Sample Preparation and Analytical Methodologies

As stated earlier, the US Federal Government has proposed the determination of 5 drug classes in hair cocaine, opiates, amphetamines, phencyclidine, and cannabinoids. Refer to Table 6.1. The analysis of drug analytes in hair is a multistep process: (1) decontamination of the surface of the hair fibers through washing; (2) sample preparation to facilitate ease of handling and release of drug analytes from the hair matrix; (3) solvent incubation or digestion to release drug analytes; (4) extraction and purification of drug analytes with liquid-liquid or solid-phase extraction techniques; (5) analysis by immunochemical or chromatographic techniques.

Workplace drug testing requires a sample to be tested by 2 separate procedures based on different scientific principles. In general, hair testing, like urine testing for workplace drug testing, uses immunoassay for the initial screening technique followed by confirmation using a mass sped rat technique. Immunoassays are chemical tests used to detect a specific drug class or a drug analyte in hair by using antibodies with defined specificity as part of an immunological reaction immunoassays are the preferred method because they are conducive to automation, require minimal sample preparation, and have relatively inexpensive labor and material costs. Immunochemical techniques currently used for hair testing include primarily radioimmunoassay (RIA) and enzyme-linked immune-sorbent assay (ELISA).

One or more of the first 3 sample preparatory steps previously listed may not be used during immunoassay testing. This time-saving choice is possible since samples identified by the immunoassay to contain a drug must be re-aliquoted and subjected to the more comprehensive confirmatory test.

The first steps often used throughout the confirmatory test protocol are decontamination procedures that are used to remove unwanted interferents (i.e., lipids, oils, cosmetics}, and exogenous analytes (e.g., environmental drug residues) potentially coating the hair surface. These decontamination procedures may use organic solvents, phosphate buffers, water, soaps, and various combinations and can take minutes to hours to perform. In additions some investigators have proposed the use of “Wash kinetic criteria” to demonstrate the effectiveness of the decontamination procedure. In this process, the drug concentrations in the last wash are determined, a multiple of the amount found is subtracted from the hair test results and this new value for the amount of drug in the hair is compared to the cutoff concentration. This procedure provides a conservative reporting process for reporting drug positives. Second, hair samples may be prepared for analyte isolation and extraction through mechanical pulverization (powdering), cutting into small segments, or left as intact strands of up to 4 cm long. The hair matrix can be further degraded by chemical or enzymatic digestion to allow access to drug that is present in the hair. Third a sample clean-up using liquid-liquid or solid-phase extraction is needed to further reduce interfering materials. Solid phase extraction is most often used because of the low concentrations of drug present in the matrix (relative to urine), the complexity of the specimen, and the need for highly efficient recovery.

In addition to the gold standard confirmatory method of gas chromatography/mass spectrometry (GC-MS) liquid chromatography/mass spectrometry (LC/MS), tandem mass spectrometry (GC/MS/MS, LC/MS/MS), and 2-dimensional gas chromatography/mass spectrometry with cryogenic focusing (GC/GC/MS), are techniques currently being used to confirm the presence of drugs in hair. Tandem mass spectrometry with increased sensitivity has allowed detection and quantification of analytes with low concentrations in hair such as 11-nor-∆9-tetrahydrocannabinol-9-carboxylic acid (THCA), fentanyl, and the metabolites of benzodiazepines. Other techniques, while promising, require further investigations to determine whether they will meet standards equivalent to current analytical techniques. For example, initial reports of a capillary zone electrophoresis-mass spectrometry ( CZE/MS) method to determine drugs of abuse and their metabolites [namely, 6-monoacetylmorphine (6-AM), morphine (MOR), amphetamine (AMP), methamphetarnine (MAMP), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BE), ephedrine, and cocaine (COC)] in human hair indicate that amount of hair ( 100 mg) required for this method is 5 to 10 times more than that needed for the sensitive tandem MS methods (10-20 mg). In addition, the limits of detection are not comparable for some analytes. Table 6-3 shows selected assays to detect drugs in hair.

Although it is important to have a general understanding of the practices used for the preparation and analysis of hair, no work would be complete without a discussion of the analytes detected in hair and the analytical methodologies used for that detection.

Cocaine

The detection of the compound cocaine in hair is not sufficient to identify drug use. Cocaine is often smoked and the cocaine released into the air may coat the hair shaft, leading to environmental continuation. In addition, cocaine may be on the surfaces of areas where it was used and can be transferred to the hair by contaminated hands. For this reason, other cocaine analytes and even ratios of the parent to metabolite are evaluated in workplace drug-testing programs in an attempt to ensure that the hair test only identifies illicit use of cocaine. There are up to 4 cocaine analytes routinely investigated for workplace drug testing. These analytes include the parent compound, cocaine, as well as BE, cocaethylene (CE), and norcocaine (NCOC). Parent cocaine is the most abundant analyte, followed by BE (10-50% of parent), CE (<20% of parent), and NCOC (<10% of parent). Not only are the analyte concentrations evaluated, but for BE the metabolite-to-parent ratio is determined to help differentiate drug ingestion from possible contamination. Cocaine’s rapid and complete metabolic disposition leads to low levels of unchanged drug in body fluids, but as previously mentioned, this is not the case for hair. Although parent cocaine may be most abundant in hair, it should not exist without the presence of metabolites because it acts as a non-acute repository. As BE is the most abundant cocaine metabolite in the hair, its concentration as well as the relationship of its concentration to the concentration of cocaine has been selected as an indicator of drug use. In this system, a BE-to-cocaine ratio that is <0.05 suggests that the hair is contaminated with cocaine and should not be reported as positive for drug use. Ratios for other cocaine analytes have also been monitored, with less success.

Procedures used to detect cocaine analytes include GC/MS, GC/MS/MS, and LC-MS /MS in APCI mode. Recently Moore et al. validated a cocaine confirmatory procedure using LC/MSMS with positive chemical ionization PCI procedure to simultaneously detect COC, BE, CE, and NCOC identifying 2 multiple reaction monitor mode (MRM) transitions, their ratio, and retention time . Deuterated internal standards were used for all analytes. This is particularly important for cocaine analytes, which have similar molecular weights and chemical properties; thus, the same product ion may also be present. Valiance in the ion ratios of 20% or less, customarily used for urinalysis was achieved in this method and reported for the first time for authentic hair specimens (Research specimens and proficiency specimens). All of these confirmatory methods and others mentioned in this section successfully detected the cocaine analytes at concentrations consistent with the SAMHSA-proposed guidelines. Refer again to Table 6.1.

One group from the United States has recently published several manuscripts detailing hair testing procedures for cocaine as well as demographic analyses of the results obtained from hair testing. Cairns et al. looked at cocaine concentration in 2 distinct populations. Results of 75 confirmed drug users and more than 6,000 workplace drug testing specimens were reviewed. The distribution of cocaine concentrations in drug user confirmed positive urinalysis and workplace populations authors do not state total workplace population, only confined positive hair testing results is listed in Table 6.4. While 86.6% of the cocaine containing hair from drug users had cocaine concentrations 2,000 pg/mg and higher, 45.8% of the workplace population, individuals seeking employment or gainfully employed, had similar concentrations. Additionally, the analyte profile for the workplace population showed the existence of BE levels greater than 5% for most of the confirmed drug users, but in some individuals (4-15%) of those with cocaine ≥500 pg/mg, the BE was <5% of the cocaine values. Even in the drug user population, 3 to 5% of the hairs containing cocaine did not contain BE at levels greater than 5% of the cocaine concentration. In addition, there were hair samples in both populations that contained cocaine >500 and BE at <5% and also contained other cocaine metabolites, such as CE and NCOC. In the workplace population, over half of the samples contained CE >50 pg/mg hair and about 78% of these contained NCOC at >50 pg/mg hair.

Similarly, Bourland et al. reported cocaine concentrations in 30 human head hair samples. These samples were randomly chosen production samples that had previously been reported positive by ELISA and GC-MS where additional hair was available for re-analysis. The cocaine analyte concentration ranges and means are listed in Table 6-5. Based on SAMHSA-proposed guidelines and SoHT guidelines listed in Table 6.1, at least 1 subject (Subject X: COC, 0.42; BE, 0.10) would have been reported as negative by SoHT guidelines and 2 subjects (Subject E COC, 21.36; BE, 0.62; Subject X) would have been reported negative for cocaine by SAMHSA-proposed guidelines.

Cocaine concentrations reported by Cairns et al. and Bourland et al. are consistent (Refer to Table 6.4 and 6.5). However, the smaller population studied by Bourland et al. had a lower percentage of subjects with concentrations (<2,000 pg/mg (20% vs 45%).

Opiates

The 3 primary opiate analytes reported for workplace drug testing include codeine, morphine, and 6-acetylmorphine. In hair, the latter 2 are normally the most abundant analytes. The screening and confirmatory cutoff concentrations for these opiate analytes are generally around 200 pg/mg. Other opiates less frequently detected include acetylcodeine, a contaminate in heroin and synthetic narcotics such as buprenorphine, fentanyl, and methadone.

Early studies demonstrated that morphine could be sufficiently removed from the hair matrix with a heated (100 °C) HCI extraction for 60 minutes, but extracting with methanol/trifluoroacetic acid produced minimum hydrolysis and maximum recovery of 6-acetylmorphine. Immunoassay based screening kits targeting morphine are available at the suggested cutoff concentration for opiate detection in both microplate and liquid reagent formats. Immunoassay kits for other opoids such as oxycodone, fentanyl, and buprenorphine are also commercially available but may require modifications when used for testing hair. Numerous procedures using GC/MS and LC/MS have been reported for the analysis of opiate analytes either alone or in combination with other drug classes.

Drug Concentrations

In 2005, Hill et al. reported morphine, codeine, and 6-acetylmorphine results for more than 471 workplace drug-testing cases consisting of an RIA screen (Cutoff 200 pg/ng). Morphine concentrations were confirmed above the cutoff in less than half (40%) of the hair samples, suggesting cross-reactivity with other opiate analytes. At morphine concentrations below 50 pg/mg, 6-acetylmorphine was present <5% of the time and present 18% of the time at concentrations below 200 pg/mg. Above the suggested cutoff of 200 pg/mg, 58.6% of the hair samples contained 6-acetylmorphine. In their study of subjects from a methadone clinic, they found that of 69 subjects whose urine samples were positive for morphine 30 did not contain 6-acetylmorphine above a 10 ng/mL cutoff. Analysis of hair samples taken from these 3 individuals found that 29 or 97% contained 6-acetylmorphine above 200 pg/mg. Similarly, analysis of paired hair samples from the 39 individuals whose urine samples contained morphine and 6-acetylmorphine showed all hair samples contained morphine and 6-acetylmorphine above the 200 pg/mg cutoff. These results suggest that the current SAMHSA cutoffs for morphine and 6-acetylmorphine in urine may be conservative and may not detect some heroin users and that proposed confirmed hair cutoff concentrations would increase the number of individuals testing positive for opiates.

In yet another study, Kauerl and Rohrich reported that the opiate concentrations and frequency of detection in 850 suspected drug users were 28.0 to 79,820 pg/mg for 6-AM in 141 cases, or 16.6%, and 11.0 to 7,800 pg/mg for morphine in 87 cases, or 10.2% of the samples. Heroin was also detected in 38 (4.5%) of the samples.

Scheidweiler et al. reported codeine concentrations in hair samples using LC-APCI-MS/MS. Hair was collected weekly from 10 volunteers after codeine administration at 60- and 120-mg/70-kg doses every other day for 1 week. The volunteers resided in a secure research ward for the Midweek study period and were at maximum (Cmax) (low dose 1291 ±182 pg/mg; 2725 ± 426 pg/mg) in 1 to 3 weeks. The concentrations were largely dose-dependent and were highly variable among individuals. Morphine and norcodeine were either not detected or detected minimally. This study also demonstrated a positive linear relationship between total melanin content of hair and codeine and corroborated other clinical studies as detailed. The first earlier study by Kronstrand et al. administered a single 100-mg codeine tablet orally to 9 volunteers. Hair collected after 7, 14, 21, and 28 days showed codeine concentrations of 24 – 176 pg/mg with peak concentrations occurring during the third week. Another study by RoperoMiller et al. used the same clinical study protocol and dosing scheme as Scheidweiler et al. and reported maximum total drug concentrations of 2,900 ± 1,600 pg/mg at the 60-mg/70-kg dose and 5100 ±2,700 pg/mg at the 120-mg/70-kg dose by taking the sum of the concentrations found in the hair and the combined wash fractions. Results were reported this way to allow for comparison to drug concentrations of nail scrapings collected at the same time to compare drug incorporation patterns of these keratinized matrices. Both investigations used GC/MS.

Other Opoids

Other opoids which are successfully detected in hair are proposed for workplace drug testing include oxycodone, hydrocodone, hydromorphone, methadone, and fentanyl. Similarly buprenorphine and tramadol have been detected in hair but are not discussed further in this chapter.

Fentanyl

Fentanyl is a synthetic opoid which is used as a narcotic analgesic for the treatment of severe pain because it can be up to 200 times more potent than morphine. The impetus to test for fentanyl in workplace drug testing has multiple origins. Health professionals are known to abuse this drug and monitoring is appropriate for these professions. Today, prescriptions for fentanyl as a self-medicating patch not requiring medical supervision are increasingly used as a pain management medication in the United States, increasing its availability for abuse. As recently as 200ff, HHS issued a warning that fentanyl was being combined with heroin and cocaine in street drug specimens in a number of urban areas in the United States. Moore et al. reported fentanyl concentrations in postmortem hair samples at concentrations ranging from 12 – 1,930 pg/mg. In all cases the hair results were confirmed by positive results in urine (n = 1) and/or blood (n = 4). These investigators used ELISA with a screening cutoff concentration of 20 pg/mg and 2-dimensional GC/MS with pulsed injections achieving a limit of quantification (LOQ) of 5 pg/mg. Without pulsed injections the LOQ was 10 times greater. Recently, Musshoff et al. also reported hair fentanyl concentrations up to 2,920 pg/mg in patients who are receiving opoids for the treatment of cancer pain.

Oxycodone

Jones et al. reported the simultaneous detection of codeine, morphine hydrocodone, hydromorphone, 6- acetylmorphine, and oxycodone in hair spiked with these drugs. However, opoid concentrations in authentic hair samples were not reported. Moore et al. reported oxycodone in two postmortem hair samples at concentrations of 10,175 and 707 pg/mg.

Hydrocodone and Hydromorphone

Hair collected from admitted opiate users was quantified by GC/MS following screening ELISA. Twenty-four hair specimens collected from volunteers showed the presence of hydrocodone ranging from 130 pg/mg to 15,933 pg/mg. Four of the hair samples also contained hydromorphone ranging in concentration from 59 pg/mg to 504 pg/mg. In the same study, hair samples from 5 self-reported codeine users showed concentrations of hydrocodone between 592 pg/mg and 15,933 pg/mg. The magnitude of the hydrocodone concentration in 1 of these 5 cases questions the reliability of the self-reports claiming use of codeine. Moore et al. also detected hydrocodone in a postmortem hair sample at a concentration of 882 pg/mg.

Methadone

For workplace drug testing, use of methadone is usually identified by the presence of only methadone. However in some cases, EDDP (2-ethylidene-1,5-dimethyl-3,3- diphenylpyrrolidine), the major metabolite of methadone, may also be included. Hair analysis performed by one laboratory in the United Kingdom over a Midyear period demonstrated a 0.03% (n = 37,217) confirmation rate for methadone. Cooper et al. used GC/MS to determine hair methadone concentrations in the hair of 46 drug users and 64 postmortem cases. Methadone concentrations ranged from 100 pg/mg to 8,300 pg/mg for methadone and 100 pg/mg to 1,200 pg/mg for EDDP. Paterson et al. determined the methadone concentrations found in hair of 50 patients receiving daily oral methadone treatment at low (20-50 mg), medium (60-100 mg), and high (110-165 mg) dosages. The ranges for the methadone concentrations ranges were 4,200 to 22,700 pg/mg, 2,600 to 63,200 pg/mg, and 9,600 to 43,900 pg/mg at the low-, medium-, and high dose regimens, respectively. Overall, there is a dose-concentration trend in that the mean for the low-dose group was lower than for the medium or high groups of similar means, but the ranges for all 3 groups overlap considerably. Hair analysis in 9 autopsy cases showed the presence of methadone in 4 cases, 2 of which could not he verified due to insufficient quantity for testing. The other 2 postmortem hair samples had methadone concentrations of 1,235 pg/mg and 2,389 pg/mg.

Amphetamines

Amphetamines are relatively easy to detect in hair using GC/MS analysis or equally sensitive methods of confirmation. Simultaneous detection of AMP, MAMP, and several amphetamine analogs including MDA, MDMA, 3,4-methylenedioxyethylamphetamine MDEA, and 3,4-methylenedioxyo-α-ethyl-N-methylphenethylamine (MBDB) were reported early in the literature. Moreover, specialized investigations of AMP and MAMP in a single hair by HPLC-chemiluminescence (HPLC-ChemLu) and segmental analysis of hair for these amphetamines has also been reported.

Drug Concentrations

In 1990, Nakahara et al. reported methamphetamine concentrations ranging from 2,000 pg/mg to 57,000 pg/mg in head hair from 12 subjects. In 2004, Cairns et al. reported amphetamine concentrations in clinical (n = 40) and large workplace populations screening samples (amphetamine, n = 2766 and MDMA, n = 617) by radioimmunoassay and confirming by LC-MS/MS. MRM was used, utilizing the following target ions: methamphetamine, m/z 91; methamphetamine d-11, m/z 96; amphetamine, m/z 91; amphetamine-d8, m/z 96; MDMA, m/z 135 and m/z 136; and MDA, m/z 135 and m/z 137. The authors used a 3.75-h wash procedure consisting of multiple steps using sequential isopropanol and phosphate buffer washes. The wash liquid from each step was subsequently analyzed as part of this laboratory’s wash kinetic criteria to evaluate the potential external contamination of samples for reporting purposes. Methamphetamine concentrations in the clinical samples (170-34,400 pg/mg hair) were similar to those observed in the workplace population (less than the cutoff of 500 pg/mg to >20,000 pg/mg hair), but the workplace population had a lower percentage of high methamphetamine concentrations. In both populations, there was no direct relationship between methamphetamine and associated amphetamine concentrations. The amphetamine concentrations were found to vary widely at all levels of corresponding methamphetamine. MDMA-positive workplace samples were also reported with concentrations ranging from below the cutoff of 500 pg/mg to >10,000 pg/mg, and the corresponding MDA concentrations varied widely from 10 pg/mg to >3,000 pg/mg hair. These results for the amphetamine analogs were comparable to results of 2 other investigative groups. Rothe et al. reported that the hair from 20 subjects taking ecstasy and speed tablets had MDMA concentrations ranging from 110 to 8,300 pg/mg and MDA concentrations ranging from 40 to 2,100 pg/mg. More recently, Miyaguchi et al. reported the toxicological analysis for MAMP and AMP with as little as 2 milligrams of hair using a rapid sample preparation coupled with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). These investigators compared micropulverized extraction (MPE), acidic methanol extraction, and alkaline digestion using actual-case hair specimens. The MPE extraction demonstrated slightly better recoveries and the results in pg/mg for each subject by this preparation were as follows: Subject 1: MAMP 52,600 ± 2,170 and AMP 2,910 ± 170; Subject 2: MAMP 11,640 ± 1,710 and AMP 300 ± 40.0; Subject 3: MAMP 63,910 ± 6730 and AMP 6,150 ± 740; Subject 4: MAMP 18,520 ± 1,460 and AMP 2,010 ± 170; Subject 5: MAMP 20,160 ± 3,760 and AMP 950 ± 150.

Phencyclidine

Phencyclidine (PCP) is a somewhat basic drug, which is readily detected in hair as the parent drug analyte. PCP was, first detected in human hair in the early 1980s. Most routine analyses detect only un-metabolized PCP, although metabolites are readily detectable in human hair PCP in hair can easily be measured by immunoassays and simple GC-MS analysis. Electron impact ionization and selective ion monitoring of 3 ions and 2 ratios is a common analytical procedure for the detection of PCP in hair.

PCP is included in routine hair analysis even though most studies indicate its prevalence rate is lower than most other drugs of abuse (Refer to Table 6-2). For instance, Swartz et al. collected urine and hair from 203 consenting schizophrenic patients. Drug use in the preceding months was detected in hair using a radioimmunoassay for PCP and other drugs of abuse. Urine was also tested for drugs of abuse using point-of care immunoassay devices. None of the 203 participants tested positive for PCP. While only 33 (16.3%) self-reported illicit substance use, 25 {jl2.4%) had a positive urine test, and 63 (31.0%) had a positive hair assay. When the results from the self-report and the urine and hair tests were combined, 78 participants (38.4%) were classified as users during the 3-month period preceding the tests. Similarly, Mieczkowski and Newel used hair and urine testing as an objective measure of drug treatment outcome in a criminal justice diversionary treatment program for first-time, nonviolent offenders. Hair samples were collected at study commencement and bimonthly intervals during the program, with random urine testing also being used. Hair analysis at intake showed 1 of the 91 subjects (1.0%) to have a positive result for PCP.

Drug Concentrations

Reported PCP concentrations in hair for any population are limited. A search of the literature resulted in the following findings.

As early as 1997, Nakahara et al. analyzed some extracts of clinical hair specimen from 8 PCP users. The hairs were digested in methanol-5N HCl (20:1), extracted using solid-phase extraction and analyzed by GC/MS after a silinated derivatization. Their results showed PCP concentrations ranging between 330 and 1,400 pg/mg, and its 2 major metabolites.

Hair samples were assayed by GC/MS and 2 patients were positive for PCP at concentrations up to 1,500 pg/mg. In 2005, Chaffer and Hill reported PCP concentrations in hair samples from a proven drug user population ranging from the confirmatory cutoff concentration of 300 pg/mg up to 15,000 pg/mg.

Cannabinoids

Marijuana, or cannabis sativa, is the most frequently used illicit drug in the United States. While the active constituent of cannabis, ∆9-tetrahydrocannabinol (THC), is the most abundant analyte in hair, other analytes, including 11-hydroxy-∆9-tetrahydrocannabinol (11-OH-THC), THCA, cannabinol (CBN), and cannabidiol (CBD) are habitually monitored for workplace drug testing. 11-OH-THC and THCA are monitored because they are metabolites of THC and their presence corroborates actual drug ingestion. However, the concentrations of THCA and 11-OH-THC in hair are extremely small because of the low rate at which acidic substances are incorporated into the hair matrix. In addition, certain washing procedures (e.g., dichloromethane) and sample preparation procedures (e.g., use of plastic containers3 can significantly decrease cannabinoid concentrations in hair). In addition, traditional GC/MS analysis is not reproducible or even possible to perform at a THCA LOQ below 1.0 pg/ng. Therefore, highly sensitive techniques must be used for the analysis of THC metabolites in hair, making it one of the most difficult analytes to detect.

Drug Concentrations

Musshoff et al. determined cannabinoid concentration in pubic hair using techniques such as headspace solid-phase microextraction and gas chromatography-mass spectrometer. The range of concentrations of these compounds found in pubic hair was as follows: THC at 100 to 4,000 pg/mg (n = 9), CBN at 100 to 400 pg/mg (n= 5), and CBD 600 to 19,000 pg/mg (n = 5). Kints et al. analyzed hair for THCA using GC-NCI-MS and reported concentrations ranging from 20 to 390 pg/mg. In a study investigating hair samples taken from 850 suspected drug users, Kauert and Rohrich reported THC concentrations ranging from 9.0 to 16,700 pg/mg in 104 (12.2%) of the cases.

Another current report by Skopp et al. studied the potential relationship between the cannabinoid concentration in hair and the cumulative dose in regular users of cannabis (0.5-2.5 grams/day). Head hair samples (vertex region) were obtained from 12 male regular users of cannabis and 10 male subjects with no experience of cannabis serving as negative controls. The concentration of THC, CBN, and CBD in hair was determined using gas chromatography-mass spectrometer and the concentration ranges for each analyte were as follows:

THC at 90 to 720 pg/mg, CBN at 50 to 260 pg/mg, and CBD at none detected to 570 pg/mg. As anticipated, cannabinoids were present in any hair sample of cannabis users, but were not detectable in control specimens. A boost in the amount of cannabinoids in hair with increasing dose was evident, but a significant relationship between THC and the amount of cannabis used could not be established. Each cannabinoid concentration alone could not correlate to a dose; though, a cumulative (sum of THC, CBD, and CBN) cannabinoid concentration was significantly correlated to either the reported cumulative cannabis dose during the last 3 months or to the cannabis use during the last 3 months estimated from the reported daily dose and the frequency per year (r = 0.68 or 0.71, P = 0.023 or 0.014). As such, the authors concluded that the sum of major cannabinoids in hair of regular users may provide a better measure of drug use than THC.

An additional clinical study investigated the deposition of cannabinoids in human hair following controlled administration of cannabis. Huestis et al. collected 53 head hair specimens from 38 males with a history of cannabis use as evidenced by self-report and con med by urinalysis. The study was conducted in accordance with in an institutional review board approved protocol that included controlled, double-blind administration of THC. Subjects were grouped as daily users (n = 18) or non-daily use, i.e., 1 to 5 cannabis cigarettes per week at (n= 20). Hair specimens were collected upon admittance and following smoking of two 2.7% TIC cigarettes (n = 13) or multiple oral doses totaling 116 mg THY (n = 2). There was a 7-day span between the smoking of each THC cigarette, and a hair specimen was collected later in the same day as the last smoked THC cigarette. Hair specimens were screened by ELISA (cutoff 5pg/mg; THC target) and confirmed by tandem GC-MS. Eighty-three percent of specimens that screened positive were consumed by at a cutoff concentration of 0.1 pg/mg hair (THCA). Surprisingly, 19 specimens (36%) contained no detectable THC, or THCA at the limits of quantification (LOQ) of 1.0 and 0.1 pg/mg hair, respectively. Two specimens 3.8% contained THC only, 14 (26%) THCA only, and 18 (34%) had both cannabinoids present. Twenty-two (62%) of the hair specimens without THC and/or THCA were collected prior to dosing from admitted marijuana users. Detection rates were significantly different (P <0.05, Fisher’s exact test) between daily cannabis users (85%) and non-daily users (52%). There was no difference in detection rates between African-American and Caucasian subjects (P > 0.3, Fisher’s exact test) corroborating, earlier preliminary findings. Detectable cannabinoid concentrations were as follows: THC, 3.4pg/mg to >100 pg/mg; THCA, 0.10pg/mg to 7.3pg/mg. The cannabinoid concentrations were positively correlated (r = 0.38, P < 0.01, Pearson’s product moment correlation). As most cannabinoid screening tests have higher activity win the metabolites than the parent drug, the relatively low amounts of the metabolites in relationship to the parent drug result in lower confirmation rates than many other drug classes. The authors acknowledged that Manufacturers may be able to change the specificity of the initial test and optimize the detection and confirmation rates, but at present immunoassays similar to that used in this study appear to have acceptable performance.” This study indicates that hair testing for marijuana is most effective for picking up chronic marijuana users and can easily miss the occasional marijuana user.

Alcohol

Ethyl alcohol cannot be straightforwardly detected in hair, which may be understandable due to the volatile nature of teds compound However, minor metabolites produced from non-oxidative biotransformation of ethyl alcohol, such as ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) are potential markers of ethyl alcohol use. Less than 0.05% of the dose of ethyl alcohol is metabolized to ethyl glucuronide. EtG is a nonvolatile metabolic marker of alcohol use and was first detected in human urine by Jaakonmaki et al. FAEEs are enzymatically formed in a minor side route of ethanol biotransformation from free fatty acids or lipids remaining in the bloodstream up to 24 h after cessation of alcohol consumption. FAEEs are typically characterized as the sum of concentrations of ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate. Once these minor metabolites are circulating in the bloodstream they can then be incorporated into human hair follicles. As the hair grows, it absorbs both FAEEs and EtG into its structure, which remains in the hair indefinitely. These markers are only produced when there is alcohol in the bloodstream, such that the more markers there are; the more alcohol one has consumed. Thus, the presence of EtG 697, 981 and FAEEs ~91 represents markers of chronic alcohol consumption. The analytical methods used for EtG determinations were GC-MS and LC MS-MS, and GC-MS was also used for FAEE detection. Janda et al. concluded that the presence of ethyl glucuronide in hair has been correlated with frequent alcohol misuse and not with social drinking.

Drug Concentrations

To make the difference of chronic alcoholism or excessive drinking by detection of one or both of these markers, a high enough cutoff or threshold level would need to be established since theoretically social drinking can produce these markers. At present, a cutoff level is not well characterized, but Bendroth et al. proposed a 30 pg/mg cutoff for EtG in hair as an indicator of alcohol abuse. A cutoff of 1,000 pg/mg (1 ng/mg) in hair has been proposed for FAEE to indicate excessive alcohol consumption, and a concentration <400 pg/mg has been proposed as indicative of teetotalers or social drinkers. Yegles et al. looked at both FAEE and EtG in the hair of 4 separate populations: (1) alcoholics in a withdrawal treatment program, (2) fatalities know to be alcoholics, (3) social drinkers, and (4) teetotalers. In this study no EtG was detected in the hair of social drinkers or teetotalers at a limit of detection LOD and LOQ of 2 and 4pg/mg respectively. However, in 4 social drinkers and in the teetotaler population, which included pooled hair from children, FLEE was detected at concentrations ranging from370 pg/mg to 500pg/mg and 50pg/mg to 370pg/mg, respectively. The analysis of hair in 10 alcoholics in a withdrawal treatment program yielded an average concentration of 159.5 Pg/mg of EtG, ranging from 30 pg/mg to 415 pg/mg, and with an average concentration of 3,614 pg/mg of FAEE (650 pg/mg to 20,480 pg/mg). The highest concentrations of EtG and FAEE were seen in the hair of postmortem specimens from 11 known alcoholics which averaged 910 pg/mg EtG (72 pg/mg to 3,380 pg/mg) and 9,391 pg/mg FAEE (1,300 pg/mg to 30,600 pg/mg). A similar study looking at 4 separate populations (alcoholics postmortem; alcoholics, psychiatric cases; social drinkers; children) yielded similar findings. No EtG was detected in the hair of social drinkers and children, whereas EtG was detected in hair of 14 of 16 alcoholic postmortem specimens and 4 of 4 psychiatric alcoholic patients. The concentrations of EtG in the 14 positive postmortem hair specimens ranged from 218 pg/mg to 4,025 pg/mg and 119 pg/mg to 388 pg/mg in the hair of the alcoholic psychiatric patients. No LOD or LOQ was characterized in the above study, so it is unclear whether EtG was present below 100pg/mg in the 2 postmortem specimens that were reported as EtG not detected. Bendroth et al. compared EtG in postmortem hair to phosphatidylethanol by path in postmortem whole blood of postmortem specimens. Bendroth et al. found measurable levels of EtG in the hair of 4^9 of 70 autopsy cases compared to 36 cases containing (PEth) in whole blood. Only 39 of the 49 positive EtG hair specimens were above the 30 pg/mg cutoff, with concentrations ranging from 7.5pg/mg to 10,400 pg/mg EtG.

Other Drugs

Other drugs that have been investigated for the purposes of workplace drug testing include benzodiazepines, pain management drugs (e.g., buprenorphine), and non-benzodiazepine hypnotics (e.g., zolpidem). Currently the most common reason to test human hair for these drugs is for drug-facilitated sexual assault (DFSA) investigations. However, a study conducted in the United Kingdom over a 5-year period from 2001 to 2005 cited diazepam as the fifth most common drug detected in human hair among workplace drug test specimens. In the above study, THC was the most common drug detected in 4% of the workplace population followed by codeine (2%), cocaine (2%), MDMA (0.2%), and diazepam (0.1%). The major analytical methods that are used for the detection of benzodiazepines and metabolites, zolpidem, and buprenorphine in human hair are GC-NCI/MS, LC-MS/MS USA, and LC/MS.

Drug Concentrations

In a French study, 115 human head hair samples were tested for “forensically relevant” benzodiazepines by CC-NCI/MS. The head hair specimens tested positive for nordiazepam (200-18,870 pg/mg, n = 42), oxazepam (100-500 pg/mg, n = 14), flunitrazepam (19-148 pg/mg, n = 31), lorazepam (31-49 pg/mg, n = 4), and alprazolam (300-1,240 pg/mg, n = 23). Benzodiazepines such as clonazepam, bromazepam, and flunitrazepam have been successfully identified in human hair even when only a single dose has been administered, which is a relevant scenario in DFSA investigations. In 6 of 10 volunteers each administered a 3-mg dose of Klonopin (clonazepam), the metabolite 7-amino-clonazepam (7-ACLO) was detected at concentrations ranging from 1.2 pg/mg to 8.4 pg/mg. However, clonzepam (CLO) was not detected in any subject. When more than a single dose was involved, CLO was successfully identified in human head hair specimens. In human head hair specimens collected from 10 psychiatric patients prescribed clonazepam, 7-ACLO was detected in 9 of 10 patients at 13.7-1,267 pg/mg and CLO was detected in 6 of 10 patients (10.7-180 pg/mg). Two volunteers were each dosed with 6 mg of bromazepam and head hair samples 1 month later resulted in bromazepam concentrations of 0.8 and 4.7 pg/mg. A single 2-mg dose of Rohypnol (flunitrazepam) was administered to 10 healthy volunteers and 7-aminoflunitrazepam (7AF) was detected in all 10 subjects (0.6-8.0 pg/mg), whereas flunitrazepam was detected in some patients, all below the LOQ using GC-NCI/MS. Cirimele et al. tested 40 postmortem head hair samples obtained from heroin overdose victims for Rohypnol use and found 14 samples positive for flunitrazepam, with a mean concentration of 60 pg/mg (31-129 pg/mg), and 26 samples contained 7-AF at an average concentration of 46 pg/mg (3-161 pg/mg). Tetrazepann was detected in the hair of 2 volunteers being administered a single dose of 50 mg after 4 weeks at 123-175 pg/mg using an LC MS/MS method 61139. However, 1 study was unable to detect lorazepam after a single exposure despite LC-MSIMS analysis with an LOQ of l.0pg/mg. Alprazolam was detected in the head hair from 2 separate criminal cases in France, involving adolescent victims, at concentration; ranging from 0.4 pg/mg to 4.9 pg/mg in hair segments either 1.0 or 2.0 cm in length. Buprenorphine was detected in head hair via LCIMS at 23 pg/mg in an adolescent victim of sexual abuse. Zolpidem was identified in human head hair of a 23 year-old victim of sexual assault at 0.75 pg/mg using LC-MS-MS.

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