Forensic Science International

ELSEVIER 70 (1995) 39-51

Forensic Science International

Testing human hair for drugs of abuse. IV. Environmental cocaine contamination and washing effects
Wen Ling Wang, Edward J. Cone*
Addiction Research Center, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA Received 10 May 1994; accepted 20 June 1994

Active cocaineuseresultsin sequestration of parent drug in hair. In addition, hair hasunique physicochemical properties that permit absorption of cocaine from the environment. When hair is testedfor evidenceof cocaine,it is important to considerwhetherthe positive test resultedfrom active drug useor environmentalcontamination.In a series of laboratory experiments, it wasfound that exposure of cut hair to cocainevapor (crack smoke) and to aqueous solutions of cocainehydrochlorideresultedin significantcontaminationof hair samples.Similar resultswereobtainedwith two subjects who wereexposedto cocainevapor in an unventilatedroom. The amount of contaminationadsorbed by hair depended upon both time and extent of exposure. Washingthe hair samples with methanolremoved> 70%of the cocainecontaminantafter cocainevapor exposure,but waslesseffective (< 50%)following contaminationwith aqueouscocaine. Shampootreatment cycles (overnight soaking)progressively removedincreasing amountsof cocainefrom the contaminatedhair, but residual cocaineremained after 10cycles.Studies were alsoperformedto determine the usefulness of benzoylecgonine asa markerof active cocaineadministration.Smallamounts of benzoylecgonine (ca. 1 ng/mg)wereformed in hair as a result of environmentalcontaminationwith cocaine. Also, it was found that benzoylecgoninecould be adsorbedfrom illicit cocaine contaminated with benzoylecgonine. It wasconcludedthat positive hair test results shouldbe interpretedcautiouslydueto the possibilityof environmental contaminationfrom cocaineand relatedconstituents. Hair analysis;Cocaine; Benzoylecgonine; Toxicology; Contamination; Decontamination; Forensics
Keywordr: * Corresponding author. Elsevier Science Ireland Ltd. SSDI 0379-0738(94)01616-D

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W.L. Wang, E.J. Cone/ Forensic Sci. ht. 70 (1995) 39-51

1. Introduction

Hair is a complex matrix consisting primarily of two or three ar-keratin chains wound into a helix forming strands called microfibrils. These microfibrils are organized into larger bundles called macrofibrils which comprise the bulk of the cortex, the inner structure of hair. The strands are stabilized by disulfide and hydrogen bonds giving the microfibrils a semi-crystalline structure. The cortex of hair is surrounded by a protective layer of epithelial cells called the cuticle. The cuticle cells overlap in a shingle arrangement, holding the cortex together and serving as a protective barrier to the environment. Without the cuticle, the cortex degenerates, becomes frayed and breaks. Usually there is a gradual change in the cuticle along the hair shaft. The cuticle closest to the root is intact, but toward the tip the cuticle shows 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 hair and adsorb onto the inner matrix to carboxyl groups, amino acid side chains and peptide bonds [2]. A network of arterial capillaries nourish the growing hair bulb (root) via the papilla. Presumably, after drug administration, lipid soluble drug molecules in blood diffuse through the papilla and into the hair bulb. There they bind to macro-

0
Fig. I. Model of drug deposition in hair.

Hair Precursors

Incorporation in New Hair

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molecules and are incorporated into the hair strand. This is one of several key mechanisms proposed for the sequestration of drugs in hair. Other possibilities for drug entry into hair include deposition on hair via sebum, sweat and from the external environment. A model illustrating drug entry into hair via these different mechanisms is shown in Fig. 1. A somewhat similar model was reported by Chittleborough and Steel [3] for the deposition of trace metals in hair from active intake and from environmental contamination. In their model, analytes gain entry into hair from blood, in vivo, by way of unidentified storage pools in the body. The significance of both models lies in the recognition of environmental contamination as a potentially important source of hair contamination. In Fig. 1, sweat and sebum transfer drug to the contributors hair and to the environment where it may become a source of environmental contamination to other individuals. A clear distinction is made in this model between environmental contamination (pseudo-contamination) of hair from the individuals sweat (I&,, pathway) and sebum (Ksh pathway) and environmental contamination (I& pathway) from the external environment arising from the myriad of possibilities presented to an individual in their everyday activities. The proposed model also recognizes that the drug depot sequestered in hair is not constant, but releases drug back to the environment. Very little is known regarding the extent, frequency or conditions necessary for environmental drug contamination of hair to occur. Many factors are likely to affect the degree of hair penetration by chemicals from the environment including the condition of the hair cuticle, amount and length of exposure, pH effects, hydration,state of the hair matrix and physicochemical properties of the drug. To evaluate some of these factors, a series of experiments were undertaken to determine if environmental drug contamination of hair would lead to false positive test results. Cocaine was chosen as the model compound because of widespread illicit use and its availability in two different chemical forms (hydrochloride salt and free base). In this report, the following environmental contamination issues were evaluated: (1) capacity of hair to adsorb cocaine base (vapor) and cocaine hydrochloride (aqueous solution); (2) rate at which environmental cocaine vapor contamination occurs; (3) effects of shampoo cycles on removal of cocaine contamination; (4) comparison of cocaine contamination of live subjects hair versus cut hair; and (5) evaluation of the stability of cocaine in contaminated hair when subjected to alkaline pH conditions.
2. Materials and methods

2.1. Chemicals, reagents and material

Cocaine hydrochloride was obtained from Mallinckrodt, Inc. (St. Louis, MO). Cocaine base (crack) was prepared from cocaine hydrochloride by treatment of an aqueous solution with sodium bicarbonate. Benzoylecgonine tetrahydrate, ecgonine methyl ester HCl, benzoylnorecgonine and norcocaine HCl were obtained from the Research 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 ester and ecgonine ethyl ester were generous gifts from Dr Andrew Allen, Addiction

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Research Center, National Institute on Drug Abuse, Baltimore, MD. [2H3]cocaethylene was purchased from Radian Corporation (Austin, TX). [ 2H3]cocaine, [Hslbenzoylecgonine tetrahydrate, and [2HJecgonine methyl ester HCl were purchased from Sigma Chemical Co. (St. Louis, MO). Methanol, methylene chloride, 2-propanol and acetonitrile (J.T. Baker Chemical Co., Phillipsburg, NJ) were HPLC grade solvents. N, 0-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (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 purchased from Worldwide Monitoring Corp. (Horsham, PA). Gas chromatography-mass spectrometry (GCYMS) autosampler microvials were purchased from Sun BrokersTM, Inc. (Wilmington, NC). Acetate buffers (pH 4.0 and pH 6.0) were prepared as varying mixtures of 0.5 M sodium acetate and 0.5 M acetic acid. Sodium fluoride 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 Mild Alkaline Wave treatment (Matrix Essentials, Solon, OH) were purchased from a local pharmacy.
2.2. Instrumentation

Quantitative analyses were performed on a Hewlett-Packard 5890A gas chromatograph with an autosampler (HP7673A) and interfaced with a HewlettPackard 5970B mass selective detector (MSD). A split-splitless capillary inlet system and a HP-l fused-silica capillary column (12 m x 0.2 mm i.d., 0.33~pm film thickness) 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-p1 samples. Ultra-pure grade helium was used as the carrier gas at a flow rate of 1 ml/mm. The initial oven temperature was 7OC, hold for 1 min, programmed to 220C at 35C/min, hold at 220C for 0.25 min, programmed to 250C at lOC/min and hold for 3 min. The injection port and transfer line temperatures were 250C and 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 parenthesis): anhydroecgonine methyl ester, m/z (152), 166, 181; [2H3]ecgonine methyl ester, 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 benzoylnorecgonine, m/z (404), 140,298. The mass ion defect of all quantitative ions for standards and internal standards were determined daily with an unextracted standard at a resolution of 0.1 amu. The GC/MS was autotuned daily according to manufacturers instruction. The electron multiplier was operated at +200 eV relative to the tune value. Daily maintenance of the GC/MS included clipping of the GC column and replacement of the injector septum, liner and seal.

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Quantitation of ecgonine methyl ester, cocaine, cocaethylene, and benzoylecgonine was based upon ratios of peak area to the corresponding deuterated internal standard analogs for each analyte. Quantitation of other compounds was based upon ratios of peak area to the nearest corresponding deuterated internal standard. Analytes were identified based upon comparison of retention time and relative abundance of confirming ions to the corresponding values of authentic standards assayed in the same run. 2.4. Hair analysis Hair samples (10 mg) were weighed in a filtration column and the bottom of the column was sealed. After adding 1 ml of methanol wash solvent, the hair was cut into fine particles with surgical scissors. The total time of cutting and washing lasted approximately 1 min. The methanol wash fraction was collected and the remaining hair was rinsed with methanol (2 x 1 ml) under vacuum. The wash fractions were combined, internal standards were added and the solution was evaporated to dryness at room temperature. The residue was treated with 3 ml of 0.5 M acetate buffer (pH 6) 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 column and adding of 1 ml of methanol. Internal standards were added and a mini magnetic stirring bar was placed in the column. The top of the column was sealed and the column was placed in a l-dram vial. The vial was incubated with stirring in a water bath at 40C for 18 h. After cooling, the extract was collected and the remaining hair sample was rinsed with 0.5 ml of methanol. The extract and rinse fractions were combined and evaporated to dryness. The residue was treated with 3 ml of 0.5 M acetate buffer (pH 6) containing 0.5% (w/v) sodium fluoride and extracted by the SPE procedure. Prior to extraction, plasma and saliva samples (1 ml) were treated with internal standards, diluted with acetate buffer (pH 6) and filtered through a filtration column. 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 acetate 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 standards were added prior to SPE extraction. The samples were drawn through the columns with vacuum-assisted elution and the columns were washed with water (2 x 1 ml) and acetate buffer (1 ml, pH 4.0). The columns were aspirated for 5 min, then washed with acetonitrile (2 x 1 ml). The columns were dried for 5 min and eluted with 3 x 2 ml of elution solvent [methylene chloride-Zpropanol (8:2) with 2% ammonium hydroxide]. Vacuum flow rate was controlled at l-2 ml/mm during processing. The eluate was collected, evaporated to dryness, and the residue was reconstituted with 20 ~1 of acetonitrile. BSTFA (20 ~1 containing 1% TMCS) was added and the mixture was heated at 60C for 30 min. The derivatized extract was transferred to an autosampler vial and an aliquot (1 ~1) was analyzed by GUMS. 2.5. Contamination of hair with cocaine vapor Cocaine 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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W.L. Wang, E. J. Cone/Forensic Sci. ht. 70 (1995) 39-51

were composed of non-porous materials. Cocaine base was vaporized in a glass beaker open to the atmosphere at a temperature of 190C. The cocaine was located in 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 height of 5 ft. An additional bundle was suspended as a control sample in the adjoining room. Careful precautions were taken to prevent contamination during handling. Contamination experiments were conducted in which the amount of vaporized cocaine was varied (0, 5, 10, 25, and 100 mg) and the time of exposure (60 min) was constant, 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 under similar conditions as the contamination experiments with cut hair. The two individuals (one male and one female), who reported no active use of cocaine, were exposed to cocaine vapor (100 mg) for 60 min. Head hair samples were collected just prior to exposure, immediately after exposure, 1 day and 8 days later. No special precautions were taken regarding care of their hair and normal hygiene and grooming practices continued during this period.
2.6. Contamination of hair with aqueouscocaine

Drug free control hair samples (N = 3) were contaminated with aqueous solutions of 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 absorbent paper and air dried. A control sample was prepared in a similar manner with tap water substituted for drug solution. In a similar experiment, control hair was contaminated with an aqueous solution of cocaine hydrochloride and benzoylecgonine present in equal concentrations (0.01 mgml).
2.7. Hair treatment and wash experiments

Hair samples contaminated with cocaine vapor and aqueous cocaine (N = 3) were divided into approximately equal portions and used in various wash and hair treatment experiments. The effects of soap (shampoo) were evaluated by soaking contaminated hair samples in a 2% (w/v) aqueous solution of Suave@ shampoo overnight at room temperature. After soaking, the hair was removed, rinsed with warm water (10 x 10 ml) followed by a similar rinse with deionized water, then airdried. 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 treatment of hair contaminated with aqueous cocaine hydrochloride (0.05 mg/ml) with Perfecto@ Mild Alkaline Wave solution. The hair was soaked in a 25% (v/v) solution for 20 min at room temperature. After soaking, the hair was removed, rinsed with distilled water and dried at room temperature. 3. Results
3.1. Cocaine vapor contamination studies Concentration effects. Drug-free control hair (cut head hair) was contaminated

exposure for 60 min to cocaine vapor in varying amounts in an unventilated

by room.

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Table 1 Contamination of drug-free control hair by vaporization of cocaine base in a small unventilated rooma

w (m8)
0 5 10 25 50 100

Cocaine (ng/mg) Wash 0 8.8 12.5 20.5 23.1 28.5 Extract 0 2.5 2.3 4.0 3.7 2.2

Benzoylecgonine (ng/mg) Wash 0 0 0 0 0 0 Extract 0 0 0 0 0 0

aData represent results of single measurements of hair samples.

Substantial amounts of cocaine were deposited on hair from all doses (Table 1). As the amount of cocaine exposure increased, cocaine contamination appeared to plateau near the 25mg cocaine vapor dose with only minor increases thereafter. Analysis of wash and extract fractions indicated that the majority of cocaine was removed in the methanol ,wash fraction (78-93%). Benzoylecgonine and other cocaine metabolites were not detected. Time course of contamination. The duration of cocaine vapor exposure and the effect of room location on hair contamination were investigated in an experiment in which 100 mg of cocaine base was vaporized in an unventilated room. Hair samples were suspended in different comers of the room and removed after various exposure times, Cocaine was detectable within 5 min of exposure and increased in a linear manner over the 60-min period (Table 2). Anhydroecgonine methyl ester was detectable on hair within 15 min. No other cocaine analytes were detected. Different locations in the room produced only minor differences in amount of cocaine and anhydroecgonine methyl ester contamination. Effect of shampoocycles on contamination. Removal of cocaine from contaminated hair 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 cocaine and anhydroecgonine methyl ester (Table 3). Cocaine was subsequently found only in the extract fraction and was consistently < 1 ng/mg. No other drug analytes were detected in the wash or extract fractions. Human exposure studies. An experiment was performed in which two volunteers without 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 contaminated with cocaine and anhydroecgonine methyl ester. Approximately 80% of the cocaine was removed in the methanol washing step performed prior to extraction. Samples that were collected after the first day following the exposure session contained smaller amounts of cocaine. During the 8-day collection period, both staff members reported practicing normal hygienic procedures including periodic shampooing of their hair on a 24-h cycle (Staff #l) or 48-h cycle (Staff #2).

Table 2 Effect of time of exposure and sample location in exposure room on amount of contamination of drug-free control hair with cocaine vapor (100 mg)a Anhydroecgonine methyl ester (ng/mg) Extract Location #l 0 0 0.8 2.9 2.5 5.9 0 0 1.4 1.8 5.3 14.8 0 0 3.6 19.7 17.1 24.2 0 0 3.1 21.0 15.2 19.5 Location #2 Location #I Wash Location %2 Extract Location #l 0 0 0.4 3.4 2.2 4.1 Location #2 0 0 0.3 1.9 3.0 6.5

Length of exposure (mm)

Cocaine (np/mg)

Wash

Location #l

Location #2

0 5 15 30 45 60

0 0.6 1.4 15.0 23.4 28.7

0 1.0 8.3 18.3 26.8 35.1

aLocations #l and #2 were in opposite comers of the exposure room.

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Table 3 Cleansing Shampoo

41

effect cyclesa

of shampoo

on cocaine (ng/mg)

contaminated

hair (cocaine

vapor,

100 mg) methyl Extract ester (ngmg)

Cocaine Wash

Anhydroecgonine Wash

Extract

19.6

1 2 3
aEach cycle consisted shampoo (pH 7.0).

0 0 0
of an overnight

7.3 0.7 0.5 0.4

soak at room temperature

39.9 0 0 0
in 2% (w/v)

10.0 0.6 0.2 0.2

aqueous solution of Suave

3.2. Aqueous cocaine contamination Concentration effects and shampoocycles. Drug-free control hair samples (cut head

hair) were contaminated by soaking overnight at room temperature in aqueous solutions of cocaine hydrochloride (Table 5). Increasing concentrations of cocaine were absorbed with increasing dose, but proportional amounts of cocaine were not bound at the highest concentration compared to lower concentrations suggesting that binding capacity of hair for cocaine was limited. Analysis of wash and extract fractions indicated that 16-41% of the cocaine contaminant was removed in the methanol wash fraction. Traces of benzoylecgonine were detected following contamination with cocaine at 0.05-mg/ml and O.l-mg/ml concentrations. Shampoo soaking overnight at room temperature removed substantial amounts of cocaine, but detectable concentrations persisted through 10 shampoo treatment cycles. Benzoylecgonine contamination. Contamination experiments were conducted in which drug-free control hair samples (N = 3) were contaminated with a mixture of

Table 4 Cocaine contamination Time of measurement

of human Cocaine Wash

volunteers (ngmg) Extract

hair as a result

of exposure Anhydroecgonine Wash

to cocaine methyl Extract

vapor ester (ngmg)

Staff#l
Pre-exposure Post-exposure 1 Day 8 Days

0.4a 21.9 0.6

0.5

0 5.6 1.2 0.6

0

0 6.2 0 0
0

0
1.8

0 0

staffw2
Pre-exposure Post-exposure 1 Day 8 Days sTIris staff member 0 0

37.1 0.4
0

8.6 0.7 0.5

with other recent

3.8 0.7 0
experiments

3.3 0 0
involving vaporized cocaine.

had been associated

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Table 5 Contamination of drug-free control hair (100 mg) with aqueous solutions of cocaine hydrochloride and cleansing effects of shampoo Cocaine HCI bs/ml) 0.01 Shampoo cycle9 0 1 2 3 10 0 I 2 3 10 0 1 2 3 10 Cocaine (ng/mg) Wash 19.2 0.4 0 0 0 209.2 1.1 0.3 0.2 0.3 216.0 5.5 3.4 1.6 0.3 Extract 98.3 12.8 5.6 4.1 0.5 302.5 76.0 21.4 15.3 2.0 352.4 143.6 59.3 48.3 3.6 Benzoylecgonine (ng/mg) Wash 0 0 0 0 0 0.5 0 0 0 0 0.7 0 0 0 0 Extract 0 0 0 0 0 0.8 0 0 0 0 1.5 0.4 0 0 0

0.05

0.1

Qch cycle consisted of an overnight soak at room temperature in 2% (w/v) aqueous solution of Suavee shampoo (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 the hair samples. Shampoo soaking overnight at room temperature removed substantial amounts of cocaine and benzoylecgonine, but detectable concentrations of cocaine and benzoylecgonine persisted through 10 shampoo treatment cycles. Stability of cocaine in hair. An experiment was conducted with cocaine contaminated hair in which samples (N = 3) were treated with an alkaline hair treatment

Table 6 Contamination 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 shampoo Shampoo cyclesa Cocaine (ng/mg) Wash 0 1 2 3 10 6.2, 0.3, 0.2, 0.2, 0.1, 8.8 0.3 0.2 0.2 0.2 Extract 24.8, 25.9 2.5, 2.9 1.5, 2.0 1.5, 1.6 0.2, 0.5 Benzoylecgonine (ng/mg) Wash 0.3, 0.5 0, 0 0, 0.2 0, 0 0.2, 0 Extract 3.0, 3.3 0.3, 0.4 0.2, 0.2 0.1, 0.3 0, 0

Data 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 Suaves shampoo (pH 7.0).

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(pH, 8.9) to determine if benzoylecgonine and ecgonine methyl ester would be formed as a result of the hair treatment. Before treatment, cocaine and metabolite concentrations 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 as follows: cocaine, 77.2 ng/mg; benzoylecgonine, 0.7 ng/mg, and ecgonine methyl ester, 0.8 ng/mg. Only the cocaine concentration was affected by the treatment. Consequently, 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 period spanning several months of an individuals recent past makes it an attractive specimen for many applications. A positive or negative drug test can have major implications concerning an individual under investigation for illicit drug abuse. The validity of hair testing results depends upon the ability of the test to correctly identify parent drug and/or metabolite following active drug use. A test that fails to distinguish between active and passive drug exposure would lack validity to the extent and frequency that false positive results were reported because of passive drug exposure. In the present studies, hair samples that were externally exposed to cocaine vapor and to aqueous solutions of cocaine hydrochloride tested positive for cocaine immediately after exposure and continued to test positive at lower concentrations after numerous shampoo wash cycles. The degree of contamination was dependent upon the amount and length of exposure. When hair samples were exposed to cocaine vapor in an unventilated room, cocaine contamination continued to increase long after the cocaine base had been vaporized (Table 2). This suggested that hair could become contaminated from cocaine vapor (smoke) long after crack cocaine smoke had been generated. Also, equivalent concentrations of cocaine were deposited on hair in a manner that was independent of room location. This indicated that diffusion of cocaine vapor occurred rapidly throughout the room in approximately equal amounts. Obviously, the ventilation conditions of the area in which cocaine vapor was produced would be an important factor in determining the extent and duration of hair contamination. There were substantial amounts of cocaine contamination deposited in hair as a result of exposure to cocaine vapor (> 20 ng/mg) or aqueous solutions of cocaine hydrochloride (> 200 ng/mg). During the processing, extraction and analysis of these contaminated hair samples, it was noted that washing with methanol removed different amounts of cocaine from hair exposed to cocaine vapor versus hair exposed to aqueous cocaine. The majority (> 70%) of cocaine contamination was removed from cocaine vapor exposed hair by a brief methanol wash and these findings were consistent 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 methanol washing of aqueous cocaine exposed hair. These findings suggested that contamination of hair by aqueous cocaine results in greater drug penetration of the hair

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matrix. It was noted that the resulting contaminant from aqueous cocaine was far more resistant to removal by various washing procedures than hair exposed to cocaine vapor. Also, substantial amounts of cocaine persisted in hair through 10 cycles of soaking overnight in shampoo solution. Presumably, penetration of hair by aqueous cocaine was facilitated by the presence of water which is known to produce considerable swelling of hair [4]. Water molecules also bind to the polypeptide chains of keratin in place of internal hydrogen bonds, thus increasing the size of the hair strand and its plasticity. The positive charge on the cocaine molecule in aqueous solution (cocaine hydrochloride) also could have influenced the affinity of hair for cocaine. Analysis of hair samples that had been contaminated by cocaine vapor exposure revealed the presence of anhydroecgonine methyl ester. This analyte arose as a result of the thermal elimination of a molecule of benzoic acid from cocaine during heating. 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 for active cocaine use because of substantial deposition from the external environment during the production of cocaine vapor. Other potential biological markers for active cocaine use include cocaethylene [6], norcocaine and benzoylecgonine. Neither cocaethylene or norcocaine were identified in any of the current contamination experiments with cocaine, Consequently, their presence in hair would appear to be valid markers for active cocaine use as long as it can be assumed that environmental contamination with these compounds did not occur, i.e. they were not present in illicit cocaine. The presence of benzoylecgonine in hair has been suggested to be indicative of active cocaine use [7]. In the present studies, only traces of benzoylecgonine were detected in hair in experiments involving high concentrations of cocaine contamination (Table 4). Very careful optimization of all assay conditions was needed to eliminate artifactual production of benzoylecgonine. Without careful precautions, benzoylecgonine can be generated during the extraction process, particularly at elevated pH conditions, and false positive results would be generated. Despite the chemical lability of cocaine toward hydrolysis, cocaine sequestered in hair appeared to exhibit unusual stability. Even when cocaine contaminated hair was subjected to severe alkaline conditions (alkaline wave treatment, pH = 8.9), there was no evidence of conversion of cocaine to benzoylecgonine or ecgonine methyl ester. Hair can become contaminated with benzoylecgonine from the environment. Substantial concentrations of benzoylecgonine were absorbed when hair was deliberately exposed to aqueous solutions of cocaine mixed with benzoylecgonine (Table 6). Consequently, the usefulness of this analyte as a marker of active cocaine use 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 has occurred. Initially, after hair becomes contaminated, a large portion of the cocaine contamination can be removed easily by bathing and shampooing. If freshly contaminated hair is analyzed, a large portion of the cocaine contaminant would be found in the wash fraction (assuming this step is employed in the assay). The

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presence of high concentrations of cocaine or benzoylecgonine in the wash fraction versus the hair extract would be highly indicative of very recent environmental contamination. Consequently, recent contamination (prior to bathing) should be readily detectable by evaluation of wash/extract ratios for the analytes of interest. After bathing or shampooing, the pattern of environmental hair contamination becomes less clear. As the bulk of surface contamination is removed through normal hygienic cleansing procedures, cocaine that has penetrated the hair matrix remains and appears primarily in the extract fraction when hair is analyzed. At this point, little contamination is removed in the wash fraction and contamination is no longer evident by evaluation of the wash/extract ratio. Analysis of environmentally contaminated hair for cocaine that has been subjected to several bathing/shampoo cycles generally produces results that are indistinguishable from patterns obtained from active cocaine users.
Acknowledgment

The authors would like to thank W.D. Darwin for technical assistance.
References
[l] V. Valkovic, In Human Hair, Volume I: Fundamentals and Methods for Measurement of Elemental Composition, CRC Press, Boca Raton, FL, 1988, pp. 39-44. [2] MM Breuer, The binding of small molecules and polymers to keratin and their effects on the physicochemical and surface properties of hair fibers. In Orfanos, Montagna and Stuttgen (eds.), Hair Research, Springer-Verlag, Berlin, 1981, pp. 96-l IS. [3] G. Chittleborough and B.J. Steel, Is human hair a dosimeter for endogenous zinc and other trace elements. Sci. Total Environ., 15 (1980) 25-35. [4] E Bums, A. Fookson and M.M. Breuer, Water in Polymers, A.C.S. Symposium Series, No. 127, 1980, p. 309. [5] P. Jacob, III, R.T. Jones, N.L. Benowitx, A.T. Shulgin, E.R. Lewis and B.A. Elias-Baker, Cocaine smokers excrete a pyrolysis product, anhydroecgonine methyl ester. Clin. Toxicol., 28 (1990) 121-125. [6] E.J. Cone, D. Yousefnejad, W.D. Darwin and T. Maguire, Testing human hair for drugs of abuse. II. Identification of unique cocaine metabolites in hair of drug abusers and evaluation of decontamination procedures. J. Anal. Toxicol., 15 (1991) 250-255. [7] G. Karen, J. Klein, R. Forman and K. Graham, Hair analysis of cocaine: differentiation between systemic exposure and external contamination. J. C/in. Pharmacol., 31 (1992) 671-675.