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
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  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 . In an entirely different set of circumstances, the finding of cocaine metabolite in an employee's urine using a cutoff of 150 ng/mL  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 . 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."  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 . 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  and (b) The Merck Index: An Encyclopedia ofChemicals, Drugs, and Biologicals . 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) . 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 . 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)  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 . 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 . 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 , amphetamines , cocaine , phencyclidine , and methadone  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 .
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 . 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 , and cocaine and BZE , have been measured in sebum. Codeine and norcodeine have also been detected and measured in sebum . 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 .
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  addresses numerous issues associated with membrane transport. A similar review may be found in reference . 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 . 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 , 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.  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 .
(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.
The basic scheme for the metabolism of the major psychoactive component of marijuana, [[DELTA].sup.9]tetrahydrocannabinol (THC), is presented in Structure 1 .
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 .
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 . 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 .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  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].
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The major metabolic cascade for cocaine is shown in Structure 2 . 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 . 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 .
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 . Thus, the commonly detected metabolites of cocaine probably are insufficient for demonstrating cocaine use through hair testing.
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In addition to the metabolites presented above, cocaine also oxidizes in vivo to meta (m)  and para (p) hydroxyl metabolites where the hydroxyl groups are on the phenyl ring of the benzoyl moiety  as shown in Structure 3. The formation of p-hydroxycocaine and m-hydroxycocaine in the microsomes of mice and other animals has been demonstrated . 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 , 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 . In addition, in the rat, dimethoxyhydroxycocaine  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 . The m-  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 .
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The m- and p-hydroxyl metabolites of BZE  and BZE and cocaine  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 .
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 . 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 . 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) .
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.  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 . 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.  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.
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 .
In vivo, the p-hydroxylation of methamphetamine (and, by extension, amphetamine), appears to be facilitated primarily by CYP2D6 . CYP2D6 is known to be highly polymorphic with over 100 allelic variants identified as of this writing . 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 . 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 , 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  using 121 amphetamines-positive urines out of approximately 1,000 Federal Mexican truck drivers.
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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) . 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. , 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 . The demethylation of MDMA to MDA is governed mainly by CYP1A2  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 . It is notable that in Ref. , 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 , 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 . CYP3A4 can demonstrate some polymorphism, but is considerably less polymorphic than CYP2D6  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) . From a separate study , 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 . 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).
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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.
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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 , 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 ) 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 . 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 . 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 .
In their study of the incorporation of codeine and metabolites into hair, Gygi et al.  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. , 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.  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. , 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. , codeine glucuronide was not found because the rat forms relatively little codeine-6-glucuronide. However, in later studies by Lee et al. , 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 , which is about equal to the conversion to hydromorphone , 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 . 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. , 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  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.
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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 , 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 . 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 .
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  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 . 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 . 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.  and Jones et al. , 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 .
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.
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Although over 50 benzodiazepines are now marketed for human consumption , metabolic cascades for only two common benzodiazepines (diazepam and alprazolam) are represented in Structure 12  or Structure 13 . 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.
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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 , it would be reasonable to use the glucuronides of oxazepam and temazepam as markers of use. However, in at least one single-dose study  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 .
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 .
Nitrazepam also forms 2-amino-5-nitrobenzophenone and 3-hydroxy-2-amino-5-nitrobenzophenone through opening of the 1,4-diazepine ring  (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  (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 . 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 . 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].
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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 . 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 .
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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 .
While ethyl glucuronide (EtG) remained stable in a control group, hair coloring and bleaching were found to decrease EtG significantly . 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 . 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 . In a separate reference , 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 . 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 . In one anecdotal case  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 .
The Society of Hair Testing has issued a consensus statement on hair testing for chronic excessive alcohol consumption .
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 . 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.  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.  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.  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 :
* Alcohol dehydrogenase,
* Aldehyde dehydrogenase,
* Esterase D,
* P450 Aryl hydrocarbon hydrolase,
* P450 Aromatase,
* P450 0-Deethylase,
* Glutathione reductase,
* NADPH P450 reductase,
* Glucosyl transferase,
* Glutathione-S-epoxide transferase, and
Work by Wolf et al. has demonstrated the presence of propionyl-CoA carboxylase and pyruvate carboxylase in hair roots .
In an article published after the works by Henderson and Potsch et al., Joseph et al.  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  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.  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.  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.  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. , 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.  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.  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.  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.  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.  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.  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.  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. , glutathione-S-epoxide transferase (GSH-T) can be detected in freshly isolated human hair follicles . By looking at the hydroxylation of dehydroepiandrosterone, Vermokken et al.  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 . 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 . Aromatase and 3[beta]-hydroxysteroid dehydrogenase have been located in both the human hair follicle and the sebaceous gland . 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  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
Research Triangle Park, North Carolina
United States of America
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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 ).
Caption: Figure 2: Development of the human hair follicle. (Reproduced with permission from Elsevier-Saunders .)
Caption: Figure 3: Anagen hair follicle. (Reproduced with permission from Elsevier-Saunders .)
Caption: Figure 4: Alignment and assembly of keratin molecules and filament packing. (Reproduced with permission for Elsevier-Saunders .)
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 .)
Caption: Figure 6: General human hair follicle and accompanying glands. (Reproduced with permission from Elsevier-Saunders .)
Caption: Figure 7: Schematic representation of the lipid bilayer comprising animal cells. (Reproduced with permission from Science and Garland Science .)
Caption: Figure 8: Cascade for reporting hair cocaine (modified from the reference). (Reproduced, following modification, with permission from Oxford Publishing .)
Caption: Figure 9: Multi-compartment model for drug incorporation into hair. (Reproduced with permission from Elsevier .)
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 .)
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 .)
Table 1. 2004 proposed hair testing cutoffs (pg/mg)  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  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  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 ) 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  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.  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  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  [alpha]-Hydroxy Yes  [alpha],4-Dihydroxy Unknown -- Estazolam 4-Hydroxy Unknown -- 1-Oxo Unknown  Etizolam 1'-Hydroxy Unknown -- [alpha]-Hydroxy Unknown -- Midazolam 1-Hydroxy Yes  4-Hydroxy Yes  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.
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|Publication:||Forensic Science Review|
|Date:||Jan 1, 2017|
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