Opiate Recidivism in a Drug-Treatment Program: Comparison of Hair and Urine Data

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1 Journal of Analytical Toxicology, Vol. 27, October 23 Opiate Recidivism in a Drug-Treatment rogram: Comparison of Hair and Bradley K. Charles 1, Jayme E. Day 1, Douglas E. Rollins 1, David Andrenyak;, Walter Ling 2, and Diana G. Wilkins,* 1Center for Human Toxicology, University of Utah, Salt Lake City, Utah and 2Integrated Substance Abuse rograms, Department of sychiatry and Biobehavioral Sciences, David Geffen School of Medicine at UCLA, Los Ange/es, California Abstract I The objective of this preliminary study was to determine whether hair can be used as an adjunct specimen for the monitoring of opiate use in a drug-treatment program. Subjects (n = 1) initiating clinical therapy for opiate addiction were monitored for up Io 17 weeks with hair and urinalysis. Questionnaires were administered weekly to document hair cuts and chemical treatments. Hair specimens were collected weekly by cutting at the scalp and segmented into 1-cm lengths prior to analysis. Codeine (COD), morphine (MOR), and 6-monoacetylmorphine (6-MAM) concentrations in hair were measured by liquid chromatography-mass spectrometry (LC-MS) [limit of detection (LOD): 2 pg/mg for COD and 6-MAM; pg/mg MOR]. Urine specimens were analyzed by semiquantitative radioimmunoassay (2-ng/mL cutoff) and LC-MS for codeine (COD), morphine (MOR), morphine-31~-glucuronide (M3G), morphine-613- glucuronide (M6G), and 6-monoacetylmorphine (6-MAM). The LOD and limit of quantitation (LOQ) in urine for COD, M3G, M6G, and 6-MAM were 1 ng/ml and 2 ng/m/for MOR. Interpretation of the segmental hair data in this study was complex and generaley was not in agreement with urine data in most cases. Evaluation of hair data suggested that 6 of 1 subjects discontinued opiate use by the end of the study, whereas 3 of 1 appeared to have reduced their use. One subject appeared not to have used opiates throughout the entire study. In contrast, evaluation of urine data suggested that only of 1 subjects significantly reduced use, and 6 of 1 continued drug use on at least an intermittent basis. Urine appeared to be a more sensitive indicator of changes in the pattern(s) of drug use during the course of clinical drug treatment. Introduction Hair has a number of characteristics that make it an attractive matrix for recidivism and compliance monitoring. In contrast to plasma and urine, once drug has been incorporated into the hair shaft it is sequestered from further metabolism and excretion, which potentially increases the time window for ' Author to whom correspondence should be addressed: University of Utah. 2 South 23 East. Room. Salt Lake City, UT 1. detection of drugs. akahara et al. (1) reported that methoxyphenamine incorporated into hair shortly after administration remained in a "drug band" without diffusion along the hair shaft. If this held true for other drugs as suggested by the authors, hair analysis could potentially be used to determine the timing of drug use. This characteristic would be attractive for a drug-rehabilitation program because it may permit therapeutic drug dosage regimen to be individualized for optimum effectiveness. Many drugs of abuse, such as heroin, A-tetrahydrocannabinol (THC), and phencyclidine (C), are generally nonpolar in nature. After ingestion, the drug is metabolized to metabolites that are typically more polar than the parent drug. The drug and/or metabolite can then readily be excreted in the urine. Hair can also be thought of as a site of excretion for drugs and metabolites. It appears that hair preferentially incorporates non-polar parent drugs and their short-lived metabolites, such as 6-monoacetylmorphine (6-MAM) (2,3). This characteristic may be used to differentiate between therapeutic drug use (i.e., detection of codeine prescribed for pain) versus drug abuse (i.e., detection of heroin or 6-MAM). Another advantage of hair analysis is that one can obtain a second sample from the original source at a later date, presumably without losing the ability to detect drug use (). However, hair analysis has a number of potential drawbacks for use in recidivism and compliance monitoring. Because hair grows relatively slowly (approximately. mm/day), sufficient hair does not grow out of the follicle to be cut for analysis until approximately seven days after drug usage; therefore, hair analysis is unable to detect very recent use (,6). Another potential limitation of hair analysis lies in the inherent variability of human hair growth. It is generally assumed that scalp hair grows 1 cm/month (approximately.33 mm/day); however, there has been a wide range of growth rates reported in the literature. For example, Saitoh et al. (7) reported that scalp hair grows at a rate of 1.32 and 1.3 cm/month (. and. ram/day for males and females, respectively); Myers and Hamilton () reports 1.2 and 1. cm/month (.3 and.36 ram/day for males and females, respectively). For two fema]e subjects, Giovanoli-Jakubczak and Berg () report 1. and.7 cm/month (.37 and.2 ram/day), whereas Uematsu et al. 1 2 Reproduction (photocopying) of editorial content of Is journal is prohibited without publisher's permission.

2 Journal of Analytical Toxicology, Vol. 27, October 23 (1) and Wilkins et al. (11) report ranges of.-1.3 and.37-. cm/month (.3-.3 ram/day and.-.32 ram/day), respectively. Therefore, the variability in hair growth estimates limits the interpretation of chronology of drug usage. Also, a number of investigators have reported that pigmentation plays a differential role in drug incorporation into hair (11-21). Several reports suggested that there is a potential for racial bias in hair testing for drugs of abuse (22,23). If participation in a recidivism- or compliance-monitoring program is court-ordered, and legal action can result from a positive (recidivism monitoring) or negative (compliance monitoring) test result, then interpretation of a test with a potential for racial bias becomes legally and ethically challenging. The effect of chemical and cosmetic treatments on hair analysis is another potential limitation. 6tsch and Skopp (2) have demonstrated that chemical and cosmetic treatments such as perming and bleaching decrease the stability of opiates in hair, and other researchers have demonstrated that it decreased concentrations of cocaine and its metabolites (2), as well as methamphetamine and amphetamine (26). Yegles et al. (27) demonstrated that bleaching also decreased the stability of benzodiazepines in hair. Despite these limitations, hair continues to be a viable matrix for many recidivism- and compliance-monitoring applications because a subject can serve as his or her own control over time. The objectives of the present preliminary study are as follows: 1. to evaluate a method of analyzing hair samples for 6-MAM, codeine (COD), and morphine (MOR) using liquid chromatography-mass spectrometry (LC-MS); 2. to evaluate whether the quantitative determination of opiates by segmental hair analysis can predict drug use during a clinical drug-treatment program; and 3. to determine if segmental hair analysis is sufficiently sensitive to detect changes in opiate usage during the course of a clinical drug-treatment program. Materials and Methods Human subjects Ten patients seeking treatment for opiate dependence at the izarro Treatment Center (Los Angeles, CA) were recruited after obtaining informed consent. Informed consent for Human Subjects Research was approved by the Institutional Review Boards of the University of Utah and Friends Research, Inc. Subjects were non-obese males and females with varying hair colors and were between 1 and 6 years of age. Subjects received intensive outpatient treatment for opiate addiction, including daily buprenorphine (BU) therapy at the clinic for 16 weeks, followed by a detoxification protocol. lasma and hair specimens were collected once a week. Urine specimens were collected three times a week. Hair cuts and/or chemical treatments were monitored by weekly interview and questionnaire. Hair cuts and chemical treatments add a variable that was not controlled in our study. Our goal was to mimic a realistic drugtreatmeflt-program setting. We believed that the typical patient in a four-month routine treatment program would not abstain from these practices. Standards and controls Drug-free hair for calibrator and quality control samples were obtained from clippings provided by human volunteers in the Center for Human Toxicology and verified to be drug and metabolite free for COD, MOR, and 6-MAM by MS. This hair was pooled, minced into approximately 1-mm pieces, and transferred into 16 1-ram silanized culture tubes. Ten milligrams (.2 rag) of drug-free hair was aliquoted for each calibrator and quality control sample. Human urine used for calibrator, quality control, and subject samples was obtained from a pool provided by volunteers in the Center for Human Toxicology and verified to be drug free by mass spectrometry. Urine was analyzed by LC-MS and determined to be negative for morphine-3~-glucuronide (M3G), morphine-6[3-glucuronide (M6G), COD, MOR, and 6-MAM. One milliliter of drug-free urine was aliquoted into 16 x 1-ram silanized tubes for each calibrator and quality control sample. Collection of hair specimens Hair samples were collected from the posterior vertex region of the scalp, as close as possible to the scalp, and each sample was secured to a piece of aluminum foil with adhesive tape. The root and tip sides were marked to facilitate subsequent orientation of the hair and sent to the laboratory via overnight courier. The samples were then segmented into 1-cm lengths (measured from the scalp) prior to analysis. The small hair segments were then minced into approximately 1-mm pieces in pre-weighed 2-mL scintillation vials on a Mettler AE 2 analytical scale and the weight recorded. Samples prepared from segments closest to the scalp were the heaviest, and segments towards the tips usually weighed less because of normal hair growth, as well as damage due to regular hair care (combing and brushing) and chemical treatments. When there were fewer than 1 individual 1-cm segments in a particular sample, the analytical balance was not sufficiently sensitive to accurately determine the mass. Because these sample weights were not accurately measurable, the segments were not analyzed by LC-MS. For example, Subject I demonstrated an apparent discrepancy in the number of hair segments between week # and #. In week #, 11 segments were analyzed; in week #, 23 segments were analyzed. This might suggest to the reader that the subject's hair grew cm in weeks. However, this was not the case; the laboratory received a total weight of less than 2 mg for week #, resulting in many 1-cm segments with less than the required 1 strands. Variability of quantitation in small segments Because of the small patient sample sizes available after segmentation, there were concerns that the reproducibility of quantitative data would be too variable to permit historical assessment of drug use. To address this potential problem, an experiment was designed to evaluate the variability of quantitation when less than 1 mg of hair was available for analysis. Hair for this assessment was obtained from a pool of rat hair (n =1) taken from male Sprague-Dawley rats (1-16 rag) that were housed at constant room temperature with alternating -h light and dark cycles with free access to food and water. Animals 13

3 Journal of Analytical Toxicology, Vol. 27, October 23 were housed individually in hanging wire cages to prevent contamination from bedding, urine, or the saliva of other rats. Codeine hydrochloride dissolved in normal saline (2 mg/ml as free-base) was administered by intraperitoneal (ip) injection once per day at a dose of mg/kg (n = 1) for days. rior to the first dose, a 1-in 2 area of hair was shaved to the skin from the animal's back with an electric animal shaver. The areas were allowed to grow hair during the days of drug administration and were reshaved 1 days after beginning the drug administration. Harvested rat hair was pooled, finely cut, mixed and aliquoted into pre-weighed 16 1 silanized tubes (., 1.,., and 1. mg; n = for each mass). Samples were extracted and analyzed as described (see reparation and extraction of hair samples and LC-MS of hair samples). reparation and extraction of hair samples Hair samples were not washed prior to analysis, but subjects were allowed to wash their hair daily with their usual hair care product. Working solutions of MOR, COD, and 6-MAM were prepared in methanol at.1,.1,.1, 1., and 1. ng/pl. Calibrator samples were fortified with the methanolic working solutions at.1,.2,.,.,.1,.2,.3,., 1., 2., 3.,., 1, 2, and ng/mg and allowed to equilibrate for 1 rain. Quality control samples were fortified with similarly prepared working solutions at.3, 2., and 1 ng/mg (n = 3 each concentration). rior to extraction, calibrator, quality control, and subject samples were fortified with 2 IJL of 1. ng/ijl MOR-d3, COD-d3, and 6-MAM-d3. The final internal standard concentration was 2. ng/mg. Reference materials were supplied by Cerilliant TM (Austin, TX) and Sigma Chemicals (St. Louis, MO). All hair samples were then directly extracted in 1. ml of acetonitrile overnight on a shaking water bath at approximately 6 cycles/min and at room temperature. The ph of each sample was adjusted to. by adding 1. ml of saturated sodium borate and drops of.tm HC[. Verification of ph was accomplished using ph indicator paper (Whatman Maidstone, England). The samples were vortex mixed briefly and ml of n-butyl chloride/acetonitrile (:1, v/v) was added to each sample. Samples were rocked for min and then centrifuged for 2 min at 26 rpm (International Centrifuge, Boston, MA). The organic layer was transferred to 13 x 1-mm silanized tubes where it was evaporated to dryness under a stream of air at ~ The residues were reconstituted in IJL of 1mM ammonium acetate (ph.)/acetonitrile (:1, v/v) and transferred to 3-1JL conical bottom autosampler vials. reparation and extraction of urine samples for M3G, M6G, COD, MOR, and 6-MAM Working solutions of M3G, M6G, MOR, COD, and 6-MAM were prepared in MilliQ water (Millipore, Bedford,.~) at.1, 1., and 1. ng/ijl. Calibrator samples were fortified with the aqueous working solutions at 1, 2,, 1,2, and ng/ml. Quality control samples were fortified with similarly prepared working solutions at 2, 7, and 2 ng/ml (n = 3 at each concentration). rior to extraction by solid-phase extraction (SE) 1. ml of calibrator, quality control sample and 1 IJL of patient sample were fortified with 1 1JL of 1. ng/tjl of M3G-d 3, MOR-d3, COD-d3, and 6-MAM-d 3 also prepared in MilliQ grade water. The final internal standard concentration was 1 ng/ml. Reference materials were supplied by Cerilliant and Sigma Chemical. Each sample was adjusted to ph. by addition of 2 ml of 1mM ammonium bicarbonate, ph and drops of 1.M ammonium bicarbonate (ph ). Verification of ph was accomplished using ph indicator paper (Whatman). The samples were briefly vortex mixed and centrifuged for 1 min at 26 rpm (International Centrifuge). SE of the samples was performed using CEC1 Clean-Up extraction columns (United Chemical Technologies, Inc., Bristol, A). The SE columns were prepared by sequentially passing 2 ml methanol, 2 ml deionized MilliQ-grade water, and 2 ml 1mM ammonium bicarbonate (ph ) through the sorbent bed. Each sample was transferred to a separate SE column and allowed to pass through the column. The columns were washed by passing 2 ml of 1mM ammonium bicarbonate (ph ) through the sorbent bed. The columns were thoroughly dried by applying a 1 mm Hg vacuum for approximately 1 min. Analytes were eluted by passing 3 ml of methanol under gravity flow. Eluates were evaporated to dryness under a stream of air at ~ The residues were reconstituted in 2 IJL.1% formic acid/methanol (:1, v/v) and transferred to 3-pL conical bottom autosampler vials. reparation and extraction of urine samples for oxycodone (OCOD), oxymorphone (OMOR), and hydrocodone (HCOD) Working solutions of OCOD, OMOR, and HCOD were prepared in MilliQ grade water at.1, 1., and 1. ng/pl. Calibrator samples were fortified with the aqueous working solutions at 1,, 1, 2, and 1 ng/ml. rior to extraction by SE 1. ml of calibrator quality control and patient sample were fortified with 1 ~JL of 1. ngfljl OCOD-d3, OMOR-d3, and HCOD-d3 also prepared in MilliQ grade water. The final internal standard concentration was 1 ng/ml. Reference materials were supplied by Cerilliant. The ph of each sample was adjusted to ph. by the addition of 1 ml of % potassium phosphate dibasic (Fisher) and 2 drops of 1M ammonium hydroxide (Fisher). Verification of ph was accomplished using ph indicator paper, ph 6-. (phydrion Brooklyn, Y). The samples were briefly vortex mixed and centrifuged for min at 26 rpm (International Centrifuge). SE of the samples was performed using CSDAU Clean Screen extraction columns (United Chemical Technologies, Inc.). The SE columns were prepared by sequentially passing 3 ml methanol and 3 ml deionized MilliQ-grade water (Millipore, Bedford, MA) through the sorbent bed. Each sample was transferred to a separate SE columns and allowed to pass through the column. The columns were washed by sequentially passing 3 ml deionized MilliQ-grade water and 2 ml of.1m acetate (ph ) through the sorbent bed. The columns were thoroughly dried by applying a 1 mm Hg vacuum for approximately min. Analytes were eluted by passing 3 ml of dichloromethane/isopropanol/concentrated ammonium hydroxide (7:2:2, v/v) under gravity flow. Eluates were evapo- 1

4 Journal of Analytical Toxicology, Vol. 27, October 23 rated to dryness under a stream of air at ~ The residues were reconstituted in 1 pl.1% formic acid/methanol (:1, v/v) and transferred to 3-pL conical bottom autosampler vials. LC-MS of hair samples Quantitation of COD, MOR, and 6-MAM concentration were performed by atmospheric pressure electrospray ionization (AI-ES) LC-MS using a Hewlett-ackard series 11 LC-MS (Agilent Technologies, aio Alto, CA). The opiate analytes were separated using a YMC ODS-AQ TM reversed-phase HLC column (2. x mm x 3 pm; Waters Corporation, Milford, MA) and a mobile phase consisting of 1raM ammonium acetate (ph.) (solvent A), and acetonitrile (solvent B). The mobile phase flow rate was.2 ml/min. The organic composition of the gradient was increased from 2: (A:B) to :1 at -1 rain, then increased to 6: by 6 min and held for rain. The total run time was 1 rain, with a 3-rain post run time. The mass selective detector (MSD) was set to selective ion monitoring (SIM) mode to detect ions at rn/z 26 (MOR), 2 (MOR-d3), 3 (COD), 33 (COD-d3), 32 (6-MAM), and 331 (6-MAM-d3). The scan time was.6 s/cycle. Additional method parameters included: drying gas flow = 1. L/rain; drying gas temperature = 3~ nebulizer pressure = 2 psig; capillary voltage = 2 V; the fragmentor voltage = V; and electron multiplier gain = 1. LC-MS of urine samples for M3G, M6G, COD, MOR, and 6-MAM The following method is an adaptation of the method by Slawson et al. (2). Quantitation of M3G, M6G, COD, MOR, and 6-MAM was performed by AI-ES LC-MS using a Hewlett- ackard series 11 LC-MS. The opiate analytes were separated using a YMC ODS-AQ reversed-phase HLC column (2. x mmx 3 pro) and a mobile phase consisting of.1% formic acid (solvent A) and methanol (solvent B). The mobile phase flow rate was.2 ml/min. The gradient composition was 7:3 (A:B) at -1 rain and was increased to 7:3 from i to rain. The total run time was 1 rain with -rain post run time. The MSD was set to selective ion monitoring (SIM) mode to detect ions at m/z 26 (MOR), 2 (MOR-d3), 3 (COD), 33 (COD-d3), 32 (6-MAM), 331 (6-MAM-d3), 62 (M3G and M6G), and 6 (M3G-d3). The scan time was.71 s/cycle. Additional method parameters included: drying gas flow = 1. Umin; drying gas temperature = 3~ nebulizer pressure = 2 psig; capillary voltage = 3 V; the fragmentor voltage = V; and electron multiplier gain = 1. Extensive validation was not reported here as this data has been previously reported (2). LC-MS of urine samples for OCOD, OMOR, and HCOD The following is an modification of the method by Slawson et al. (2). Quantitation of OCOD, OMOR, and HCOD was performed by AI-ES LC-MS using a Hewlett-ackard series 11 LC-MS. The opiate analytes were separated using a YMC ODS- AQ reversed-phase HLC column (2. x mmx 3 pro) and a mobile phase consisting of.1% formic acid (solvent A) and methanol (solvent B). The mobile phase flow rate was.2 ml/min. The gradient composition was 7:3 (A:B) at -1 rain and was increased to 7:3 from 1 to rain. The totai run time was 1. rain. The MSD was set to selective ion monitoring (SIM) mode to detect ions at m/z 3 (HCOD), 33 (HCOD-d3), 32 (OMOR), 3 (OMOR d3), 316 (OCOD), and 322 (OCOD d6). The scan time was 1. s/cycle. Additional method parameters included: drying gas flow = 1. L/rain; drying gas temperature = 3~ nebulizer pressure = 2 psig; capillary voltage = 2 V; the fragmentor voltage = V for HCOD and OMOR and 7 V for OCOD; and electron multiplier gain = 1.. Radioimmunoassay () of urine Urine specimens were semi-qualitatively analyzed by Coat-A- Count Opiate Screen (Diagnostic roducts Corporation, Los Angeles CA), using the manufacturer's instructions. The tubes were measured with a ackard Auto-Gamma Cobra II TM Gamma Counter (acker Instrument Co., Meriden, CT). Resuits were reported in both a semiquantitative (ng/ml) and qualitative (positive/negative) format, with a cutoff of 2 ng/ml The samples were quantitated with the Cobra TM II software factory-installed software package using linear curve fits, with a Iog-logit x/y transform. Buprenorphine-3-glucuronide for estimation of potential cross-reactivity with this kit was obtained from the IDA Research Drug Supply, Research Triangle Institute (RTI) (Research Triangle ark, C). Single analyte control study 6-MAM, a metabolite of heroin, is deacetylated to morphine under hydrolytic conditions. There was some concern as to how much 6-MAM was hydrolyzed during the hair extraction method described. To address this concern, 1 mg of drug-free human hair (n = ) was fortified with 3 pl of 1 ng/pl 6-MAM and 3 pl of 1 ng/pl 6-MAM-d3. The 6-MAM and 6-MAM-d3 stock solutions were freshly prepared and were found to contain less than 2% morphine impurity. The final concentrations for the standard and internal standard were 3 ng/mg and 3 ng/mg, respectively. These samples were then extracted and analyzed as described (see reparation and extraction of hair samples and LC-MS of hair samples). Any morphine detected during analysis would be a result of hydrolysis during the extraction or analytical method. As a reference against which the extracted samples would be compared, the same amount of methanolic 6-MAM and 6-MAM-d3 stock was added to silanized 13 x 1-ram culture tubes and evaporated to dryness under a stream of air at ~ The residues were reconstituted in pl of 1raM ammonium acetate (ph.)/acetonitrile (:1, v/v) and transferred to 3-pL conical bottom autosampler vials, as described in reparation and Extraction of Hair Samples. These reference samples were then analyzed according to the method described (see LC-MS of hair samples). Statistical analysis Differences between means were analyzed by Student's t-test using rimer of Biostatistics (Vs.., The McGraw-Hill Companies, Inc.) software. Differences were considered significant and the null hypothesis rejected if thep value was less than.. 1

5 Journal of Analytical Toxicology, Vol. 27, October 23 Results Analytical method LC-MS method for hair. Quantitation of MOR, COD, and 6-MAM was accomplished by calculating the peak-area ratios for the molecular ions of each analyte and its respective deuterated internal standard. Linear fits for the analytes yielded the following linear ranges -, pg/mg for COD and 6-MAM, and -, pg/mg for MOR (r ~ >. for all analytes). Intra-assay precision and accuracy were determined by analyzing samples (n = ) of drug-free hair fortified with known amounts of MOR, COD, and 6-MAM prior to analysis (Table I). The coefficients of variation for intra-assay precision were determined to be less than 7% for MOR, COD, and 6-MAM at 3, 2, and 1, pg/mg. The interassay precision and accuracy was determined by analyzing drug-free human hair fortified with known concentrations of MOR, COD, and 6-MAM (Table Table I. Intra-assay recision and Accuracy for Hair Analysis (1-rag Specimen Weight)* Morphine Codeine 6-MAM 3 pg/mg Mean = 27.7 Mean = 2. Mean = (n = ) STD Dev =.1 STD Dev =.11 STD Dev = C.V. =.6 C.V. = 1.2 C.V. = % Target = 2. % Target = 6.61 % Target = 2 pg/mg Mean = 1.7 Mean = 1.72 Mean = (n = ) STD Dev = 11.1 STD Dev = STD Dev = C.V. =. C.V. =. C.V. = % Target =.2 % Target = 7.7 % Target = 1, pg/mg Mean = Mean = 2. Mean = (n = ) STD Dev = 3.6 STD Dev = 1.2 STD Dev = C.V. = 2.2 C.V. =.1 C.V. = % Target = 1.62 % Target =.3 % Target = II). The coefficients of variation for interassay precision were determined to be less than % for MOR, COD, and 6-MAM at 3, 2, and 1, pg/mg. The interassay accuracy was determined to be within 13% of the target values for MOR, COD, and 6-MAM. Recoveries of COD and 6-MAM from extraction were determined to be 2% or greater at 3, 2, and 1, pg/mg by method of internal standard addition (n = at each concentration). The recovery of MOR was determined to be between 32% and 3% at the same concentrations. Despite the low recovery of MOR, it was sufficient to permit quantitation of samples down to pg/mg with precision of less than 1% and accuracy within 13% of the theoretical target concentration. The stability of the extracted samples was also evaluated by comparing quantitative results from two separate analytical runs of the same subject samples, days between initial and final re-injection (individual data not shown). The samples were frozen at-2~ between analyses and reconstituted prior to reanalysis. Samples were within 1% of original value over this -day period. Variability of quantitation in small seg- ments. The developed method for opiates was based upon extraction of 1 mg of hair. However, it was noted that once segmented, nu merous samples consisted of less than 1 mg of.73 hair. Therefore, an assessment of the accuracy 2.1 and precision of quantitative measurement with smaller sample sizes was performed. For 1.1 the purposes of this evaluation, a source of 6.1 hair known to contain drug incorporated into 3.36 the hair shaft structure was required. To ac- 1. complish this, a pool of hair was obtained from 17.1 rats (n =1), that had been administered COD 1.7 (see Methods: Variability of quantitation in loo.o6 recision and accuracy ior MOR, COD, and 6-MAM in hair fortified with a known concentration of drug. Quality control specimens were analyzed in a single analytical batch over an 1-h period. Table II. Interassay recision and Accuracy for Hair Analysis (1-rag Specimen Weight) Morphine Codeine 6-MAM 3 pg/mg Mean = Mean = Mean = 21.3 (n = 32) STD Dev = 17.1 STD Dev = 17.7 STD Dev = C.V. = 6.2 CV. = 6. C.V. =. % Target = 2.22 % Target =.7 % Target = pg/mg Mean =.61 Mean = 1. Mean = (n=31) STD Dev =.2 STD Dev = 1.1 STD Dev = 1.3 C.V. = 6.7 C.V. = 7.1 C.V. = 6. % Target = 6.2 % Target =. % Target = , pg/mg Mean = Mean = 7. Mean = 7. (n = 32) STD Dev = 62.7 STD Dev = STD Dev = 6.21 C.V. = 6.32 C,V. = 7.3 C.V. =.1 % Target = % Target = 7. % Target = 7.1 " recision and accuracy for MOR, COD, and 6-MAM were evaluated in hair fortified with a known concentration of drug. Quality control specimens were accumulated for 11 analytical batches over an -month period of time. One 2 p&/rng specimen was not analyzed and could not be included in the interassay precision and accuracy calculations. 16 small segments). Using the validated hair weight of 1 mg of hair, the concentrations of COD and MOR in unwashed hair were 21.2 and 2.6 pg/mg, respectively (Table III). These concentrations served as the control, or reference value, to which other data were compared. The mean concentration of codeine measured in the 2.- and.-mg segments demonstrated no significant difference in quantitative values from control hair (t = 2.6 with degrees of freedom; p =.6, and t = 2.71 with degrees of freedom;p =.72, respectively). However, the mean concentration of codeine measured in the segments of 1. and. mg demonstrated a significant difference from the control (t =. with degrees of freedom; p =., and t =.6 with 7 degrees of freedom; p =., respectively). With respect to the MOR metabolite, there were significant differences between the mean concentrations of morphine measured in the 2.-mg samples (t =.1 with degrees of freedom; p =.), as well as the.-rag samples (t = 3.66 with degrees of freedom; p =.). o MOR was detected in the samples that were smaller than 2.

6 Journal of Analytical Toxicology, Vol. 27, October 23 Table IlL Variability of Quantitation in Small Segment Sample STD % Drug Amount Mean Dev C.V. Targel* n Control Codeine 1 mg Morphine 1 mg Experimental Codeine Morphine*.rag mg mg mg t. mg rag * Calculated by comparing to 1 mg quantitation. * One O.S-mg sample was not able to be quantitated and was deleted. * Morphine 1.O-rag and.-rag samples were not detected. rag. It should be noted that the LOQ for MOR was pg/mg, and there was only 2.6 pg/mg of MOR detected in the 1-rag sample. Therefore, it is likely that there was insufficient drug in the smaller hair samples. Generally, 2. mg of hair was required to accurately quantitate (COD) or detect (MOR) in hair segments with acceptable precision. LC--MS of urine samples for M3G, M6G, COD, MOR, and 6- /1/AM. Quantitation of M3G, M6G, MOR, COD, and 6-MAM was accomplished by calculating the peak-area ratios for the molecular ions of each analyte and its respective deuterated internal standard. M3G-d3 were used as the internal standard for M6G. Linear fits for the analytes yielded the following linear ranges: 1- ng/ml for M3G, M6G, COD, and 6-MAM, and 2- ng/ml for MOR (r 2 >. for all analytes). Intra-assay precision and accuracy were determined by analyzing samples (n = ) of drug-free urine fortified wfth known amounts of M3G, M6G, MOR, COD, and 6-MAM prior to analysis (Table IV). The coefficient of variation for intra-assay precision Table IV. Intra-assay recision and Accuracy for Urine* M3G M6G MOR COD 6-MAM 2 ng/ml Mean = 26.6 Mean = 27.3 Mean = Mean = Mean = 2.2 n = STD Dev =.6 STD Dev = 1. STD Dev = 1.37 STD Dev = 1. TD Dev = 1. C.V. = 2.% C.V. = 6.% C.V. =.2% C.V. =.6% C.V. = 6.% % Target = 16. % Target = 1. % Target = % Target = 2. % Target = 6. 7 ng/ml Mean = 7.2 Mean = Mean =. Mean = Mean = 71.6 n = STD Dev =. STD Dev = 7.7 STD Dev = 3. STD Dev = 3.13 STD Dev =.77 C.V = 1.1% C.V. =.6% C.V. =.2% C.V. =.66% C.V. =.% % Target = 1.26 % Target =.6 % Target = 1.6 % Target =.7 % Target =.7% 2 ng/ml Mean = 1.6 Mean =. Mean = Mean = 11.7 Mean = 17.1 n = STD Dev = 2.1 STD Dev = 2.3 STD Dev = 6.76 STD Dev =.7 STD Dev =.71 C.V. = 1.21% C.V = 1.6% C.V. = 3.% C.V. =.1% C.V. =.% % Target =.7 % Target = 6.7 % Target = 1.33 % Target =.7 % Target = 7.7 * Quality control specimens were analyzed in a single analytical batch over an 1-h period. Table V. Interassay recision and Accuracy for Urine M3G M6G MOR COD 6-MAM 2 ng/ml Mean = 2.2 Mean = 27. Mean = 2.2 Mean = 27. Mean = 2.3 STD Dev = 1.2 STD Dev = 2.6 STD Dev = 1.2 STD Dev = 2.6 STD Dev = 1.63 C.V. =.% C.V. = 7.3% C.V. =.% C.V. = 7.3% C.V. = 6.% % Target = % Target = % Target = % Target = 111.2% % Target = 11.3% n=3 n=3 n=3 n=3 n=2 7 ng/ml Mean = Mean = 1.21 Mean = 7. Mean = Mean = 71.6 STD Dev = 2.6 STD Dev =.6 STD Dev =.2 STD Dev = 6.3 STD Dev =.77 C.V. = 3.7% C.V. = 11.% C.V. = 11.3% C.V. =.2% C.V. =.% % Target =.63 % Target = 1.2 % Target =.3 % Target = 6.3 % Target =.7 n= 3 n=3 n= 3 n= 32 n= 26 2 ng/ml Mean = 1.2 Mean = Mean = 1. Mean = 1. Mean = 11. TD Dev = 7.2 STD Dev = TD Dev = 22.3 STD Dev = STD Dev = 13. C.V. = 3.% C.V. =.% C.V. = 11.6% C.V. = 7.1% C.V = 7.2% % Target =.21 % Target = 16.3 % Target =. % Target = 2.% % Target =.72% n=3 n=3 n=3 n=3 n=2 * Interassay precision and accuracy for M3G, M6G, MOR, COD, and 6-MAM were evaluated in urine fortified with a known concentration of drug. Quality control specimens were accumulated for 11 analytical batches over a 7-month period of time. There were a number of outliers for 6-MAM and COD because of problems with working solutions. These outliers were deleted and not included in calculation of precision or accuracy. One 7-ng/mL MOR quality control sample was also deleted because of an unusually high do/d3 ratio. 17

7 Journal of Analytical Toxicology, Vol. 27, October 23 was determined to be less than 7% for M3G, MOR, and COD and less than 11% for M6G and 6-MAM at 2, 7, and 2 ng/ml. The interassay precision and accuracy was determined by analyzing drug-free urine samples fortified with known concentrations of M3G, M6G, MOR, COD, and 6-MAM on 11 separate analytical batches (Table V). The coefficients of variation for interassay precision were determined to be less than % for M3G, M6G, MOR, COD, and 6-MAM at 2, 7, and 2 ng/ml. The interassay accuracy was determined to be within % of the theoretical values for M3G, M6G, MOR, COD, and 6-MAM at 2, 7, and 2 ng/ml. Recoveries of M3G, M6G, MOR, COD, and 6-MAM extracted from urine were analyzed at 2, 7, and 2 ng/ml by method of internal standard addition (n = at each concentration). The recoveries for M3G were determined to be greater than %; M6G recoveries ranged from 7 to 76%; MOR recoveries were greater than 7%; and COD and 6- MAM recoveries exceeded 2%. Single analyte control study. Because of the possibility for conversion of 6-MAM to MOR during the analytical process, a brief assessment was made using fortified control materials. According to our analysis, there was less than 2% conversion of 6-MAM to MOR at 3 ng/mg in the analysis procedure. This is consistent with our previously published data for conversion of 6-MAM to MOR with an enzymatic (rotease VIII) digestion procedure (2). These conversions rates were comparable to those of the acetonitrile method used for the current study, the acetonitrile extraction was much simpler and takes less time. This was an important practical consideration since we were analyzing up to 1 samples per analytical batch. Clinical study Opiate use-assessment by analysis of hair only. To evaluate whether the quantitative determination of opiates by segmental hair analysis can predict drug use during a clinical drug-treatment program, hair quantitation data from the first three 1-cm segments of each sample was compared to data collected from both and LC-MS analysis of the urine samples collected from the subjects. Complete data for each subject is shown in Figures 1-1. Each figure is comprised of three main sections: 1. hair quantitative data, 2. urine data, and 3. subject hair treatment profile. Although hair samples were collected weekly, quantitative data for hair segments is presented for three specific time periods only, representing the beginning, middle and end of the study period. This data, along with identification of the specific sample (Week o.), is shown in each figure. Also, the column labeled "Segment o." corresponds to the number of the 1-cm segment on the intact hair shaft in relation to its proximity to the scalp. The lowest segment numbers correspond with the segments that lie closest to the scalp, and represents the most Subject A Segment Baseline Mid- End- o.t Week #1 Week # Week #17 6-MAM Codeine Morphine 6-MAM Codcine I Morphine 6-MAM Codeine Morphine (p~/mb) (pg/m~) (p~/mg) (.p~mg) (og/mg) (p~/mg) l ) 2 o 3 ) t r.d, D ~ :~.D, 7 7, ,2 31.1,D. D ~.D, D. I D.,D * D ),D. 7,13 r,d ,D, , D. 36.7,D ' D, ',,D, 1 6,13. ) * * * * * * * * * Subject Hair Treatment rofile Sex atural Hair Hair Treatments Hair Treatments Hair Cuts During Color rior to During the Female Light Brown Weave/Bleach one Week I =ot Detected.A.-ot Analy'z--d - < LOQ but > LOD *- o Segment f " 1 cm segmenu measured from scalp end to Up "-< LOQ but LOD for morphlne-3-glucuronlde, and morphlne-6-glucuronlde =osltive =egotlve Figure 1. Subject A. Week o. LC/M$ (n~/mlp (n~/mi.) RUt (>2 r~/ml.) (~ng/ml.) I M3G r M6G 1. 2 D D D >lo D l 1.A >1OO II D O >too M3G) M6G >1OOO >11 1 M3G M3G v M6G A. 1 D. 1 D

8 Journal of Analytical Toxicology, Vol. 27, October 23 recent hair growth, whereas the higher numbered segments represent older growth. 6-MAM was the most common analyte found in the hair sampies, followed by MOR and COD, respectively. Concentrations of 6-MAM in hair ranged from to,33 pg/mg, MOR ranged from to 2773 pg/mg, and COD ranged from to 21 pg/mg. Based on hair data only, six subjects (A, B, D, G, H, and J) appeared to significantly reduce ingestion of COD, 6-MAM, or MOR by the end of the study. Three subjects appeared to decrease use (E, F, and I). Subject C never had analyte concentrations above the LOQ throughout the duration of the study, despite substantial clinical evidence of opiate abuse (i.e., positive pre-study urine screening and physician evaluation at the start of the study). Hair treatment profile. The subject hair treatment profile describes the gender, and subject-reported natural hair color, pre- and during study cosmetic hair treatments, as well as a record of hair cuts for the duration of the study. Six subjects (A, B, C, F, G, and I) were female and four (D, E, H, and J) were male. Seven often subject (A, B, C, E, G, I, and J) cut their hair at least once during the study. Six out of 1 subjects (A, B, C, D, G, and J) performed a cosmetic treatment prior to the study, and subjects (B, C, F, and G) performed at least 1 cosmetic treatment during the study. Four subjects (A, D, H, and J) described their natural hair color as either light brown or blonde; two subjects (F and G) had medium brown hair; and four subjects (C, B, E and I) had either dark brown or black hair color. Opiate use-assessment by analysis of urine only. Where sufficient urine volume was available, specimens were analyzed by both LC-MS and. However, there were several instances when insufficient volume was available for LC-MS analysis. For example, subject H had samples analyzed by and only 3 samples available for LC-MS (see Figures 1-1). Qualitative and quantitative urine data is also shown in each figure. The quantitative data includes that obtained by LC-MS, as well as semiquantitative. The LC-MS data represents a sum (ng/ml) of the five opiate compounds analyzed in our assay: MOR, M3G, M6G, COD, and 6-MAM. Total opiate concentrations found in urine ranged from to 12,66 ng/ml over the course of the study. Based on urine data only, five subjects (B, E, F, I, and H) appeared to have continued to ingest MOR, M3G, M6G, COD, and 6-MAM throughout the course of the study. ~vo sub- Segment Bamnne Mkl-StUW o,r Week #1 Week #7 6-MAM Codeine Morphine 6-MAM Codeine Morphine 6-MAM (p~/mg) (pft/mg) (pg/mr) I ,D. 6 7 ".D, *,D. * * * * 1,D. * * Subject B Sex Female atural Hair Color Black -ot Detected but > LOD " e Segment t" 1 cm segments measured from scalp end to tip -ositive -egative Figure 2. Subject. Subject Hair Treatment rofile ~ttt~ Wook g17 Hair Hair Hair Cuts Treatments Treatments During the rior to During Black Hair Darker Week 3,17 Dye Weeks 2,6, 17 Lighter Week Codeine Morphine (pr/mg) 66. D. Week o. LC~r~ (~/ml~ I g s , I O (ng/ml) (>2 ng/ml) (>3og/mL) >1(3 >1(3 >I >looo >1~3 >I >looo >I >I >I >I >I >lo >I( >I(XX) >1(3 >I( >I >I >1(3 >I(X~O >16 >logo >I.23.1 >I >I >I >I >lo(y) >logo 71,6.73 >I >IO(X) 1

9 Journal of Analytical Toxicology, Vol. 27, October 23 jects (A and D) appeared to decrease use by the end of the study. q'~vo subjects (C and G) appeared to stop using by the end of the study, and one subject (J) appeared to discontinue ingesting MOR, M3G, M6G, COD, and 6-MAM, but resumed use toward the end of urine monitoring. Urine data was also categorized qualitatively as positive or negative with cutoffs greater than 2 ng/ml, and greater than 3 ng/ml. In addition, semi-quantitative data for opiate content (ng/ml) was also determined for each sample. The column labeled "Week o.", refers to the time when the urine sample was obtained. The sampling date of the hair Was used as the reference for when a week began. The kit used for this analysis consists of an antibody directed to M3G, but is also highly cross-reactive with many other opiate-like compounds (see Table VI). Four subjects (B, E, F and I) continued to ingest MOR, M3G, M6G, COD, and 6-MAM throughout the course of the study. Four subjects (A, D, G, and J) intermittently used throughout the course of the study and one subject continued using through the mid-point of the study, then discontinued use toward the end of the study period. Comparison of and LC-MS urine data. Table VII demonstrates that 63% of the urine samples positive at the cutoff greater than 2 ng/ml were also positive by LC-MS and 13.% were negative by both methods. There were interindividual differences in agreement between the and LC-MS results. The agreement ranged from a high of 1% (Subject I) to a low of 1% (Subject C). Because of interindividual differences between the LC-MS and results and the observation that the analysis indicated a higher opiate concentration compared with the LC-MS analysis when there were discrepancies, the possibility of cross-reactivity to buprenorphine (BLI) or one of its metabolites was considered. To test this hypothesis, blank urine was fortified with BU, norbuprenorphine (B), or buprenorphine-3g-glucuronide (B3G) at 1, 1,, 1, 2, and ng/ml (n = 2 at each level for each analyte). These standards were analyzed by the DC Coat-A-Count Hair Quantitative Segment ~ o.t Week #1 Week #7 Data r:ne.eter Wink #17 Subject C Urine Dam Week ~ Ilia O. ~ ~l/ll~ (>2 ng/ml) (>3ng/m~ 6-MAM Codeine Motphim: 6-MAM Codeine Moq~hine 6-MAM (piing) (r (gg/rag) , * * * 1 " * * 1 * * * 16 " * " 17 ' * 1 * * * * " * Sex Female atural Hair Color Dark Brown Subject Hair Treatment rofile Hair Hair Treatments Treatments rior to During All-over Lighter- Color- lighter Week 1 (Bleach) (Roots Only') - ot Detected = < LOQ but > LOD * - O Segment t " 1 cm segments measured from scalp end to tip "-< LOQ but LOB for morphine-3-gloeuronlde, and morphlne-6-glaeuronlde.-< LOQ but LOD for morphlne-3-glueurooide -ositive -egative Figure 3. Subject C. Codeine Mo~hinr (l~/mg) (pwmg) Hair Cuts During the 23,D,,D Week I 1( M3G 1 t.72 1 D D. 3,D M3GtM6G >1O 217. >11 >I.6.D, D 6 M3G D >1( ] 1 M3G)M6G >1( A D D. 1,1 1 3,22 1,D, D D 1,D, 1 1 D. 1 2

10 Journal of Analytical Toxicology, Vol. 27, October 23 Opiate Screen kit according to the manufacturers instructions and as described in methods. one of the replicates, for any of the concentrations had ratios (% bound/maximum binding) that were within in the dynamic range of the assay of the calibrators, 2-1 ng/ml. These results demonstrate that the DC Coat-A-Count Opiate screen had very little, if any, cross-reactivity to BU, B, or B3G within the 2-1 ng/ml range of calibrators. The possibility that the subjects were ingesting some other opiates that were cross-reactive with the kit, but were not detected in the LC-MS protocol, was also considered. According to the Coat-A-Count Opiate Screen kit insert, "results are expressed in nanograms of morphine-3-glucuronide per milliliter (ng/ml)." For some compounds the kit showed greater cross-reactivity than to morphine-3- glucuronide (Table VI). A urine screen for HCOD, OCOD, and OMOR was performed by extracting eight urine samples that showed the greatest discrepancy between the and the LC-MS results. The extracts were then analyzed by LC-MS AI-ES in SIM mode. o HCOD, OCOD, or OMOR were detected in these urine specimens. Discussion Hair extraction method We successfully developed and validated a sensitive and specific LC-MS analysis for MOR, COD, and 6-MAM in hair samples. Rothe and ragst compared a number of different solvents, including acetonitrile, for use in direct extraction of opiates from hair (3). They found that acetonitrile, a polar aprotic solvent, had a medium extraction yield when compared to nonpolar, hydrophobic solvents (low extraction yield), and more polar alcohol solvents (high extraction yield). However, the method used was a short (2 h), rigorous (heating samples to -~ method that could lead to hydrolysis of heroin and 6- MAM, which was not evaluated in their report. To minimize hydrolysis of 6-MAM, the acetonitrile extraction was performed overnight at room temperature. Under these conditions hydrolysis of 6-MAM to MOR was less than 2% at 3 ng/mg. It is possible that this extraction in acetonitrile does not completely remove all incorporated drug. If so, our quantitative results may be lower than the true value. However, because reference materials containing known amounts of opiates incorporated Segme~ Baaellno Mkl,-St~ Fnd-Btudy o,t Week ilo Week #7 Week #16 Subject D 6-MAM Codeine Mmphioc 6-MAM Codeine Morphine 6-MAM Codeine Moq~hine (pwmx) (palms) (pg/ms) (pa/mg) (pwma,) (pwme) (pg/mt,) Cog/mR) I I.1 2 * " " ,D t D- I} D, 6,17 * * " %6 _D_ * * * 17.2 hid. 1 * * " * * * * * * * * *.2 Subject Hair Treatment profile Sex atural Hair Hair Hair Cuts Hair Color Treatments Treatments During the rior to During Male Light Brown All-over one one Color-lighter (bleach) " ot Detected.A. - ot Analyzed -< LOQ but > LOD * - o Segment t" - I cm g.gmentt mcuurcd from scalp end to tip.a. = ot Analyzed "-< LOQ bat LOD for ceddue -ositive -egative Figure. Subject D. Week LO/M RUt o. (nu/mlp (r~l/ml) (>2 ng/ml..) (>~ml) I >IOQ I,D, I(D A >1~ >1(3 >I~( >I 6 D A >IOG >1~ 137. >1(} >IOQ I I.D, 7, >1~ II 2.1.1, , 13.D D D, 16.D, 16 COD.3 17 COD ,A. 21

11 Journal of Analytical Toxicology, Vol. 27, October 23 (rather than fortified) in hair were unavailable, this could not be assessed. Variability of quantitation in small segment sample When sample weights were less than 2. mg (COD) or less than 1 mg (MOR) there were significant concentration differences when compared to controls. Even though hair weights were small, the analytical method appeared to be sufficiently sensitive (LODs: 2 pg/mg for COD and 6-MAM, and pg/mg MOR) to reliably detect and estimate the pattern of drug use for COD, MOR, and 6-MAM. Comparison of and LC-MS urine data There were concerns about the lack of agreement between the urine data produced by and LC-MS analyses (Table VII). In particular it was noted that of specimens were negative by LC-MS and positive by. cross-reactivity with BU and its metabolites was considered as a possible cause for this disagreement. This possibility was eliminated as we did not detect any cross-reactivity above the lowest calibrator provided in the kit at concentrations of 2 ng/ml BU, B, or B3G. The possibility that the subjects were ingesting some other opiate(s) that were cross-reactive, but were not detected in the LC-MS protocol, was also considered. Eight urine samples that showed the greatest dichotomy between and LC-MS were analyzed by LC-MS, specifically for OCOD, OMOR, and HCOD. We did not detect the presence of these compounds. In a second LC-MS analytical run of these samples in full scan mode, there were no significant peaks that could be identified as possible opiate-like compounds. Thus, the discrepancy in these results cannot be readily explained; however, it is possible these urine samples contained other opiates that were not included in the LC-MS protocol. It was noted while preparing these eight samples for extraction that all of them had significant amounts of particulate matter in them. It is unknown whether this may have interfered with the expected performance of the. Comparison of hair and urine data To evaluate whether the quantitative determination of opiates by segmental hair analysis can predict drug use during a clinical drug treatment program, the subjects were divided into four broad categories based upon the agreement between hair and urine data, as well as the subjects opiate use patterns. Urine analysis was used as the frame of reference by which drug use was estimated. The categories are as follows: 1. continued use Segment Baseline Mid- End- o.t Week #1 Week #7 Week #17 6-MAM Codeine Morphine 6-MAM Codeine Morphine 6-MAM {,pg/mt,) (pg/mr) tg/mr) (pg/ml{) 1 362A D D D. * ,D. D. * D. * * * * * * 7.7 * * * * Subject Hair Treatment rofile Sex atural Hair Hair llair Cuts itair Color Treatments Treatments During the rior to During Male Black one one Weeks 3, 1, 16 =ot Detected = < LOQ but LOD *- o Segment t - I cm segments measured from scalp end to tip r LOQ but > LOD for morphlnc-3-glucurnnidc i-< LOQ but > LOD for morphlne-6-glucuronlde =-osltlve =egstive Figure. Subject E. Subject E Codeine Morphine ~pi/mg) tpg/m~) s Week LC/M$ o, (ng/ml)~ (ng/ml) (>2 ng/ml) (>3(X)ng/mL) 6,31 F I11. >1(3 p A >I(XX) 3 M3G D >1(3 1.27,A >1( M6G 16 7 D M3G 23.7 D I >1(X) 1 D > >1( M3G M3G > >1C~

12 Journal ofanalytical Toxicology, Vol. 27, October 23 gested continued drug use and was in poor agreement with the hair data. Three subjects (D, G, J) demonstrated a decrease in hair drug concentrations to non-detectable by the end of the study, again suggesting discontinued or reduced opiate use. Urines collected and tested by were positive in an "intermittent" pattern throughout the study (3 of 7; 26 of 2; 26 of 31). The LC-MS testing of the same urine samples were less frequently positive (17 of ; 11 of 3; of 2), but still suggested that these subjects "occasionally" ingested opiates; however, this was not reflected in the hair data. 3~vo subjects (A, C) had hair drug and metabolite concentrations that were either non-detectable or below the LOQ at the beginning of the study, despite substantial clinical evidence of drug abuse and positive urine opiate screens prior to enrollment. At the conclusion of the study, analytes were not detectable in the hair of either subject; however, urine specimens and good agreement; 2. continued use and poor agreement; 3. intermittent use with poor agreement; and. mixed usage and poor agreement. Three subjects (E,F,I)demonstrated little quantitative change in detectable analyteconcentrations in hair segments closest to the scalp over time, suggesting continued opiate abuse. Urine opiate immunoassayswere positive for all urines collected from these subjects (1 of 1) and were in good agreement with quantitative hair data. LC-MS data was not quite as conclusive (131 of 1), but nevertheless, data for these three subjects suggested they continued to ingest opiates throughout the study. For two subjects (H, B), drug concentrations measured in hair segments decreased over time to below the LOD/LOQ,suggesting discontinued/reduced opiate use. However, the frequency of -positive urines ( of and of ) and LC-MS positive urines ( of 3 and 7 of ) strongly sug- Subject F S,gnat MkJ-Stu~ week #7 w,ek #1 o.t 6-MAM Codeine Moq~hineI 6-MAM (p~nr} 1.7 (pe/mr) (vwml I IvWma) ;!1S,7p 21, $ ~ ,i , t , I I 6. $6= D , 7. F.nd-atu~ wocx #17 Week O. Codeine Moq~hineI 6-MAM Codeine Moq~hincJ (pg/ma) 7C ~ 27 ( ~ 16 6( 27 t 3 ] 1 (pwmg) I (pwmg) (l~mg) (vwmg) I , , [ i , , ,1 72.,D , , , Subject Hair Treatment rofile I I I Sex Female atural Hair Color Medium Brown Hair Treatments rior to one Hair Treatments During Darker Week II (Color Shampoo) -o < LO~ b.i > LOD *- o Segment - I cm segments minuted from1r I~< I. ~ but> LOD for morphine -ox'dive -egative end to Up Hair Cuts During the one 1 1 I I II II I 1 (ng/ml) MOR , >I >I >1(3 >I13 >I >1(3 >I(X} >1{3 >I(X} >1(3 >I >I >I >1(3 >1( lisa,d, Figure 6. Subject F. LO/M (ng/l~.)* >I >1(3 6.1 (>"~ n g / m l. ) (>3ng/mL) >I~ >I(X) 17,7 >looo 1 >I(XX) 23

13 Journal of Analytical Toxicology, Vol. 27, October 23 for subject A were occasionally (-1 of 1; LC-MS-1 of 3) positive throughout the study. Subject C demonstrated the greatest discrepancy between the and LC-MS results (- 1 of 3; LC-MS 1 of ). The urine data suggests that the subject frequently ingested drugs and continued to do so until the middle of week #1 which would be inconsistent with the hair data. In contrast, LC-MS urine data indicates that the subject only occasionally took drugs and completely quit by the end of week #, which would be more consistent with the hair data. To evaluate whether the quantitative determination of opiates by segmental hair analysis can establish sufficient evidence to determine the efficacy of a clinical drug treatment program, the number of subjects that discontinued use by the end of the study according to hair data were compared to the number that actually discontinued use, according to urine data. According to the hair data 7 of 1 subjects were judged to have discontinued use, while according to the urine data of 1 subjects were judged to have significantly reduced use. This would suggest that hair analysis is not as accurate an indicator as is urine. It should be noted that many of the subjects ( of 1) described themselves as having light or medium colored hair. Seven of 1 colored their hair before and/or during the course of the study. Both characteristics are factors that could lead to lower opiate concentrations. This might contribute to the decreased detection of 6-MAM, COD, and MOR in the hair specimens. The potential effects of hair color and chemical treatment on quantitative hair data was considered. Subjects demonstrating good agreement between hair and urine data had dark brown or black hair and reported no chemical treatments before or during the study. Subjects with moderate-to-poor agreement between hair and urine data had various hair colors (blonde to dark brown). Some, but not all, of these subjects reported using hair color (darker) during the study. Interestingly, the two subjects (A, C) with low or non-detectable baseline hair concentrations reported using bleaching agents just prior to study enrollment. Segmental hair analysis for changes in opiate use One of the stated purposes of this study was to determine if segmental hair analysis is sufficiently sensitive to detect changes in opiate usage during the course of a clinical drug treatment program. Urine analysis was used as the frame of reference to which hair was compared, because of its long history as the standard of testing for drugs of abuse. Three broad categories of subject drug use were established based on urine results (see Figures 1-1): continued use (subjects B, E, F, H, and I), intermittent use (A and J), and significant reduction of opiate use (C, D, and G). Even if one takes into consideration the delay between ingestion of the drug and its appearance above the scalp, there is generally poor agreement between the patterns demonstrated in the urine data and those in the hair data. Specific examples, based on urine results, that demonstrate three types of Segment Baseline Mid- End-1udy o.t Week I Week # Week 1t Subject G 6-MAM Codeine Morphine 6-MAM Codeine Morphine 6-MAM Codeine Morphine ~g/mg) (pg/mb) ~a/rnlt) (p~/mg) I D ~,D D ,D.,D ),D..D 6 3 -t 21. * * * D " * * * * * * * * ,6 * * * * * * I 1.6.D, 72.2 * * * " * * l I 17,3. * * ' " * * " * * * * * Subject Hair Treatment rofile Sex atural Hair Hair Hair Cuts Hair Color Treatments Treatments During the rior to During Female Medium Color Darker- Color Darker- Week Brown all over all over Week 2, ot Dotected.A. - ot Analyzed -< LOQ but > LOD * - o Segment t - 1 cm segments meajured from scalp end to tip /-6-MAM not Included In opiate total quantltot on "-< LOQ but LOD for morphiae-3-glucuroalde, and morph uc-6-glucuronlde -ositive -egative Figure 7. Subject G. Week o. LO/II ~mlf" 31. I 76.2 I D, 3 M3G 13. M3G D. 7,D. 1 D. I D. I I II 11 D D 13.D, 1,D, D (no/ml) (>~ ng/ml) (>3]Ong/mL) >Ir >IO >I >I~ >I.A hi , p o 11, >1(3 2

14 Journal of Analytical Toxicology, Vol. 27, October 23 findings are described here: 1. continued opiate use; 2. intermittent use; and 3. significant reduction of opiate use. According to the urine analysis data in Figure, subject H continued to ingest an opiate throughout the duration of the study. In contrast, hair data for week # suggests the subject had reduced the frequency or intensity of drug usage as evidenced by the drug concentrations in segment #1 being below the LOQ (6-MAM), or not detected (COD and MOR). By week #17, analyte concentrations were below the LOQ, or not detected in all segments. One possible explanation for this discrepancy between urine and hair data may be that the self-report of chemical treatments may not be accurate. Alternatively, this subject may have altered their opiate use such that an opiate other than heroin, COD or MOR was used but was not analyzed for in the hair protocol. Subject J, shown in Figure 1, exhibited an intermittent pattern of drug use according to the urine analysis data. During week #, #2, and #, this subject ingested opiates as evidenced by both LC-MS and urine data. However, according to hair analysis data, drug concentrations in segments #1-# of week # were below the LOQ, or not detected for all analytes. During weeks #11 and #, this same subject tested positive by urine, but the hair analysis for segment #1-#3 of week #16, did not detect any drug. If one allows for the time required for detection of drug in hair above the scalp, and assuming that hair grows 1 cm/month, one would expect that all three segments would quantitate within the dynamic range of the assay. One possibility for this discrepancy between hair and urine data may be that the self-report of chemical treatments may not be accurate. Another possibility is that this subject may not have used drug in high enough doses or frequently enough to produce hair that had incorporated sufficient drug (above the LOD) to be detected. The minimum dose and frequency of administration of opiates required to produce a detectable concentration in hair is not currently known. Alternatively, this subject may have altered their opiate use such that an opiate other than heroin, COD or MOR was used but was not analyzed for in the hair protocol. Subject G is an example of a subject whose urine data suggested significantly reduced drug usage. During weeks #-#2, this subject ingested an opiate according to both LC-MS and data. If one allows for the time required for detection of drug in hair above the scalp, and assuming that hair grows 1 cm/month, one would expect that opiates would be detected in segment numbers 2 and/or 3 of week #. However, according to hair analysis, the concentration of all analytes (in all segments) for week # were below the LOQ, or were not detected. Overall, hair data for week #16, in agreement with LC-MS data for urine, suggests that opiate use was significantly reduced. One possibility for this discrepancy between hair and urine data may be that the hair data accurately reflects a decrease in opiate Segment BiseUne o3 Week # 6-MAM Codeine I IIII, A..A * * * * * 1 * M~b'tnay Wink # Week #17 Subject H Morphine 6-MAM Codeine! Morphine 6-MAM Codeine Morphine 3.1 D. D 3.22.A, 7..D 711.t 2.3 * 3.72 * D. D. * D. * * Subject Hair Treatment rofile Sex atural Hair Hair Hair Cuts Hair Color Treatments Treatments During the rior to During Male Dirty Blonde one one one : ot Detected.A. = ot Analyzed - < LOQ but > LOD * : o Searaent i' : 1 r legments measured from scalp end to tip /--MAM not Included In opiate total quantltatioo *-< LOQ but > LOD for morphlne-3-glucoronldr and morphlne-6-glucuronldc I~oaitlve :egauve Figure. Subject H. Week LC/MS o. (ng/ml)~ (ng/ml) i >].D >I D 1 M3G' I A. >1O~ 2. >1OO >I >I 71,7.A, l I I > >I , >I ,3 >1~1 (>2 ng/ml) {>3ng/mL) 2

15 Journal of Analytical Toxicology, Vol. 27, October 23 Subject I S~rn*nt ~ o.t M~d- Week # 6-MAM ! I Codeine Cog/rag) (173.2) {23.3) D. *" *~ E a ~ Week # Week #16 rahinel 6-MAM g/rag)! 27.1 ~ : D I ~r, I 2. n I 2.6 ~,, I 7. ~113 ] ~ I.2 D I ~D! 72.1 Codeine Morphine 6-MAM I Codeine (g/mg) (pwmg)! (Rt/mg) 11, 26 unu.~ n [7~"~ [ ~ i O I 67. 7,61 I,D. u.so u n,a~ sa I no]~;~u I isoa I m~~ I 36~! 11'1[11111 I ~ l S l i~lsl 1'[OIC./I~l;1 " [ I, l. ' l I ~ l S l i ~ l I * 1 l t # ~ J [ ~ I ~l l i l t : t O, l ' ~! I ~,llj i l t I t i I [:I..~,1.[I I ~,l o l ~l Ihllltgl I~lsllZl ol IO] I~lsl "" l ~ l H lr! i1 [ l ~ l il~][~ll I ~i l el l ~ e l I :1:[[;$11~1 I l l ~ l Sl [ l ~ l t l l ~ l D l n l ~ m ~ l Ilkl~#il~l l :;.~t#l I E~gfllla~mme,,1 i I[r p~..]lgi;i l ~ IIS l II'~'] I ] [.]J[Egl I ~ J I I D I I ~ l l I DgtgXOI I~lSl i~idi Morphine ~.D i~ll I~l I ItAa I~li IOlli t= [ ~11Sl [.. ~ '. a : m l e n, l ~ m D1 n I ~ 1,'n ~l il~ Subject Hair Treatment rofile Sex Female atural Hair Color Hair Tmtme.ts Dark Brown rior to one Hair Treatments During one Hair Cuts During the Weeks, 17. D. " ot Detected - < L O Q but LOD = o Segment t " I cm segments measured from scalp end to tip ~ o segment, difference in hair length noted. See text for explanation -ositive -egative Week o. ~ (rig/mid R~ (ng/ml,} I II , , , >1(3 >1(3 >1OO >I >1(3 >1~ >1OO >1(3 >1~ >1(3 >IG(3 >1(3 >1(3 >1(3 (>2 ng/ml) ~ m L ) Figure. Subject I. Subject J Segment o.t Baseline Week # 6-MAM l " Codeine *.D D D. D. D. * Mid- Week # Morphinc D. D, D. D. * 6-MAM ~ Codeine D. D.,D. End- Week #1 Morphinc (p~/mg).d, D. D. D..D, 6-MAM Codeine D, ,D. * * Subject Hair Treatment rofile Sex atural Hair Color Male Light Brown Hair Treatments r i o r to Stud~' All-over Color-lighter Hair Treatments During one Hair Cuts During the Weeks.1 - ot Detected.A.- ot Analyzed - < LOQ but > L e D * - o Segment t " I em segmenu measured from scalp end to tip "-< LOQ but > LOD for morphjne-3-glucuronlde, and morphlne-6-glucuronlde -ositive -egative Figure 1. Subject J. 26 Morphine (pg/rn~) D.D * Week o. LC/MS (ng/ml) a (ng/ml) M3G~d6G 31. I.A. I , A..A > (>2,ng/mL) (:fflng/ml) l I.A. A. A >13 II I

16 Journal of Analytical Toxicology, Vol. 27, October 23 Table Vl. Coat-A-Count Opiate Screen Cross-Reactivity Data* ng/ml Added ercent Cross-Reactivity Morphine I % I 62% Codeine I % alorphine I 36% 1 27% Dihydrocodeine I 2% I 223% ormorphine I 2% I 6% Hydromorphone 1 13% 1 > 1% Hydrocodone 1 1% 1 116% 1, > 1% Morphine-6~-glucuronide 1 % 1 11% 1 3% I, % Oxycodone 1 63% 1 % I % I, 3% * From kit insert, published March 17, 17, by Coat-A-Count Opiate Screen (Diagnostic roducts Corporation, Los Angeles, CA). Table VII. Comparison of LC-MS versus LC-MS Ratio % 2: 63.6% - - 6: 13.1% - 2:.% - : 22.7% = ositive - = egative use, but is not sensitive enough to document minor changes in dosage history. Another possibility is that the chemical treatments reported by the subject during weeks #2 and # could have destabilized any opiates in the hair causing a decrease in the drug content in the hair. Conclusions Interpretation of the hair data in this study was complex and generally was not in agreement with urine data in most cases. This would suggest that hair was a poor matrix for routine use in recidivism monitoring at the present time. The effects of hair color and chemical treatments on interpretation of hair data requires further investigation. Urine appeared to be a more sensitive indicator of changes in the pattern(s) of drug use during the course of clinical drug treatment. Acknowledgments This research was supported by IH grant no. DA6. The authors would like to thank Christian aulsen for his assistance in hair sample preparation. We would also like to gratefully acknowledge Dr. Walter Ling and the staff of the izarro Treatment Center for their advice and assistance in the clinical portion of this study. References I. Y. akahara, M. Shimamine, and K. Takahashi. Hair analysis for drugs of abuse. III. Movement and stability of methoxyphenamine (as a model compound of methamphetamine) along hair shaft with hair growth. J. Anal. Toxicol. 16:23-27 (). 2. B.A. Goldberger and Y.A. Caplan. Testing human hair for drugs of abuse.ill. Identification of heroin and 6-acetylmorphine as indicators of heroin use. J. Anal Toxicol. 1: (11). 3. D.E. Rollins, D.G. Wilkins, S.. Gygi, M.H. Slawson, and.r. agasawa. Testing for drugs of abuse in hair--experimental observations and indications for future research. Forensic ci. Rev. : 2-36 (17).. W.A. Baumgartner and V.A. Hill. Drug Testing In Hair,. Kintz, Ed. CRC ress, Inc, Boca Raton, FL, 16, pp M. Saitoh, M. Uzuka, and M. Sakamoto. Rate of hair growth. In Advances in Biology of Skin. VoL IX. Hair Growth. roceedings of the University of Oregon Medical School Symposium on the Biology of Skin, 167. W. Montagna and Ri. Dobson, Eds. ergamon ress, Oxford, England, 16, pp R.L. Dupont and W.A. Baumgartner. Drug testing by urine and hair analysis: complementary features and scientific issues. Forensic cl Int. 7:63-6 (1). 7. M. Saitoh, M. Uzuka, and M. Sakamoto. Rate of hair growth. In Advances in Biology of Skin. Vol. IX. Hair Growth. roceedings of the University of Oregon Medical School Symposium on the Biology of Skin, 167. W. Montagna and R.L. Dobson, Eds. ergamon ress, Oxford, England, 16, pp R.J. Myers and J.B. Hamilton. Regeneration and rate of growth of hairs in man. Ann. YAcad. Sci. 3:62-6 (11).. T. Giovanoli-Jakubczak and G.G. Berg. Measurement of mercury in human hair. Arch. Environ. Health 2:13-1 (17). 1. T. Uematsu,. Miyazawa, and M. akashima. The measurement of ofloxacin in hair as an index of exposure. Eur. Clin. harmacol :1- (11 ). 11. D.G. Wilkins, A. Mizuno, C.R. Borges, M.H. Slawson, and D.E. Rollins. Ofloxacin as a reference marker in hair of various colors. ]. Anal Toxicol. 27: (23).. S.. Gygi, R.E. Joseph, Jr., E.J. Cone, and D.G. Wilkins. Incorporation of codeine and metabolites into hair: role of pigmentation. Drug Metab. Dispos. 2:-1 (16). 13. S.R Gygi, D.G. Wilkins, and D.E. Rollins. A comparison of phenobarbital and codeine incorporation into pigmented and nonpigmented rat hair. J. harm. Sci. 6:2-21 (17). 1. D.G. Wilkins, A.S. Valdez,.R. agasawa, S.R Gygi, and D.E. Rollins. Incorporation of drugs for the treatment of substance abuse into pigmented and nonpigmented hair. J. harm. cl 7:3- (17). 1. M.H. Slawson, D.G. Wilkins, and D.E. Rollins. The incorporation of drugs into hair: relationship of hair color and melanin concen- 27

17 Journal of Analytical Toxicology, Vol. 27, October 23 tration to phencyclidine incorporation. J. Anal. Toxicol. 22: 6-13 (1). 16. R. Kronstrand, S. FOrstberg-eterson, B. K~gedal, J. Ahlner, and G. Larson. Codeine concentration in hair after oral administration is dependent on melanin content. Drug Monit. Toxicol. : 1-1 (1). 17. D.L. Hubbard, D.G. Wilkins, and D.E. Rollins. The incorporation of cocaine and metabolites into hair: effects of dose and hair pigmentation. Drug Metab. Dispos. 2:16-16 (2). 1. C.R. Borges, D.G. Wilkins, and D.E. Rollins. Amphetamine an n-acetylamphetamine incorporation into hair: an investigation of the potential role of drug basicity in hair color bias. J. Anal. Toxicol. 2: (21). 1. T. Uematsu,. Miyazawa, O. Okazaki, and M. akashima. ossible effect of pigment on the pharmacokinetics of ofloxacin and its excretion in hair. J. harm. ScL 1: - (). 2. Y. Takiguchi, R. Ishihara, R. Kato, S. Kamihara, M. Yokota, and T. Uematsu. Measurement of flecainide in hair as an index of drug exposure. J. harm. $ci. :11-16 (21). 21. D.E. Rollins, D.G. Wilkins, and G.G. Krueger. Codeine disposition in human hair after single and multiple doses. Eur. ]. Clin. harmacol. :31-37 (16). 22. G.L. Henderson, M.R. Harkey, and C. Zhou. Incorporation of isotopically labeled cocaine into human hair: race as a factor. J. Anal ToxicoL 22: (1) Kintz, V. Cirimele, and B. Ludes. harmacological criteria that can affect the detection of doping agents in hair. Forensic ci. Int. 17:32-33 (2). 2. L. tsch and G. Skopp. Stability in hair fibers after exposure to cosmetic treatment. Forensic Sci. Int. 1:- (16). 2. M.J. Welch, L.T. Sniegoski, C.C. AIIgood, and M. Habram. Hair analysis for drugs of abuse: evaluation of analytical methods, environmental issues, and development of reference materials. J. Anal Toxicol. 17:3-3 (13) Takayama, S. Tanaka, R. Kizu, and K. Hayakama. High-performance liquid chromatography study on effects of permanent wave, dye and decolorant treatments on methamphetamine and amphetamine in hair. Biomed. Chromatogr. 13: (1). 27. M. Yegles, Y. Marson, and R. Wennig. Influence of bleaching on stability of benzodiazepines in hair. Forensic Sci. Int. 17:7-2 (2OOO). 2. M.H. Slawson, D.J. Crouch, D.A. Andrenyak, D.E. Rollins, J.K. Lu, and RL. Bailey. Determination of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in plasma after intravenous and intrathecal morphine administration using HLC with electrospray ionization and tandem mass spectrometry. J. Anal ToxicoL 23:6-73 (1). 2. K.M. H1d, D.G. Wilkins, and D.E. Rollins. Simultaneous quantitation of cocaine, opiates, and their metabolites in human hair by positive ion chemical ionization gas chromatography-mass spectrometry. J. Chromatogr. Sci. 36:-13 (1). 3. M. Rothe and E ragst. Solvent optimization for the direct extraction of opiates from hair samples. J. Anal. ToxicoL 1:236-2 (1). 2

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