]. Abbreviations used in this review are listed in Table 2.

I. HAIR DEVELOPMENT AND STRUCTURE

Before discussing the presence of drugs and/or drug metabolites in hair, a brief discussion of hair as a biological matrix was done.

A. Development of Hair and Structure of Hair

Like teeth and nails, hair is an appendage of skin, which is the largest organ of the human body. Briefly, follicle formation in the embryo is initiated by signals from the dermis that direct the epidermis to form dermal condensates, as shown in Figure 2, following epidermal placode (focal thickening) formation. Placodes are first seen on the scalp and face within 10-11 weeks EGA (estimated gestational age) [110]. The epidermal placodes direct dermal cells to condense (dermal condensate) and form dermal papilla. Placodes subsequently develop in a caudal and ventral direction. Keratinocytes are directed by dermal papilla to proliferate and extend deeper into the dermis. Within 12-14 weeks EGA, the base of the developing hair follicle surrounds the presumptive dermal papilla forming the hair peg, which is the second stage shown in Figure 2. There are bulges on the superficial portion of the developing hair follicle as shown in the third stage in Figure 2. Where an apocrine gland is not formed, there are only two bulges seen. The deepest bulge is the insertion point of the future arrector pili muscle and the location of the presumptive follicular stem cells. The middle bulge becomes the sebaceous gland, while the upper bulge may become the apocrine gland if formed. As discussed below, the apocrine gland is of very limited topology in the adult human.

Normally, human hair morphogenesis occurs only once. Lanugo (the fine hair on the body of the fetus), vellus (the fine hair that succeeds the lanugo over most of the body), and terminal (the coarse hair growing on various areas of the body during the adult years) hairs follow essentially the same principles. The first "coat" that is formed is fine, long, and is a variably pigmented lanugo hair that is shed in an anterior to posterior wave in the seventh and eighth month of gestation. A second coat of shorter, fine unpigmented lanugo hair then grows in all areas except the scalp and is shed 3-4 months after birth. Pigmented hairs on the scalp are shed postnatally in an anterior to posterior wave. The first two patterns of growth are synchronous. After the first two cycles, hair starts to grow in an asynchronous or "mosaic" pattern. As a skin appendage comparison, nail growth is continuous throughout life. In pre-pubertal children, hair is generally terminal (large, usually pigmented, and medullated) on the scalp, eyelashes and eyebrows. On the other hand, it is vellus (very short, non-pigmented, and usually non-medullated) on the face, trunk, and extremities. During puberty, vellus hairs in some sites start to enlarge under the influence of androgens. Conversely, some terminal hairs on the scalp can revert to miniaturized vellus-like hairs in post-pubertal individuals with androgenetic alopecia.

Once a mature hair follicle is generated during embryogenesis, it begins to cycle and does so autonomously throughout its lifetime. The phases of the hair cycle include active growth (anagen, I-VI, 2-6 years in terminal scalp hair), regression (catagen, I-VIII, 2-3 weeks in terminal scalp hair), and rest (telogen, 3 months for human scalp hair). Kenogen references a telogen follicle with no club hair (a hair whose root is surrounded by a bulbous enlargement composed of keratinized cells, usually just before normal loss of the hair from the follicle). A fourth stage, exogen, was termed around 1998 and indicates active hair shedding during anagen IV where the new hair pushes out the old hair.

The primary function of the anagen follicle is hair production. Although macroscopically simple and homogeneous, a proximal hair follicle is complex as shown in Figure 3.

The germination center around the hair bulb papilla is formed by matrix cells, which are melanocytes and keratinocytes [171]. The germination zone is in the basement membrane. Rapid mitotic cell division forces movement of the layers above the germination center toward the outer epidermis. The genes for keratin (scleroproteins) [6] expression are above the papilla. In the same area as keratin gene expression, pigment, if there is any, is incorporated. Although there are numerous layers in a developing hair, fundamentally there are three hair shaft layers--cuticle, cortex, and medulla. During hair development, cortex cells change from spherical at the germination level to spindle-like. During the change from spherical to spindle-like, protein filaments are produced. The filaments fill the cell and are fused together as depicted in Figure 4. Above the zone in which keratin is formed and pigment is incorporated, but still below the infundibulum, the hair is hardened by disulfide cross-linking and dehydration. Cuticle cells originate from the outer shell of the papilla. In addition, cuticle cells, which contain amorphous protein, change to a shingle-like structure.

The sebaceous gland is above the zone in which hair hardening occurs. It is in the area of the exit of the sebaceous gland that the inner root sheath, which initially forms a mechanically supportive tube around the developing hair fiber, is degraded. After the degradation of the inner root sheath, the mature hair emerges from the skin.

From the above discussion, it should be apparent that hair is a complex appendage of skin and that hair from different areas of the adult human can exhibit different properties and be exposed to different physiological environments.

At the time of this writing, 54 functional keratin genes have been identified [55]. Also, there is a complex pattern of expression of hair keratins in the human anagen follicle. The pattern is shown schematically in Figure 5.

Although keratin is considered an impediment to hair drug testing, as a protein complex, keratin plays an important role in the incorporation of drugs and their metabolites into hair.

B. Glands Adjacent to Human Hair Follicles

Several mechanisms by which drugs and/or their metabolites are incorporated into hair will be presented. Incorporation will be discussed in the following section of this review. However, prior to discussing incorporation mechanisms, a brief presentation on apocrine sweat, eccrine sweat, and sebum, all of which may contact hair prior to harvesting it for analysis, is essential and is described in the short summary sections below.

1. Eccrine Sweat Glands

With exception to the external auditory canals, the vermillion lips, and some areas of the female genitalia, about 1.5-4 million eccrine sweat glands, are distributed over the entire cutaneous surface. The highest concentrations of eccrine sweat glands are found on the palmoplantar surfaces. Eccrine sweat glands are developmentally and functionally different from the apocrine glands. An illustration of the eccrine sweat gland and its physical relation to the hair follicle, which in the diagram contains an apocrine gland, is shown in Figure 6.

Eccrine sweat is a sterile, dilute electrolyte solution that contains primarily sodium chloride, potassium, and bicarbonate. Other components include antimicrobial peptides such as dermcidin, proteolytic enzymes, glucose, pyruvate, lactate, urea, ammonia, calcium, amino acids, epidermal growth factor, cytokines, and immunoglobulins.

A multitude of studies on drugs and drug metabolites in sweat has been summarized in a 2013 review article by de Giovanni and Fucci [66]. It was noted in the review that sweat is one of the few biological matrices in which heroin is readily detected. Anumber of drugs such as ethyl alcohol [34], amphetamines [218], cocaine [196], phencyclidine [165], and methadone [78] have been detected in sweat; many times, they are found in concentrations greater than in blood. Since hair for analysis is often in close, long-term contact with eccrine sweat, sweat as a fluid from which drugs and their metabolites may be transferred is and has been considered [92,93]. Thus, eccrine sweat must be considered as a potential source of drug/drug metabolite found in hair.

2. Apocrine Glands

As can be visualized easily from the above discussion on the development of the hair follicle, apocrine glands may be associated with a hair follicle. In the human embryo, apocrine glands are present over the entire skin surface. However, in the adult human, apocrine glands are restricted to the axillae, anogenital region, periumbilical region, areolae, nipples, and the vermillion border of the lip [186].

Apocrine glands secrete very small amounts of an oily fluid or sweat. The apocrine sweat is sterile, odorless, and viscous with a pH of 5.0-6.5. The apocrine sweat contains precursors of odiferous substances such as cholesterol, triglycerides, fatty acids, cholesteryl esters, and squalene. Apocrine sweat also contains androgens, carbohydrates, ammonia, and ferric ion. Although initially sterile and odorless, skin bacteria modify the sweat to give it its typical rancid (Corynebacterium spp.) or sweaty (micrococcus spp.) odor referenced as bromhidrosis or body odor.

Apocrine glands are secreted by a "decapitation" or "pinching off' method which releases the luminal portion of the cell. Apocrine glands also use merocrine or exocytosis of vesicles and holocrine or plasma membrane rupture for their secretory process.

Where apocrine glands are present, the deposition of drugs/drug metabolites into apocrine sweat should be considered as stated above for eccrine sweat.

3. Apoeccrine Sweat Glands

Apoeccrine sweat glands have been described in some studies, but not in all studies [186]. Since the existence of apoeccrine glands is questionable and if present, their function appears similar to that of apocrine glands. Thus, apoeccrine glands will be considered no further in this brief review.

4. Sebaceous Glands

The development and distribution of the sebaceous glands has been discussed above. Except for ectopic or free sebaceous glands of the vermillion lips, anogenital mucosae, areolae, and eyelids, sebaceous glands are associated with hair follicles. Sebaceous glands produce sebum via holocrine secretion. Consequently, approximately 14 hours of transit time is required for sebum to pass through the follicular canal to the skin [59,60]. Sebum consists of free fatty acids, wax and sterol esters, triglycerides, and squalene. Sebum has multiple functions including hormonal and immune modulation. Sebum production is a sensitive indicator of androgenic activity i.e. it increases at the time of puberty and decreases in later adulthood; especially in postmenopausal women. Sebaceous follicles are rich in microorganisms including Malassezia spp., Staphylococcus epidermis, and Propionobacterium spp.

Cocaine and its metabolites BZE and ecgonine methyl ester [93], and cocaine and BZE [125], have been measured in sebum. Codeine and norcodeine have also been detected and measured in sebum [93]. Although less is known about the drug/drug metabolite composition of sebum than is known about eccrine sweat, drug and drug metabolite can certainly be detected in sebum. Thus, sebum also needs to be considered when investigating the incorporation of drug and metabolite into hair [92].

II. METABOLISM AND PHARMACOKINETICS

As a general subject, the metabolism and pharmacokinetics of xenobiotics in humans are well-covered in numerous comprehensive references [35,164]. Thus, only a brief synopsis of metabolism and pharmacokinetics as it applies to commonly encountered drugs that may be abused will be presented. As the goal is to review drugs and their metabolites in human hair as viable markers of use and abuse, metabolism will be discussed only briefly, followed by a short presentation on the post-absorptive distribution of drugs and metabolites. This is with emphasis on distribution into hair. Following the general presentation, individual drugs/drug classes will be covered mostly as their metabolism applies to the potential or actual incorporation of metabolites and the parent substance into hair.

A. Drug/Drug Metabolite Distribution into Hair

Referring to Figures 3 and 7, it is readily apparent that in order for a drug and/or its metabolite in blood to be incorporated into a growing hair shaft, the drug or metabolite must cross several membranes or a protein barrier unless transport is through the interstitial fluid between layers or columns of cells (paracellular). Should a drug or its metabolite be present in sebum and incorporate into hair by contact of the sebum with the hair shaft, even a membrane and/or a protein barrier must be crossed for incorporation to take place. The same applies to the incorporation of drug and/or metabolite when the hair shaft is bathed with eccrine or apocrine sweat that contains the drug and/or drug metabolite. Reference [35] addresses numerous issues associated with membrane transport. A similar review may be found in reference [123]. Although both references cover the topic of membranes and xenobiotic passage through membranes, a brief synopsis is in order before proceeding with drug/drug metabolite membrane passage and ultimately incorporation into hair.

The lipid bilayer is the primary feature of the animal cell membrane [2]. The majority of the lipids in a cell membrane are phospholipids, glycolipids, and cholesterol. The phospholipids are mostly phosphatidylethanolamine and phosphatidylglycerol, which are amphipathic lipids. The lipids in a cell membrane are arranged so that the hydrophobic tails of the lipids face inward, forming a non-polar inner region. On the other hand, the hydrophilic heads of the lipids face outward into the primarily aqueous environment on both the inside and the outside of the cell. Therefore, an illustration of the lipid bilayer for a red cell and in a rafting or specialized area is presented in Figure 7.

The overall thickness of the lipid bilayer is about 7-9 nM. As shown in Figure 7, the lipids all have the same straight-chain, saturated hydrocarbon tail. However, when lipids with an unsaturated tail are inserted, there is some disruption of the membrane allowing more facile passive (no energy expended; follows Fick's law of diffusion) or active (requires an expenditure of cellular energy for transport) transport across the membrane. Numerous proteins are inserted into the lipid bilayer. Some proteins traverse the entire lipid bilayer allowing them to function as receptors or allowing the formation of aqueous pores, ion channels, and transporters. The lipid bilayer is differentially permeable and controls what enters and exits individual cells.

Most drugs and toxicants cross the lipid bilayer membrane by passive diffusion (transcellular diffusion). Such diffusion depends primarily on the drug's or toxicant's octanol-water partition coefficient (P). The greater is P; the more facile is diffusion. If the drug or toxicant is permanently charged, very little, if any, can cross the lipid bilayer by passive diffusion. Ifthe drug or toxicant can exist in both ionized and un-ionized forms; usually depending on the pH of the medium in which it is dissolved, it will be the neutral or un-ionized form that crosses the lipid bilayer by passive diffusion. Thus, when looking at the passive diffusion of an ionizable molecule across the lipid bilayer, the pH on both sides of the membrane needs to be considered. Passive transport, which is where the substance passes from an area of high concentration to an area of lower concentration, can be simple. Passive transport also can be facilitated by a carrier-mediated transport process in which there is no input of energy and therefore actually enhanced movement is down a chemical gradient as in simple passive diffusion. In addition to simple and carrier mediated diffusion through a lipid bilayer, some small (approximately 600 Da) hydrophilic organic molecules may penetrate cells through aqueous pores in the cell membrane using a process referenced as filtration.

When looking at a layer of cells rather than an individual cell membrane, small organic molecules may also cross the cellular layer by passive diffusion between cells using interstitial fluid (paracellular transport), as stated above, eliminating the need to cross the lipid bilayer twice.

In addition to diffusion into a cell through its membrane, active transport also exists, which may play an important role in some instances. Active transport has a direct requirement for energy, movement against a gradient, saturability, selectivity, and competitive inhibition by co-transported compounds. Membrane transport that directly couples with ATP (adenosine triphosphate) hydrolysis is termed as primary active transport. In secondary active transport, the transport of one solute against its concentration gradient across a biological membrane is driven by the transport of another solute in accordance with its concentration gradient. Symporters, also termed co-transporters, transport the two solutes in the same direction. Antiporters or exchangers transport the two solutes in opposite directions. Transporters are critical to cell homeostasis. Consistent with the critical role of transporters, there are about 2000 transporter genes in the human genome or approximately 7% of the total number of human genes.

In one reference directed at hair drug testing [170], the authors summarized the main variables important to the transport of drug molecules into the matrix cells and the melanocytes of the hair follicle, which gives some idea of the complexity of the processes involved in the incorporation of drugs and their metabolites into hair. These variables are:

* Nature of the membrane

* Blood flow (sink vs. non-sink)

* Plasma protein binding of the drug/metabolite molecule

* Lipid solubility of the drug/drug metabolite (log P)

* Ratio of the ionized to non-ionized drug/drug metabolite molecules ([pK.sub.a], Henderson-Hasselbalch equation)

* Molecular size and geometry of the drug/drug metabolite molecule

* Micro-climate (pH, temperature)

* Concentration gradient

* pH gradient

B. Basic Metabolism

With only a few notable exceptions, almost all drugs that enter the human body by any number of available pathways are metabolized or modified chemically. As was true for drug/drug metabolite distribution above, there are numerous references that describe drug metabolism in great detail [69,164,191]. Thus, for this review, only the metabolism of selected drugs and general points of toxicology which are useful for hair analyses will be addressed.

In the formative years (1950s to the 1990s) of modern toxicology, very little theory was available and most information was anecdotal. However, Parkinson et al. [164] have organized principles or rules that apply in a majority of cases into points. Only the first 7 points appear to be applicable to the incorporation of drugs and their metabolites into hair and, thus, are the only points listed out of the original 29 [164].

(1) Although exceptions to this rule exist, xenobiotic (drug) transformation or drug metabolism is the process of converting lipophilic or fat-soluble chemicals, which are readily absorbed, into hydrophilic or water-soluble chemicals and which are readily excreted into urine or bile.

(2) The biotransformation of xenobiotics can be divided into four categories by the reaction they catalyze: hydrolysis, reduction, oxidation, and conjugation.

(3) In general, individual xenobiotic-transforming enzymes are located on a single organelle.

(4) In general, xenobiotic transformation is accomplished by a limited number of enzymes with broad substrate specificity.

(5) Hydrolysis, reduction, and oxidation (Phase I) expose or introduce a functional group that can be converted to a water-soluble conjugate (Phase II).

(6) Oxidation, reduction, hydrolysis, methylation, and acetylation generally produce only a moderate increase in water-solubility while glucuronidation, sulfonation, glutathionylation, and amino acid conjugation generally cause a marked increase in hydrophilicity.

(7) Not all biotransformation reactions are catalyzed by mammalian enzymes. Some are catalyzed by gut microflora.

In addition to these general points, the metabolism of selected drugs (cannabinoids, cocaine, amphetamines, opiates and opioids, phencyclidine, benzodiazepines, and ethanol) is reviewed below.

1. Cannabinoids

The basic scheme for the metabolism of the major psychoactive component of marijuana, [[DELTA].sup.9]tetrahydrocannabinol (THC), is presented in Structure 1 [29].

Although the glucuronide conjugate of 11-nor-[[DELTA].sup.9]tetrahydrocannabinol-9-carboxylic acid (THCA) is shown in the above reaction cascade as a terminal product, conjugation may occur with the parent substance, the active 11-hydroxy metabolite, and the 8,11-dihydroxy metabolite. The metabolite 11-hydroxy-THC is conjugated through UDP-glucuronosyltransferase (UGT) 1[[DELTA].sup.9] and 1A10, while THCA is conjugated by UGT 1A3 and 1A1 [133].

The metabolism of the parent drug THC exemplifies Points (1), (2), (5), and (6) above. In the metabolic cascade for THC, polarity and, thus, water-solubility generally increases from top to bottom; especially in the formation of THCA-glucuronide. Likewise although not shown, when any one of the compounds in the cascade above THCA forms a glucuronide, water-solubility is markedly increased (Point 5).

If the active parent substance, THC, is the analyte chosen for hair analysis, it has been shown that donor or patient hair can be contaminated in vitro by marijuana smoke [207]. Even though concentrations of THCA are low in hair, THCAhas been shown to be a reliable indicator of marijuana use; especially if the hair has been washed properly [80].Although the metabolite of THC, THCA, has been shown to be a reliable indicator of marijuana use and devoid of issues associated with external contamination, Pichini has recently demonstrated that THCA-glucuronide [166] can be detected in hair and used as a biomarker. In several cases in the Pichini paper where parent THC was present, but THCA (10/20) could not be detected or was less than the LOQ, THCA-glucuronide (16/20) was detectable and quantifiably reportable. It is notable that THCA does not appear to be significantly formed or present in the smoke produced when marijuana is combusted [141,145].

[FORMULA NOT REPRODUCIBLE IN ASCII]

2. Cocaine

The major metabolic cascade for cocaine is shown in Structure 2 [14]. It is notable that the metabolite/ impurity picture with cocaine is far more complicated than the simplistic sequence outlined. Impurities in cocaine include but are not limited to cinnamoylcocaines, formylcocaine, truxillic acids, truxillines, pseudoecgonine, tropacocaine and its derivatives, cuscohygrine, hygrine, hydroxycocaines, trimethoxycocaines, and the metabolites shown in the above cascade [148]. Solvent residues and cutting agents may further cloud the cocaine picture. Refining crude cocaine may lead to compounds such as N-formylnorcocaine, N-benzoylnorecgonine methyl ester, and N-norecgonine methyl ester, which may arise from permanganate used in the process [149].

Cocaine can contaminate hair in vitro either through contact with cocaine as the free base or hydrochloride salt or through contact with smoke that contains free base cocaine. Although metabolites of cocaine would appear to be a solution to the external contamination issue for cocaine, benzoylecgonine (BZE), ecgonine methyl ester, norcocaine, have all been detected as impurities in illicit cocaine [150]. Thus, the commonly detected metabolites of cocaine probably are insufficient for demonstrating cocaine use through hair testing.

[FORMULA NOT REPRODUCIBLE IN ASCII]

In addition to the metabolites presented above, cocaine also oxidizes in vivo to meta (m) [129] and para (p) hydroxyl metabolites where the hydroxyl groups are on the phenyl ring of the benzoyl moiety [197] as shown in Structure 3. The formation of p-hydroxycocaine and m-hydroxycocaine in the microsomes of mice and other animals has been demonstrated [220]. In the same study, p-hydroxycocaine was shown to be as pharmacologically active in mice as the parent cocaine. Since the hydroxyl metabolites of cocaine may exist as glucuronide or sulfate conjugates [228], Zhang & Foltz used hydrolysis although hydrolysis was not used in at least three urine hydroxybenzoylecgonine methodologies [49,108,176] and isolation was still successful. Also, hydroxymethoxybenzoylmethylecgonines have been identified as cocaine metabolites [199]. In addition, in the rat, dimethoxyhydroxycocaine [89] can be identified as a cocaine metabolite. In the same study on rat urine, N-hydroxynorcocaine, m-hydroxycocaine, and m-hydroxy-p-methoxycocaine were found to be present as conjugates and was extracted after treatment with glusulase. When ethyl alcohol is used with cocaine, ethyl analogues of the hydroxycocaines were also found [198]. The m- [61] and p-hydroxybenzoylecgonine along with norbenzoylecgonine [108,176] have been used as conclusive evidence that a cocaine-metabolite positive urine specimen is positive due to the actual use of cocaine rather than due to the addition of cocaine to urine followed by in vitro hydrolysis to BZE. m-Hydroxycocaine has been identified as a cocaine impurity, while p-hydroxycocaine and o-hydroxycocaine have not [212].

[FORMULA NOT REPRODUCIBLE IN ASCII]

The m- and p-hydroxyl metabolites of BZE [167] and BZE and cocaine [62] have been used to improve the detection rate of cocaine in meconium. It is of interest that out of 28 subjects with usable data in the Elsohly study, no ortho-hydroxycocaine, ortho-hydroxybenzoylecgonine, or para-hydroxycocaine was reported.

The ring hydroxylated cocaines and the corresponding BZE hydrolysis products are only a small fraction of a dose of cocaine regardless of the route of administration as summarized in Table 3 [49].

In saliva, which reflects plasma, which may influence hair levels directly, a mean maximum concentration ([C.sub.max]) of p-HOCOC of 367 [+ or -] 80 ng/mL was observed at an average of 1.3 [+ or -] 0.6 hours ([T.sub.max]) in 6 individuals after a multi-week protocol of single and multiple cocaine doses [94]. In a separate study that involved individuals with recent high-dose cocaine experience, m-HOCOC, p-HOCOC, m-HOBZE, and p-HOBZE were observed in the plasma of some of the study volunteers prior to the start of the study [144]. Following low (75 mg/70 kg) and high (150 mg/70 kg) subcutaneous dosing with cocaine hydrochloride, the hydroxyl metabolite pattern was found (see Table 4) [109].

During hydrolysis of the methyl ester group of the hydroxycocaines, the corresponding hydroxylbenzoylecgonines can be formed. Whether phenyl ring hydroxylation occurs with the parent cocaine, the hydrolysis product BZE, or both is somewhat inconsequential. The important analytical observation is that ring hydroxylated cocaines and BZEs can also be found in illicit and, sometimes, licit cocaine samples [37,149] as well as mammalian metabolites of cocaine. Hydroxycocaines also may be formed by the action of hydrogen peroxide and other bleaching/oxidizing agents on cocaine in user hair which lowers the amount of cocaine originally present. Also, aqueous agents such as [H.sub.2][O.sub.2] remove non-oxidized cocaine and cocaine oxidation products from hair, further reducing the total amount of cocaine originally present [40,53,95,205,227].

The o-, m-, and p-hydroxycocaines have been shown to be able to demonstrate that cocaine in a hair sample is due to use, external contamination, and/or both. Schaffer et al. [185] used an extensive hair wash. The final wash was retained and analyzed for cocaine. Any cocaine found in the final 60-minute wash was multiplied times five deliberately to overestimate the amount of drug that would be removed by actually continuing to wash for another 5 hours. If a sample was found contaminated and/or excessively damaged, the result could be reported as invalid or unsuitable [81]. The extended wash was found to completely remove hydroxycocaines formed by the in vitro action of peroxide. A value of [greater than or equal to] 2 for the ratio of p-hydroxycocaine to o-hydroxycocaine and m-hydroxycocaine to o-hydroxycocaine was proposed to provide additional protection against a false positive hair cocaine result due to in vitro contamination by cocaine. In a separate study that examined drug chemists' hair, Morris-Kukoski et al. [153] analyzed the extensive washing of hair, and also employed the following formula to determine an adjusted, final cocaine value:

[Cocaine.sub.final] = [Cocaine.sub.hair] - (5 x [Cocaine.sub.wash])

In the above formula, which is similar to that used by Schaffer et al. and Hill et al., [Cocaine.sub.hair] is the measured cocaine value in hair and [Cocaine.sub.wash] is the cocaine measured in the final hair wash. One outcome of the study was that no hydroxycocaine metabolites were detected in any of the hair washes. None of the drug chemists' hair digests contained norcocaine, cocaethylene, or any of the hydroxycocaine metabolites. Based on their results and previous work, the cascade presented in Figure 8 for the reporting and interpretation of hair cocaine results was developed.

3. Amphetamines

The basic metabolic cascade for both amphetamine (AMP) and methamphetamines (MAMP) [12,22,23] is shown in Structure 4. Hydroxylated compounds in the above scheme also may be conjugated. Specifically, p-hydroxymethamphetamine forms sulfate and glucuronide conjugates [192,193,204]. It is notable that p-hydroxymethamphetamine along with the o- and m-positional isomers can be produced by the reaction of methamphetamine with hydrogen peroxide ([H.sub.2][O.sub.2]) which is a main component of hair dye and decolorant treatments [205].

In vivo, the p-hydroxylation of methamphetamine (and, by extension, amphetamine), appears to be facilitated primarily by CYP2D6 [115]. CYP2D6 is known to be highly polymorphic with over 100 allelic variants identified as of this writing [84]. Variants such as CYP2D6*3A and *4A show no activity in vivo or in vitro. CYP2D6 lacks activity in 5-14% of Caucasians, 0-5% of Africans, and 0-1% of Asians [229]. In addition to polymorphic variants that demonstrate little or no activity, some drugs such as paroxetine may inhibit a normally active CYP2D6 leading to a phenotype that demonstrates reduced activity [87,164]. Thus, unless other enzymes that catalyze the oxidation of AMP and MAMP to the corresponding p-hydroxy metabolites can be substituted for CYP2D6, the p-hydroxy metabolites may be reliable indicators of AMP and/or MAMP use in only about 90-95% of donors tested regardless of the matrix employed. Although the sulfate or glucuronide conjugates might be chosen as metabolites to eliminate the possibility of the identification of p-hydroxy compounds formed from bleach and amphetamines present due to environmental contamination [206], the p-hydroxy derivative must be formed and present before the sulfate or glucuronide can be produced. In urine, which may not reflect the metabolite pattern in hair, the distribution of 4-hydroxyamphetamine (4-HOAMP) presented in Table 5 was found [142] using 121 amphetamines-positive urines out of approximately 1,000 Federal Mexican truck drivers.

[FORMULA NOT REPRODUCIBLE IN ASCII]

The incorporation of p-hydroxymethamphetamine (p-HOMAMP) (ICR; [Hair]/AUC, which is the ratio of drug concentration in hair divided by the area under the curve in plasma expressed as [micro]g min/mL) into rat hair was significantly worse than the ICR for MAMP itself (0.07 for p-HOMAMP vs. 0.13 for MAMP) [155]. The poor ICR for para-HOMAMP relative to MAMP was thought to be a combination of the rapid disappearance of p-HOMAMP post-injection relative to MAMP and the two compounds' melanin affinity and lipohilicity (Table 6). HOAMP and AMP showed a similar pattern (Table 6) [156,158].

The presence of hydroxyamphetamines in hair is commonly viewed as being the result of HOMAMP and/ or HOAMP, passing from blood into the developing hair shaft and/or passing from the stratum corneum into the cuticle of the hair shaft (see "Incorporation of Xenobiotics Into Hair"). However, from the work of Potsch et al. [170], the hydroxycompounds may be the result of oxidation of the precursor drugs (MAMP and AMP) in the hair follicle. Likewise, HOAMP possibly may be formed from HOMAMP in the hair follicle.

The major metabolic cascade for methylenedioxymethamphetamine (MDMA) is given in Structure 5 [115,127, 188,189,193,194]. The demethylenation of MDMA to 3,4-dihydroxymethamphetamine (HHMA) is regulated by CYP2B6, CYP2D6, CYP1A2, CYP3A4, and other CYP-independent mechanisms [36,112,132]. Other CYP450 enzymes such as CYP2C19 may also be involved [175]. The demethylation of MDMA to MDA is governed mainly by CYP1A2 [132] or possibly CYP2B6 [36,175]. 0-Methylation is catalyzed by catechol-O-methyltransferase or COMT. Furthermore, HMMA, HMA, 3,4-dihydroxymethamphetamine, and 3,4-dihydroxyamphetamine are all subject to conjugation prior to elimination [175]. It is notable that in Ref. [24], the time course of the appearance of dihydroxymethamphetamine and hydroxymethoxymethamphetamine is opposite that described in Structure 5.

It is worth noting that the hydroxyamphetamines are structurally similar to several of the naturally produced catecholamines [179], which may become important analytically. The structures of the most similar compounds are displayed in Structure 6 although numerous other like compounds occur naturally.

Based on the above brief presentation, HHMA and HMMA and/or their sulfate and glucuronide conjugates would appear to be good candidates for metabolites specific to the use of the MDMA. Likewise, the MDA metabolites HHAand HMAas conjugates appear to be good candidates.

4. Opiates and Opioids

The basic metabolic cascade for codeine, morphine, and diacetylmorphine [19,56] is shown in Structure 7.

Morphine also forms a 3,6-diglucuronide and a 3-ethereal sulfate. Normorphine also forms a 3-glucuronide and a 6-glucuronide. As minor pathways, codeine can be converted into hydrocodone [44,56,163] and morphine can metabolize to hydromorphone [46,56,83].

The transformation of morphine to normorphine, which is a minor pathway, is catalyzed mostly by CYP3A4 and, to a lesser extent, CYP2C8 [173]. CYP3A4 can demonstrate some polymorphism, but is considerably less polymorphic than CYP2D6 [84] which shows substantial polymorphism. In one publication, morphine was stated to be 6-glucuronidated not only by UGT2B7, but also by UGT1A1 and 1A8, which also catalyzed morphine-6glucuronidation at relatively low morphine concentrations (<100 m [micro]M) [161]. From a separate study [200], UGT1A1, 1A3, 1A6, 1A8, 1A9, 1A10, and 2B7 all catalyzed the formation of morphine-3-glucuronide, but only UGT2B7 formed morphine-6-glucuronide.

When reviewing morphine as a parent drug incorporating it into rat hair, morphine is a fairly poor incorporator. In one reference, morphine's incorporation rate or ICR (concentration in hair ([hair]) in ng/mL/area under curve (AUC) in plasma in [micro]gmin/mL) was stated as 0.03 [158]. As a reference point, cocaine's ICR in the same reference is 3.6 or 120x that of morphine. Cocaine has one of the highest ICR's of any common drug. In contrast, BZE, which has one of the worst ICR's except for THCA (vide supra), has an ICR of 0.003. Thus, morphine's ICR is only 10x that of one of the worst ICR's (BZE), but less than 1% that of the best ICR (parent cocaine).

[FORMULA NOT REPRODUCIBLE IN ASCII]

Regardless of morphine's ICR, morphine has been detected in hair in numerous references [1,3,10,33,50, 51,64,70,72,82,86,111,113,121,124,128,136,137,138,140, 143,154,168,177,181,195,203,224]. However, morphine associated with hair may be present as an environmental contaminant or due to actual use of morphine or one of morphine's precursors such as codeine or 6-acetylmorphine as was true for amphetamines, cocaine, and THC, above. Thus, it would be prudent to search for a suitable morphine metabolite that can serve as a surrogate.

[FORMULA NOT REPRODUCIBLE IN ASCII]

[FORMULA NOT REPRODUCIBLE IN ASCII]

Referring to the metabolic scheme for morphine above, normorphine might be a good candidate to demonstrate use rather than just exposure since normorphine is not marketed as a drug. However, one drawback to choosing normorphine would be that its polarity should be slightly greater than morphine's polarity. An increased polarity might lead to a lessened ICR relative to morphine itself. If conversion of morphine to normorphine occurs in the hair follicle, the increased polarity should not be an issue. Another potential shortcoming associated with the use of normorphine is that the conversion of morphine to normorphine requires CYP3A4. As stated above, although CYP3A4 is not nearly as polymorphic as CYP2D6 [84], there is some potential for polymorphism resulting in decreased enzymatic activity. Such reduction might result in a decreased production of normorphine from morphine unless other enzymes are present in sufficient activity to compensate for the CYP3A4 deficiency. Likewise, any substance that inhibits CYP3A4 (e.g., ketoconazole or erythromycin [164]) could result in a phenotype that resembles a genotypic CYP3A4 deficiency. In actual practice, in a study of five subjects who received three doses of cocaine hydrochloride (75 mg/70 kg, subcutaneous) and three doses of codeine sulfate (60 mg/70 kg, oral) on alternating days in week 4 of the study (low-dose week) with a repeat double-dose in week 8 (high-dose week), normorphine (0.2 ng/mg) was present in only one specimen collected one week after administration of high doses of codeine [92]. In another study of five individuals admitted to an addiction research center, cocaine and BZE were positive, but codeine was not detected; in addition, morphine was found in 3 out of the 5 participants with no norcodeine and no normorphine detected [82]. In a study of a female who cultivated opium poppies in her garden and 3 heroin users, morphine and codeine were found in the hair of all 4 subjects, but normorphine was only found in the hair of 2 out of the 3 drug offenders [122].

In their study of the incorporation of codeine and metabolites into hair, Gygi et al. [75] found morphine glucuronide levels of 0.67 [+ or -] 0.08, 1.04 [+ or -] 0.37, and 13.80 [+ or -] 3.60 ng/mg hair in Sprague-Dawley, Dark Agouti, and Long-Evans rats, respectively. The corresponding free morphine levels were 0.34 [+ or -] 0.04, 0.51 [+ or -] 0.11, and 14.4 [+ or -] 1.81 ng/mg hair. Morphine glucuronide levels were obtained by difference (total morphine minus free morphine) using acid hydrolysis. In a separate, but related study by Gygi et al. [74], acid hydrolysis of glucuronide conjugates was compared to enzymatic hydrolysis and was found to be similar. The increase in morphine after acid hydrolysis was found to be due to the hydrolysis of glucuronide conjugates rather than increased extraction of morphine from hair after acid treatment. In a separate study, Toyo'oka et al. [211] directly determined morphine and the 3-[beta]-D-and 6-[beta]-D-glucuronides incorporated into Dark Agouti rat hair using reversed-phase high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS).

The metabolic cascade for codeine is incorporated into the same scheme used for morphine above. Codeine can be converted into norcodeine, which is not a marketed drug, primarily under the influence of CYP3A4. Like normorphine, norcodeine can also form the 6-glucuronide, which is not a marketed drug. Codeine itself can form the 6-glucuronide, which is not also a marketed drug. Codeine also can be transformed into morphine, which is a highly marketed drug and is not helpful to demonstrate use as opposed to simple exposure.

Although the ICR for codeine was not stated in the study of Nakahara et al. [158], which included morphine, codeine would be expected to have a higher ICR than morphine due to covering of the phenolic 3-hydrogen with a methyl group. Thus, this makes the codeine molecule much less polar than morphine and less acidic.

Regardless of codeine's ICR, codeine also has been detected in hair in numerous references which are essentially the same as the references that cite morphine [3,10,33,50,51,64,70,72,82,86,111,113,121,124,128,136, 137,138,140,143,154,168,177,181,195,203,223]. However, codeine which is associated with hair may be present as an environmental contaminant or due to actual use of codeine, as was true for amphetamines, cocaine, morphine, and THC above. Thus, it would be prudent to search for a suitable codeine metabolite that can serve as a surrogate.

In the studies of Gygi et al. [74], codeine glucuronide was not found because the rat forms relatively little codeine-6-glucuronide. However, in later studies by Lee et al. [121], a method was developed that included norcodeine, which was demonstrated in rat hair.

Hydrocodone and hydromorphone [20,21,56] are interrelated through the scheme shown in Structure 8. The transformation of hydrocodone to norhydrocodone [45], which is about equal to the conversion to hydromorphone [48], is catalyzed mostly by CYP3A4 [85,134]. The transformation of hydrocodone to hydromorphone is catalyzed mostly by CYP2D6 [85,136]. CYP3A4 can demonstrate some polymorphism, but is considerably less polymorphic than CYP2D6 [84]. The reduction of hydrocodone yields 6[alpha]- & 6[beta]-hydrocodol. The product 6[alpha]-hydrocodol is also known as dihydrocodeine, which is a marketed drug.

Hydrocodone has been detected in hair and reported in several publications [1,9,91,130,146,203,225]. Hydromorphone, which is a separately marketed drug, was included as a standard in the majority of the methods for hydrocodone [91,130,146,225]. Norhydrocodone, which is not a separately marketed drug, was not noted as a standard in any of the methods for hydrocodone. As was true for the previously discussed opiates, a metabolite of hydrocodone that is not a separately marketed drug (e.g., hydromorphone, dihydrocodeine) needs to be identified. It is notable that in one of the studies by Moore et al. [146], hydrocodone was identified at 130-15,933 pg/mg hair in 24 volunteers who stated previous use of hydrocodone. However, only 4 of the 24 hydrocodone-positive hair samples were positive for hydromorphone (59-504 pg/ mg hair). It was also noted in the Moore et al. study [146] that hair concentrations of hydrocodone from five self-reported codeine users (codeine = 575-20,543 pg/ mg hair), hydrocodone, was present at concentrations of 592-15,933 pg/mg hair. However, neither morphine nor hydromorphone was present.

[FORMULA NOT REPRODUCIBLE IN ASCII]

[FORMULA NOT REPRODUCIBLE IN ASCII]

Even though norhydrocodone is not mentioned in current hair drug testing publications, it is a natural choice. Although parent hydrocodone is reported to conjugate only to a minor extent [48], the hydrocodone glucuronide and/ or sulfate may be worth investigating.

When administered as a separate drug, hydromorphone's major metabolic pathway involves the formation of hydromorphone-3-glucuronide via UGT2B7 and, to a lesser extent, UGT1A2 [56]. A minor metabolic route for hydromorphone is the reduction to the 6[alpha]- and 6[beta]-hydromorphols, which are further conjugated as the 3-glucuronides. Another minor metabolic pathway for hydromorphone is A-demethylation, which has been reported to be catalyzed primarily by CYP3A4, 3A5, 2C9 and 2D6, but not CYP1A2 [31].

Fruitful pursuits for the identification of unique metabolites of hydromorphone that may show the use of the drug as opposed to simple environmental exposure probably lie in hydromorphone-3-glucuronide although norhydromorphone may be present in sufficient quantity in hair to make it a useful metabolite. Hydromorphone-3sulfate is a metabolite of hydromorphone [114] and may be worth investigating as well.

As was true for hydrocodone, there is a metabolic scheme for oxycodone and oxymorphone [26,27,56] that is separate from codeine and morphine. This is shown in Structure 9.

As would be anticipated from previously described studies on hydrocodone and codeine, the most prominent metabolite of oxycodone in blood is noroxycodone, which is produced primarily through the actions of CYP3A4/5 [56]. Oxymorphone, which is a separately marketed drug, is a relatively minor metabolite of oxycodone produced primarily by the actions of CYP2D6, which can show considerable polymorphism. The noroxymorphone seen after ingestion of oxycodone appears to be derived mainly from noroxycodone with a negligible amount coming from the metabolism of oxymorphone [56,118,119]. Also, as anticipated, oxycodone can be reduced to 6[alpha]- and 6[beta]-oxycodol [8]. Phase II conjugation occurs for oxymorphone and noroxymorphone, but not to any appreciable extent for oxycodone or noroxycodone [47,118,119].

Oxymorphone is essentially at the end of the metabolic chain for oxycodone. Metabolically, there is very little that can be done to oxymorphone other than N-demethylate, which is minimal, form a conjugate, or be reduced to oxymorphols which are then conjugated.

Other than the analytical methods of Moore et al. [147] and Jones et al. [91], little exists in the open literature on oxycodone and oxymorphone in hair. The primary metabolite of oxycodone, noroxycodone, should be a prime candidate to demonstrate the use of oxycodone as opposed to simple exposure to the drug. Noroxymorphone might be considered, but since it can arise from the metabolism of oxycodone or oxymorphone, it is probably not a viable choice to show oxycodone use.

Fentanyl, which is a powerful analgesic, has a completely different structure and metabolism from opiate analgesics. Fentanyl's metabolic cascade [17,56] is shown in Structure 10.

Not surprisingly, the conversion of fentanyl to norfentanyl is catalyzed by CYP3A4 and, possibly, CYP3A5 [73,88,116]. None of the metabolites of fentanyl is marketed as a separate drug.

Norfentanyl has been included in at least one chromatographic-mass spectrometric hair methodology for opioid analgesics [154].

5. Phencyclidine

The metabolic cascade for phencyclidine or PCP is shown in Structure 11 [28,182]. In human hair containing PCP, the findings of PCPdiol and PCHP are important indicators of active drug use [159,183]. Although t-PCPdiol was a minor metabolite in the hair of rats intoxicated with PCP, it was determined that t-PCPdiol was the major metabolite detected in human PCP users' hair. Whether PCHP and/or t-PCPdiol can be produced by the actions of hair bleaching and coloring formulae on PCP has not been demonstrated at the time of this writing.

[FORMULA NOT REPRODUCIBLE IN ASCII]

[FORMULA NOT REPRODUCIBLE IN ASCII]

6. Benzodiazepines

Although over 50 benzodiazepines are now marketed for human consumption [222], metabolic cascades for only two common benzodiazepines (diazepam and alprazolam) are represented in Structure 12 [15] or Structure 13 [11]. This is primarily because many 1,4-benzodiazepines used medicinally follow either the diazepam or the alprazolam metabolic pathway. Several major exceptions exist as will be noted in this section when they occur. Numerous other 1,4-benzodiazepines which are currently considered as only research chemicals also exist in addition to the benzodiazepines approved for medicinal use.

[FORMULA NOT REPRODUCIBLE IN ASCII]

[FORMULA NOT REPRODUCIBLE IN ASCII]

Due to the extent of metabolism of diazepam, it would appear that finding a metabolite that can be detected in hair to show use rather than environmental exposure should be a simple task. However, as can be seen from Table 7, the metabolites of diazepam are themselves marketed as separate drugs. Thus, the finding of diazepam and nordiazepam in a hair sample might be interpreted as detection of the parent drug and the major metabolite, or it could be interpreted as the finding of two separate drugs present due to environmental exposure.

Since the glucuronides of morphine and other opiates have been detected in hair [75], it would be reasonable to use the glucuronides of oxazepam and temazepam as markers of use. However, in at least one single-dose study [219] where oxazepam and temazepam glucuronides were included as analytes, the glucuronides were not detected (LOQ = 10 pg/mg hair for glucuronides). The failure to detect the glucuronides may be due to the small amount incorporated in a single dose. Thus, the determination of environmental exposure to diazepam and/or one of its separately marketed metabolites versus actual use of the drug may depend on ratios of the parent drug to metabolites rather than a unique metabolite.

Other common non-triazolo, non-nitro 1,4-benzodiazepines also follow oxidative metabolic pathways similar to that seen with diazepam. As shown in Table 8, opportunities for identifying a unique metabolite may exist in some but not all cases.

For the so-called trizolo 1,4-benzodiazepines, opportunities for the formation of unique metabolites increase. This is a result of the possibilities of the hydroxylation of the methyl group on the triazolo ring, oxidation at the 1-position, diazepine ring opening, and hydroxylation similar to that seen with the basic benzodiazepine nucleus as shown in Structure 13 and Table 9 for alprazolam [11].

When a 1,4-benzodiazepine contains a nitro group on the benzophenyl ring (clonazepam, flunitrazepam, nitrazepam), further opportunities for unique metabolites exist due to primarily the reduction of the nitro group to an amino group with subsequent formation of an acetamide. Clonazepam forms 7-aminoclonazepam by reduction of the nitro group. The metabolite 7-aminoclonazepan further forms 7-acetamidoclonazepam. As shown in Structure 14, the parent drug and the two metabolites in the metabolic cascade can be 3-hydroxylated and conjugated [13].

Nitrazepam also forms 2-amino-5-nitrobenzophenone and 3-hydroxy-2-amino-5-nitrobenzophenone through opening of the 1,4-diazepine ring [25] (see Structure 15).

Flunitrazepam demethylates on the 1,4-diazepine ring, forms a hydroxyl at the 3-position on the diazepine ring, and forms an amine by the reduction of the nitro group. The amine group formed by reduction of the nitro group of the benzodiazepine phenyl ring further forms an acetamide [18] (see Structure 16).

None of the metabolic products of the nitro benzodiazepines appears to be a separately marketed drug. The metabolic products of nitrobenzodiazepines which appear in the hair of those who have ingested the parent drug have been demonstrated in several studies [41,42,160,180].

7. Ethyl Alcohol (Ethanol, "Alcohol")

The primary metabolic scheme for ethyl alcohol is given in Structure 17 [16]. In addition to the major metabolic scheme for the biodegradation of ethanol presented above, ethanol is non-oxidatively converted into small amounts of ethyl glucuronide, ethyl sulfate, phosphatidyl ethanol, and fatty acid ethyl esters [32]. About 0.02-0.06% of consumed alcohol is transformed into ethyl glucuronide by activated glucuronic acid (UDP-GA or uridine diphosphate-glucuronic acid) and the enzyme UDP-glucuronosyl transferase [54,68,172].

[FORMULA NOT REPRODUCIBLE IN ASCII]

[FORMULA NOT REPRODUCIBLE IN ASCII]

As noted above, ethanol can form esters with free fatty acids (fatty acid ethyl esters or FAEE). Approximately 20 ethanol-fatty acid combinations, primarily from myristic, oleic, stearic and palmitic acids, can exist in natural human products. FAEEs are thought to be deposited in hair by three mechanisms--incorporation of the esters from systemic blood circulation or surrounding tissues into the cells of the hair root, diffusion of ethanol into the cells of the hair root where esterifi cation occurs, and synthesis of the ester in the sebaceous glands with subsequent distribution of the sebum onto the hair and incorporation into the hair. The main route of incorporation appears to be through sebum. The analysis of FAEEs in hair has been refined to make possible the distinction among alcoholics, social drinkers, and teetotalers [7,201]. Exposure of hair to ethanol in vitro can produce increases in FAEE [65]. Although shampooing, permanent waving, dyeing, bleaching or shading with routine frequency do not appear to affect FAEEs by causing false negative results, fatty acid esters which may be a component of some lotions and gels may result in false elevations of FAEEs. Also, ethanol in hair sprays, lotions, and deodorants will cause false elevations of FAEEs presumably through reacting with any free fatty acids that might be present or through transesterification of glycerides [76].

[FORMULA NOT REPRODUCIBLE IN ASCII]

Although less frequently cited in the open literature on hair testing, ethyl sulfate, which is another minor ethanol metabolite, has been proposed as a potential biomarker of intrauterine exposure to ethanol [151].

While ethyl glucuronide (EtG) remained stable in a control group, hair coloring and bleaching were found to decrease EtG significantly [53]. When hair was treated with alkaline hydrogen peroxide, especially when ammonia was present, EtG was shown to be significantly degraded. Even before significant degradation of EtG due to peroxide and ammonia began, the formation of cysteic acid from the cysteine in keratin was noted through the use of Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy or ATR-FTIR [4]. In a separate in vitro bleaching experiment, EtG was found to be a sensitive and specific marker of alcoholism, but strong bleaching was shown to cause false negatives. In addition, a bleaching solution was found to lead to heavy ion suppression that could be avoided using an appropriate solid phase extraction (SPE) procedure. In the same study, the results indicated that it is more likely that EtG is washed out of the hair rather than hydrolyzed with hydrogen peroxide [152]. In a separate reference [104], it was noted that chemical treatment or excessive washing can lead to the removal of some EtG, while FAEE are less sensitive to frequent washing.

EtG in hair has been compared to phosphatidylethanol in the whole blood [30]. EtG in hair was found to be a better post mortem marker of chronic alcohol abuse than phosphatidyl ethanol in blood. However, it was noted that an ethyl glucuronide level in hair below the cutoff does not completely eliminate previous alcohol abuse.

The combined use of EtG and FAEE for the determination of chronic alcohol consumption status has been compared in several studies [104,172,201]. In general, an elevated FAEE in the presence of a normal EtG is due to hair care products that contain FAEEs and/or ethanol. It is notable that body hair is useful for EtG and FAEE testing except pubic hair, which in orders of magnitude is higher probably due to the proximity of pubic hair to urine [172]. In one anecdotal case [102] where EtG was excessively elevated in the presence of normal FAEEs and normal blood chemistries, the elevated EtG was determined due to the use of an herbal hair lotion that was an ethanolic plant extract. In one study, there was no significant effect of bleaching and dyeing on FAEEs. Hair gel and wax, oil or grease showed no significant effect on either EtG or FAEEs, while bleaching showed a decrease of 80% over non-bleached hair. Likewise, dyed hair showed a 63% reduction of EtG over undyed hair. The regular use of hairspray containing ethanol approximately doubled FAEEs relative to hair not treated with an alcohol-containing hairspray [201].

The Society of Hair Testing has issued a consensus statement on hair testing for chronic excessive alcohol consumption [100].

III. INCORPORATION OF XENOBIOTICS INTO HAIR

Although the actual mechanisms by which drugs and/ or their metabolites are incorporated into human hair is not the main subject of this review, a brief discussion is in order.

From Figure 6, it is quite apparent that the human hair follicle is well-vascularized, which should help facilitate the transfer of drugs and/or drug metabolites from blood to the growing hair shaft. However, Henderson noted that the finding of drugs and/or their metabolites in human hair is not simply the result of passive transfer from blood to the hair bulb where keratins are synthesized to form the hair shaft that eventually breaks through the surface of the skin [77]. Specifically, it was noted by the author that drug-to-metabolite ratios in hair are markedly different from those determined in blood, the time for drugs to appear in hair varies considerably from subject to subject, the distribution of drug along the hair shaft is not always consistent with that predicted from hair growth, and inter-subject drug and metabolite concentrations in hair differ considerably when the same dose of drug is administered. In response to the known scientific observations at the time of the writing of his review, he developed the multi-compartment model shown below in Figure 9.

As stated above, reference to apocrine gland secretion is applicable only in a limited topology in antenatal humans.

Immediately following the publication by Henderson, Kalasinsky et al. [96] examined the distribution of hydromorphone in hair from a chronic user. Hair that had been microtomed both cross-sectionally and laterally or lengthwise was studied using infrared microscopy. From their results, it was concluded that there exist two different drug entry routes into the hair. The first route, which is the primary route, occurs at hair root formation with binding to the medulla. In the second mechanism, drug enters the cuticle and it is distributed across the hair. The medulla was not always found to be continuous. Kalasinsky et al. also investigated the incorporation of spiked drug into hair. Spiked drug was found to incorporate into the hair in a fashion just opposite that of the drug that entered the hair i.e., spiked drug did not incorporate in the medulla as did drug that was incorporated through the ingested route. In various studies of approximately five years after that of Kalasinsky et al., Kimura et al. [99] suggested that MAMP tends to concentrate more in the medulla, by employing laser microscopy and the head hair of five deceased chronic MAMP users. The authors further concluded that it was not clear whether there was a direct correlation between the distributions of melanin and MAMP in hair, but stained colloidal gold particles coated with anti-MAMP were found agglutinated mainly in the area of hair sections rich in melanin.

Potsch et al. [170] described four general routes of drug uptake into the hair. The routes listed were:

* The endogenous pathway which is the result of drug substances in the systemic circulation due to intentional drug consumption taken up during hair formation or histogenesis,

* The endogenous-exogenous pathway which is illustrated by drug molecules absorbed or otherwise transferred into keratinized hair from perspiration (sweat, sebum, and transdermal excretion via the stratum corneum),

* The exogenous pathway which results from drug molecules deposited from the environment (e.g., pollution, hair treatments, pharmaceutical formulations) and entering the hair by absorption, and

* The exogenous-endogenous shunt which is drug unintentionally entering the systemic circulation by respiration or transdermal absorption and being endogenously incorporated during hair fiber histogenesis.

Although incorporation of drug/drug metabolite into hair is commonly perceived to be from the systemic circulation of the tissue surrounding the hair shaft into the hair shaft, it is most notable that the hair follicle itself contains numerous enzymes, some of which are shown below [170]:

* Alcohol dehydrogenase,

* Aldehyde dehydrogenase,

* Carboxylases,

* Esterase D,

* P450 Aryl hydrocarbon hydrolase,

* P450 Aromatase,

* P450 0-Deethylase,

* Glutathione reductase,

* NADPH P450 reductase,

* Glucosyl transferase,

* Glutathione-S-epoxide transferase, and

* Sulfotransferase.

Work by Wolf et al. has demonstrated the presence of propionyl-CoA carboxylase and pyruvate carboxylase in hair roots [223].

In an article published after the works by Henderson and Potsch et al., Joseph et al. [93] found that after dosing, codeine and parent cocaine were the primary analytes in sebum and stratum corneum. The drugs appeared in sebum post-dose within 1-2 hours and were detected for 1-2 days. Peak drug concentrations in stratum corneum occurred 1 day after the completion of dosing while elimination continued over the next 1-2 weeks. Thus, it appears that the skin can indeed act as a deep compartment for drugs as implied by both Henderson and Potsch.

Still, later work by Nakahara and Hanajiri [155] defined a relationship between drug polarity and the incorporation rate (ICR) of drug into hair. The authors concluded that ICR could be related to two factors--melanin affinity and lipophilicity.

In more recent work, Porta et al. [169] utilized matrix-assisted laser desorption ionization-mass spectrometric imaging (MALDI-MSI) to visualize the incorporation of cocaine into the washed single hairs of chronic cocaine users. Atracing in which the concentration decreased from the tip of the hair to the scalp (6-cm length segment) was presumed to indicate reduced consumption by individual H7 in the 6 month period preceding sampling and analysis as illustrated in Figure 10.

Conversely, Kamata et al. [97] used matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry along with dosing of an over-the-counter drug (o-methoxyphenamine; MOP; Asukuron) to follow the time course of drug incorporation. Using hair sectioning and GC/MS, the actual movement (2.8-3.2 mm/ week contemporaneously with hair growth and without diffusion) and stability (approximately 50% decrease in drug 5 months post-dose) of the drug had been followed by Nakahara et al. [157] in 1992. Using plucked hair from three subjects (A-C), the time courses shown in Figure 11 (MALDI-MS/MS and LC-MS/MS) were obtained.

Based on their studies with plucked hair, the authors concluded that a substantial concentration of the MOP is deposited first onto the hair bulb after dosing. Post-deposition, only a small amount appears to be incorporated into the hair matrix forming a 2-3 mm band. A comparable amount of drug also appears to be incorporated into the keratinized hair shaft in the upper dermis zone forming another distinct band of about 2 mm. Thus, at least for MOP, there are two major drug incorporation sites which cause overlap and deteriorate chronological resolution down to about 11 days or perhaps longer.

Although the incorporation of xenobiotics may have originally been envisioned as a simple transfer from blood to the hair bulb or hair follicle, it now appears to be fairly complex. Indeed, a portion of a drug found in hair may have been transferred from the bloodstream to the hair bulb or follicle. However, there also appear to be significant contributions from passage across the stratum corneum and transfer from sebum and sweat depending on the polarity of the molecule in question. Also, the absence of an active transfer mechanism or filtration, and the presence of highly polar metabolites such as glucuronides in hair is not easily explained by transfer and actually may be present by de novo synthesis in the hair follicle itself. Although non-Phase II metabolites of drugs are viewed as having been transferred from blood like the parent drug, the non-Phase II metabolites may be formed, at least, in part in the hair follicle by metabolism of the parent drug in the hair follicle.

The finding of metabolites of drugs in hair may be thought of as the transfer of the parent drug (lipophilic) into hair along with the transfer of the less lipophilic metabolite accounting for the finding of more parent drug than metabolite incorporated into hair. However, as suggested by Potsch et al. [170], developing hair and the tissue surrounding the hair follicle may contain the right combination of enzymes to produce metabolites from drug incorporated hair or hair into which drug is in the process of entering. Xenobiotics, specifically drugs, which may be metabolized by enzymes in skin have been demonstrated in numerous publications. Manevski et al. [131] showed the presence of aldehyde oxidase, which is a molybdoflavoenzyme that usually oxidizes azaheterocycles in therapeutic drugs, in fresh human skin. Although several xenobiotic metabolizing enzymes such as the CYPs (cytochrome P450), FMOs (flavin monooxygenase), ADH (alcohol dehydrogenase), and UGTs (uridine glucuronosyl transferase) were less than detectable or only weakly expressed, Fabian et al. [63] did find that NAT1 (S-acetyl transferase) was well-conserved and highly active along with UGT1A10 and GST p1 (glutathione S-transferase) being moderately expressed in keratinocytes. ALDH (aldehyde dehydrogenase) was also found to be quantifiable and present with relatively high activities in keratinocytic cell lines and NADPH (reduced nicotinamide adenine dinucleotide phosphate) dependent cytochrome c activity was above the LOQ. In looking at Phase II metabolic enzymes, Hewitt et al. [79] also found glutathione S-transferases, N-acetyltransferase 1, and UDP-glucuronosyltransferases to be readily measurable in the whole skin and in levels comparable to those found in the liver. Likewise, Gotz et al. [71] found that all three of the Phase II enzymes they investigated (glutathione S-transferase, UDP-glucuronosyltransferase, and VV-acetyltransferase) were present and highly active in skin as compared to phase I enzymes. In a separate study, Wiegand et al. [221] noted generally better expression of Phase I and Phase II enzymes in the epidermis of human foreskin when compared to the dermis. Among the CYPs, CYP2D6 and 2B6 were noted not to be found, while 1A1, 1B1, 2A6, 2E1, 2S1, and 3A4 were variably expressed weakly to highly. The flavin monooxygenases (1, 3, and 5) were variably expressed weakly to highly with no FMO3 found in the epidermis. With only exception to one dermis specimen, NAT1, UGT1A10, and GSTP1 were expressed moderately to highly in the dermis and epidermis. While investigating the stereoselective hydrolysis of ketoprofen ethyl ester, Zhu et al. [230] noted hCE-2, a carboxylesterase of toxicological importance, was present in abundance in HaCaT keratinocytes.

Focusing on enzymes in the hair follicle or bulb, from the observation that hair, which is largely keratin and which is a set of specific proteins, de facto numerous active enzymes for the production and assembly of proteins exist within the follicle itself. Goedde et al. [67] were able to investigate ADH (alcohol dehydrogenase) and ALDH (aldehyde dehydrogenase) activity in hair roots as part of the study of genetic polymorphisms. As noted in the work of Potsch et al. [170], glutathione-S-epoxide transferase (GSH-T) can be detected in freshly isolated human hair follicles [57]. By looking at the hydroxylation of dehydroepiandrosterone, Vermokken et al. [217] concluded that a monooxygenase system comparable to the corresponding system in the liver operates in the hair follicle. Propionyl-CoA carboxylase and pyruvate carboxylase activity have been detected in hair roots as part of an investigation of biotin-dependent enzyme deficiencies [221]. Likewise, arylhydrocarbonhydroxylase (AHH) activity has been found in hair follicles as part of a separate study of a test to see whether the AHH-controlling gene is of significance in causing cancer [135]. Aromatase and 3[beta]-hydroxysteroid dehydrogenase have been located in both the human hair follicle and the sebaceous gland [184]. As noted above, the sulfate conjugate of p-hydroxyamphetamine and/ or p-hydroxymethamphetamine may be important to finding a metabolite that demonstrates use rather than simple exposure to the amphetamines. Indeed, at least in rats, a sulfotransferase has been identified for minoxidil [58] in the portion of the outer root sheath that undergoes differentiation in a pattern dissimilar to the epidermis. The finding of less metabolite than parent drug in hair might be explained by the increased polarity of metabolites compared to parent drugs resulting in less incorporation of a more polar metabolite relative to its parent. Conversely, although not every exact combination of enzymes to complete the metabolic cascade into which a drug might enter has been characterized and located where its catalytic activity would be required, enough enzymes have been identified and localized to suggest quite strongly that the finding of drug metabolites in hair is due in large part if not exclusively to metabolism in the hair follicle or a closely associated functionality such as the sebaceous gland.

Hair testing that targets just the parent drugs depends solely on incorporation of the drug into hair regardless of the mechanism of incorporation. Hair testing that further targets drug metabolites needs another physiological step, which is the metabolism of the parent drug. Depending on a drug's metabolic cascade, metabolism may or may not produce a required metabolite in every individual. Additionally, metabolism normally does not convert a drug completely into a single end product. Regardless of whether they are produced metabolically in the hair follicle itself, incorporated as metabolites into the formed hair, or a combination of the two processes, metabolites appear in hair by mechanisms that are possibly different from or in addition to those used to incorporate the parent substance. Thus, targeting metabolites of drugs in hair testing may have to be performed with the understanding that a small percentage of donors that are also drug users will become transparent to the drug metabolite testing system if they do not genetically and/or phenotypically possess the enzyme(s) required to produce the metabolite or do not have the transport mechanism(s) to incorporate the metabolite into hair.

R. M. White

Center for Forensic Sciences

RTI International

Research Triangle Park, North Carolina

United States of America

REFERENCES

[1.] Achilli G, Cellerino GP, Melzi d'Eril GV, Tagliaro F: Determination of illicit drugs and related substances by high performance liquid chromatography with an electrochemical coulometric-array detector; J Chromatogr A 729:273; 1996.

[2.] Alberts B, Johnson A, Lewis JH, Morgan D, RaffM, Roberts K, Walter P: Molecular Biology of the Cell, 6th ed; Garland Science: New York, NY; Chap 10; 2015.

[3.] Aleksa K, Walasek P, Fulga N, Kapur B, Gareri J, Koren G: Simultaneous detection of seventeen drugs of abuse and metabolites in hair using solid phase micro extraction (SPME) with GC/MS; Forensic Sci Int 218:31; 2012.

[4.] Ammann D, Becker R, Kohl A, Hanisch J, Nehls I: Degradation of the ethyl glucuronide content in hair by hydrogen peroxide and a non-destructive assay for assay oxidative hair treatment using infra-red spectroscopy; Forensic Sci Int 244:30; 2014.

[5.] Anderson DM: Borland's Illustrated Medical Dictionary, 32nd ed; Elsevier: Philadelphia, PA; 2012.

[6.] Arin MJ, Roop DR, Koch PJ, Koster MI: Biology of Keratinocytes; In Bolognia JL, Jorizzo JL, Schaffer JV (Eds): Dermatology, 3rd ed; Elsevier Saunders: Philadelphia, PA; 2012.

[7.] Auwarter V, Sportkert F, Hartwig S, Pragst F, Vater H, Diffenbacher A: Fatty acid ethyl esters in hair as markers of alcohol consumption. Segmental hair analysis of alcoholics, social drinkers, and teetotalers; Clin Chem 47:2114; 2001.

[8.] Baldacci A, Thormann W: Analysis of oxycodol and noroxycodol stereoisomers in biological samples by capillary electrophoresis; Electrophoresis 26:1969; 2005.

[9.] Balikova MA, Habrdova V: Hair analysis for opiates: evaluation of washing and incubation procedures; J Chromatogr B, Anal Technol Biomed Life Sci 789:93; 2003.

[10.] Barroso M, Dias M, Vieira DN, Lopez-Rivadulla M, Queiroz JA: Simultaneous quantitation of morphine, 6-acetylmorphine, codeine, 6-acetylcodeine and tramadol in hair using mixed-mode solid-phase extraction and gas chromatography-mass spectrometry; Anal Bioanal Chem 396:3059; 2010.

[11.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 74; 2014.

[12.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 122; 2014.

[13.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 493; 2014.

[14.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 511; 2014.

[15.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 618; 2014.

[16.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 781; 2014.

[17.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 846; 2014.

[18.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 869; 2014.

[19.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man 10th ed; Biomedical Publications: Seal Beach, CA; p 992; 2014.

[20.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 1011; 2014.

[21.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 1017; 2014.

[22.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 1263; 2014.

[23.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 1266; 2014.

[24.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 1318; 2014.

[25.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 1466; 2014.

[26.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 1528; 2014.

[27.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 1534; 2014.

[28.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 1596; 2014.

[29.] Baselt RC (Ed): Disposition of Toxic Drugs and Chemicals in Man, 10th ed; Biomedical Publications: Seal Beach, CA; p 1948; 2014.

[30.] Bendroth P, Kronstrand R, HelanderA, Greby J, Stephanson N, Krantz P: Comparison of ethyl glucuronide in hair with phosphatidylethanol in whole blood as post-mortem markers of alcohol abuse; Forensic Sci Int 176:76; 2008.

[31.] Benetton SA, Borges VM, Change TKH, McErlane KM: Role of individual human cytochrome P450 enzymes in the in vitro metabolism of hydromorphone; Xenobiotica 34:335; 2004.

[32.] Bertholf RL, Bazydlo LAL: Biomarkers of acute and chronic alcohol ingestion; In Caplan YH, Goldberger BA (Eds): Garriott's Medicolegal Aspects of Alcohol, 6th ed; Lawyers & Judges: Tucson, AZ; 2015.

[33.] Boojaria A, Masrournia M, Ghorbani H, Ebrahimitalab A, Miandarhoie M: Silane modified magnetic nanoparticles as a novel adsorbent for determination of morphine at trace levels in human hair samples by high-performance liquid chromatography with diode array detection; Forensic Sci Med Pathol 11:497; 2015.

[34.] Brown DJ: The pharmacokinetics of alcohol excretion in human perspiration; Methods Find Exp Clin Pharmacol 7:539; 1985.

[35.] Buxton ILO, Benet LZ: Pharmacokinetics: The dynamics of drug absorption, distribution, metabolism, and elimination; In Brunton L, Chabner B, Knollman B (Eds): Goodman & Gilman's The Pharmacological Basis of Therapeutics, 12th ed; McGraw-Hill Books; 2011.

[36.] Carmo H, Brulport M, Hermes M, Oesch F, Silva R, Ferreira LM, Branco PS, de Boer D, Remiao F, Carvalho F, et al.: Influence of CYP2D6 polymorphism on 3,4-methylenedioxymethamphetamine ("Ecstasy") cytotoxicity; Pharmacogenet Genom 16:798; 2006.

[37.] Casale JF, Moore JM: Lesser alkaloids of cocaine-bearing plants. II. 3-Oxo-substituted tropane esters: Detection and mass spectral characterization of minor alkaloids found in Soutth American Erythroxylum coca var. coca; J Chromatogr A 749:173; 1996.

[38.] Cheze M, VfllainM, Pepin G: Determination of bromazepam, clonazepam and metabolites after a single intake in urine and hair by LC-MS/MS. Application to forensic cases of drug facilitated crimes; Forensic Sci Int 145:123; 2004.

[39.] Cirimele V, Kintz P, Ludes B: Screening for forensically relevant benzodiazepines in human hair by gas chromatography-negative ion chemical ionization-mass spectrometry; J Chromatogr B 700:119; 1997.

[40.] Cirimele V, Kintz P, Mangin P: Drug concentrations in human hair after bleaching; J Anal Toxicol 19:331; 1995.

[41.] Cirimele V, Kintz P, Mangin P: Determination of chronic flunitrazepam abuse by hair analysis using GC-MS-NCI; J Anal Toxicol 20:596; 1996.

[42.] Cirimele V, Kintz P, Staub C, Mangin P: Testing human hair for flunitrazepam and 7-amino-flunitrazepam by GC/ MS-NCI; Forensic Sci Int 84:189; 1997.

[43.] Cone EJ; Personal correspondence; December, 2016.

[44.] Cone EJ, Darwin WD, Gorodetzky CW: Comparative metabolism of codeine in man, rat, dog, guinea-pig and rabbit: Identification of four new metabolites; J Pharmacy Pharmacol 31:314; 1979.

[45.] Cone EJ, Darwin WD, Gorodetzky CW, Tan T: Comparative metabolism of hydrocodone in man, rat, guinea pig, rabbit, and dog; DrugMetab Dispos 6:488; 1978.

[46.] Cone EJ, Heit HA, Caplan YH, Gourlay D: Evidence of morphine metabolism to hydromorphone in pain patients chronically treated with morphine; J Anal Toxicol 30:1; 2006.

[47.] Cone EJ, Heltsley R, Black DL, Mitchell JM, LoDico CP, Flegel R: Prescription opioids. I. Metabolism and excretion patterns of oxycodone in urine following controlled single dose administration; J Anal Toxicol 37:255; 2013.

[48.] Cone EJ, Heltsley R, Black DL, Mitchell JM, LoDico CP, Flegel R: Prescription opioids. II. Metabolism and excretion patterns of hydrocodone in urine following controlled single dose administration; J Anal Toxicol 37:486; 2013.

[49.] Cone EJ, Tsadik A, Oyler J, Darwin WD: Cocaine metabolism and urinary excretion after different routes of administration; Ther Drug Monit 20:556; 1998.

[50.] Di Corcia D, D'Urso F, Gerace E, Salomone A, Vincenti M: Simultaneous determination in hair of multiclass drugs of abuse (including THC) by ultra-high performance liquid chromatography-tandem mass spectrometry; J Chromatogr B 899:154; 2012.

[51.] Cordero S, Paterson S: Simultaneous quantification of opiates, amphetamines, cocaine and metabolites and diazepam and metabolite in a single hair sample using GC-MS; J Chromatogr B 850:423; 2007.

[52.] Crunelle CL, Yegles M, De Doncker M, Dom G, Cappelle D, Maudens KE, van Nuijs AL, Covaci A, Neels H: Influence of repeated permanent coloring and bleaching on ethyl glucuronide concentrations in hair from alcohol-dependent patients; Forensic Sci Int 247:18; 2015.

[53.] Cuypers E, Flinders B, Bosman IJ, Lusthof KJ, Van Asten AC, Tytgat J, Heeren RMA: Hydrogen peroxide reactions on cocaine in hair using imaging mass spectrometry; Forensic Sci Int 242:103; 2014.

[54.] Dahl H, Stephanson N, Beck O, Helendar A: Comparison of urinary excretion characteristics of ethanol and ethyl glucuronide; J Anal Toxicol 26:201; 2002.

[55.] de Berker D, Higgins CA, Johoda C, Christiano AM: Biology of hair and nails; In Bolognia JL, Jorizzo JL, Schaffer JV (Eds): Dermatology, 3rd ed; Elsevier Saunders: Philadelphia, PA; 2012.

[56.] DePriest AZ, Puet BL, Holt AC, Roberts A, Cone EJ: Metabolism and disposition of prescription opioids: A Review; Forensic Sci Rev 27:115; 2015.

[57.] Dijkstra AC, Misdom LW, Goos CMAA, Hukkelhoven MWAC, Vermorken AJM: Glutathione-S-epoxide transferase in human hair follicles; Cancer Lett 31:105; 1986.

[58.] Dooley TP, Walker CJ, Hirshey SJ, Falany CN, Diani AR: Localization of minoxidil sulfotransferase in the rat liver and the outer root sheath of anagen pelage and vibrissa follicles; J Investig Dermatol 96:65; 1991.

[59.] Downing DT, Strauss JT: On the mechanism of sebaceous secretion; Arch Dermatol Res 272:343; 1982.

[60.] Downing DT, Strauss JT, Ramasastry M, Abel M, Lees CW, Pochi PE: Measurement of the time between synthesis and surface excretion of sebaceous lipids in sheep and man; J Invest Dermatol 64:215; 1975.

[61.] ElSohly MA, Kopycki WJ, Murphy TP, Lukey BJ: GC/MS analysis of m-hydroxybenzoylecgonine in urine. Forensic implications in cocaine use; Clin Lab Med 18:699; 1998.

[62.] Elsohly MA, Kopycki W, Feng S, Murphy TP: Identification and analysis of the major metabolites of cocaine in meconium; J Anal Toxicol 23:446; 1999.

[63.] Fabian E, Vogel D, Blatz V, Ramirez T, Kolle S, Eltze T, van Ravenzwaay B, Oesch F, Landsiedel R: Xenobiotic metabolizing enzyme activities in cells used for testing skin sensitization in vitro; Arch Toxicol 87:1683; 2013.

[64.] Fernandez P, Lago M, Lorenzo RA, Carro AM, Bermejo AM, Taberno MJ: Optimization of a rapid microwave assisted extraction method for the simultaneous determination of opiates, cocaine and their metabolites in human hair; J Chromatogr B 877:1743; 2009.

[65.] de Giovanni N, Donadio G, Chiarotti M: Ethanol contamination leads to fatty acid ethyl esters in hair samples; J Anal Toxicol 32:156; 2008.

[66.] de Giovanni N, Fucci N: The current status of sweat testing for drugs of abuse: A review; CurrMedChem 20:1; 2013.

[67.] Goedde HW, Agarwal DP, Harada S: Genetic studies on alcohol-metabolizing enzymes: Detection of isozymes in human hair roots; Enzyme 25:281; 1980.

[68.] Goll M, Schmitt G, GanBmann B, Aderjan RE: Excretion of ethyl glucuronide in human urine after internal dilution; J Anal Toxicol 26:262; 2002.

[69.] Gonzalez FJ, Coughtrie M, Tukey RH: Drug metabolism; In Brunton L, Chabner B, Knollman B (Eds): Goodman & Gilman's The Pharmacologic Basis of Therapeutics, 12th ed; McGraw-Hill Books; 2011.

[70.] Gottardo R, Fanigliulo A, Bortolotti F, De Paoli G, Pascali JP, Tagliaro F: Broad-spectrum toxicological analysis of hair based on capillary zone electrophoresis-time-of-flight mass spectrometry; J Chromatogr A 1159:190; 2007.

[71.] Gotz C, Pfeiffer R, Tigges J, Ruwiedel K, Huberthal U, Merk HF, Krutmann J, Edwards RJ, Abel J, Pease C, et al.: Xenobiotic metabolism capacities of human skin in comparison with a 3D-epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: Phase II enzymes; Exp Dermatol 21:364; 2012.

[72.] Gouveia CA, Oliveira A, Pinho S, Vasconcelos C, Carvalho F, Moreira RF, Dinis-Oliveira RJ: Simultaneous quantification of morphine and cocaine in hair samples from drug addicts by GC-EI/MS; Biomed Chromatogr: BMC 26:1041; 2012.

[73.] Guitton J, Buronfosse T, Desage M, Lepape A, Brazier JL, Beaune P: Possible involvement for multiple cytochrome P450's in fentanyl and sufentanil metabolism as opposed to alfentanil; Biochem Pharmacol 53:1613; 1997.

[74.] Gygi SP, Colon F, Raftogianis RB, Galinsky RE, Wilkins DG, Rollins DE: Dose-related distribution of codeine and its metabolites into rat hair; Drug Metab Disp 24:282; 1996.

[75.] Gygi SP, Joseph RE Jr, Cone EJ, Wilkins DG, Rollins DE: Incorporation of codeine and metabolites into hair; Drug Metab Disp 24:495; 1996.

[76.] Hartwig S, Auwarter V, Pragst F: Effect of hair care and hair cosmetics on the concentrations of fatty acid ethyl esters in hair as markers of chronically elevated alcohol consumption; Forensic Sci Int 131:90; 2003.

[77.] Henderson GL: Mechanisms of drug incorporation into hair; Forensic Sci Int 63:19; 1993.

[78.] Henderson GL, Wilson BK: Excretion of methadone and metabolites in human sweat; Res Commun Chem Pathol Pharmacol; 5:1; 1973.

[79.] Hewitt NJ, Edwards RJ, Fritsche E, Goebel C, Aeby P, Scheel J, Reisinger K, Ouedraogo G, Duche D, Eilstein J, et al.: Use of human in vitro skin models for accurate and ethical risk assessment: Metabolic considerations; Toxicol Sci 133:209; 2013.

[80.] Hill VA, Schaffer MI, Stowe GN: Carboxy-THC in washed hair: Still the reliable indicator of marijuana ingestion; J Anal Toxicol 40:345; 2016.

[81.] Hill V, Loni E, Cairns T, Sommer J, SchafferM: Identification and analysis of damaged or porous hair; Drug Test Anal 42; 2014.

[82.] Hold KM, Wilkins DG, Rollins DE, Joseph RE Jr, Cone EJ: Simultaneous quantitation of cocaine, opiates, and their metabolites in human hair by positive ion chemical ionization gas chromatography-mass spectrometry; J Chromatogr Sci 36:125; 1998.

[83.] Hughes MM, Atayee RS, Best BM, Pesce AJ: Observations on the metabolism of morphine to hydromorphone in pain patients; J Anal Toxicol 36:250; 2012.

[84.] [The] Human Cytochrome P450 (CYP) Allele Nomenclature Committee: The Human Cytochrome P450 (CYP) Allele Nomenclature Database; www.cypalleles.ki.se (accessed September 15, 2016).

[85.] Hutchinson MR, Menelaou A, Foster D J, Coller JK, Somogyi AA: CYP2D6 and CYP3A4 involvement in the primary oxidative metabolism of hydrocodone by human liver microsomes; Br J Clin Pharmacol 57:287; 2004.

[86.] Imbert L, Dulaurent S, Mercerolle M, Morichon J, Lachatre G, Gaulier J-M: Development and validation of a single LC-MS/MS assay following SPE for simultaneous hair analysis of amphetamines, opiates, cocaine and metabolites; Forensic Sci Int 234:132; 2014.

[87.] Ingelman-Sundberg M: Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): Clinical consequences, evolutionary aspects and functional diver-sity; Pharmcogenomics J 5:6; 2005.

[88.] Jin M, Gock SB, Jannetto PJ, Jentzen JM, Wong SH: Pharmacogenomics as molecular autopsy for forensic toxicology: Genotyping cytochrome P450 3A4*1B and 3A5*3 for 25 fentanyl cases; J Anal Toxicol29:590; 2005.

[89.] Jindal SP, Lutz T: Ion cluster techniques in drug metabolism: Use of a mixture of labelled and unlabeled cocaine to facilitate metabolite identification; J Anal Toxicol 10:150; 1986.

[90.] Johansen SS, Dahl-Sorensen RA: Drug rape case involving triazolam detected in hair and urine; IntJ Leg Med 126:637; 2012.

[91.] Jones J, Tomlinson K, Moore C: The simultaneous determination of codeine, morphine, hydrocodone, hydromorphone, 6-acetylmorphine, and oxycodone in hair and oral fluid; J Anal Toxicol 26:171; 2002.

[92.] Joseph RE Jr, Hold KM, Wilkins DG, Rollins DE, Cone E: Drug testing with alternative matrices. II. Mechanism of cocaine and codeine deposition in hair; J Anal Toxicol 23:396; 1999.

[93.] Joseph RE Jr, Oyler JM, Wstadik AT, Ohuoha C, Cone EJ: Drug testing with alternative matrices I. Pharmacological effects and disposition of cocaine and codeine in plasma, sebum, and stratum corneum; J Anal Toxicol 22:6; 1998.

[94.] Jufer RA, Wstadik A, Walsh SL, Levine BS, Cone EJ: Elimination of cocaine and metabolites in plasma, saliva, and urine following repeated oral fluid administration to human volunteers; J Anal Toxicol 24:467; 2000.

[95.] Jurado C, Kintz P, Menendez M, Repetto M: Influence of the cosmetic treatment of hair on drug testing; Int J Legal Med 110:159; 1997.

[96.] Kalasinsky KS, Magluilo J Jr, Schaefer T: Study of drug distribution in hair by infrared microscopy visualization; J Anal Toxicol 18:337; 1994.

[97.] Kamata T, Shima N, Sasaki K, Matsuta S, Takei S, Katagi M, Miki A, Zaitsu K, Nakanishi T, Sato T, et al.: Time-course mass spectrometry imaging for depicting drug incorporation into hair; Anal Chem 87:5476; 2015.

[98.] Kim J, Lee S, In S, Choi H, Chung H: Validation of a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair using LC-MS/MS and its application to human and rat hair; J Chromatogr B 879:878; 2011.

[99.] Kimura H, Mukaida M, Mori A: Detection of stimulants in hair by laser microscopy; J Anal Toxicol 23:577; 1999.

[100.] Kintz P: Consensus of the Society of Hair Testing on hair testing for chronic excessive alcohol consumption 2009; Forensic Sci Int 196:2; 2010.

[101.] Kintz P, Cirimele V, Vayssette F, Mangin P: Hair analysis for nordiazepam and oxazepam by gas chromatography-negative-ion chemical ionization mass spectrometry; J Chromatogr B 677:241; 1996.

[102.] Kintz P, Mangin P: Determination of gestational opiate, nicotine, benzodiazepine, cocaine and amphetamine exposure by hair analysis; J Forensic Sci Soc 33:139; 1993.

[103.] Kintz P, Nicholson D: Interpretation of a highly positive ethyl glucuronide result together with negative fatty acid ethyl esters result in hair and negative blood results; Forensic Toxicol 32:176; 2014.

[104.] Kintz P, Nicholson D: Testing for ethanol markers in hair: Discrepancies after simultaneous quantification of ethyl glucuronide and fatty acid ethyl esters; Forensic Sci Int 243:44; 2014.

[105.] Kintz P, Tracqui A, Mangin P: [Tobacco, drug and narcotic abuse during pregnancy. Evaluation of in utero exposure by analysis of hair of the neonate]; Presse Medicale 21:2139; 1992.

[106.] Kintz P, Villain M, Cirimele V, Pepin G, Ludes B: Windows of detection of lorazepam in urine, oral fluid and hair, with a special focus on drug-facilitated crimes; Forensic Sci Int 145:131; 2004.

[107.] Kintz P, Villain M, Ludes B: Testing for the undetectable in drug-facilitated sexual assault using hair analyzed by tandem mass spectrometry as evidence; Ther Drug Monit 26:211; 2004.

[108.] Klette KL, Poch GK, Czarny R: Simultaneous GC-MS analysis of meta- and para-hydroxybenzoylecgonine and norbenzoylecgonine: A secondary method to corroborate cocaine ingestion using nonhydrolytic metabolites; J Anal Toxicol 24:482; 2000.

[109.] Kolbrich EA, Barnes AJ, Gorelick DA, Boyd SJ, Cone EJ, Huestis MA: Major and minor metabolites of cocaine in human plasma following controlled subcutaneous cocaine administration; J Anal Toxicol 30:501; 2006.

[110.] Koster MI, Loomis CA, Koss T, Chu D: Skin development and maintenance; In Bolognia JL, Jorizzo JL, Schaffer JV (Eds): Dermatology, 3rd ed; Elsevier Saunders: Philadelphia, PA; 2012.

[111.] Koster RA, Alffenaar JW, Greijdanus B, VanDernagel JE, Uges DR: Fast and highly selective LC-MS/MS screening for THC and 16 other abused drugs and metabolites in human hair to monitor patients for drug abuse; Ther Drug Monit 36:234; 2014.

[112.] Kreth K-P, Kovar K-A, Schwab M, Zanger UM: Identification of human cytochromes P450 involved in the oxidative metabolism of "Ecstasy"-related designer drugs; Biochem Pharmacol 59:1563; 2000.

[113.] Kronstrand R, Nystrom I, Strandberg J, Druid H: Screening for drugs of abuse in hair with ion spray LC-MS-MS; Forensic Sci Int 145:183; 2004.

[114.] Kurogi K, Chepak A, Hanrahan MT, Liu MY, Sakakibara Y, Suiko M, Liu, MC: Sulfation of opioid drugs by human cytosolic sulfotransferases: metabolic labeling study and enzymatic analysis; Eur J Pharm Sci 62:40; 2014.

[115.] Kuwayama K, Tsujikawa K, Miyaguchi H, Kanamori T, Iwata YT, Inoue H: Interaction of 3,4-methylenedioxymethamphetamine and methamphetamine during metabolism by in vitro human metabolic enzymes and in rats; J Forensic Sci 57:1008; 2012.

[116.] Labroo RB, Paine MF, Thummel KE, Kharasch ED: Fentanyl metabolism by human hepatic and intestinal cytochrome P450 3A4: Implications for interindividual variability in disposition, efficacy, and drug interactions; DrugMetab Disp 25:1072; 1997.

[117.] Laloup M, Fernandez Mdel M, Wood M, Maes V, De Boeck G, Vanbeckevoort Y, Samyn N: Detection of diazepam in urine, hair and preserved oral fluid samples with LC-MS-MS after single and repeated administration of myolastan and valium; Anal Bioanal Chem 388:1545; 2007.

[118.] Lalovic B, Kharasch E, Hoffer C, Risler L, Liu-Chen LY, Shen DD: Pharmacokinetics and pharmacodynamics of oral oxycodone in healthy human subjects: Role of circulating active metabolites; Clin Pharmacol Ther 79:461; 2006.

[119.] Lalovic B, Phillips B, Risler LL, Howald W, Shen DD: Quantitative contribution of CYP2D6 and CYP3A to oxycodone metabolism in human liver and intestinal microsomes; Drug Metab Disp 32:447; 2004.

[120.] Lee S, Han E, In S, Choi H, Chung H, Chung KH: Determination of illegally abused sedative-hypnotics in hair samples from drug offenders; J Anal Toxicol 35:312; 2011.

[121.] Lee S, Han E, Kim E, Choi H, Chung H, Oh SM, Yun YM, Jwa SH, Chung KH: Simultaneous quantification of opiates and effect of pigmentation on its deposition in hair; Arch Pharm Res 33:1805; 2010.

[122.] Lee S, Park Y, Han E, Choi H, Chung H, Oh SM: Thebaine in hair as a marker for chronic use of illegal opium poppy substances; Forensic Sci Int 204:115; 2011.

[123.] Lehman-McKeeman LD: Absorption, Distribution, and Excretion of Toxicants; In Klasssen CD (Ed); Cassarett & Doull's Toxicology. The Basic Science of Poisons, 8th ed; McGraw-Hill Education; 2013.

[124.] Lendoiro E, Quintela O, de Castro A, Cruz A, Lopez-Rivadulla M, Concheiro M: Target screening and confirmation of 35 licit and illicit drugs and metabolites in hair by LC-MSMS; Forensic Sci Int 217:207; 2012.

[125.] Lester L, Uemura N, Ademola J, Harkey MR, Nath RP, Kim SJ, Jerschow E, Henderson GL, Mendelson J, Jones RT: Disposition of cocaine in skin, interstitial fluid, sebum, and stratum corneum; J Anal Toxicol 26:547; 2002.

[126.] Levine B, CaplanY: Pharmacology and toxicology of ethyl alcohol; In Caplan YH, Goldberger BA (Eds): Garriott's Medicolegal Aspects of Alcohol, 6th ed; Lawyers & Judges: Tucson, AZ; 2015.

[127.] Lin LY, Di Stefano EW, Schmitz DA, Hsu L, Ellis SW, Lennard MS, Tucker GT, Cho AK: Oxidation of methamphetamine and methylenedioxymethamphetamine by CYP2D6; Drug Metab Disp 25:1059; 1997.

[128.] Liu H-C, Liu RH, Lin D-L: Simultaneous quantitation of amphetamines and opiates in human hair by liquid chromatography-tandem mass spectrometry; J Anal Toxicol 39:183; 2015.

[129.] Lowery WT, Lomonte JN, Hatchett D, Garriott JC: Identification of two novel cocaine metabolites in bile by gas chromatography and gas chromatography/mass spectrometry in a case of acute intravenous cocaine overdose; J Anal Toxicol 3:91; 1979.

[130.] Madry MM, Bosshard MM, Kraemer T, Baumgartner MR: Hair analysis for opiates: Hydromorphone and hydrocodone as indicators of heroin use; Bioanalysis 8:953; 2016.

[131.] Manevski N, Balavenkatraman KK, Berschi B, Swart P, Walles M, Carmenisch G, Schiller H, Kretz O, Ling B, Wettstein R, et al.: Aldehyde oxidase activity in fresh human skin; Drug Metab Disp 42:2049; 2014.

[132.] Maurer HH, Bickeboeller-Friedrich J, Kraemer T, Peters FT: Toxicokinetics and analytical toxicology of amphetamine-derived designer drugs ("Ecstasy"); Toxicol Lett 112-113:133; 2000.

[133.] Mazur A, Lichti CF, Prather PL, Zielinska AK, Bratton SM, Gallus-Zawada A, Finel M, Miller GP, Radominska-Pandya A, Moran JH: Characterization of human hepatic and extrahepatic UDP-glucuronosyltransferase enzymes involved in the metabolism of classic cannabinoids; Drug Metab Disp 37:1496; 2009.

[134.] Menelaou A, Hutchinson MR, Quinn I, Christensen A, Somogyi AA: Quantification of the O- and N-demethylated metabolites of hydrocodone and oxycodone in human liver microsomes using liquid chromatography with ultraviolet absorbance detection; J Chromatogr B 785:81; 2003.

[135.] Merk H, Rumpf M, Bolsen K, Wirth G, Goerz G: Inducibility of arylhydrocarbon-hydroxylase activity in human hair follicles by topical application of liquor carbonis detergens (coal tar); Br J Dermatol 111:279; 1984.

[136.] Miguez-Framil M, Cabarcos P, Tabernero MJ, Bermejo AM, Bermejo-Barrera P, Moreda-Pineiro A: Matrix solid phase dispersion assisted enzymatic hydrolysis as a novel approach for cocaine and opiates isolation from human hair; J Chromatogr A 1316:15; 2013.

[137.] Miguez-Framil M, Moreda-Pineiro A, Bermejo-Barrera P, Alvarez-Freire I, Tabernero MJ, Bermejo AM: Matrix solid-phase dispersion on column clean-up/pre-concentration as a novel approach for fast isolation of abuse drugs from human hair; J Chromatogr A 1217:6342; 2010.

[138.] Miguez-Framil M, Moreda-Pineiro A, Bermejo-Barrera P, Cocho JA, Tabernero MJ, Bermejo AM: Electrospray ionization tandem mass spectrometry for the simultaneous determination of opiates and cocaine in human hair; Anal Chim Acta 704:123; 2011.

[139.] Miller EI, Wylie FM, Oliver JS: Detection of benzodiazepines in hair using ELISA and LC-ESI-MS-MS; J Anal Toxicol 30:441; 2006.

[140.] Miller EI, Wylie FM, Oliver JS: Simultaneous detection and quantification of amphetamines, diazepam and its metabolites, cocaine and its metabolites, and opiates in hair by LC-ESI-MS-MS using a single extraction method; J Anal Toxicol 32:457; 2008.

[141.] Milman G, Schwope DM, Gorelick DA, Huestis MA: Cannabinoids and metabolites in expectorated oral fluid following controlled smoked cannabis; Clin Chim Acta 413:765; 2012.

[142.] Miranda-G E, Sordo M, Salazar AM, Contreras C, Bautista L, Garcia AER, Ostrosky-Wegman P: Determination of amphetamine, methamphetamine, and hydroxyamphetamine derivatives in urine by gas chromatography-mass spectrometry and its relation to CYP2D6 phenotype of drug users; J Anal Toxicol 31:31; 2007.

[143.] Moller M, Aleksa K, Walasek P, Karaskov T, Koren G: Solid-phase microextraction for the detection of codeine, morphine and 6-monoacetylmorphine in human hair by gas chromatography-mass spectrometry; Forensic Sci Int 196:64; 2010.

[144.] Moolchan ET, Cone EJ, Wstadik A, Huestis MA, Preston KL: Cocaine and metabolite elimination patterns in chronic cocaine users during cessation: Plasma and saliva analysis; J Anal Toxicol 24:458; 2000.

[145.] Moore C, Coulter C, Uges D, Tuyay J, van der Linde S, van Leeuwen A, Garnier M, Orbita J Jr: Cannabinoids in oral fluid following passive exposure to marijuana smoke; Forensic Sci Int 212:227; 2011.

[146.] Moore C, Feldman M, Harrison E, Rana S, Coulter C, Kuntz D, Agrawal A, Vincent M, Soares J: Disposition of hydrocodone in hair; J Anal Toxicol 30:353; 2006.

[147.] Moore C, Marinetti L, Coulter C, Crompton K: Analysis of pain management drugs, specifically fentanyl, in hair: Application to forensic specimens; Forensic Sci Int 176:47; 2008.

[148.] Moore JM, Casale JF: Detection and characterization of cocaine and related tropane alkaloids in coca leaf, cocaine, and biological specimens; Forensic Sci Rev 7:77; 1995.

[149.] Moore JM, Casale JF: In-depth chromatographic analyses of illicit cocaine and its precursor, coca leaves; J Chromatogr A 674:165; 1994.

[150.] Moore JM, Cooper DA: The application of capillary gas chromatography-electron capture detection in the comparative analyses of illicit cocaine samples; J Forensic Sci 38:1286; 1993.

[151.] Morini L, Marchei E, Vagnarelli F, Algar OG, Groppi A, Mastrobattista L, Pichini S: Ethyl glucuronide and ethyl sulfate in meconium and hair--potential biomarkers of intrauterine exposure to ethanol; Forensic Sci Int 196:74; 2010.

[152.] Morini L, Zucchella A, Polettini A, Politi L, Groppi A: Effect of bleaching on ethyl glucuronide in hair: An in vitro experiment; Forensic Sci Int 198:23; 2010.

[153.] Morris-Kukoski C, Montgomery MA, Hammer RI: Analysis of extensively washed hair from cocaine users and drug chemists to establish new reporting criteria; J Anal Toxicol 38:628; 2014.

[154.] Musshoff F, Lachenmeier K, Trafkowski J, Madea B, Nauck F, Stamer U: Determination of opioid analgesics in hair samples using liquid chromatography/tandem mass spectrometry and application to patients under palliative care; Ther Drug Monit 29:655; 2007.

[155.] Nakahara Y, Hanajiri R: Hair analysis for drugs of abuse. XXI. Effect of para-substituents on benzene ring of methamphetamine on drug incorporation into rat hair; Life Sci 66:563; 2000.

[156.] Nakahara Y, Kikura R: Hair analysis for drugs of abuse XIII. Effect of structural factors on incorporation of drugs into hair: The incorporation rates of amphetamine analogs; Arch Toxicol 70:841; 1996.

[157.] Nakahara Y, Shimamine M, Takahashi K: Hair analysis for drugs of abuse. III. Movement and stability of methoxyphenamine (as a model compound of methamphetamine; J Anal Toxicol 16:253; 1992.

[158.] Nakahara Y, Takahashi K, Kikura R: Hair analysis for drugs of abuse. X. Effect of physicochemical properties of drugs on the incorporation rates into hair; Biol Pharm Bull 18:1223; 1995.

[159.] Nakahara Y, Takahashi K, Sakamoto T, Tanaka A, Hill VA, Baumgartner WA: Hair analysis for drugs of abuse XVII. Simultaneous detection of PCP, PCHP, and PCPdiol in human hair for confirmation of PCP use; J Anal Toxicol 21:356; 1997.

[160.] Negrusz A, Bowen AM, Moore CM, Dowd SM, Strong MJ, Janicak PG: Deposition of 7-aminoclonazepam and clonazepam in hair following a single dose of klonopin; J Anal Toxicol 26:471; 2002.

[161.] Ohno S, Kawana K, Nakajin S: Contribution of UDP-glucuronosyltransferase 1A1 and 1A8 to morphine-6glucuronidation and its kinetic properties; Drug Metab Disp 36:688; 2008.

[162.] O 'Neill MJ (Ed): The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 15th ed; Royal Society of Chemistry: Cambridge, UK; 2013.

[163.] Oyler JM, Cone EJ, Joseph RE Jr, Huestis MA: Identification of hydrocodone in human urine following controlled codeine administration; J Anal Toxicol 24:530; 2000.

[164.] Parkinson A, Ogilvie BW, Buckley DB, Kazami F, Czerwinski M, Parkinson O: Biotransformation of xenobiotics; In Klasssen CD (Ed), Cassarett & Doull's Toxicology. The Basic Science of Poisons, 8th ed; McGraw-Hill Education; 2013.

[165.] Perez-Reyes M, di Guiseppi S, Brine DR, Smith H, Cook CE: Urine pH and phencyclidine excretion; Clin Pharmacol Ther 32:635; 1982.

[166.] Pichini S, Marchei E, Martello S, Gottardi M, Pellegrini M, Svaizer F, Lotti A, Chiarotti M, Pacifici R: Identification and quantification of 11-nor-A-tetrahydrocannabinol-9-carboxylic acid glucuronide (THC-COOH-glu) in hair by ultra-performance liquid chromatography tandem mass spectrometry as a potential hair biomarker of cannabis use; Forensic Sci Int 249:47; 2015.

[167.] Pichini S, Marchei E, Pacifici R, Pellegrini M, Lozano J, Garcia-Algar O: Application of a validated high-performance liquid chromatography-mass spectrometry assay to the analysis of m- and p-hydroxybenzoylecgonine in meconium; J Chromatogr B 820:151; 2005.

[168.] Pichini S, Pacifici R, Altieri I, Pellegrini M, Zuccaro P: Determination of opiates and cocaine in hair as trimethylsilyl derivatives using gas chromatographytandem mass spectrometry; J Anal Toxicol 23:343; 1999.

[169.] Porta T, Grivet C, Kraemer T, Varesio E, Hopfgartner G: Single hair cocaine consumption monitoring by mass spectrometric imaging; Anal Chem 83:4266; 2011.

[170.] Potsch L, Skopp G, Moeller MR: Biochemical approach on the conservation of drug molecules during hair fiber formation, Forensic Sci Int 84:25; 1997.

[171.] Pragst F, Balikova MA: State of the art in hair analysis for detection of drug and alcohol abuse; Clin Chim Acta 370:17; 2006.

[172.] Pragst F, Rothe M, Moench B, Hastedt M, Herre S, Simmert D: Combined use of fatty acid ethyl esters and ethyl glucuronide in hair for diagnosis of alcohol abuse: Interpretation and advantages; Forensic Sci Int 196:101; 2010.

[173.] Projean D, Morin PE, Tu TM, Ducharme J: Identification of CYP3A4 and CYP2C8 as the major cytochrome P450 is responsible for morphine A-demethylation in human liver microsomes;Xenobiotica 33:841; 2003.

[174.] Ramirez Fernandez Mdel M, Wille SM, di Fazio V, Kummer N, Hill V, Samyn N: Detection of benzodiazepines and z-drugs in hair using an UHPLC-MS/MS validated method: Application to workplace drug testing; Ther Drug Monit 37:600; 2015.

[175.] Rietjens SJ, Hondebrink L, Westerink RHS, Meulenbelt J: Pharmacokinetics and pharmacodynamics of 3,4-methylenedioxymethamphetamine (MDMA): interindividual differences due to polymorphisms and drug-drug interactions; CritRev Toxicol 42:854; 2012.

[176.] Robandt PP, Reda LJ, Klette KL: Complete automation of solid-phase extraction with subsequent liquid chromatography-tandem mass spectrometry for the quantification of benzoylecgonine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine, and norbenzoylecgonine in urine--Application to a high-throughput urine analysis laboratory; J Anal Toxicol 32:577; 2008.

[177.] Romolo FS, Rotolo MC, Palmi I, Pacifici R, Lopez A: Optimized conditions for simultaneous determination of opiates, cocaine and benzoylecgonine in hair samples by GC-MS; Forensic Sci Int 138:17; 2003.

[178.] Ropero-Miller JD: A decade revisited--Forensic and clinical applications of hair testing; Forensic Sci Rev 19:50; 2007.

[179.] Rosano TG, Eisenhofer G, Whitley RJ: Catecholamines and serotonin; In Burtis CA, Ashwood ER, Bruns DE, (Eds): Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th ed; Elsevier Saunders: Philadelphia, PA; 2006.

[180.] Rust KY, Baumgartner MR, Meggiolaro N, Kraemer T: Detection and validated quantification of 21 benzodiazepines and 3 "z-drugs" in human hair by LCMS/MS; Forensic Sci Int 215:64; 2012.

[181.] Sabzevari O, Abdi K, Amini M, Shafiee A: Application of a simple and sensitive GC-MS method for determination of morphine in the hair of opium abusers; Anal Bioanal Chem 379:120; 2004.

[182.] Sakamoto T, Tanaka A: Hair analysis for drugs of abuse XII. Determination of PCP and its major metabolites, PCHP and PPC, in rat hair after administration of PCP; J Anal Toxicol 20:124; 1996.

[183.] Sakamoto T, Tanaka A, Nakahara Y: Incorporation of phencyclidine and its hydroxylated metabolites into hair; Life Sci 62:561; 1998.

[184.] Sawaya ME, Penneys NS: Immunohistochemical distribution of aromatase and 3[beta]-hydroxysteroid dehydrogenase in human hair follicle and sebaceous gland; J Cutan Pathol 19:309; 1991.

[185.] Schaffer M, Cheng C-C, Chao O, Hill V, Matsui P: Analysis of cocaine and metabolites in hair: Validation and application of measurement of hydroxycocaine metabolites as evidence of cocaine ingestion; Anal Bioanal Chem 408:2043; 2016.

[186.] Schaller M, Plewig G: Structure and function of eccrine, apocrine and sebaceous glands; In Bolognia JL, Jorizzo JL, Schaffer JV (Eds); Dermatology, 3rd ed; Elsevier Saunders: Philadelphia, PA; 2012.

[187.] Scott KS, Nakahara Y: Astudy into the rate of incorporation of eight benzodiazepines into rat hair; Forensic Sci Int 133:47; 2003.

[188.] Segura M, Farre M, Pichini S, Peiro AM, Roset PN, Ramirez A, Oituno J, Pacifici R, Zuccaro P, Segura J, et al.: Contribution of cytochrome P450 2D6 to 3,4-methylenedioxymethamphetamine disposition in humans; Clin Pharmacokinet 44:649; 2006.

[189.] Segura M, Ortuno J, Farre M, McLure JA, Pujadas M, Piarro N, Liebaria A, Joglar J, Roset PN, Segura J, de la Torre R: 3,4-Dihydroxymethamphetamine (HHMA). A major in vivo 3,4-methylenedioxymethamphetamine (MDMA) metabolite in humans; Chem Res Toxicol 14:1203; 2001.

[190.] Senczuk-Przybylowska M, Florek E, Piekoszewski W, Merritt TA, Lechowicz E, Mazela J, Kulza M, Breborowicz GH, Krzyscin M, Markwitz W, et al.: Diazepam and its metabolites in the mothers' and newborns' hair as a biomarker of prenatal exposure; J Physiol Pharmacol 64:499; 2013.

[191.] Shen DD: Toxicokinetics; In Klasssen CD (Ed): Cassarett & Doull's Toxicology. The Basic Science of Poisons, 8th ed; McGraw-Hill Education; 2013.

[192.] Shima N, Kamata HT, Kamata T, Nishikawa M, Katagi M, Tscuchihashi H: The concentrations of glucuronide and sulfate of p-hydroxymethamphetamine in methamphetamine users' urine, and optimization of their hydrolysis conditions; Jpn J Forensic Sci Tech 12:73; 2007.

[193.] Shima N, Kamata HT, Katagi M, Tsuchihashi H: Urinary excretion of the main metabolites of methamphetamine, including p-hydroxymethamphetamine-sulfate and p-hydroxymethamphetamine-glucuronide, in humans and rats; Xenobiotica 36:259; 2006.

[194.] Shima N, Kamata H, Katagi M, Tsuchihashi H, Sakuma T, Nemoto N: Direct determination of glucuronide and sulfate of 4-hydroxy-3-methoxymethamphetamine, the main metabolite of MDMA, in human urine; J Chromatogr B 857:123; 2007.

[195.] Skender L, Karacic V, Brcic I, Bagaric A: Quantitative determination of amphetamines, cocaine, and opiates in human hair by gas chromatography/mass spectrometry; Forensic Sci Int 125:120; 2002.

[196.] Smith FP, Liu RH: Detection of cocaine metabolite in perspiration stain, menstrual blood stain, hair; J Forensic Sci 31:1269; 1986.

[197.] Smith RM: Arylhydroxy metabolites of cocaine in the urine of cocaine users; J Anal Toxicol 8:35; 1984.

[198.] Smith RM: Ethyl esters of arylhydroxy- and arylhydroxymethoxycocaines in the urines of simultaneous cocaine and ethanol users; J Anal Toxicol 8:38; 1984.

[199.] Smith RM, Poquett MA, Smith PJ: Hydroxymethoxybenzoylecgonines: New metabolites of cocaine from human urine; J Anal Toxicol 8:29; 1984.

[200.] Stone AN, Mackenzie PI, Galetin A, Houston JB, Miners JO: Isoform selectivity and kinetics of morphine 3- and 6-glucuronidationby humanudp-glucuronosyltransferases: evidence for atypical glucuronidation kinetics by UGT2B7; Drug Metab Disp 31:1086; 2003.

[201.] Suesse S, Pragst F, Mieczkowski T, Selavka CM, Elian A, Sachs H, Hastedt M, Rothe M, Campbell J: Practical experiences in application of hair fatty acid ethyl ester and ethyl glucuronide for detection of chronic alcohol abuse in forensic cases; Forensic Sci Int 218:82; 2012.

[202.] Sun Q, Xiang P, Shen B, Yan H, Shen M: Determination of triazolam and alpha-hydroxytriazolam in guinea pig hair after a single dose; J Anal Toxicol 34:89; 2010.

[203.] Sun YY, Xiang P, Shen M: Simultaneous determination of 11 opiates in hair by liquid chromatography-tandem mass spectrometry; YaoXueXue Bao 46:1501; 2011.

[204.] Takayama N, Lio R, Tanaka S, Chinaka S, Hayakawa K: Analysis of methamphetamine and its metabolites in hair; Biomed Chromatogr 17:74; 2003.

[205.] Tanaka S, Lio R, Chinaka S, Takayama N, Hayakawa K: Analysis of reaction products of cocaine and hydrogen peroxide by high performance liquid chromatography/ mass spectrometry; Biomed Chromatogr 16:390; 2002.

[206.] Tanaka S, Lio R, Chinaka S, Takayama N, Hayakawa K: Identification of reaction products of methamphetamine and hydrogen peroxide in hair dye and decolorant treatments by high-performance liquid chromatography; Biomed Chromatogr 15:45; 2001.

[207.] Thorspecken J, Skopp G, Potsch L: In vitro contamination of hair by marijuana smoke; Clin Chem 50:596; 2004.

[208.] Toyo'oka T, Kumaki Y, Kanbori M, Kato M, Nakahara Y: Determination of hypnotic benzodiazepines (alprazolam, estazolam, and midazolam) and their metabolites in rat hair and plasma by reversed-phase liquid-chromatography with electrospray ionization mass spectrometry; J Pharm Biomed Anal 30:1773; 2003.

[209.] Toyo'oka T, Kanbori M, Kumaki Y, Nakahara Y: Determination of triazolam involving its hydroxy metabolites in hair shaft and hair root by reversed-phase liquid chromatography with electrospray ionization mass spectrometry and application to human hair analysis; Anal Biochem 295:172; 2001.

[210.] Toyo'oka T, Kanbori M, Kumaki Y, Oe T, Miyahara T, Nakahara Y: Detection of triazolam and its hydroxy metabolites in rat hair by reversed-phase liquid chromatography with electrospray ionization mass spectrometry; J Anal Toxicol 24:194; 2000.

[211.] Toyo'oka T, Yano M, Kato M, Nakahara Y: Simultaneous determination of morphine and its glucuronides in rat hair and rat plasma by reversed-phase liquid chromatography with electrospray ionization mass spectrometry; Analyst 126:1339; 2001.

[212.] United Nations Office on Drugs and Crime: Methods for mpurity Profiling of Heroin and Cocaine, UN Document Number ST/NAR/35; 2005; https://www.unodc.org/pdf/ publications/report_st-nar-35.pdf(accessed December 9, 2016).

[213.] US Department ofHealth and Human Services: Mandatory guidelines for Federal workplace drug testing; Fed Reg 73:71857; 2008.

[214.] US Department of Health and Human Services: Proposed mandatory guidelines for Federal workplace drug testing; Fed Reg 69:19673; 2004.

[215.] US Department of Justice: Proposed Language Regrading Expert Testimony and Lab Reports in Forensic Science; www.justice.gov/forensics (accessed December 9, 2016).

[216.] Vayssette F, Cirimele V, Kintz P, Mangin P: [Detection of nordiazepam in the hair of drug addicts]; Ann pharm Fr 54:211; 1996.

[217.] Vermorkken AJM, Goos CMAA, Henderson PTh, Bloemendal H: Hydroxylation of dehydroepiandrosterone in human scalp hair follicles; Br J Dermatol 100:693; 1979.

[218.] Vree TB, Muskens ATJM, van Rossum JM: Excretions of amphetamines in human sweat; Arch Int Pharmacodyn 199:311; 1972.

[219.] Wang X, Johansen SS, Zhang Y, Jia J, Rao Y, Jiang F, Linnet K: Deposition of diazepam and its metabolites in hair following a single dose of diazepam; Int J Leg Med in press.

[220.] Watanabe K, Hida Y, Matsunaga T, Yamamoto I, Yoshimure H: Formation ofp-hydroxycocaine from cocaine by hepatic microsomes of animals and its pharmacological effects in mice; Biol Pharm Bull 16:1041; 1993.

[221.] Wiegand C, Hewitt NJ, Reisinger K: Dermal xenobiotic metabolism: A comparison between native human skin, four in vitro skin test systems and a liver system; Skin Pharmacol Physiol 27:263; 2014.

[222.] Wikipedia--Benzodiazepines; https://en.wikipedia.org/ wiki/Benzodiazepine (accessed September 8, 2016).

[223.] Wolf B, Raetz H: The measurement of propionyl-CoA carboxylase and pyruvate carboxylase activity in hair roots; Its use in the diagnosis of inherited biotin-dependent enzyme deficiencies; Clin Chim Acta 130:25; 1983.

[224.] Wu YH, Lin KL, Chen SC, Chang YZ: Integration of GC/ EI-MS and GC/NCI-MS for simultaneous quantitative determination of opiates, amphetamines, MDMA, ketamine, and metabolites in human hair; J Chromatogr B 870:192; 2008.

[225.] Xiang P, Shen M, Shen BH, Ma D, Bu J, Jiang Y, Zhou XY: [Determination of opiates inbiological human samples by liquid chromatography-tandem mass spectrometry]; Fa YiXue Za Zhi 22:52; 2006.

[226.] Yegles M, Mersch F, Wennig R: Detection of benzodiazepines and other psychotropic drugs in human hair by GC/MS; Forensic Sci Int 84:211; 1997.

[227.] Yegles M, Wennig MR: Influence of bleaching on stability of benzodiazepines in hair; Forensic Sci Int 107:87; 2000.

[228.] Zhang JY, Foltz RL: Cocaine metabolism in man: Identification of four previously unreported cocaine metabolites in human urine; J Anal Toxicol 14:201; 1990.

[229.] Zhou SF, Liu JP, Chowbay B: Polymorphism of human cytochrome P450 enzymes and its clinical impact; Drug Metab Rev 41:89; 2009.

[230.] Zhu Q-G, Hu J-H, Liu J-Y, Lu S-W, Liu Y-X, Wang J: Stereoselectivity characteristics and mechanisms of epidermal carboxylesterase metabolism observed in HaCaT keratinocytes; Biol Pharm Bull 30:532; 2007.

Robert M. White, Sr. received his B.A. in chemistry from Vanderbilt University (Nashville, TN) in 1967 and Ph.D. degree in physical organic chemistry from the University of Florida (Gainesville, FL) in 1972. Dr. White is currently working for the Center for Forensic Sciences, RTI International (Research Triangle Park, NC).

Upon completing his education, Dr. White served two and a half years of active duty in the US Army. After then, he undertook a post-doctoral in clinical chemistry with Dr. John Savory at the University of North Carolina (Chapel Hill, NC). Dr. White has served as the biochemist at Presbyterian Hospital (Charlotte, NC); Director of Life Insurance Testing at SmithKline-BioScience (Nashville, TN); Co-director for Chemistry and Toxicology at Naples Community Hospital and Diagnostic Services (Fort Myers, FL); Blood Bank Director at North American Biologicals, Inc. (Fort Myers, FL); and Health Care Risk Manager for Montgomery Eye Center (Naples, FL). Dr. White was also the scientific director for the Diagnostic Services State of Florida Toxicology Laboratory and Responsible Person for the NLCP-certified laboratory (Fort Myers, FL). Dr. White also served as an Adjunct Professor at Florida Gulf Coast University and Hodges University in Naples/Fort Myers, FL. Dr. White was a full member of the Naples Community Hospital Department of Medicine (Naples, FL). Dr. White has qualified as an expert in toxicology in the criminal, administrative, and civil courts of the State of Florida and Federal Administrative proceedings. Dr. White's major research interests include oral fluid drug testing, biological matrix stabilization for drug testing, urine substitution and adulteration, hair drug testing, and method development.

Dr. White is licensed as a Clinical Laboratory Director in Florida with specialties in Chemistry, Serology/ Immunology, and Molecular Pathology. Dr. White is also licensed in the State of Tennessee as a Clinical Laboratory Director and licensed by the State of Florida as a Health Care Risk Manager. Dr. White is a member of the American Chemical Society, the American Association for Clinical Chemistry, the American Academy of Forensic Sciences, and the Society of Forensic Toxicologists. Dr. White is a fellow in the American Board of Forensic Toxicologists and a diplomate to the American Board of Clinical Chemistry in Clinical Chemistry, Toxicological Chemistry, and Molecular Diagnostics.

* Corresponding author: Dr. Robert M. White, RTI International Center for Forensic Sciences, 3040 Cornwallis Road, Research Triangle Park, NC 27709; + 1 919 541 6508 (voice); bwhite@ rti.org.

Caption: Figure 1: General windows of detection for xenobiotics in common biomatrices (Courtesy of E. J. Cone [43]).

Caption: Figure 2: Development of the human hair follicle. (Reproduced with permission from Elsevier-Saunders [55].)

Caption: Figure 3: Anagen hair follicle. (Reproduced with permission from Elsevier-Saunders [171].)

Caption: Figure 4: Alignment and assembly of keratin molecules and filament packing. (Reproduced with permission for Elsevier-Saunders [6].)

Caption: Figure 5: Schematic representation of keratin distribution in human hair ((1) This protein weakly expressed at this site; (2) To date, expression of this protein has only been detected in single cortex cells; (3) To date, this protein has only been detected invellus hairs.) (Reproduced with permission from Elsevier-Saunders [6].)

Caption: Figure 6: General human hair follicle and accompanying glands. (Reproduced with permission from Elsevier-Saunders [5].)

Caption: Figure 7: Schematic representation of the lipid bilayer comprising animal cells. (Reproduced with permission from Science and Garland Science [2].)

Caption: Figure 8: Cascade for reporting hair cocaine (modified from the reference). (Reproduced, following modification, with permission from Oxford Publishing [153].)

Caption: Figure 9: Multi-compartment model for drug incorporation into hair. (Reproduced with permission from Elsevier [77].)

Caption: Figure 10: MALDI-MSI tracing of cocaine in a single human hair from a chronic cocaine user. (Reproduced with permission from the American Chemical Society [169].)

Caption: Figure 11: Time course of the incorporation of MOP into the plucked hair of three subjects dosed with 50 mg of MOP dihydrochloride. (Reproduced with permission from the American Chemical Society [97].)
Table 1. 2004 proposed hair testing cutoffs (pg/mg) [214]

Initial Test                     Confirmatory Test

Analyte                 Cutoff   Analyte                Cutoff

Marijuana metabolites     1      THCA (a)                0.05
Cocaine metabolites      500     Cocaine                 500
                                 Cocaine metabolites      50
Opiate metabolites       200     Codeine                 200
                                 Morphine                200
                                 6-Acetylmorphine (b)    200
Phencyclidine            300     Phencyclidine           300
Amphetamines             500     Amphetamine             300
                                 Methamphetamine (c)     300
MDMA                     500     MDMA                    300
                                 MDA                     300
                                 MDEA                    300

(a) 11-Nor-[[DELTA].sup.9]-tetrahydrocannabinol-
9-carboxylic acid.

(b) Specimen must also contain morphine at a
concentration greater than or equal to 200 pg/mg.

(c) Specimen must also contain amphetamine at a
concentration greater than or equal to 50 pg/mg.

Table 2. Abbreviations of drugs and terms

Abbreviation   Full name

ADH            Alcohol dehydrogenase
AHH            Arylhydrocarbon hydroxylase
ALDH           Aldehyde dehydrogenase
ATP            Adenosine triphosphate
AUC            Area under curve
BZE            Benzoylecgonine
CYP            Cytochrome P450
Da             Dalton
ESI-MS         Electrospray ionization mass spectrometry
EtG            Ethyl glucuronide
FAEE           Fatty acid ethyl ester
GC/MS          Gas chromatography-mass spectrometry
HOBZE          Hydroxybenzoylecgonine (phenyl ring)
HOCOC          Hydroxycocaine (phenyl ring)
HPLC           High-performance liquid chromatography
ICR            Incorporation rate; [Hair]/AUC, which is the ratio of
                 drug concentration in hair divided by the area under
                 the curve in plasma expressed as [micro]g min/mL
IN             Insufflation
IV             Intravenous
LC-MS/MS       Liquid chromatography-mass spectrometry/mass
                 spectrometry or liquid chromatography-tandem mass
                 spectrometry
LOD            Limit of detection
LOQ            Limit of quantification
MALDI          Matrix-assisted laser desorption ionization
MDA            Methylenedioxyamphetamine
MDMA           Methylenedioxymethamphetamine
MOP            Methoxyphenamine
NADPH          Reduced nicotinamide adenine dinucleotide
P              Partition coefficient
PCP            Phencyclidine
PCR            Polymerase chain reaction
RIA            Radioimmunoassay
S              Smoked
THC            [[DELTA].sup.9]-Tetrahydrocannabinol
THCA           11-Nor-[[DELTA].sup.9]-tetrahydrocannabinol-
                 9-carboxylic acid
UDP            Uridine diphosphate
UGT            Uridine-diphosphoglucuronosyl transferase

Table 3. Percent urine cocaine metabolites post-dose [49]

OH-Compound    Route and amount of administration (b)
observed (a)

               IV (25 mg)             IN (32 mg as HCl)

m-HOCOC        0.081 [+ or -] 0.024   0.080 [+ or -] 0.027
p-HOCOC        0.006 [+ or -] 0.003   0.007 [+ or -] 0.004
m-HOBZE        0.454 [+ or -] 0.160   0.477 [+ or -] 0.158
p-HOBZE        0.433 [+ or -] 0.105   0.486 [+ or -] 0.119
Total % of     57.1                   45.4
  dose (c)

OH-Compound    Route and amount of administration (b)
observed (a)

               S (42 mg as free base)

m-HOCOC        0.044 [+ or -] 0.013
p-HOCOC        0
m-HOBZE        0.186 [+ or -] 0.083
p-HOBZE        0.244 [+ or -] 0.065
Total % of     24.9
  dose (c)

(a) m-HOBZE: meta-hydroxybenzoylecgonine;
p-HOBZE: para-hydroxybenzoylecgonine.

(b) IV: intravenous; IN: insufflation; S: smoked.

(c) Total percent of dose accounted for in urine specimen (3 day).

Table 4. Minor cocaine metabolites in plasma
post-subcutaneous dose [109]

OH-Compound   Dose   % Positive (a)      [C.sub.max]
observed                                   (ng/mL)

m-HOCOC       Low         12.5        3.9 [+ or -] 0.1
              High        35.7        6.3 [+ or -] 1.8
p-HOCOC       Low         13.3        3.0 [+ or -] 0.4
              High        50.0        6.5 [+ or -] 1.3
m-HOBZE       Low         60.0        5.2 [+ or -] 0.5
              High        57.1        10.2 [+ or -] 1.2
p-HOBZE       Low         86.7        9.8 [+ or -] 1.0
              High        78.6        25.9 [+ or -] 4.6

OH-Compound   Dose     [T.sub.max]        Conc.
observed                  (hour)          (ng/mL)
                                        in positive
                                          samples

m-HOCOC       Low    2.0 [+ or -] 1.0     3.7-4.0
              High   3.2 [+ or -] 0.5    2.6-13.1
p-HOCOC       Low    1.5 [+ or -] 0.5     2.6-3.3
              High   2.1 [+ or -] 0.3    2.5-13.2
m-HOBZE       Low    6.9 [+ or -] 1.0    2.6-10.1
              High   4.6 [+ or -] 1.0    2.5-16.7
p-HOBZE       Low    3.5 [+ or -] 0.3    2.6-16.3
              High   3.8 [+ or -] 0.2    2.5-57.7

(a) Percent of specimen positive for metabolite at 1 or
more times.

Table 5. Concentration (ng/mL) of 4-hydroxyamphetamine (in
urine) among amphetamines-positive samples (Reproduced with
permission from J Anal Toxicol [142])

Amphetamine     n     Methamphetamine   n    4-OH-AMP (a)    n
Conc. (ng/mL)         Conc. (ng/mL)          Conc. (ng/mL)

1,000-5,000     19    1,000-5,000       1    0-500           26
5,000-10,000    44    5,000-10,000      4    500-1,000       25
10,000-20,000   33    10,000-20,000     4    1,000-2,000     30
20,000-50,000   12    20,000-50,000     2    2,000-5,000     18
>50,000         1     >50,000           1    >5,000          10
Total           109                     12                   109

(a) LOD: 15 ng/mL.

Table 6. Relationship of melanin, lipophilicity and
hair incorporation rate (ICR) for
amphetamines and hydroxyamphetamines. [155,156,158]

Compound   Melanin affinity   Lipophilicity
           (x [10.sup.-4])
                              HPLC retention

MAMP       0.880-1.25         -0.80
OH-MAMP    0.20-0.360         -1.43
AMP        0.800              --
OH-AMP     0.320              --

Compound   Lipophilicity               ICR
                                   ([Hair]/AUC)
                   Log
           [P.sub.octanol/water]

MAMP       0.86                    0.13-0.3
OH-MAMP    0.51                    0.07-0.09
AMP        0.82                    0.10-0.20
OH-AMP     0.41                    0.03-0.08

Table 7. Diazepam and metabolites as separately
marketed drugs [222]

Drug          Trade name

Diazepam      Antenex, apaurin, apzepam, apozepam,
                diazepan, hexalid, pax, stesolid,
                stedon, valium, vival, valaxona
Nordiazepam   Madar, stilny
Oxazepam      Seresta, serax, serenid, serepax,
                sobril, oxabenz, oxapax, pamox
Temazepam     Restoril, normison, euhypnos,
                temaze, tenox

Table 8. Common 1,4-benzodiazepines and their major metabolites

Parent Drug                Metabolite

                   Name                         A marketed drug

Bromazepam         Hydroxybromazepam                  No
                   Cleavage Product                   No
                   Hydroxylated Product               No
Brotizolam         Hydroxymethyl                      No
                   4-Hydroxy                          No
Chlordiazepoxide   Norchlordiazepoxide                No
                   Demoxepam                          No
                   Nordiazepam                        Yes
                   Oxazepam                           Yes
Clobazam           4'-Hydroxy                         No
                   N-Desmethyl                        No
                   4'-Hy droxydesmethy l              No
Clorazepate        Nordiazepam                        Yes
                   Oxazepam                           Yes
Flunitrazepam      7-Hydroxy                          No
                   N-Desmethyl                        No
                   7-Amino                            No
Flurazepam         Aldehyde                           No
                   N-1-Hydroxy ethyl                  No
                   Desalkyl                           No
                   N-1-Desalkyl-3-hydroxy             No
Halazepam          Nordiazepam                        Yes
                   3-Hydroxy                          No
Loprazolam         Acetamido                          No
                   A-Oxide                            No
                   Hydroxy                            No
Lorazepam          Glucuronide                        No
Lormetazepam       Lorazepam                          Yes
                   Glucuronide                        No
Medazepam          Normedazepam                       No
Phenazepam         3-Hydroxy                          No
Prazepam           Nordiazepam                        Yes
                   3-Hydroxy                          No
                   Oxazepam                           Yes
Quazepam           2-Oxo                              No
                   A-Desalkyl-2-oxo                   No
                   3-Hydroxy-2-oxo                    No
                   3-Hydroxy-N-desalkyl-2-oxo         No
Tetrazepam         3-Hydroxy                          No
                   3-Hydroxy-nortetrazepam            No
                   Nortetrazepam                      No
                   3'-Hydroxy                         No
                   3'-Hy droxynortetrazepam           No
                   Diazepam                           Yes
                   Nordiazepam                        Yes

Parent Drug         Metabolite

                   Found in hair   Ref.

Bromazepam         Yes.            [38]
                   Unknown         --
                   Unknown         --
Brotizolam         Unknown         --
                   Unknown         --
Chlordiazepoxide   Unknown         --
                   Unknown         --
                   Yes             [98,101,117,139,187,208,216]
                   Yes             [98,102,105,139]
Clobazam           Unknown         --
                   Unknown         --
                   Unknown         --
Clorazepate        Yes             [98,101,117,139,192,217]
                   Yes.            [98,101,102,105,139]
Flunitrazepam      Unknown         ---
                   Unknown         ---
                   Yes             [41,42]
Flurazepam         Unknown         --
                   Yes             [190]
                   Yes             [174,187]
                   Unknown         --
Halazepam          Yes             [98,101,117,139,216]
                   Unknown         --
Loprazolam         Unknown         --
                   Unknown         --
                   Unknown         --
Lorazepam          Unknown         --
Lormetazepam       Yes             [39,102,106,226]
                   Unknown         --
Medazepam          Unknown         --
Phenazepam         Unknown         --
Prazepam           Yes             [98,101,117,139,187,216]
                   Unknown         --
                   Yes             [98,102,105,107,139]
Quazepam           Unknown         --
                   Unknown         --
                   Unknown         --
                   Unknown         --
Tetrazepam         Unknown         --
                   Unknown         --
                   Unknown         --
                   Unknown         --
                   Unknown         --
                   Yes             [102,107,117,120,139,187]
                   Yes             [98,101,117,139,187,216]

Table 9. Common triazolobenzodiazepines and their major metabolites

Parent drug   Metabolite

              Metabolite (a)        Found in hair   Ref.

Alprazolam    4-Hydroxy             Yes             [208]
              [alpha]-Hydroxy       Yes             [208]
              [alpha],4-Dihydroxy   Unknown         --
Estazolam     4-Hydroxy             Unknown         --
              1-Oxo                 Unknown         [208]
Etizolam      1'-Hydroxy            Unknown         --
              [alpha]-Hydroxy       Unknown         --
Midazolam     1-Hydroxy             Yes             [208]
              4-Hydroxy             Yes             [208]
              1,4-Dihydroxy         Unknown
Triazolam     1-Hydroxy             Yes             [90,180,202
                                                      209,210]
              4-Hydroxy             Yes             [202,209,210]
              1,4-Dihydroxy         Yes             [209,210]

(a) None of these metabolites are marketed as a separate drug.
COPYRIGHT 2017 Central Police University
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2017 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:White, R.M.
Publication:Forensic Science Review
Article Type:Report
Date:Jan 1, 2017
Words:20759
Previous Article:Principles of Toxicology, 3rd ed.
Next Article:Forensic SNP genotyping with SNaPshot: technical considerations for the development and optimization of multiplexed SNP assays.
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters