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ELSEVIERForensic ciencenternational

70 (1995) 39-51

ForensicScienceInternational

Testing human hair for drugs of abuse. IV.Environmental cocaine contamination andwashing effectsWen Ling Wang, Edward J. Cone*

Addiction Research Center, National Institute on Drug Abuse, National Institutes of Health, Baltimore,MD 21224, USAReceived 10 May 1994; accepted 20 June 1994

Active cocaineuse esults n sequestration f parent drug n hair. In addition, hair hasuni-que physicochemical roperties that permit absorption of cocaine from the environment.When hair is tested or evidenceof cocaine, t is important to considerwhether he positivetest resulted rom active drug useor environmentalcontamination. n a series f laboratoryexperiments,t was ound that exposure f cut hair to cocainevapor (crack smoke) nd toaqueous olutions f cocainehydrochlorideresulted n significantcontaminationof hair sam-ples.Similar resultswereobtainedwith two subjectswho wereexposed o cocainevapor inan unventilatedroom. The amount of contaminationadsorbed y hair depended pon bothtime and extent of exposure.Washing he hair samples ith methanol emoved> 70 of thecocainecontaminantafter cocainevapor exposure,but was esseffective (< 50 ) ollowingcontaminationwith aqueouscocaine. Shampoo reatment cycles (overnight soaking)pro-gressively emoved ncreasing mountsof cocaine rom the contaminatedhair, but residualcocaine emained fter 10cycles.Studieswere alsoperformed o determine he usefulnessfbenzoylecgonine sa markerof active cocaineadministration.Smallamounts f benzoylecgo-nine (ca. 1 ng/mg)were ormed n hair as a result of environmentalcontaminationwith co-caine. Also, it was found that benzoylecgoninecould be adsorbed rom illicit cocainecontaminatedwith benzoylecgonine.t wasconcluded hat positive hair test results houldbeinterpretedcautiouslydue o the possibilityof environmental ontamination rom cocaineandrelatedconstituents.Keywordr: Hair analysis;Cocaine; Benzoylecgonine;Toxicology; Contamination; Decon-tamination; Forensics

* Corresponding author.Elsevier Science Ireland Ltd.SSDI 0379-0738(94)01616-D

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40 W.L. Wang, E.J. Cone/ Forensic Sci. ht. 70 (1995) 39-511. Introduction

Hair is a complex matrix consisting primarily of two or three ar-keratin chainswound into a helix forming strands called microfibrils. These microfibrils areorganized into larger bundles called macrofibrils which comprise the bulk of the cor-tex, the inner structure of hair. The strands are stabilized by disulfide and hydrogenbonds giving the microfibrils a semi-crystalline structure. The cortex of hair is sur-rounded by a protective layer of epithelial cells called the cuticle. The cuticle cellsoverlap in a shingle arrangement, holding the cortex together and serving as a pro-tective barrier to the environment. Without the cuticle, the cortex degenerates,becomes frayed and breaks. Usually there is a gradual change in the cuticle alongthe hair shaft. The cuticle closest to the root is intact, but toward the tip the cuticleshows signs of damage and in cases of unusually long hair, may be totally missing[l]. Water, organic molecules, and trace metals can penetrate the structure of hairand adsorb onto the inner matrix to carboxyl groups, amino acid side chains andpeptide bonds [2].A network of arterial capillaries nourish the growing hair bulb (root) via thepapilla. Presumably, after drug administration, lipid soluble drug molecules in blooddiffuse through the papilla and into the hair bulb. There they bind to macro-

Hair0recursors

0ncorporationin New HairFig. I. Model of drug deposition in hair.

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W.L. W ang, E.J . Cone/ Forensic Sri. ht. 70 (1995) 39-51 41

molecules and are incorporated into the hair strand. This is one of several keymechanisms proposed for the sequestration of drugs in hair. Other possibilities fordrug entry into hair include deposition on hair via sebum, sweat and from the exter-nal environment. A model illustrating drug entry into hair via these differentmechanisms is shown in Fig. 1. A somewhat similar model was reported by Chit-tleborough and Steel [3] for the deposition of trace metals in hair from active intakeand from environmental contamination. In their model, analytes gain entry into hairfrom blood, in vivo, by way of unidentified storage pools in the body. The signifi-cance of both models lies in the recognition of environmental contamination as apotentially important source of hair contamination. In Fig. 1, sweat and sebumtransfer drug to the contributors hair and to the environment where it may becomea source of environmental contamination to other individuals. A clear distinction ismade in this model between environmental contamination (pseudo-contamination)of hair from the individuals sweat (I&,, pathway) and sebum (Ksh pathway) andenvironmental contamination (I& pathway) from the external environment arisingfrom the myriad of possibilities presented to an individual in their everyday activi-ties. The proposed model also recognizes that the drug depot sequestered in hair isnot constant, but releases drug back to the environment.Very little is known regarding the extent, frequency or conditions necessary forenvironmental drug contamination of hair to occur. Many factors are likely to affectthe degree of hair penetration by chemicals from the environment including the con-dition of the hair cuticle, amount and length of exposure, pH effects, hydration,stateof the hair matrix and physicochemical properties of the drug. To evaluate some ofthese factors, a series of experiments were undertaken to determine if environmentaldrug contamination of hair would lead to false positive test results. Cocaine waschosen as the model compound because of widespread illicit use and its availabilityin two different chemical forms (hydrochloride salt and free base). In this report, thefollowing environmental contamination issues were evaluated: (1) capacity of hairto adsorb cocaine base (vapor) and cocaine hydrochloride (aqueous solution); (2)rate at which environmental cocaine vapor contamination occurs; (3) effects ofshampoo cycles on removal of cocaine contamination; (4) comparison of cocainecontamination of live subjects hair versus cut hair; and (5) evaluation of thestability of cocaine in contaminated hair when subjected to alkaline pH conditions.2. Materials and methods2.1. Chemicals, reagents and materialCocaine hydrochloride was obtained from Mallinckrodt, Inc. (St. Louis, MO).Cocaine base (crack) was prepared from cocaine hydrochloride by treatment of anaqueous solution with sodium bicarbonate. Benzoylecgonine tetrahydrate, ecgoninemethyl ester HCl, benzoylnorecgonine and norcocaine HCl were obtained from theResearch Technology Branch, National Institute on Drug Abuse (Rockville, MD).Cocaethylene and norcocaethylene were generous gifts from Dr Ivy Carroll,Research Triangle Institute, Research Triangle, NC. Anhydroecgonine methyl esterand ecgonine ethyl ester were generous gifts from Dr Andrew Allen, Addiction

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42 W.L. Wang. E.J. Cone/Forensic Sci. ht. 70 (1995) 39-51Research Center, National Institute on Drug Abuse, Baltimore, MD. [2H3]coca-ethylene was purchased from Radian Corporation (Austin, TX). [ 2H3]cocaine,[Hslbenzoylecgonine tetrahydrate, and [2HJecgonine methyl ester HCl were pur-chased from Sigma Chemical Co. (St. Louis, MO). Methanol, methylene chloride,2-propanol and acetonitrile (J.T. Baker Chemical Co., Phillipsburg, NJ) were HPLCgrade solvents. N, 0-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% tri-methylchlorosilane (TMCS) was purchased from Pierce Chemical Co. (Rockford,IL). All other chemicals were reagent grade. Solid phase extraction (SPE) columns(Clean Screen@ DAU, 200 mg-10 ml) and filtration columns (4 ml) were purchasedfrom Worldwide Monitoring Corp. (Horsham, PA). Gas chromatography-massspectrometry (GCYMS) autosampler microvials were purchased from SunBrokersTM, Inc. (Wilmington, NC). Acetate buffers (pH 4.0 and pH 6.0) wereprepared as varying mixtures of 0.5 M sodium acetate and 0.5 M acetic acid. Sodiumfluoride was added to the pH 6.0 buffer to give a final concentration of 0.5% (w/v).Suave@ Full Body Shampoo (Helene Curtis, Inc, Chicago, IL) and Perfectoe MildAlkaline Wave treatment (Matrix Essentials, Solon, OH) were purchased from alocal pharmacy.2.2. InstrumentationQuantitative analyses were performed on a Hewlett-Packard 5890A gaschromatograph with an autosampler (HP7673A) and interfaced with a Hewlett-Packard 5970B mass selective detector (MSD). A split-splitless capillary inlet systemand a HP-l fused-silica capillary column (12 m x 0.2 mm i.d., 0.33~pm filmthickness) were utilized for the analyses.2.3. Chromatographic conditions

The splitless injection mode with a purge-off time of 0.5 min was used for l-p1samples. Ultra-pure grade helium was used as the carrier gas at a flow rate of 1ml/mm. The initial oven temperature was 7OC, hold for 1 min, programmed to220C at 35C/min, hold at 220C for 0.25 min, programmed to 250C at lOC/minand hold for 3 min. The injection port and transfer line temperatures were 250Cand 280C respectively.The mass selective detector was operated in the selective ion monitoring mode.Three ions for each component were monitored. The ions for each compound were

monitored in the following elution order (quantitative ion is indicated in paren-thesis): anhydroecgonine methyl ester, m/z (152), 166, 181; [2H3]ecgonine methylester, m/z (99), 85; ecgonine methyl ester, m/z (96), 82, 271; [2HJcocaine, m/z (185),85; cocaine, m/z (182), 82, 303; [2Hs]cocaethylene, m/z (199), 85; cocaethylene, m/z(196), 82, 317; [2HJbenzoylecgonine, m/z (243), 85; benzoylecgonine, m/z (240), 82,361; norcocaine, m/z (140), 240, 346; norcocaethylene, m/z (254), 140, 360; and ben-zoylnorecgonine, m/z (404), 140,298. The mass ion defect of all quantitative ions forstandards and internal standards were determined daily with an unextracted stan-dard at a resolution of 0.1 amu. The GC/MS was autotuned daily according tomanufacturers instruction. The electron multiplier was operated at +200 eV relativeto the tune value. Daily maintenance of the GC/MS included clipping of the GC col-umn and replacement of the injector septum, liner and seal.

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W.L. Wang, E.J. Cone I Forensic Sci. ht. 70 (1995) 39-51 43Quantitation of ecgonine methyl ester, cocaine, cocaethylene, and benzoylecgo-nine was based upon ratios of peak area to the corresponding deuterated internalstandard analogs for each analyte. Quantitation of other compounds was basedupon ratios of peak area to the nearest corresponding deuterated internal standard.Analytes were identified based upon comparison of retention time and relative abun-dance of confirming ions to the corresponding values of authentic standards assayedin the same run.

2.4. Hair analysisHair samples (10 mg) were weighed in a filtration column and the bottom of thecolumn was sealed. After adding 1 ml of methanol wash solvent, the hair was cutinto fine particles with surgical scissors. The total time of cutting and washing lastedapproximately 1 min. The methanol wash fraction was collected and the remaininghair was rinsed with methanol (2 x 1 ml) under vacuum. The wash fractions werecombined, internal standards were added and the solution was evaporated to drynessat room temperature. The residue was treated with 3 ml of 0.5 M acetate buffer (pH6) containing 0.5% (w/v) sodium fluoride and extracted by the SPE procedure.The remaining hair sample was extracted by resealing the bottom of the columnand adding of 1 ml of methanol. Internal standards were added and a mini magneticstirring bar was placed in the column. The top of the column was sealed and the col-umn was placed in a l-dram vial. The vial was incubated with stirring in a water bathat 40C for 18 h. After cooling, the extract was collected and the remaining hairsample was rinsed with 0.5 ml of methanol. The extract and rinse fractions were com-bined and evaporated to dryness. The residue was treated with 3 ml of 0.5 M acetatebuffer (pH 6) containing 0.5% (w/v) sodium fluoride and extracted by the SPEprocedure.Prior to extraction, plasma and saliva samples (1 ml) were treated with internalstandards, diluted with acetate buffer (pH 6) and filtered through a filtration col-umn. SPE columns were conditioned with methanol (2 x 2 ml), water (2 x 2 ml)and acetate buffer (1 ml, pH 6.0). Vacuum was removed prior to addition of the ace-tate buffer to prevent the column from going to dryness. Hair washes, hair extracts,and plasma, urine and saliva filtrates were added to the wet column. Internal stan-dards were added prior to SPE extraction. The samples were drawn through the col-umns with vacuum-assisted elution and the columns were washed with water (2 x 1ml) and acetate buffer (1 ml, pH 4.0). The columns were aspirated for 5 min, thenwashed with acetonitrile (2 x 1 ml). The columns were dried for 5 min and elutedwith 3 x 2 ml of elution solvent [methylene chloride-Zpropanol (8:2) with 2% am-monium hydroxide]. Vacuum flow rate was controlled at l-2 ml/mm during process-ing. The eluate was collected, evaporated to dryness, and the residue wasreconstituted with 20 ~1 of acetonitrile. BSTFA (20 ~1 containing 1% TMCS) wasadded and the mixture was heated at 60C for 30 min. The derivatized extract wastransferred to an autosampler vial and an aliquot (1 ~1) was analyzed by GUMS.2.5. Contamination of hair with cocaine vaporCocaine vapor contamination studies were performed in a closed room(7 x 8 x 8 feet, length x width x height) without ventilation. Interior surfaces

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44 W.L. Wang, E. J. Cone/Forensic Sci. ht. 70 (1995) 39-51were composed of non-porous materials. Cocaine base was vaporized in a glassbeaker open to the atmosphere at a temperature of 190C. The cocaine was locatedin the center of the room at a height of 2.5 feet. Bundles of cut drug-free control hair(ca. 50 mg) were suspended with string in the four comers of the room at a heightof 5 ft. An additional bundle was suspended as a control sample in the adjoiningroom. Careful precautions were taken to prevent contamination during handling.Contamination experiments were conducted in which the amount of vaporized co-caine was varied (0, 5, 10, 25, and 100 mg) and the time of exposure (60 min) wasconstant, and experiments in which the time of exposure was varied (0,5, 15,30,45,60 min) and the amount (100 mg) was constant.Head hair from two volunteers was contaminated with cocaine vapor undersimilar conditions as the contamination experiments with cut hair. The two in-dividuals (one male and one female), who reported no active use of cocaine, wereexposed to cocaine vapor (100 mg) for 60 min. Head hair samples were collected justprior to exposure, immediately after exposure, 1 day and 8 days later. No special pre-cautions were taken regarding care of their hair and normal hygiene and groomingpractices continued during this period.2.6. Contamination of hair with aqueouscocaineDrug free control hair samples (N = 3) were contaminated with aqueous solutionsof cocaine hydrochloride (0.01,0.05, and 0.1 mg/ml) by soaking bundles of hair (ca.200 mg) overnight. Following soaking, hair bundles were removed, blotted on absor-bent paper and air dried. A control sample was prepared in a similar manner withtap water substituted for drug solution. In a similar experiment, control hair wascontaminated with an aqueous solution of cocaine hydrochloride and benzoylecgo-nine present in equal concentrations (0.01 mgml).2.7. Hair treatment and wash experimentsHair samples contaminated with cocaine vapor and aqueous cocaine (N = 3) weredivided into approximately equal portions and used in various wash and hair treat-ment experiments. The effects of soap (shampoo) were evaluated by soaking con-taminated hair samples in a 2% (w/v) aqueous solution of Suave@ shampooovernight at room temperature. After soaking, the hair was removed, rinsed withwarm water (10 x 10 ml) followed by a similar rinse with deionized water, then air-dried. Repeated shampoo and rinse steps were performed in the same manner.The effects of a high pH alkaline wave treatment (pH, 8.9) was evaluated by treat-ment of hair contaminated with aqueous cocaine hydrochloride (0.05 mg/ml) withPerfecto@ Mild Alkaline Wave solution. The hair was soaked in a 25% (v/v) solutionfor 20 min at room temperature. After soaking, the hair was removed, rinsed withdistilled water and dried at room temperature.3. Results3.1. Cocaine vapor contamination studiesConcentration effects. Drug-free control hair (cut head hair) was contaminated byexposure for 60 min to cocaine vapor in varying amounts in an unventilated room.

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W.L. Wang, E. J. Cone /Forensic Sci. ht. 70 (1995) 39-51 45Table 1Contamination of drug-free control hair by vaporization of cocaine base in a small unventilated roomaw (m8) Cocaine (ng/mg) Benzoylecgonine (ng/mg)

Wash Extract Wash Extract0 0 0 0 05 8.8 2.5 0 0

10 12.5 2.3 0 025 20.5 4.0 0 050 23.1 3.7 0 0100 28.5 2.2 0 0

aData represent results o f single measurements of hair samples.

Substantial amounts of cocaine were deposited on hair from all doses (Table 1). Asthe amount of cocaine exposure increased, cocaine contamination appeared toplateau near the 25mg cocaine vapor dose with only minor increases thereafter.Analysis of wash and extract fractions indicated that the majority of cocaine wasremoved in the methanol ,wash fraction (78-93%). Benzoylecgonine and other co-caine metabolites were not detected.Time course of contamination. The duration of cocaine vapor exposure and theeffect of room location on hair contamination were investigated in an experiment inwhich 100 mg of cocaine base was vaporized in an unventilated room. Hair sampleswere suspended in different comers of the room and removed after various exposuretimes, Cocaine was detectable within 5 min of exposure and increased in a linearmanner over the 60-min period (Table 2). Anhydroecgonine methyl ester was detec-table on hair within 15 min. No other cocaine analytes were detected. Different loca-tions in the room produced only minor differences in amount of cocaine andanhydroecgonine methyl ester contamination.Effect of shampoocycles on contamination. Removal of cocaine from contaminatedhair was evaluated by soaking samples overnight at room temperature in 2% (w/v)Suave@ shampoo solution. The first cycle of soaking removed the majority of co-caine and anhydroecgonine methyl ester (Table 3). Cocaine was subsequently foundonly in the extract fraction and was consistently < 1 ng/mg. No other drug analyteswere detected in the wash or extract fractions.Human exposure studies.An experiment was performed in which two volunteerswithout a history of active cocaine use were exposed to cocaine vapor in a closed,unventilated room during the vaporization of 100 mg of cocaine base (Table 4).Head hair samples collected immediately after exposure were highly contaminatedwith cocaine and anhydroecgonine methyl ester. Approximately 80% of the cocainewas removed in the methanol washing step performed prior to extraction. Samplesthat were collected after the first day following the exposure session containedsmaller amounts of cocaine. During the 8-day collection period, both staff membersreported practicing normal hygienic procedures including periodic shampooing oftheir hair on a 24-h cycle (Staff l) or 48-h cycle (Staff 2).

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W.L. Wang, E.J. Cone/Forensic Sci. Int. 70 (1995) 39-51 41Table 3Cleansing effect of shampoo on coca ine contaminated hair (cocaine vapor, 100 mg)

Shampoo cyclesa

0123

Cocaine (ng/mg)Wash Extract19.6 7.30 0.70 0.50 0.4

Anhydroecgonine methyl ester (ngmg)Wash Extract39.9 10.00 0.6

0 0.20 0.2aEach cycle consis ted of an overnight soak at room temperature in 2 (w/v) aqueous solution of Suaveshampoo (pH 7.0).

3.2. Aqueous cocaine contaminationConcentration effects and shampoo ycles. Drug-free control hair samples (cut headhair) were contaminated by soaking overnight at room temperature in aqueous solu-tions of cocaine hydrochloride (Table 5). Increasing concentrations of cocaine wereabsorbed with increasing dose, but proportional amounts of cocaine were not boundat the highest concentration compared to lower concentrations suggesting that bind-ing capacity of hair for cocaine was limited. Analysis of wash and extract fractionsindicated that 16-41% of the cocaine contaminant was removed in the methanolwash fraction. Traces of benzoylecgonine were detected following contamination

with cocaine at 0.05-mg/ml and O.l-mg/ml concentrations. Shampoo soaking over-night at room temperature removed substantial amounts of cocaine, but detectableconcentrations persisted through 10 shampoo treatment cycles.Benzoylecgonine contamination. Contamination experiments were conducted inwhich drug-free control hair samples (N = 3) were contaminated with a mixture of

Table 4Cocaine contam ination of human volunteers hair as a result of exposure to cocain e vaporTime ofmeasurement

Staff lPre-exposurePost-exposure1 Day8 Days

Cocaine (ngmg)Wash Extract

0.4a 021.9 5.60.6 1.20.5 0.6

Anhydroecgonine methyl ester (ngmg)Wash Extract

0 06.2 1.80 00 0

staffw2Pre-exposure 0 0 0 0Post-exposure 37.1 8.6 3.8 3.31 Day 0.4 0.7 0.7 08 Days 0 0.5 0 0sTI ris staff member had been assoc iated with other recent experiments involving vaporized cocaine .

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48 W.L. Wang, E.J. Cone/Forensic Sci. ht. 70 (1995) 39-51Table 5Contamination of drug-free control hair (100 mg) with aqueous solutions o f cocaine hydrochloride andcleansing effects of shampooCocaine HCI Shampoobs/ml) cycle9

Cocaine (ng/mg) Benzoylecgonine (ng/mg)Wash Extract Wash Extract

0.01 0 19.2 98.3 0 01 0.4 12.8 0 02 0 5.6 0 03 0 4.1 0 010 0 0.5 0 00.05 0 209.2 302.5 0.5 0.8I 1.1 76.0 0 02 0.3 21.4 0 03 0.2 15.3 0 010 0.3 2.0 0 00.1 0 216.0 352.4 0.7 1.51 5.5 143.6 0 0.42 3.4 59.3 0 03 1.6 48.3 0 010 0.3 3.6 0 0

Qch cycle consisted of an overnight soak at room temperature in 2% (w/v) aqueous solution of Suaveeshampoo (pH 7.0).

cocaine hydrochloride (0.01 mg/ml) and benzoylecgonine (0.01 mg/ml) (Table 6).Approximately 10 times more cocaine than benzoylecgonine was absorbed by thehair samples. Shampoo soaking overnight at room temperature removed substantialamounts of cocaine and benzoylecgonine, but detectable concentrations of cocaineand benzoylecgonine persisted through 10 shampoo treatment cycles.Stability of cocaine in hair. An experiment was conducted with cocaine con-taminated hair in which samples (N = 3) were treated with an alkaline hair treatment

Table 6Contamination of drug-free control hair with aqueous solutions of cocaine hydrochloride (0.01 mp/ml)and benxoylecgonine (0.01 mg/ml) and cleansing effects of shampooShampoo cyclesa Cocaine (ng/mg) Benzoylecgonine (ng/mg)

Wash Extract Wash Extract0 6.2, 8.8 24.8, 25.9 0.3, 0.5 3.0, 3.31 0.3, 0.3 2.5, 2.9 0, 0 0.3, 0.42 0.2, 0.2 1.5, 2.0 0, 0.2 0.2, 0.23 0.2, 0.2 1.5, 1.6 0, 0 0.1, 0.3

10 0.1, 0.2 0.2, 0.5 0.2, 0 0, 0Data represent results of duplicate hair samples measured in two separate assays.Each cycle consisted of an overnight soak at room temperature in 2% (w/v) aqueous solution of Suavesshampoo (pH 7.0).

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W.L. Wang, E.J. Cone/Forensic Sci. Int. 70 (1995) 39-51 49(pH, 8.9) to determine if benzoylecgonine and ecgonine methyl ester would be form-ed as a result of the hair treatment. Before treatment, cocaine and metabolite con-centrations in the extract fraction of contaminated hair were as follows: cocaine,140.2 ng/mg; benzoylecgonine, 0.6 ng/mg; and ecgonine methyl ester, 0.4 ng/mg.Following alkaline wave treatment, cocaine and metabolite concentrations were asfollows: cocaine, 77.2 ng/mg; benzoylecgonine, 0.7 ng/mg, and ecgonine methylester, 0.8 ng/mg. Only the cocaine concentration was affected by the treatment. Con-sequently, it appeared that benzoylecgonine and ecgonine methyl ester, if formed,were also removed in the process.4. Discdon

Forensic hair testing for drugs of abuse has grown in popularity in recent years.The potential ability to test for drug exposure in hair samples that grew over a periodspanning several months of an individuals recent past makes it an attractive speci-men for many applications. A positive or negative drug test can have major implica-tions concerning an individual under investigation for illicit drug abuse. The validityof hair testing results depends upon the ability of the test to correctly identify parentdrug and/or metabolite following active drug use. A test that fails to distinguish be-tween active and passive drug exposure would lack validity to the extent and fre-quency that false positive results were reported because of passive drug exposure.In the present studies, hair samples that were externally exposed to cocaine vaporand to aqueous solutions of cocaine hydrochloride tested positive for cocaine im-mediately after exposure and continued to test positive at lower concentrations afternumerous shampoo wash cycles. The degree of contamination was dependent uponthe amount and length of exposure. When hair samples were exposed to cocainevapor in an unventilated room, cocaine contamination continued to increase longafter the cocaine base had been vaporized (Table 2). This suggested that hair couldbecome contaminated from cocaine vapor (smoke) long after crack cocaine smokehad been generated. Also, equivalent concentrations of cocaine were deposited onhair in a manner that was independent of room location. This indicated that diffu-sion of cocaine vapor occurred rapidly throughout the room in approximately equalamounts. Obviously, the ventilation conditions of the area in which cocaine vaporwas produced would be an important factor in determining the extent and durationof hair contamination.There were substantial amounts of cocaine contamination deposited in hair as aresult of exposure to cocaine vapor (> 20 ng/mg) or aqueous solutions of cocainehydrochloride (> 200 ng/mg). During the processing, extraction and analysis of thesecontaminated hair samples, it was noted that washing with methanol removed dif-ferent amounts of cocaine from hair exposed to cocaine vapor versus hair exposedto aqueous cocaine. The majority (> 70%) of cocaine contamination was removedfrom cocaine vapor exposed hair by a brief methanol wash and these findings wereconsistent for cut hair studies (Tables l-3) and the studies with two human subjects(Table 4). In contrast, less than 50% of cocaine contamination was removed by meth-anol washing of aqueous cocaine exposed hair. These findings suggested that con-tamination of hair by aqueous cocaine results in greater drug penetration of the hair

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50 W.L. Wung, E.J. Cone/Forensic Sci. ht. 70 (1995) 39-51matrix. It was noted that the resulting contaminant from aqueous cocaine was farmore resistant to removal by various washing procedures than hair exposed tococaine vapor. Also, substantial amounts of cocaine persisted in hair through 10cycles of soaking overnight in shampoo solution. Presumably, penetration of hair byaqueous cocaine was facilitated by the presence of water which is known to produceconsiderable swelling of hair [4]. Water molecules also bind to the polypeptidechains of keratin in place of internal hydrogen bonds, thus increasing the size of thehair strand and its plasticity. The positive charge on the cocaine molecule in aqueoussolution (cocaine hydrochloride) also could have influenced the affinity of hair forcocaine.Analysis of hair samples that had been contaminated by cocaine vapor exposurerevealed the presence of anhydroecgonine methyl ester. This analyte arose as a resultof the thermal elimination of a molecule of benzoic acid from cocaine during heat-ing. Its presence in urine has been suggested to be indicative of crack smoking [5];however, its presence in hair would not appear to be a valid biological marker foractive cocaine use because of substantial deposition from the external environmentduring the production of cocaine vapor.

Other potential biological markers for active cocaine use include cocaethylene [6],norcocaine and benzoylecgonine. Neither cocaethylene or norcocaine were identifiedin any of the current contamination experiments with cocaine, Consequently, theirpresence in hair would appear to be valid markers for active cocaine use as long asit can be assumed that environmental contamination with these compounds did notoccur, i.e. they were not present in illicit cocaine. The presence of benzoylecgoninein hair has been suggested to be indicative of active cocaine use [7]. In the presentstudies, only traces of benzoylecgonine were detected in hair in experiments involv-ing high concentrations of cocaine contamination (Table 4). Very careful optimiza-tion of all assay conditions was needed to eliminate artifactual production ofbenzoylecgonine. Without careful precautions, benzoylecgonine can be generatedduring the extraction process, particularly at elevated pH conditions, and false posi-tive results would be generated.Despite the chemical lability of cocaine toward hydrolysis, cocaine sequestered inhair appeared to exhibit unusual stability. Even when cocaine contaminated hair wassubjected to severe alkaline conditions (alkaline wave treatment, pH = 8.9), therewas no evidence of conversion of cocaine to benzoylecgonine or ecgonine methylester.Hair can become contaminated with benzoylecgonine from the environment.Substantial concentrations of benzoylecgonine were absorbed when hair wasdeliberately exposed to aqueous solutions of cocaine mixed with benzoylecgonine(Table 6). Consequently, the usefulness of this analyte as a marker of active cocaineuse is also limited by the possibility of environmental contamination.In summary, environmental contamination of hair by cocaine can occur rapidly,in high amounts and without the knowledge of the individual that contamination hasoccurred. Initially, after hair becomes contaminated, a large portion of the cocainecontamination can be removed easily by bathing and shampooing. If freshly con-taminated hair is analyzed, a large portion of the cocaine contaminant would befound in the wash fraction (assuming this step is employed in the assay). The

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