What constitutes a positive result in hair analysis: proposal for the establishment of cut-off values

Forensic Science International 70 (1995) 3-11

ELSEVIER

Forensic Science International

What constitutes a positive result in hair analysis: proposal for the establishment of cut-off values Pascal Kintz*, Patrice Mangin htitut

de Meahhe

Legale, II Rue Hwnann, 67000 Strasbourg, France

Received 5 May 1994; accepted 24 June 1994

Abstract Hair is still a seldomusedspecimenin mostlaboratoriesbut its analysishasthe potential of makinga valuablecontribution. Despitethe many worthwhile reports, the scientificcommunity at large still hasreservationsabout the validity of hair analysis.Someof this is due to a lack of consensus amongthe active investigatorson how to interpret the resultsfrom an analysisof hair. In USA, passiveexposureseemsto be a major problem,which can only be eliminatedwith difficulty. On the other hand, in Europe, scientistsare performing standard decontaminationprocedures.It would be very helpful if a group of active researchers on hair analysis,representativeof academic,governmentand private laboratoriescould definewhat arethe areasof agreementand what are the issuesthat requirefurther efforts to get a consensus.We proposethe following guidelines:(1) a completedecontaminationprocedure,including the analysisof the washsolution; (2) two distinct analytical methods(immunoassay andGC/MS, or two different GC/MS methods);(3) the establishment of cut-off values(using 30-mghair samples), 0.5 ng/mgof 6-MAM in the caseof heroinabuse,and 1ng/mgof cocaine in the caseof cocaineabuse,which can be decreasedto 0.5 ng/mgwhenuseis supportedby other evidenceof drug intake. Keywordr:

Hair; Consensus; Cut-off values

1. Introduction

In the 1960s and 197Os, hair analysis was used to evaluate exposure to toxic heavy metals, such as arsenic, lead or mercury; this was due to atomic absorption that allowed detection in the picogram range. At this time, examination of hair for organic substances, especially drugs, was not possible because analytical methods were not sensitive enough. l

Corresponding author.

0379-0738/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0379-0738(94)01621-B

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Examination by means of drugs marked with radioactive isotopes, however, established that these substances can move from blood to hair and are deposited there. Ten years after these first investigations for drugs in hair, it was possible to detect various organic drugs by means of the radioimmunologic assay (RIA) technique; this achievement occurred simultaneously in the United States and West Germany. In 1979, Baumgartner and colleagues [l] published the first report on using RIA to detect morphine in the hair of heroin abusers. They found that differences in the concentration of morphine along the hair shaft correlated with the time of drug use. This was followed shortly thereafter by other studies on the analysis of drugs of abuse. Initially, these analytical methods based on RIA did not allow differentiation between individual opiates. A few years later however, RIA sets, that enabled better identification using sensitive cross-reactions, became available. Generally, the courts of justice only recognize the results of chemical-toxicological analyses when they are confirmed by a second independent method. For this reason, gas chromatography coupled with mass spectrometry (CC/MS) is now the method of choice for hair analysis. Since the mid-1980s, this method has made it possible to carry out hair analysis with no legal obstacles to recognition of the results. At present, hair analysis is routinely used as a tool of detection for drug use in forensic sciences, traffic medicine, occupational medicine and clinical toxicology. Despite the many worthwhile reports, the scientific community has reservations about the validity of hair analysis. Some of this is due to a lack of consensus among the active investigators on how to interpret the results of an analysis of hair and what are the optimal analytical parameters. In 1989, the Society of Forensic Toxicologists (SOFT) and National Institute on Drug Abuse (NIDA), in collaboration with the National Institute of Justice (NIJ), convened a Conference on Hair Analysis for Drug of Abuse. As a result of this conference, a Consensus Opinion was published by SOFT [2] which contained the following points among others: 1. The use of hair analysis for employee and pre-employment drug testing is premature and cannot be supported by the current information on hair analysis for drugs of abuse. Too many critical questions remain to be answered before the results can be accurately interpreted. 2. Hair may be a useful specimen in forensic investigations when supported by other evidence of drug use (e.g. analysis of blood, urine or other tissues) and when performed under generally accepted guidelines for forensic drug testing. Since this Opinion had a stifling effect on the application of hair drug testing in workplace testing, SOFT was asked to reevaluate the Opinion. The recommended Revised Consensus Opinion on Applicability of Hair Analysis for Drugs of Abuse [3] did not change item 1 but softened item 2 in that ‘competent evidence of drug use’ was now required, as opposed to more specific supporting information like ‘analysis of blood, urine or other tissues’ in the 1989 version. The scientific community has pointed out some unanswered questions remaining to be explored with regard to hair drug testing. Some of these questions might be: 1. How should specimens be prepared for analysis? (a) Digestion or extraction?

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(b) Prior washing? (c) If washed, is there a preferred washing technique? (d) Is there a preferable technique for the analysis? 2. To what extent is passive exposure a problem? If it is, can it be eliminated? 3. What conhastitutes a positive or negative result? It would be very helpful if the scientific community could define what are the areas of agreement and what are the issues that require further efforts. This commentary addresses each one of these questions and discusses interpretations of the opinions reported in the literature. 2. Sample preparation Collection procedures for hair analysis for drugs have not been standardized. Hair is best collected from the area at the back of the head, called the vertex posterior. Compared with other areas of the head, this area has less variability in hair growth rate, the number of hairs in the growing phase is more constant and the hair is less subject to age and sex-related influences [4]. The sample size varies considerably among laboratories and depends on the drug to be analyzed and the test methodologies. Sample sizes reported in the literature range from a single hair [5] to 200 mg [6]. In our laboratory, samples of 30-50 mg are currently used. Beard, public or axillae hair have no advantage over head hair as they can also be contaminated. One must also determine which segment has to be analysed: the whole hair, even in the case of long hair, a segment obtained from the proximal zone (root), or several segments? In an experiment conducted with methoxyphenamine given to five subjects over 7 days, it was demonstrated that the drug moved along the hair shaft at a rate of 2.8-3.2 mm/week, according to hair growth, without diffusion [7]. Nevertheless, the drug level in hair had decreased approximately 50%, 5 months later. One can suppose that normal hygiene washing had removed some of the drug. For screening procedures, we analyzed two segments: a 3-cm section from the root and the rest of the hair. From this, we obtained a retrospective calendar of an individual’s drug use. Information is available on a period of 3 months followed by a previous drug history. Recent abstinence can therefore be proved. The switch from heroin to another drug, like codeine or dihydrocodeine can be established with accuracy. Given the variation in hair growth rates, results from a multisectional analysis should not be used to determine a precise period of drug abuse or to compare individuals. The further away from the hair root, the more cautious the interpretation of quantitative findings of the individual hair sections has to be. The passive drug absorption by hair must be considered a possible source of false positive results. Thus, before proceeding with the analysis, a step to remove external contamination must be included. At the present time, it is possible to find literature references on hair drug testing which use no rinsing [8], rinsing with methanol or ethanol alone [9], or with aqueous buffers [lo], rinses with methylene chloride [l 11, with surfactants [7] or with water and acetone [12]. Sometimes these rinses are tested, many times they are not.

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Washing procedures must be effective in removing external contamination but not the interior drug components. Aqueous buffers, or organic solvents used in decontamination procedures are also used to solubilize drug from hair. This paradox can lead to underestimated concentrations. Our decontamination procedure involves one rinse with methylene chloride over 2 min, one rinse with water for 2 min, followed by a second rinse with methylene chloride for 2 min and a test of the last rinse by GC/MS. When tested, the rinse with water generally exhibited low concentrations of the target drugs. Drug solubilization can be achieved by chemical (acid or alkaline) hydrolysis [ 111, enzymatic hydrolysis [ 121 or direct solvent extraction [6]. Solubilization must be such that analytes are not altered or lost. Care is necessary to prevent the conversion of cocaine to benzoylecgonine or 6-monoacetylmorphine to morphine. Differences of efficiencies between enzymatic and acid hydrolysis are not statistically significant u31. A variety of analysis techniques have been applied to hair testing (RIA, FPIA, GC, GUMS, MS/MS, HPLC, capillary electrophoresis, etc.). Chromatographic procedures seem to be the most powerful tool for the identification and quantitation of drugs in hair, owing to their separation ability and their detection sensitivity [14]. The reporting of a positive result based upon immunoassay is not acceptable. All positive presumptive tests must be confirmed by a second independent test. RIA followed by GUMS or two GUMS procedures seem to be acceptable to prevent false positive analytical results. In our laboratory, positive samples obtained after acid hydrolysis are confirmed by a second GUMS analysis after enzymatic hydrolysis. 3. Passive exposure

Incorporation during hair growth from the blood stream and incorporation after hair growth from sweat and external contamination have both been proposed to account for drugs appearing in hair 1151. In determining drug use, it is essential to exclude external contamination. Various experimental procedures, including hair samples exposed to vapors or to aqueous solutions of drugs have shown that it was not possible to completely remove the incorporated drug by washing procedures [ 13,16,17]. Regardless of the washing technique, a substantial amount of drug could still be found in the final hair digest. As sweat is a vehicle by which drugs are incorporated into the hair, it can be potentially contaminated by external sources of drugs, generating false positives. To avoid these problems, Baumgartner and Hill [ 181 have suggested the use of three criteria to distinguish passive exposure from active ingestion. These criteria were based upon the kinetics of drug removal from the hair under specific conditions. Other authors [ 16,191 have proposed the presence of metabolites in documenting drug consumption. Although these procedures are used by many scientists, Blank and Kidwell [ 17,201 recently reported that external contamination of hair by drugs of abuse cannot be totally removed and that the interpretation of forensic results remains problematical.

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When contaminated, either by vapor or aqueous solutions, hair samples exhibit very high drug concentrations which are not consistent with concentrations reported in drug addicts, Therefore, these contamination experiments don’t seem to reflect what is really occurring during passive exposure. For example, the concentration of drugs measured in the hair of neonates is in the range of the concentrations of adults, even after contamination by the amniotic fluid over at least 3 months [21]. Reported concentrations of benzoylecgonine in amniotic fluid were in the range 0.04-3.06 mg/I [22], and cocaine was detected in hair in the range 0.71-2.47 ng/mg [21]. The problem of passive contamination of hair is similar to the one which occurred with urinalysis after passive inhalation of cannabis smoke. Although it was demonstrated that cannabis metabolites are present in the urine of subjects passively exposed to the smoke of marijuana cigarettes [23], the problem was solved by the use of appropriate cut-off values. This has to be done for hair testing. 4. Cut-off values in hair testing In this section, particular attention will be focused on heroin and cocaine. Due to the lack of literature, it is premature to propose cut-off values for cannabis, although there is a need to characterize positive samples. Today, it is generally accepted that the parent drug exceeds the metabolites in hair [6,9,12,13,16,19]. Moreover, the differentiation of heroin users from individuals exposed to other sources of opiates has to be achieved by identifying 6monoacetylmorphine directly. Therefore, the detection of cocaine or heroin chronic abuse was reported by evaluating cocaine or 6-monoacetylmorphine, respectively, in hair. Published ranges of drug concentrations are given Table 1. The reported concentrations are in constant evolution. For example, we recently measured 216.5 ng/mg of cocaine. The scientific establishment of cut-off values is different for cocaine and heroin. They are proposed using 30-mg hair samples.

Table 1 Published

ranges of drug concentrations

Reference

(ngmg)

6-MAM

Cocaine Range

Kintz Kauert Cone Moeller Cone Sachs Number

in hair

and Mangin [29] et al. [6] et al. (161 et al. (251 et al. [27] [28] of positive

0.4-78.4 0.1-95.1 6.4-19.2 0.3-127.0 0.41-76.0 -

(14) (10) (34) (20)

cases in parentheses.

Mean

Range

Mean

8.3 10.05 10.8 20.6 -

0.0-84.3 0.09-56.9

(57)

2.0-74.2 0.0-0.78 2.0-74.2

(34) (20)

11.3 6.96 15.3 -

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4.1. Cocaine One of the more striking findings of the analysis of cocaine in hair, is that in 30-50’S of the positive samples, cocaine is detected in the absence of any of its major metabolites. Our observations are in accordance with those of Selavka [24] by not with those of Moeller et al. [25] who reported detection of benzoylecgonine in all cases of positive cocaine. Therefore, the use of one or more metabolites as the only true markers of cocaine intake does not seem to be a viable approach to differentiating endogenous cocaine from possible cocaine contamination on the exterior of the hair. Moreover, benzoylecgonine, the primary metabolite, can be formed by cocaine hydrolysis in normal personnel hygiene with basic detergents. It appears that detecting metabolites of cocaine in positive cocaine hair increases with the cocaine concentration. In our data, when parent cocaine was the only analyte identified in a hair sample, the typical quantitative findings were relatively low. Cocaethylene was never detected when cocaine was lower than 2 ngfmg. In general, cocaine tends to predominate over benzoylecgonine by a factor of 2-10. The relationship between the analyte concentrations is cocaine > benzoylecgonine > ecgonine methylester. It should also be noted that in no cases were metabolites detected in the absence of cocaine. To prevent false positives due to external contamination, it is necessary to detect cocaine in adequate concentratiqn in the absence of metabolite. The distribution of findings for cocaine in positive hair (Fig. 1) suggests that a cut-off of 1 ng/mg of cocaine is realistic. Using this cut-off, four of the 48 positive analyses will be considered as negative. To prevent this, one can decrease this value to 0.5 ng/mg when cocaine use is supported by another evidence of drug intake, like analysis of blood or urine. In that case, only two samples remained negative in our data. To be more comfortable with regard to passive exposure, one can associate cocaine determination with the quantification of its metabolites, particularly cocaethylene. In the case of 216.5 ng/mg of cocaine, high consumption was proved by a concentration of 10.3 ng/mg for cocaethylene. nb of cases 3 2 2 1 1

Fig. 1. Distribution 90-April 94.

of quantitative

findings

for hair

in which

cocaine

was detected

from

February

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As proposed by Cone et al. [26], the ratio of benzoylecgonine to cocaine can be used to differentiate environmental drug use from active drug use. A ratio greater than 0.05 seems consistent with drug use. 4.2. Heroin

As heroin street samples always contain codeine, this is also detected in hair in cases of heroin abuse. Morphine is a metabolite of codeine and can be detected when codeine is abused. Differentiation between codeine and heroin abuse was previously achieved by quantification of morphine and codeine. A significant advance in proving heroin abuse was the detection of 6-monoacetylmorphine in hair and the integration of its analysis into routine procedures. In most cases, the 6-MAM concentrations were higher than the morphine concentrations. Ratios of 6-MAM to morphine and (6-MAM + morphine) to codeine were published to document heroin abuse [25]. These were in the range 1.3-10.0 and 4.4-155.6, respectively. However, based on our experience, these proposals are not enough. Heroin addicts can switch to codeine or other antitussives as the drugs are easier to find, and of lower cost. In such a case, codeine can reach concentrations that will reverse the ratio (6-MAM + morphine) to codeine to a value lower than 1. In 129 cases where 6-MAM was detected, 33 times (26%) the latter ratio was lower than 1. In cases where no 6-MAM can be found, generally when the sum of all the opiates detected is below 1 ng/mg, statements must be given very carefully. The distribution of our findings for 6-MAM in positive hair (Fig. 2) suggests that a cut-off of 0.5 ng/mg of 6-MAM is compatible with heroin abuse. In that case, only one sample out of 129 with a 6-MAM concentration of 0.42 ng/mg will be considered as negative. Naturally, the simultaneous determination and quantification of morphine and codeine represents the state-of-the-art to avoid analytical errors or risk of external contamination. nb of cases 5

Fig. 2. Distribution of quantitative findings for hair in which Gmonoacetylmorphine February 90-April94.

was detected from

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These criteria to establish a positive hair drug testing result have to be discussed in the light of the data from other scientists. Hair testing is a useful tool in studying drug abuse. To some degree, it has been negatively affected by various problems and controversies. The ability to determine long-term retrospective drug use with high reliability can prove useful in a variety of investigative settings including the clinical medical community, the drug of abuse treatment community and the criminal justice system. It is time to direct our research energies towards solving the remaining unanswered questions. References [I] A.M. Baumgartner, P.F. Jones, W.A. Baumgartner and C.T. Black, Radioimmunoassay of hair for determining opiate-abuse histories. J. Nucl. Med., 20 (1979) 749-752. [2] R.O. Bost, Consensus opinion summarizing the current applicability of hair analysis to testing for drugs of abuse. SOFT ToxTalk, 14 (1990). [3] W.L. Heam, Report of SOFT advisory committee on hair analysis for drugs of abuse. SOFT ToxTalk, 16 (1992) l-11. [4] MR. Harkey, Anatomy and physiology of hair. Forensic Sci. Int., 63 (1993) 9-18. [5] 0. Suzuki, H. Hattori and M. Asano, Detection of methamphetamine and amphetamine in a single human hair by gas chromatography/chemical ionization mass spectrometry. J. Forensic Sci., 29 (1984) 611-617. [6] G. Kauert, L.V. Meyer and I. Herrle, Drogen- und Medikamentennachweis im Kopthaar ohne Extraktion des Haaraufschlusses, mittels GC-MS. Z. Rechtsmed., 38 (1992) 33. [7] Y. Nakahara, M. Shimamine and K. Takahashi, Hair analysis for drugs of abuse. III. Movement and stability of methoxyphenamine (as a model compound of methamphetamine) along hair shaft with hair growth. J. Anal. Toxic& 16 (1992) 253-257. [8] T. Nagai, M. Sato, S. Kamiyama and Y. Miura, A new analytical method for stereoisomers of methamphetamine and amphetamine and its application to forensic toxicology. Clin. Biochem., 22 (1989) 439-442. [9] R. Martz, B. Donnelly, D. Fetterolf, L. Lasswell, G.W. Hime and W.L. Heam, The use of hair analysis to document a cocaine overdose following a sustained survival period before death. J. Anal. Toxicol., 15 (1991) 279-281. [IO] W.A. Baumgartner, V.A. Hill and H. Blahd, Hair analysis for drugs of abuse. J. Forensic Sci., 34 (1989) 1433-1453. [I l] P. Kintz, Determination of buprenorphine and its dealkylated metabolite in human hair. J. Anal. Toxicol., 17 (1993) 443-444. [12] M.R. MBller, P. Fey and S. Rimbach, Identification and quantitation of cocaine and its metabolites, benzoylecgonine and ecgonine methyl ester, in hair of Bolivian coca chewers by gas chromatography/mass spectrometry. J. Anal. Toxicol., 16 (1992) 291-296. [13] M.J. Welch, L.T. Sniegoski, C.C. Allgood and M. Habram, Hair analysis for drugs of abuse: evaluation of analytical methods, environmental issues, and development of reference materials. J. Anal. Toxicol., 17 (1993) 389-398. [14] M.R. Moeller, Drug detection in hair by chromatographic procedures. J. Chromatogr., 580 (1992) 125-134. [IS] G.L. Henderson, Mechanisms of drug incorporation into hair. Forensic Sci. Inr., 63 (1993) 19-29. [16] 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. [17) D.L. Blank and D.A. Kidwell, External contamination of hair by cocaine: an issue in forensic interpretation. Forensic Sci. Int., 63 (1993) 145-156. (181 W.A. Baumgartner and V.A. Hill, Hair analysis for drugs of abuse: decontamination issues. In I. Sunshine (ed.), Recent Developments in Therapeutic Drug Monitoring and Clinical Toxicology, Marcel Dekker, New York, 1992, pp. 577-597.

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G. Koren, J. Klein, R. Forman and K. Graham, Hair analysis of cocaine: differentiation between systemic exposure and external contamination. J. Clin. Pharmacol., 32 (1992) 671-675. [20] D.A. Kidwell and D.L. Blank, Comments on the paper by W.A. Baumgartner and V.A. Hill: Sample preparation techniques. Forensic Sci. Int., 63 (1993) 137-143. [21] P. Kintz and P. Mangin, Determination of gestational opiate, nicotine, benzodiazepine, cocaine and amphetamine exposure by hair analysis. J. Forensic Sci. Sot., 33 (1993) 139-142. [22] C. Moore, S. Browne, I. Tebbett, A. Negrusz, W. Meyer and L. Jain, Determination of cocaine and benzoylecgonine in human amniotic fluid using highflow solid-phase extraction columns and HPLC. Forensic Sci. Int., 56 (1992) 177-181. [23] E.J. Cone, R.E. Johnson, W.D. Darwin, D. Yousefnejad, L.D. Mel], B.D Paul and J. Mitchell, Passive inhalation of marijuana smoke: urinalysis and room air levels of delta-9-tetrahydrocannabinol. J. Anal. Toxicol., 11 (1987) 89-96. [24] C. Selavka, Discussion of forensic hair testing casework results and hair rinsing procedures. Workshop on the Analysis of Hair for Drugs of Abuse, Gaithersburg, USA, 21 May 1993. [25] M.R. Moeller, P. Fey and H. Sachs, Hair analysis as evidence in forensic cases. Forensic Sci. Int., 63 (1993) 43-53. (261 E.J. Cone, W.D. Darwin and W.L. Wang, Differentiation of environmental drug exposure from active drug use in hair analysis for drugs of abuse. 2nd International Meeting on Clinical and Forensic Aspects of Hair Analysis, Genoa, Italy, 6-8 June 1994, abstract, p. 29, [27] E.J. Cone, W.D. Darwin and W.L. Wang, The occurence of cocaine, heroin and metabohtes in hair of drug abusers. Forensic Sci. Int., 63 (1993) 55-68. 1281 H. Sachs, Drogennachweis in Haaren, Symposium Aktuelle Aspekte des Drogennachweises, Mosbach, Germany, 15 April 1993. [29] P. Kintz and P. Mangin, Simultaneous determination of opiates and cocaine and its major metabolites in human hair by GC/MS. Forensic Sci. Int (submitted).