Anal Bioanal Chem DOI 10.1007/s00216-016-9354-x

RESEARCH PAPER

Analysis of cocaine and metabolites in hair: validation and application of measurement of hydroxycocaine metabolites as evidence of cocaine ingestion Michael Schaffer 1 & Chen-Chih Cheng 1 & Oscar Chao 1 & Virginia Hill 1 & Paul Matsui 1

Received: 23 September 2015 / Revised: 4 January 2016 / Accepted: 21 January 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract An LC/MS/MS method to identify and quantitate in hair the minor metabolites of cocaine—meta-, para-, and ortho-hydroxy cocaine—was developed and validated. Analysis was performed on a triple quadrupole ABSciex API 3000 MS equipped with an atmospheric pressure ionization source via an IonSpray (ESI). For LC, a series 200 micro binary pump with a Perkin Elmer Model 200 autosampler was used. The limit of detection (LOD) and limit of quantification (LOQ) were 0.02 ng/10 mg hair, with linearity from 0.02 to 10 ng/10 mg hair. Concentrations of the para isomer in extensively washed hair samples were in the range of 1–2 % of the cocaine in the sample, while the concentrations of the ortho form were considerably less. The method was used to analyze large numbers of samples from two populations: workplace and criminal justice. In vitro experiments to determine if deodorants or peroxide-containing cosmetic treatments could result in the presence of these metabolites in hair showed that this does not occur with extensively washed hair. Presence of hydroxycocaines, when detected after aggressive washing of the hair samples, provides a valuable additional indicator of ingestion of cocaine rather than mere environmental exposure.

Parts of this work were presented at the 20th Meeting of the Society of Hair Testing (SoHT). * Virginia Hill [email protected]

1

Psychemedics Corporation, 5832 Uplander Way, Culver City, CA 90230, USA

Keywords Cocaine . Hydroxycocaine . Hair . Extended washing

Introduction In 2004, SAMHSA published proposed guidelines for federal workplace drug testing that included guidelines for hair testing [1]. At that time, the proposed guidelines for cocaine (COC) testing in hair recommended a 5 ng/10 mg hair cutoff, with benzoylecgonine (BE) ≥ 0.5 ng/10 mg hair and in a ratio to COC of ≥0.05, or with ≥0.5 ng/10 mg hair of cocaethylene (CE) or norcocaine (NOR). No hair washing or decontamination requirements were included. A study by Stout et al. in 2006 showed that various washing methods did not remove all COC or even all CE in samples experimentally contaminated with pharmaceutical cocaine containing CE [2]. However, extended aqueous washing and application of a wash criterion, developed by Baumgartner and Hill [3–5], and further demonstrated and described by Cairns et al. [6, 7], Schaffer et al. [8], and Hill et al. [9] was shown to identify the samples as contaminated rather than positive on the basis of the wash criterion and COC and BE criteria. Nonetheless, controversy ensued over the possible (although rare) presence and likely levels of CE and NOR in street cocaine that would invalidate their use as definitive indicators of ingestion [2]. Because washing methods were not included in the guidelines, but only the above metabolite criteria, analysis of cocaine in hair using only the guideline requirements has been criticized as unsafe insofar as the possibility of contamination contributing to a positive result was not ruled out with certainty. Although the reliability of the wash criterion was validated in the Stout et al. study and since then in further studies by that group [10], this laboratory proceeded to develop and validate methods to identify additional metabolites, namely the three

M. Schaffer et al.

isomers, para-hydroxycocaine (p-OH-COC), metahydroxycocaine (m-OH-COC), and ortho-hydroxycocaine (o-OH-COC), that could provide definitive indication of cocaine ingestion. The p-, m-, and o-OH-COC or OH-BE isomers, formed by hepatic microsomal oxidative metabolism of cocaine, have been identified in urine, blood, oral fluid, and meconium [11–22]. A method to analyze the OH-COC isomers in hair was developed by this laboratory [23] and also by Morris-Kukoski et al. [24] and Franz et al. [25]. The present report demonstrates the levels of the OH-COC metabolites in hair and the effects of the extended wash methods on the p-, m-, and o-OH-COC findings. The possibility of any of the compounds being produced in vitro by hair cosmetic procedures or deodorants is investigated. We also investigated whether additional certainty of ingestion is gained by determining the relative levels, or ratios, of the three isomers of hydroxycocaine to one another.

Materials and methods Chemicals and reagents Reagents used in the washing of hair samples, obtained from Sigma-Aldrich, included bovine serum albumin (BSA) (Product A8022) and monobasic sodium phosphate (P5655). Reagents for digestion of hair, obtained from Sigma-Aldrich, included Bis-Tris (B9754), proteinase K (P6556), dithiothreitol (D0632), and sodium cholate (C1254). The solvents methylene chloride, isopropanol, hexane, and ethyl acetate were HPLC grade and were Burdick and Jackson brand. Ammonium hydroxide (ACS grade) was Macron brand. Water for reagents was deionized and monitored to be at least 18 megohm-cm. Cocaine (COC), cocaine-d3 (COC-d3), benzoylecgonine (BZE), BZE-d3, cocaethylene (CE), CE-d3, norcocaine (NCOC), and NCOC-d3 were obtained from Cerilliant. Para-OH-COC, p-OH-COC-d3, m-OH-COC, m-OH-COCd3, o-OH-COC, and o-OH-COC-d3 were obtained from ElSohly Laboratories. Dimethylformamide dipropyl acetal was obtained from Sigma-Aldrich. Specimens The workplace and criminal justice cocaine-positive samples were hair samples from prior testing stored in cardboard envelopes at room temperature. Sample Preparation: washing and Digestion Samples identified as presumptive positive for COC in an FDA-cleared screening assay are reweighed for confirmation. Approximately 12 mg of hair is placed in a 12 × 75 mm polystyrene test tube for extended washing prior to digestion. For the wash procedure, 2 mL of dry isopropanol is added to the

test tube, which is then placed in a 37 °C waterbath with shaking (120 oscillations/min). After 15 min, this wash is removed and discarded, and 2 mL of 0.01 M PO4 buffer, pH 6, containing 0.1 % BSA is added to the tube which is placed in the 37 °C waterbath with shaking for 30 min. This is repeated twice more. After the isopropanol and three 30-min washes, 2 mL of 0.01 M PO4 buffer containing 0.1 % BSA is added to the tube and a 60-min wash performed at 37 °C with shaking; this is repeated once more. The wash medium from the final 1-hour wash is saved for analysis to determine the wash criterion, as follows: The amount of COC (per 10 mg of hair) in the last wash is multiplied by 5, and this value is subtracted from the value for cocaine in the hair, when it has been determined. If the result is less than 5 ng/10 mg hair (the cutoff for a positive cocaine result), this suggests that the hair may be contaminated and the hair result at least partly reflects contamination rather than drug inside the hair due to ingestion. The wash criterion, a mathematical extension of the last wash for another 5 h, is deliberately an overestimate of the amount of drug that would be removed by actually continuing to wash for another 5 h. Excessively damaged (porous) hair can also result in a failed wash criterion. These two causes can be distinguished by integrity testing and alternate wash procedures [9]. Except where stated otherwise, all of the samples in this report were washed by the above described extended wash procedure. If a sample is determined to be contaminated rather than porous, the result is reported as invalid due to contamination. After washing, 1 mL/10 mg of hair of a digestion solution is added to the tubes containing the hair samples. The digestion solution consists of 0.1 M BIS-Tris buffer with 0.2 % sodium cholate, pH 5.5, containing 1.2 % dithiothreitol and 2 units of proteinase K per milliliter. The digestion of the hair is for 8 h in a 37 °C waterbath with shaking to recover the cocaine analytes from the hair for MS analysis. All standard and control materials were spiked in negative hair. Extraction of cocaine and metabolites from the hair digest One milliliter of centrifuged digested hair is added to each conditioned SPE column preconditioned as per the manufacturer’s recommendations. The columns are Biotage, ISOLUTE (130 mg, HCX) mixed-mode sorbent. Six milliliters of deionized water (DI), followed by 3 ml of 0.1 N HCl, followed by 9 ml of methanol is added to each column. After a drying period, samples are eluted with 2 ml of methylene chloride/isopropanol/ammonium hydroxide (80:20:2), followed by evaporation of the eluate and bringing up in 150 μL of methylene chloride. Twenty-five microliters of N, N-dimethylformamide dipropyl acetal is added, with heating at 120 °C for 5 min to effect the derivatization of benzoylecgonine, to ensure separation of norcocaine from the benzoylecgonine. Sulfuric acid (0.1 N) is added as well

Hydroxycocaines in hair

as hexane/ethyl acetate (9:1). The sample is then made basic, and the hexane/ethyl acetate is evaporated. The residue is brought up in 50 μL of the mobile phase for the LC/MS/MS analysis. A 10-20-μL sample volume is injected into the LC/ MS/MS. Instrumentation Analysis for OH-COC metabolites was performed on an Applied Biosystems API 3000 (Thornhill, On, Canada) liquid chromatographic-tandem mass spectrometric (LC-MS-MS) system. The OH-COC metabolites were analyzed with a separate analytical run from the analysis of COC, BE, CE, and NOR. For LC, binary pumps LC-20AD (Shimazu) equipped with degasser DGU-20A were used. The instrument was operated in multiple ion monitoring (MRM) mode with positive electrospray ionization mode (ESI). Chromatography separations were performed using HPLC column Thermofisher Scientific BETASIL C8 (2.1 × 50 mm, 5-μm particle size). The LC flow was gradient at 0.300 ml/ min with HPLC grade acetonitrile and water (1:1) with 0.1 % formic acid as mobile phase. The MS source desolvation temperature was 350 °C. Nitrogen gas was also used as collision gas along with optimized voltages to monitor all masses. The instrument was operated under unit resolution for both Q1 and Q3 with a peak width at half height of 0.7 amu. The molecular weight of protonated hydroxymetabolites, m/z 320, was the same for p-, m-, and o-OH-COC. The isotopically labeled internal standards were measured at m/z 323. The transition measured in the multiple reaction for p-, m-, and o-OH-COC was m/z 320 ≫ m/z 182 for parent compound ion and product ion, and m/z 323 ≫ m/z 185 for isotopically labeled internal standard parent compound ion and product ion, respectively. Analyst software was used for all calculations, after calibration of the instrument with a single cutoff calibrator. The concentration of the calibrator was set at 0.04 ng/10 mg hair. Open controls were negative and at 50 and 125 % of cutoff. Analysis time was 8 min. The retention times of chromatography for the three isomers were 1.55, 1.77, and 2.51 min, respectively, for p-, m-, and o-OH-COC. A typical chromatogram is shown in Fig. 1. Method validation The following parameters were included in the method validation: linearity, accuracy (bias), intra-assay and interassay precision, lower limit of quantification (LLOQ), upper limit of quantification (ULOQ), limit of detection (LOD), carryover, and uncertainty. Intra-assay precision and linearity were determined by analyzing five duplicates of the concentrations 0.01 through 10 ng/10 mg hair spiked into negative hair; the data is shown in Table 1 for 0.01–0.06 ng/10 mg hair and

10 ng/10 mg hair for the three isomers. The LLOQ was 0.02 ng/10 mg hair, the LOD was 0.01 ng/10 mg hair, and the upper limit of linearity (ULOL) was 10 ng/10 mg hair (Table 1). The carryover limit was set at 10 ng/10 mg hair, after determining that a negative sample following the ULOL contained no detectible analytes. Interassay precision was determined with five runs of five replicates each of the concentrations 0.01 through 10 ng/10 mg hair spiked into negative hair; the data is shown in Table 1 for 0.02, 0.04, and 0.06 ng/ 10 mg hair. The uncertainty of measurement at the cutoff, determined from the %CV of 15 determinations, multiplied by 2, was ±11.6, ±9.3, and ±19.6 %, for p-OH-COC, m-OHCOC, and o-OH-COC, respectively. Reproducibility of OH-COC analytic results in the same hair samples was studied by performing repeat analyses of 10 samples for p-OH-COC, m-OH-COC, and o-OH-COC. The mean percent differences of the duplicate analyses for p-OH-COC, m-OH-COC, and o-OH-COC were 5.5, 1.3, and 6.5, respectively, with corresponding SDs of 18.4, 13.4, and 20.1. The mean percent differences for the ratios p:o, p:m, and m:o were 20.5, 17.7, and 15.1, respectively, with corresponding SDs of 12.9, 13.5, and 14.2.

In vitro effects of peroxide and deodorants on hydroxycocaines One of the most common hair cosmetic treatments worldwide is coloring, whether dyeing or bleaching, both of which employ hydrogen peroxide. For this reason, we studied the effects of peroxide on in vitro COC-contaminated hair and also on COC users’ cocaine-positive hair. The peroxide used for the experiments was ALVA brand 40-volume (12 %) white crème developer. For the in vitro contamination experiment, we first permed previously determined negative hair so that the hair would be porous and absorb large amounts of cocaine [8, 9]. The perming product was Ogilvie Precisely Right, used according to package instructions. After perming, the hair was dried before soaking in 10 μg/mL cocaine for 2 h. After soaking, the samples were rinsed with water and dried, and then submerged in 40-volume (12 %) hydrogen peroxide for 1 h at room temperature, after which they were rinsed with water to remove the peroxide, dried, reweighed, and analyzed with and without washing. In another study, to test effects of hydrogen peroxide on cocaine-positive hair samples, the samples were first washed by the wash method described in BMethod validation,^ then dried. The dried hair was divided into two aliquots, and one of these was analyzed at that stage, and the other was submerged in 40-volume (12 %) peroxide for 1 h, rinsed with deionized water, dried, and reweighed for extended washing and confirmation.

M. Schaffer et al.

Fig. 1 Chromatogram of p-OH-COC, m-OH-COC, and o-OH-COC at the cutoff of 0.04 ng/10 mg hair

In workplace testing, a frequent substitute for head hair is underarm hair, which is likely to be exposed to deodorants or anti-perspirants. To test for effects of these products on hydroxycocaines, the product brands tested were Gillette Triple Protection System containing aluminum zirconium trichlorohydrex gly (19 %), Right Guard Sport Fresh Solid containing aluminum zirconium pentachlorohydrex gly (17.8 %), and Ultra Clear Degree Extreme Blast containing aluminum zirconium tetrachlorohydrex gly (14.8 %). Previously determined COC-positive hair was washed before treatments with the deodorants. The washed hair was divided into four aliquots of at least 20 mg each. One aliquot of each hair was not treated, and each of the others was smeared with one of the three deodorants, making sure of full contact with

all the strands of the sample, and then left at ambient conditions overnight. Samples were then rinsed with deionized water, dried, and weighed for washing, digestion, extraction, and analysis by LC/MS/MS.

Results Studies of potential peroxide effects on hydroxycocaine levels in cocaine-positive hair from users Results of treating 20 COC-positive head hair samples from COC users with hydrogen peroxide are shown in Table 2. Levels of OH-COC isomers in COC-positive samples

Hydroxycocaines in hair Table 1

Validation of hydroxycocaine in hair assay by LC/MS/MS

Limit of detection (LOD), limit of quantitation (LLOQ), and intra-assay precision m-OH-Cocaine o-OH-Cocaine 0.01 ng/10 mg hair (LOD)

p-OH-Cocaine

Average (n = 5) SD

0.0098 0.00045

0.0108 0.00179

0.0126 0.00055

%CV

4.56

16.56

4.35

Average (n = 5)

0.02 ng/10 mg hair (LLOQ) 0.02 0.022

0.021

SD

0.00084

0.000894

%CV

4.14 10.14 0.04 ng/10 mg hair

0.0021

4.18

Average (n = 5)

0.041

0.04

0.042

SD %CV

0.0012 2.99

0.0019 4.95

0.0011 2.69

Average (n = 5) SD

0.06 ng/10 mg hair 0.06 0.062 0.0019 0.0035

0.0596 0.0023

%CV 3.17 5.65 Upper limit of linearity (ULOL) 10 ng/10 mg hair Average (n = 5) 9.715 9.781 SD 0.171 0.352 %CV 1.76 3.6

3.86

8.845 0.182 2.05

Interassay precision Average (n = 25 in five assays) SD %CV Average (n = 25 in five assays) SD %CV Average (n = 25 in five assays) SD %CV Average (n = 25 in five assays)

0.02 ng/10 mg hair 0.02 0.02

0.02

0.0015 0.0021 7.19 10.06 0.04/10 mg hair 0.04 0.04

0.0017 7.61

0.0019 0.0039 4.66 9.79 0.06 ng/10 mg hair

0.0024 5.84

0.06

0.06

0.06

0.0033 5.34 0.02

0.0068 11.23 0.02

0.0047 8.00 0.02

0.04

containing 49–557 ng cocaine/10 mg hair are shown on the left side of the table. The amounts of p-, m-, and o-OH-COC in these samples ranged from 0.28 to 6.33, 0.42 to 5.65, and 0.1 to 0.34 ng/10 mg hair, respectively. The ranges for the OHCOC as percent of the cocaine concentrations in the hair samples were 0.22–7.12, 0.25–4.53, and 0.04–0.3 for p-, m-, and o-OH-COC isomers in hair, respectively. These percentages reveal the great sensitivity required for the measurement of

OH-COC in hair, particularly o-OH-COC which may not be detected in samples containing lower levels of COC. In evaluating the OH-COC results, we have studied the utility of determining ratios of one isomer relative to another, namely p:o, p:m, and m:o, anticipating that ratios might help to distinguish in vitro formation, if it occurs, from metabolic deposition. In this group of 20 samples with high levels of COC, all samples contained all three OH-COC isomers. Before treatment with peroxide, the ratios for p:o ranged from 1.47 to 27.5, with an average of 9.82 and a median of 9.25; for p:m, the range was 0.32 to 2.41 with average 1.08 and median 0.85; and for m:o, the ratios ranged from 2.19 to 63.56, with an average of 11.02 and a median of 7.87. All but two samples had p:o ratios of 2 or more, and all had m:o ratios greater than 2. Thus, no samples would have been without a ratio of ≥2 for at least one of the ratios p:o or m:o. The right side of Table 2 shows the results of treating COC positives with hydrogen peroxide, followed by application of the extended wash procedure to observe the effects of peroxide on the levels of OH-COC in hair— either generation of these compounds from cosmetic treatments or loss of them due to the effects of cosmetic treatments that damage the hair. Some losses of analytes may be expected from the increased porosity resulting from the peroxide treatment as well as from the additional extended washing of the hair which, after the peroxide, was more porous than the original hair samples that were subjected to only one wash procedure Most samples showed some loss of COC (average of 9 % loss) and also of OH-COC. Both the p- and m-OH-COC concentrations showed loss (average 26 and 27 %, respectively) from the peroxide and washing process, while the o-OH-COC did not. These different responses to peroxide of p- and m- vs o- are reflected in the ratios after peroxide treatment: both the p:o and the m:o ratios were decreased (27.7 and 22.5 %, respectively), while the p:m ratio stayed nearly the same since these two isomers were similarly affected by the process. The p:o ratio of one sample (#8) fell below 2, and two m:o ratios (#1, 8) fell below 2 after treatment. These data show loss of OH-COC isomers and decrease of ratios after peroxide and extended washing rather than evidence of in vitro production of any of the OH-COC metabolites due to exposure of washed COC-positive hair samples to peroxide. Use of ratios ≥2 to indicate ingestion would thus err on the side of protecting a subject from a false-positive result, even though the effects of hydrogen peroxide on these 20 subjects do not suggest that this is likely. The wide ranges of both concentrations and ratios among the 20 samples before treatment, much larger than the duplication error presented in BMethod validation,^ demonstrate the large variations among authentic samples and may suggest a wide range in the metabolic response to cocaine ingestion.

113.6 84.9 49.2 122.4 83.5

147.3 123.4 197.7 66.2 89.9

146.4 88.8 224.6

156.9 112.6 168.2 557.7

263.1 151.7

109.6 122.9

3 4 5 6 7

8 9 10 11 12

13

16 17 18 19

20 Average

SD Median

14 15

90.9

145.9

2

1.59 (1.50) 1.70 (1.24)

3.55 (1.34) 2.04 (1.54)

3.78 (2.40) 2.25 (1.99) 3.84 (2.28) 4.06 (0.72)

2.16 (1.47) 6.33 (7.12) 1.18 (0.52)

0.33 (0.22) 1.80 (1.45) 2.35 (1.18) 0.61 (0.92) 0.57 (0.64)

0.92 (0.81) 2.19 (2.58) 1.19 (2.42) 1.60 (1.30) 0.28 (0.33)

1.42 (0.97)

0.28 (0.31)

1.58 (1.25) 1.92 (1.19)

2.08 (0.79) 2.13 (1.64)

1.57 (0.47) 4.22 (3.74) 1.85 (1.10) 2.22 (0.39)

2.83 (1.93) 3.18 (03.58) 2.91 (1.29)

0.37 (0.25) 5.59 (4.53) 5.65 (2.85) 1.00 (1.51) 0.63 (0.70)

0.95 (0.84) 2.72 (3.20) 0.73 (1.49) 1.99 (0.47) 0.88 (1.05)

0.87 (0.60)

0.42 (0.47)

ng/10 mg hair (% of COC)

0.057 (0.06) 0.20 (0.15)

0.22 (0.08) 0.20 (0.15)

0.21 (0.13) 0.21 (0.18) 0.23 (0.14) 0.34 (0.06)

0.21 (0.14) 0.23 (0.26) 0.10 (0.04)

0.15 (0.10) 0.18 (0.15) 0.31 (0.16) 0.13 (0.21) 0.13 (0.15)

0.20 (0.17) 0.16 (0.19) 0.14 (0.30) 0.21 (0.17) 0.17 (0.20)

0.20 (0.13)

0.19 (0.21)

o-OH

6.44 9.25

16.14 9.82

18.00 10.71 16.70 11.94

10.29 27.52 11.80

2.20 10.00 7.58 4.69 4.38

4.60 13.69 8.50 7.62 1.65

7.10

1.47

p:o

Ratios

0.65 0.85

1.71 1.08

2.41 0.53 2.08 1.83

0.76 1.99 0.41

0.89 0.32 0.42 0.61 0.90

0.97 0.81 1.63 0.80 0.32

1.63

0.67

p:m

8.30 7.87

9.45 11.02

7.48 20.10 8.04 6.53

13.48 13.83 29.10

2.47 31.06 18.23 7.69 4.85

4.75 17.0 5.21 9.48 5.18

4.35

2.19

m:o

m-OH

105.1 99.7

415.2 138.4

143.5 98.1 104.8 406

125.4 77.7 223.6

118.1 90.6 238.4 58.7 75.9

95.8 77.3 57.9 61.6 101.2

135.2

63.6

1.33 (1.14) 1.17 (0.92)

5.22 (1.26) 1.49 (1.21)

2.39 (1.67) 1.33 (1.36) 1.79 (1.71) 2.77 (0.68)

1.53 (1.22) 4.33 (5.57) 1.04 (0.47)

0.18 (0.15) 1.18 (1.30) 1.73 (0.73) 0.56 (0.95) 0.52 (0.69)

0.54 (0.56) 1.56 (2.02) 0.93 (1.61) 0.55 (0.89) 0.47 (0.46)

1.16 (0.86)

0.13 (0.20)

1.08 (0.86) 1.34 (1.11)

2.83 (0.68) 1.57 (1.34)

1.37 (0.95) 2.35 (2.40) 1.04 (0.99) 1.54 (0.38)

1.83 (1.46) 1.87 (2.41) 2.47 (1.10)

0.25 (0.21) 3.11 (3.43) 4.59 (1.93) 1.32 (2.25) 0.73 (0.96)

0.75 (0.78) 1.91 (2.47) 0.65 (1.12) 0.77 (1.25) 1.17 (1.16)

0.73 (0.54)

0.21 (0.33)

ng/10 mg hair (% of COC)

p-OH

COC

m-OH

COC

p-OH

After peroxide treatment

Before peroxide treatment

Para-, meta-, and ortho-hydroxycocaine in cocaine-positive hair samples before and after hydrogen peroxide exposure

1

Table 2

0.11 (0.06) 0.17 (0.16)

0.53 (0.13) 0.21 (0.17)

0.23 (0.16) 0.16 (0.16) 0.13 (0.12) 0.33 (0.08)

0.17 (0.14) 0.19 (0.24) 0.15 (0.07)

0.13 (0.11) 0.24 (0.26) 0.39 (0.16) 0.15 (0.26) 0.17 (0.22)

0.23 (0.24) 0.14 (0.18) 0.12 (0.21) 0.12 (0.19) 0.33 (0.33)

0.17 (0.13)

0.11 (0.17)

o-OH

5.13 6.87

9.85 7.11

10.3 8.31 13.7 8.39

9.00 22.7 6.93

1.38 4.92 4.44 3.73 3.06

2.35 11.1 7.75 4.58 1.42

6.82

1.18

p:o

Ratios

0.61 0.72

1.84 1.00

1.74 0.57 1.72 1.80

0.84 2.32 0.42

0.72 0.38 0.38 0.42 0.71

0.72 0.82 1.43 0.71 0.40

1.59

0.62

p:m

4.43 6.18

5.34 7.69

5.96 14.69 8.00 4.67

10.76 9.84 16.47

1.92 12.96 11.77 8.80 4.29

3.26 13.64 5.42 6.42 3.55

4.29

1.91

m:o

−2.6 −25.7 1.4 −12.2 −11.2 26.3 −19.3 17.8 −9.4 −30.5 −21.3 9.5 16.3 3.8 −27.5 6.1 −17.1 −1.6 8.1 −1.81

−3.9 −49.0 −18.6 −8.8 −39.8 −13.5 −37.1 −50.8 −41.5 −20.4 −30.2 −12.5 −17.2 −41.2 −42.3 −22.4 −17.5 −29.7 −39.0 −27.7

16.07 −4.85

−7.1

−19.8

14.0 −26.0

p:m

p:o

% Change in ratios

17.15 −24.5

−43.5 −22.5

−20.3 −26.9 −0.5 −28.5

−20.2 −28.8 −43.4

−22.2 −58.3 −35.4 14.4 −11.5

−31.4 −19.8 3.9 −32.3 −31.4

−1.4

−13.6

m:o

M. Schaffer et al.

Hydroxycocaines in hair

Studies of potential peroxide effects on hydroxycocaine levels in hair contaminated with cocaine To further study the possibility of in vitro production of hydroxycocaines, four samples negative for COC were permed to make them highly porous. As described in BMaterials and methods,^ the permed samples were contaminated with COC and then the dried hair was exposed to peroxide. The samples were analyzed for COC and all metabolites with and without the wash procedure. Results for the unwashed samples, shown in Table 3, demonstrate that the unwashed samples had absorbed large amounts of COC, from 419.2 to 1417.6 ng/10 mg hair. It is known that pharmaceutical COC preparations contain CE, and this is reflected in the presence of CE in these samples before washing, at levels of about 1.5 % of the COC. BE was present at about 0.6 % of the COC, and NOR at .07–0.3 % of the COC. Hydroxycocaines were detected in these unwashed samples after the peroxide exposure, at levels from 0.29 to 1.99 ng/10 mg hair; the three OH-COC isomers formed in similar amounts and did not show the low levels of o-OH-COC relative to p-OH-COC and m-OH-COC that are seen in COC users’ hair. None of the three ratios were above 2.0. This experiment demonstrates that although cosmetic agents such as bleach may cause formation of OH-COC analytes from contaminating COC that is accessible to external chemicals, the OH-COC isomers were completely removed by the extended wash. Further, the resulting ratios p:o and m:o were less than 2 even before

Table 3

washing, suggesting possible utility of these ratios in the application of OH-COC metabolites in hair as indicative of ingestion rather than in vitro formation. After applying the extended wash procedure to these COCcontaminated and peroxide-treated samples, only COC remained present in any of the samples, at concentrations of 0.5, 0.2, 12.6, and 6.1. The wash criterion indicated that the two samples above the cutoff of 5 ng/10 mg hair were contaminated rather than positive samples (Table 3). With external COC contamination on the hair readily available to the action of peroxide (Table 3), OH-COC isomers were produced in fairly equal concentrations that do not result in the higher ratios of p:o and m:o seen in most of the COC-positive samples from identified users. Finally, all of the in vitro formed OH-COC isomers were completely removed by the washing procedure, demonstrating that OH-COC analysis must be performed only after extended washing. Effects of deodorants on hydroxycocaine levels In workplace testing, many of the submitted samples are underarm samples that may have been exposed to an array of chemicals in deodorants. It was of concern whether these chemicals might either produce hydroxycocaines of nonmetabolic origin or destroy hydroxycocaines already deposited in the hair. Results of exposing four cocaine-positive hair samples to three different brands of underarm deodorant are shown in Tables 4 and 5. There were some changes in analyte

Cocaine contamination of hair followed by hydrogen peroxide exposure

Unwashed hair samples Sample no.

ng/10 mg hair COC

1 419.2 2 442.1 3 1417.6 4 963.2 Mean 1 SD Median Washed hair samples* Sample no. 1 0.5 2 0.2 3 12.6a 4 6.1a

Ratios

BE

CE

NOR

p-OH

% p-OH

m-OH

% m-OH

o-OH

% o-OH

p:o

p:m

m:o

2.5 2.5 7.7 4.4

6.5 6.4 18.6 14.2

1.4 0.7 1.4 0.7

0.29 1 0.94 0.95 0.79 0.34 0.94

0.07 0.23 0.07 0.1 0.11 0.08 0.08

0.56 1.99 1.29 1.44 1.32 0.59 1.37

0.13 0.45 0.09 0.15 0.21 0.16 0.14

0.34 1.15 0.68 1.04 0.8 0.37 0.86

0.08 0.26 0.05 0.11 0.12 0.09 0.09

0.84 0.86 1.38 0.91 1 0.26 0.89

0.51 0.5 0.73 0.66 0.6 0.11 0.59

1.65 1.73 1.9 1.38 1.51 0.4 1.65

NDb ND ND ND

ND ND ND ND

ND ND ND ND

ND ND ND ND

ND ND ND ND

ND ND ND ND

The last washes for no. 3 and no. 4 samples were 23.9 and 11.6 ng/10 mg hair, respectively. The COC wash criterion results for these were −106.9 and −51.8, respectively, indicating that these are contaminated samples

a

b

ND not detected

106.2 119.5 129.8 90.4 106.7

100.6 455.3 418.2

398.4 479.5

Right Guard Degree None Gillette Right Guard

Degree None Gillette

Right Guard Degree

2

3

b

a

125.4 119 98.8

110.1

None Gillette Right Guard

Degree

87.8

94.9 78.8

87.5 105.3

91.9

77.5

69.6 82.2

97.6 109.8

126.0

35.6

39.2 38.5 32

60.6 72.1

18.3 83.2 71.7

20.9 25 20 15.6 18.8

25.7 34.6

90.8

98.2 81.6

72.8 86.7

86.2

91.5

78.0 94.0

81.3 97.3

134.6

Percent of analyte in the sample with no deodorant applied

Nanograms/10 mg hair

4

108.8 137.1

None Gillette

% of none

0.5

0.8 0.7 0.5

ND ND

4 ND ND

ND ND 4.3 3.8 3.9

ND ND

ng

CE

1

ng

nga % of noneb

BZE

Treatment

Hair sample

Cocaine

Effect of deodorants on cocaine and metabolites in hair

Table 4

62.5

87.5 62.5

NA NA

NA

NA

NA NA

NA NA

NA

% of none

2.6

3.6 3.3 2.3

13.8 15.9

2.7 14.9 14.5

0.8 1 3.1 2.2 2.7

0.8 1

ng

NOR

72.2

91.7 63.9

92.6 106.7

97.3

87.1

71.0 87.1

100.0 125.0

125.0

% of none

3.67

3.87 3.68 3.18

6.05 6.92

0.42 6.74 6.55

0.37 0.4 0.57 0.36 0.43

0.42 0.45

ng

94.8

95.1 82.2

89.8 102.7

97.2

73.7

63.2 75.4

88.1 95.2

107.1

% of none

p-OH-COC

0.95

1.13 1.08 0.92

2.71 3.25

0.63 3.26 2.91

0.26 0.3 0.92 0.56 0.66

0.25 0.31

ng

84.1

95.6 81.4

83.1 99.7

89.3

68.5

60.9 71.7

104.0 120.0

124.0

% of none

m-OH-COC

0.17

0.27 0.21 0.14

0.53 0.61

0.17 0.61 0.53

0.11 0.12 0.22 0.16 0.17

0.11 0.13

ng

63.0

77.8 51.9

86.9 100.0

86.9

77.3

72.7 77.3

100.0 109.1

118.2

% of none

o-OH-COC

21.59

14.33 17.52 22.71

11.42 11.34

2.47 11.05 12.36

3.36 3.33 2.59 2.25 2.53

3.82 3.46

ng

p:o ratio

150.7

122.3 158.5

103.3 102.6

111.9

95.4

86.9 97.7

88.0 87.2

90.6

% of none

3.86

3.42 3.41 3.46

2.23 2.13

0.67 2.07 2.25

1.42 1.33 0.62 0.64 0.65

1.68 1.45

ng

112.9

99.7 101.2

107.7 102.9

108.7

108.1

103.2 104.8

84.5 79.2

86.3

% of none

p:m ratio

5.59

4.19 5.14 6.57

5.11 5.33

3.71 5.34 5.49

2.36 2.5 4.18 3.5 3.88

2.27 2.38

ng

133.4

122.7 156.8

95.7 99.8

102.8

88.8

83.7 92.8

104.0 110.1

104.8

% of none

m:o ratio

M. Schaffer et al.

Hydroxycocaines in hair Table 5

Summary of effects of deodorant treatments

Analytes after deodorant treatments as percent of untreated sample [Avg (SD)]

m-OH-COC

Ratios after treatments as percent of untreated [(Avg (SD)]

COC

BZE

NOR

p-OH-COC

Gillette

95.6 (23.2)

99.3 (25.0)

96.2 (22.3)

90.6 (19.1)

92.4 (25.9)

88.9 (20.4)

102.9 (17.0)

99.5 (9.5)

103.5 (16.0)

Right Guard

86.5 (8.2)

82.4 (8.7)

85.9 (15.6)

83.9 (6.5)

85.1 (13.6)

79.0 (20.4)

111.9 (31.7)

99.6 (10.4)

112.3 (30.0)

Degree All Products

95.1 (15.1) 92.4 (15.7)

91.6 (4.4) 91.1 (15.7)

97.8 (23.0) 93.3 (19.4)

91.6 (12.5) 88.7 (12.9)

93.1 (22.0) 90.2 (19.5)

87.3 (21.0) 85.1 19.2)

110.9 (28.5) 107.9 (24.3)

97.9 (15.0) 99.9 (10.7)

113.9 (19.0) 108.0 (20.7)

concentrations, either from the deodorant exposure or the additional extended washing of the treated samples or a combination of the two processes. However, after the deodorant treatments and extended washing, the p:o and m:o ratios did not change significantly and no increases in OH-COC metabolites were observed.

o-OH-COC

p:o ratio

p:m ratio

m:o ratio

With the in vitro experiments suggesting that presence of OH-COC metabolites in extensively washed hair is a result of metabolic formation, and not derived from peroxide or deodorants, analysis of the OH-COC isomers in large populations of COC-positive samples was undertaken. Tables 6, 7, and 8 show the ranges of p-OH-COC, m-OH-COC, and o-OH-COC, respectively, in a set of 1800 workplace-testing cocaine-positive samples. These tables reveal that 7.4, 4.9, and 35.9 % of cocaine-positive samples did not contain detectible levels of p-OH-COC, m-OH-COC, and o-OH-COC, respectively. Increasingly

higher levels of OH-COC isomers correlate with increasingly higher levels of cocaine, but the relationship is very wide ranging. Since the experimental results described above show no in vitro-produced OH-COC after washing, and the agreement of duplicate analyses of samples shows that analytic variation cannot account for the wide-ranging results, it seems likely that the variation in OH-COC in hair, whether as nanograms perweight of hair or as percent of COC, reflects metabolic processes. The levels of OH-COC isomers in samples from two distinct populations are presented in Tables 9 and 10. These tables present the data differently from Tables 6, 7, and 8, which showed levels of OH-COC isomers associated with their respective ranges of COC in the same hair sample. In Tables 9 and 10, all OH-COC isomer data from the entire range of samples is shown for head hair samples from criminal justice settings (Table 9) and workplace testing (Table 10). For the samples from criminal justice settings, the mean concentrations of p-, m-, and o-OH-COC were 1.56, 1.39, and 0.25 ng/10 mg hair, respectively (Table 9). Expressed as

Table 6 Ranges of p-OH-cocaine in cocaine-positive hair samples from workplace testing

Table 7 Ranges of m-OH-cocaine in cocaine-positive hair samples from workplace testing

Para-OH-COC

Meta-OH-COC

Surveys of hydroxycocaines in head hair samples from different populations of cocaine users

ng/10 mg hair Not detected 0.02–0.049 0.05–0.099 0.1–0.199 0.2–0.299 0.3–0.499 0.5–0.999 1.0–1.999 2.0–4.999 5.0–9.999 10–45

Cocaine n

Average

Median

134 147 205 214 148 206 243 202 202 70 29

ng/10 mg hair 9.0 12.1 16.5 12.2 19.7 12.3 27.4 17.2 35.2 20.3 52.6 28.3 90.3 54.4 167.1 100.2 399.6 286.2 644.2 558.6 678.8 593.3

Range

5.0–46.7 5.0–82.2 5.1–174.4 5.1–210 5.2–318.8 5.0–546.2 5.2–598.3 5.2–1238.4 6.5–1772.2 23.2–2670.4 170.2–2311.2

Cocaine n

Average

Median

ng/10 mg hair Not detected 0.02–0.049 0.05–0.099 0.1–0.199 0.2–0.299

88 132 163 221 166

ng/10 mg hair 10.0 8.3 14.2 11.8 19.2 14.4 22.3 13.5 31.8 18.9

5.0–46.2 5.0–46.7 5.1–79.6 5.1–214.7 5.2–337.8

0.3–0.499 0.5–0.999 1.0–1.999 2.0–4.999 5.0–9.999 10–45

230 297 221 191 67 24

45.5 84.4 149.3 399.6 635.4 611.9

5.1–641.3 5.1–601.1 8.0–1037.8 8.6–2670.4 62.8–2311.2 57.2–1625.1

27.5 52.5 99.6 286.2 520 574.9

Range

M. Schaffer et al. Table 8 Ranges of o-OH-cocaine in cocaine-positive hair samples from workplace testing Ortho-OH-COC

Table 10 samples

Hydroxycocaines in hair of cocaine users—workplace

Cocaine n

ng/10 mg hair

Average

Median

p-OH-COC ng/10 mg hair

m-OH-COC

o-OH-COC

n

1304

1415

501

Mean

1.7

1.44

0.4

SD Median

3.09 0.65

2.36 0.61

0.42 0.25

Range

0.1–33.1 Percent of cocaine

0.1–32.9

0.05–8.34

Range

ng/10 mg hair

Not detected

647

13.9

10.6

5.0–81.6

0.02–0.049 0.05–0.099

415 244

30.5 72.4

25.3 58.8

5.0–104.9 9.8–379.8

0.1–0.199 0.2–0.299

198 79

134.7 249.2

115.9 230.6

9.8–598.3 39.4–898.8

Mean

1.82

1.80

0.14

0.3–0.499

97

392.7

344.7

87.7–977.3

SD

1.87

1.78

0.09

0.5–0.999 1.0–1.999

87 30

695.7 969.8

632.8 826.8

632.8–2086.4 206.0–2670.3

Median

1.21

1.30

0.12

Range

0.06–12.51

0.019–11.4

0.2–0.79

2.0–4.999 5.0–9.999

2 1

1373.4 1625.2

N/A N/A

1371–1375 .7 1625.2

10–45

0

N/A

N/A

N/A

percent of cocaine, the results for p-, m-, and o-OH-COC were 2.06 (range 0.08–13.62), 1.95 (range 0.07–16.2), and 0.15 (range 0.02–.45), respectively. The ratios of the isomers to one another, shown in Table 11, averaged 13.25 (range 1.46–38.44), 1.41 (range 0.06–6.95), and 10.32 (range 1.53– 41.26) for p:o, p:m, and m:o, respectively. In workplace samples, the mean concentrations of p-, m-, and o-OH-COC were 1.7, 1.44, and 0.4 ng/10 mg hair, respectively (Table 10). As expected with a wide range of cocaine concentrations in these hair samples, the range of OH-COC values was also large. Even when expressed as percent of cocaine, the ranges for p-, m-, and o-OH-COC were large: 06–12.51, 0.019–11.4, and 0.2–0.79. The ratios of the isomers to one another, shown in Table 11, averaged 11.17 (range 0.93–66.05), 1.21 (range 0.5–8.34), and 9.64 (range 1.27–40.54) for p:o, p:m, and m:o, respectively. The means shown in these tables were not significantly different for the two populations.

Discussion Meta-OH-BE and p-OH-BE have previously been identified in urine [11, 14, 15, 17, 18, 23], meconium [12, 19], and blood [13, 16, 21]. Para-OH-COC and m-OH-COC in plasma after controlled administration were reported by Kolbrich et al. [21]. Para-OH-COC and m-OH-COC in plasma [21] and urine [23] after controlled administration have been reported. All three OH-COC metabolites have now been identified in hair by two laboratories, in this report and by Morris-Kukoski et al. [24]. Franz et al. have also reported the presence of pOH-COC and m-OH-COC in hair [25]. The need to combine the extended wash procedure with analysis of the OH-COC metabolites is clearly shown in the study in this report of the action of peroxide in in vitro formation of OH-COC metabolites, which are completely removed by the extended washing. Also, after exposure to deodorants, washed hair showed no Table 11 Ratios of hydroxycocaine isomers in two populations of cocaine-positive hair samples Ratios of hydroxycocaine isomers in cocaine-positive samples

Table 9 subjects

Hydroycocaine in hair of cocaine users—criminal justice Population p-OH-COC

m-OH-COC

o-OH-COC

n Mean SD Median

175 1.51 2.01 0.70

181 1.39 2.5 0.73

57 0.25 0.20 0.20

Range

0.1–23.9 Percent of cocaine 2.06 1.84 1.84 0.08–13.6

0.1–25.9

0.1–0.95

Mean SD Median Range

Criminal justice

Workplace 1.95 2.34 1.35 0.07–16.2

0.15 0.09 0.14 0.02–0.45

Statistical parameters

Ratios para:ortho

para:meta

meta:ortho

n Average SD Median

54 13.25 10.49 15.52

154 1.41 0.97 1.11

54 10.32 8.61 8.28

Range n Average SD Median Range

1.46–38.44 493 11.17 12.32 6.80 0.93–66.05

0.06–6.95 1263 1.21 0.87 0.99 0.05–8.34

1.53–41.26 493 9.64 8.04 7.34 1.27–40.54

Hydroxycocaines in hair

evidence of production of OH-COC analytes. The treatments applied to study in vitro OH-COC production in COCpositive samples, combined with extended washing, actually resulted in limited losses of analytes in COC users’ hair. Such losses are acceptable in comparison with achieving certainty that a result indicates ingestion vs. contamination. It is well known that there is drug in sweat that can deposit on hair, and extended washing removes much of the sweat-deposited drug, providing additional protection from higher values for some subjects due to sweat-deposited drug combined with variable hygienic habits such as frequency of shampooing. Possible significant loss of drug from porous hair during extended washing can be averted by determining the condition of the hair and washing of damaged or porous hair with 90 % ethanol [9]. Presently, OH-COC metabolite cutoffs are dependent on the levels of sensitivity of the mass spectrometry. We are currently moving our method from the AB Sciex 3000 to the 3200 LC-MS-MS in search of lower levels of detection, in order to detect all three metabolites in all positive samples including those with lower levels of COC. To do this will require a lower cutoff than our current 0.04 ng/10 mg hair. When sufficient sensitivity is achieved, detection of other metabolites such as CE or NOR may be superfluous. Morris-Kukoski et al. suggested that BE has little utility in establishing ingestion and proposed a reporting scheme such that a positive sample would contain COC ≥ 500 pg/mg, show presence of two of the hydroxymetabolites or NOR or CE, and pass the wash criterion [24]. Likewise, Franz et al. note that use of BE is problematic because it can be present in hair due to hydrolysis of COC as well as by metabolic process [25]. The present authors suggest that a positive may be reported if COC ≥ 500 pg/mg and the wash criterion passes, or (1) norcocaine or cocaethylene are present above levels reported in street cocaine or (2) if COC ≥ 500 pg/mg, the wash criterion fails, and two of the hydroxycocaines are present, with the additional safeguard of requiring a ratio ≥2 for p:o or m:o. This report is the first to provide a wealth of quantitative data to demonstrate the levels of all three metabolites in washed hair, both relative to the levels of COC and relative to one another. The possible utility of using ratios of the amounts of p-OH-COC and m-OH-COC to the amount of oOH-COC is exhaustively explored and a proposed value of 2 for either of these can provide extra protection, in addition to that of extended washing, against mis-interpreting possible presence of these metabolites that could result from in vitro formation, although we were unable to demonstrate in vitro formation of OH-COC metabolites after extended washing. Detection of OH-COC metabolites in hair subjected to the extended wash, which removed all in vitro OH-COC analytes in our studies, plus the use of the ratios, may be the Bholy grail^ for definitive interpretation of hair COC results indicating ingestion.

Acknowledgments The authors wish to thank Dr. Mahmoud A. ElSohly for his generous and most helpful collaboration. Compliance with ethical standards Ethical standards The authors declare that no human subjects were involved in the studies; the data presented in the paper were prior testing data stored anonymously.

Conflict of interest The authors declare that they have no competing of interests.

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Analysis of cocaine and metabolites in hair: validation and application of measurement of hydroxycocaine metabolites as evidence of cocaine ingestion.

An LC/MS/MS method to identify and quantitate in hair the minor metabolites of cocaine-meta-, para-, and ortho-hydroxy cocaine-was developed and valid...
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