Potential problems with the interpretation of hair analysis results

Due to differences in hair growth rate depending on anatomical region, age, gender, ethnicity and interindividual variability, interpretation of parent drug or/and metabolite concentrations in hair is not easy. Furthermore, as drug incorporation mechanisms into hair matrix is not yet fully understood, it is rather difficult to extrapolate details on time and dose from hair segment analysis. If incorporation sources other than from bloodstream (skin secretions and/or external/environmental contamination) are considered, interpretation becomes even more complicated. For evaluating possible passive contamination, it is essential to consider specific identification of metabolites, use of metabolite-to-parent drug ratios, assays of decontamination washes and analysis of specimens collected from other body parts. Cosmetic hair treatment, natural and artificial hair colour, differences in hair structure and specificity of analytical methodology may represent other bias sources affecting concentrations of drugs in hair. A suitable cut-off level related to the LOD will allow correct identification of drugs or metabolites in hair. Regarding the performance of different hair testing laboratories, little information is available at this time to what extent test results are comparable and their interpretation is consistent. Frequency of drug consumption and time intervals between multiple consumption or lag time between consumption and appearance in the hair has not been fully investigated and needs further research.     

Detection of antidepressant and antipsychotic drugs in human hair

The presence of therapeutic drugs and their metabolites in the hair of psychiatric patients was investigated using gas chromatography (GC)-mass spectroscopy (MS)-electron ionization (EI) and GC-MS-chemical ionization (CI). In hair samples tested from 35 subjects, carbamazepine, amitriptyline, doxepin, trihexyphenidyl, chlorpromazine, chlorprothixene, trifluoperazine, clozapine and haloperidol were detected, with maximal concentrations of 22.5, 57.7, 183.3, 15.6, 68.2, 30.0, 36.8, 59.2 and 20.1 ng/mg of hair sample, respectively. Chlorpromazine and clozapine concentrations in the hair were found to be dependent on the dosage used and their correlation coefficients were 0.8047 (P<0.001, n=16) and 0.7097 (P<0.001, n=16), respectively. Segmental analysis demonstrated that there was a correlation between the history of subject's drug exposure and the distribution of drug along the hair shaft. Our results also show that drug analysis in hair may provide useful information about drug treatment and the history of usage, and that drugs can be detected in normally kept hair for at least 16 months after intake.     

氯胺酮滥用的毛发分析研究

目的建立毛发中氯胺酮及其代谢物的分析方法并探索氯胺酮进入毛发的机理。方法通过建立豚鼠连续给药(不同剂量)实验模型获取阳性头发和采集氯胺酮滥用者头发,经处理后用GC/MSscan和SIM法分析,以鉴别、确认毛发中氯胺酮及其代谢物。结果豚鼠毛发中氯胺酮的质量分数与给药剂量存在明显的正相关性。毛发中氯胺酮质量分数依白色、棕色、黑色毛发顺序随毛发中黑色素的质量分数增加而增加。豚鼠毛发中氯胺酮与代谢物NK质量分数之比为2.33～12.94,仅在高剂量组的豚鼠毛发中才检测到DHNK,其质量分数与NK接近。15名氯胺酮滥用者黑色头发中均检出原体和代谢物NK,但DHNK少见。豚鼠毛发中代谢物相对质量分数明显高于人。结论本实验结果很好地反映了药物进入毛发代谢过程与药物和黑色素亲和力以及药物的亲脂性密切相关这一规律,但人和动物在药物代谢及进入毛发的难易程度上存在差异。本方法可以用于法庭毒物分析领域头发中氯胺酮的检测。     

Correlation of the incidence of cocaine and cocaethylene in hair and postmortem biologic samples

Hair samples are useful as a matrix for drug testing because drugs can be detected in hair for longer periods than in blood or urine. The authors report a prospective comparison of the detection of cocaine and cocaethylene in routine postmortem biologic specimens to the detection of cocaine and cocaethylene in hair. The authors collected hair samples from various areas of the head in 53 autopsy cases, prepared them, and analyzed them by gas chromatography/mass spectrometry (GC/MS) for cocaine and cocaethylene. The authors compared the results of hair analysis with the results of toxicologic analysis performed on routine postmortem samples by enzyme multiplied immunoassay technique and GC/MS. Cocaine was found in either biologic fluids or in hair in 16 of 53 samples tested. Nine samples were positive for cocaine in both biologic fluids and hair. Five samples contained cocaine only in biologic fluids, and two contained cocaine only in hair. Cocaethylene was present in two cases. Drug screening of hair provides additional information in some autopsy cases, but the authors have not made hair analysis a routine practice. It may prove useful to save hair samples in all cases for later analysis if warranted by additional history or autopsy findings.     

毒品与毛发的结合及其影响因素

毛发毒品的分析在毒品检验中具有独特优势,而毛发与毒品的结合状况及毒品聚集于毛发的机制影响其检测的准确性和灵敏性。对毒品与毛发的结合位点、毒品与毛发结合的差异性因素以及外界因素对毒品与毛发结合的影响进行了综述。     

Use of MDA (the "love drug") and methamphetamine in Toronto by unsuspecting users of ecstasy (MDMA)

It has recently been reported that purity of illicit tablets of ecstasy (MDMA) is now high. Our objective was to confirm whether hair of drug users, who request only ecstasy from their supplier, contains MDMA in the absence of other drugs. GC-MS analysis of scalp hair segments disclosed the presence of MDMA in 19 of 21 subjects and amphetamine/methamphetamine in eight subjects. Surprisingly, seven subjects had hair levels of the MDMA metabolite, MDA, equal to or greater than those of MDMA, suggesting use of MDA in addition to that of MDMA. These amphetamine derivatives might be included by clandestine laboratories to enhance effects of the drug cocktail or because of a perception that MDA synthesis might be simpler than that of MDMA. Drug users and investigators examining possible brain neurotoxic effects of MDMA need to consider that "ecstasy" tablets can contain MDA and methamphetamine despite no demand for the drugs.     

Determination of tramadol in hair using solid phase extraction and GC-MS

Tramadol is a centrally acting synthetic analgesic with mu-opioid receptor agonist activity, it is a widely prescribed analgesic used in the treatment of moderate to severe pain and as an alternative to opiates. Tramadol causes less respiratory depression than morphine at recommended doses. Its efficacy and low incidence of side effects lead to its unnecessary prescribing in patients with mild pain. Tramadol was classified as a "controlled drug" long after its approval for use in Jordan. Analysis of drugs of abuse in hair has been used in routine forensic toxicology as an alternative to blood in studying addiction history of drug abusers. A method for the determination of tramadol in hair using solid phase extraction and gas chromatography-mass spectrometry (GC-MS) is presented, the method offers excellent precision (3.5-9.8%, (M)=6.77%), accuracy (6.9-12%, M=9.4%) and limit of detection 0.5 ng/mg. The recovery was in the range of 87-94.3% with an average of 90.75%. The calibration curve was linear over the concentration range 0.5-5.0 ng/mg hair with correlation coefficient of 0.998. The developed method was tested on 11 hair samples taken from patients using tramadol as prescribed by their physician along with other different drugs in treating chronic illnesses. Tramadol was detected in all hair samples at a concentration of 0.176-16.3 ng/mg with mean concentration of 4.41 ng/mg. The developed method has the potential of being applied in forensic drug hair testing. In Jordan, hair drug testing started to draw the attention of legal authorities which stimulated forensic toxicologists in recent years to develop methods of analysis of drugs known or have the potential to be abused.     

Hair analysis for drug abuse, Part II. Hair analysis for monitoring of methamphetamine abuse by isotope dilution gas chromatography/mass spectrometry

Sectional analysis of methamphetamine abuser's hair was performed by using stable-isotope dilution GC/MS method. Drug concentrations of hair shaft cut into 2-cm sections from the root side were compared with the self-reported drug histories of 11 cases and the results of experiments on monkeys. It was found that in nine of the 11 cases, the relationship between the results of sectional analysis and drug histories coincided, but the sectional analyses of two cases were not consistent with self-reported drug history. The difference in drug concentrations between the regions of scalp hair was also investigated. Our study suggests that hair analysis, especially sectional analysis, may be useful in determining past drug history even though it is not exact.     

Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC x GC-TOFMS) for drug screening and confirmation

Comprehensive two-dimensional gas chromatography (GC x GC) is applied to analysis of drug standard mixtures containing 78 drugs of interest in forensic samples. For this study, underivatised drugs were employed. While several of the drugs were not detected at the low concentrations employed in the samples, most could be satisfactorily assigned their first and second dimension retentions in the GC x GC retention plane. For this study, time-of-flight mass spectrometry (TOFMS) detection was used. The enhanced separation possible in GC x GC is demonstrated, and typical linearity and apparatus precision are shown for tramadol, diazepam, olanzapine and desipramine using selected qualifier ions. Mass spectral library search quality for the detection of drugs in a selection of authentic forensic cases, along with retention position in the 2D retention plane, is used to support positive identification of the presence of the drugs. The analysis of 'difficult' drugs paracetamol and phenytoin is shown to produce anomalous chromatographic peak shape in the 2D plane, whereas most drugs gave acceptable peak shapes. The GC x GC technique was applied to screening drugs in forensic samples, with either flame ionisation (FID) or TOFMS detection, and compared favourably with conventional single column GC-MS analysis when tested for diazepam in an authentic forensic study.     

毛发中毒品分析

毛发分析在法庭毒品分析领域有其独特的优势,很多国家的法化学实验室,毛发分析已成为毒品检测的常规操作,并已得到了法庭的承认、采纳。本文对毒品进入毛发的机制、毛发的现场勘查、毛发中毒品分析程序、毛发中毒品分析结果的评判进行了综述。简单地介绍了毛发在现场勘查中采取、包装、送检的基本方法和技术人员在操作过程中的注意事项,以及针对毛发检验中的特殊技术处理;另外,介绍了毛发中毒品分析的特点,通过分析毛发毒品的药理机制,总结出了一些高效、便利、快速的毒品分析方法,并对各种方法进行了介绍,得出了毛发中毒品分析结果的一些特点。     

Clinical applications of hair testing for drugs of abuse--the Canadian experience

During the last 2 decades there has been a substantial increase in illicit drug consumption in North America. It has been repeatedly shown that the personal history of drug use is far from being accurate. Fearing legal consequences and embarrassment of admitted illicit substance use, most users tend to deny or, to under-report illicit drug consumption. These facts have stressed an urgent need for a biological marker which does not lose its sensitivity within a few days after the end of exposure and which may yield a cumulative reflection of long term exposure to illicit drugs. Hair analysis has emerged as such a marker. A variety of illicit and medicinal compounds have been shown to be incorporated into hair including trace metals, barbiturates, amphetamines, opiates, phencyclidine, cocaine, nicotine and cannabis. Hair analysis for drugs of abuse provides long-term information on an individual's drug use; its window of detection is limited only by the length of the hair and typically, ranges from a week to several months. After establishing and validating several hair tests during the last decade, we have analyzed over 1000 hair samples for different drugs of abuse. We used RIA for screening and GC-MS for confirmation of positive results. The aim of this report is to illustrate the diagnostic usefulness of hair testing in different age groups (newborns, children, adults) and circumstances: (criminal cases, athletes, child custody cases, etc.).     

Analysis of drugs of abuse in hair by automated solid-phase extraction, GC/EI/MS and GC ion trap/CI/MS

In our laboratory, analysis of human hair for the detection of drugs of abuse was first performed in 1995. Initially, requests for hair analysis were few, and it is only since 1997 that these analyses have become routine. As demand grew, we developed an automatic solid-phase extraction method; the use of a robot ASPEC allowed us to drop certain fastidious manipulations, and to treat a large number of samples at a time. This method is described, along with analysis by gas-chromatography-mass spectrometry (GC/MS) in selected ion monitoring mode (SIM), for the following drugs: codeine, 6-monoacetylmorphine (6-MAM), morphine, cocaine, methadone, ecstasy (MDMA) and Eve (MDE). This requires prior derivatization with propionic anhydride. The different validation parameters, linearity, repeatability, recovery and detection limits are described, as well as the application of this method to some real cases. Analysis of these cases is also performed by an ion trap GC/MS in chemical ionization mode (GC/IT/CI/MS) in order to demonstrate the usefulness of this technique as a complement to routine analysis. Analysis by GC/IT/CI/MS indeed avoids the risk of false-positive results by the identification of metabolites.     

Evidence for bias in hair testing and procedures to correct bias

A number of in vitro experiments show that different hair samples incorporate differing amounts of drugs under identical conditions. Incorporation of cocaine and morphine tends to be correlated with race, in that the hair of African American females incorporates higher concentrations of cocaine than does the hair of Caucasian males or females. Extrapolation of these data into populations has been fraught with difficulties because the dosages of drugs and their use patterns are unknown. Cosmetic treatments and hygiene alter drug binding, which must be considered in comparing populations because cosmetic treatments are often group dependent. Four reasons are proposed that account for the uptake and retention of drugs by hair and that may differ among groups: (1) permeability and other characteristics of the hair due to genetic influences, (2) cosmetic hair treatments and hair care habits (which may be culturally influenced), (3) drug removal during personal hygiene, and (4) manner and route of drug administration which can affect passive exposure to residual drugs in the environment. The data supporting bias in hair testing are reviewed and methods are proposed that use either the uptake of dyes or the incorporation of drug homologs to reduce bias.     

Hair analysis for drugs of abuse. Hair color and race differentials or systematic differences in drug preferences?

There is currently a debate in the literature on chemical drug analysis concerning the contribution of biophysical attributes associated with specimens and specimen donors to assay outcome. In recent years this debate has focused on hair analysis, but has in the past also been raised in urinalysis interpretation. In this article we examine several aspects of that controversy. First, we present data regarding the effects of hair color on the distribution of positive hair testing results for three drug classes. We compare these results to negative hair samples from comparable donors. This data is derived from head hair from preemployment donors that was classified according to seven visual color categories. We determined the distribution of colors for hair samples devoid of any of three assayed drugs (amphetamines, cocaine, and cannabinoids). Subsequently, this distribution was compared with the distributions for hairs that had tested positive for amphetamines, cocaine or cannabinoids. We examined a total of 2000 randomly selected samples; 500 negative hair samples and 500 positive samples for each of three drugs: cannabinoids, cocaine, and amphetamine. We also evaluated ethnic/racial factors in relation to positive urinalyses for various ethnic/racial groups. We examined approximately 4000 urine specimens from two different groups, each constituting around 2000 specimens. In addition to ethnicity/race and urinalysis outcome, we also examined the relationship between the hair color distributions of urine donors and the corresponding urinalysis results for the three drug classes. We also compared them to drug-negative samples. Our summary impression is that the observed outcome patterns were largely consistent with differences in drug preferences among the various societal groups. There was little evidence of a pattern attributable to hair color bias alone or selective binding of drugs to hair of a particular color. Likewise, there was no discernible pattern associated with race or ethnicity that would lend support to a "race effect" in drug analysis.     

Blood, brain, and hair GHB concentrations following fatal ingestion

Despite the increasing incidence of illicit use of gamma-hydroxybutyrate (GHB), little information is available documenting levels of the drug in GHB fatalities. We measured GHB levels in postmortem blood, brain and hair specimens from a suspected overdose case by gas chromatography/mass spectrometry (GC/MS) following solid phase extraction (SPE) and derivatization with bis(trimethyl-silyl) trifluoroacetamide (BSTFA). Examination found 330 microg/mL GHB in femoral blood and 221 ng/mg GHB in frontal cortex brain tissue, values higher than those typically reported in the literature. The hair shaft was negative for GHB whereas the plucked root bulbs with outer root sheath attached (2,221 ng/mg) and root bulbs after washing and removal of the outer root sheath (47 ng/mg) contained the drug. Our results are consistent with an acute single dose of GHB and, as the toxicology screen was negative for other drugs of abuse, emphasize the significant danger of this drug.     

Detection and diagnostic interpretation of amphetamines in hair

A review with 22 references on detection and incorporation of amphetamines in hair is presented. This review deals with the detection, incorporation into hair, behavior in the hair shaft, confirmation of past drug use and diagnosis of dependence mainly regarding amphetamine and methamphetamine, along with methoxyphenamine, methylenedioxymethamphetamine, bromomethamphetamine, deprenyl, benzphetamine, fenproporex and mefenorex. First, pretreatment, extraction and analytical methods for amphetamines in hair using immunoassay, HPLC and GC/MS are discussed. This is followed by sections describing the animal experiments, incorporation rates of amphetamines from blood to hair and relationship between drug history and drug distribution in hair. Finally, the diagnosis of amphetamine dependence and confirmation of methamphetamine baby by hair analysis is discussed. The paper concludes with a brief outlook.     

Hair analysis of opiates in mothers and newborns for evaluating opiate exposure during pregnancy

The increasing interest in toxicological hair analysis as a marker of human exposure to xenobiotics such as illicit substances or therapeutic drugs, has been made feasible by the extension of mass spectrometry, a highly sensitive method of detection. A newborn exposed to drugs in utero can suffer from a varying degree of withdrawal syndrome, a few days after birth. If of opiate origin, the withdrawal syndrome can be treated with morphine, among other therapeutics, but it is not easy to diagnose because of atypical symptoms presented by neonates and especially when maternal drug addiction has not been revealed. To assess and measure toxicological factors linked with the appearance and the severity of this syndrome, maternal and neonatal matrices such as urine, meconium and hair were collected during a protocol approved by the ethical committee. Opiates in particular were measured with GC-MS and potential combined dependences (cannabis, cocaine, amphetamine, LSD and benzodiazepines) and/or substitutive therapeutics (methadone or buprenorphine) were also assessed in 17 mother/neonate couples. Gestational opiate exposure profiles were drawn up and linked with the observed withdrawal syndromes. A withdrawal syndrome seems to appear more frequently after foetal exposure to an association of opiates/substitutive molecules (8 out of 10 withdrawal syndromes observed in this study), although the impact of cocaine and benzodiazepines must also be taken into account. The results obtained in neonatal hair make it possible to affirm foetal drug exposure and are in accordance, for the majority, with the appearance of a neonatal withdrawal syndrome (NWS). Neonatal hair analysis could contribute to assess in utero exposure to opiates, particularly when results in urine and meconium are negative or when these matrices are not available.     

Pharmacological criteria that can affect the detection of doping agents in hair

When positive drug results are reported, a common interpretive question posed is whether or not it is possible to put a quantitative finding into context. A standard answer to this inquiry is that a positive hair testing result can be interpreted as meaning that the donor has chronically or repetitively used the drug identified in the hair, but that chronic or repetitive are not defined in the same way for all individuals. The Society of Hair Testing published on June 16, 1999, a consensus opinion on the use of hair in doping situations. However, although accepted in most courts of justice, hair analysis is not yet recognised by the International Olympic Committee. To be considered as a valid specimen for doping control, some issues still need to be addressed. The scientific community has demonstrated significant concern over the proper role that hair drug testing should serve in toxicological applications. Among the unanswered questions, five are of critical importance: (1) What is the minimal amount of drug detectable in hair after administration? (2) What is the relationship between the amount of the drug used and the concentration of the drug or its metabolites in hair? (3) What is the influence of hair color? (4) Is there any racial bias in hair testing? (5) What is the influence of cosmetic treatments? The present report documents scientific findings on these questions, with particular attention to the applications of hair in doping control.     

Value of the concept of minimal detectable dosage in human hair

The influence on drug incorporation of melanin affinity, lipophilicity, and membrane permeability is of paramount importance. Despite their high lipophilicity, some drugs have quite low incorporation rate into hair, suggesting that the higher incorporation rates of basic drugs (cocaine, amphetamines.) than neutral (steroids, benzodiazepines, cannabinoids…) or acidic ones are strongly related to the penetrating ability of the drug to break through the membrane based on the pH gradient between blood and the acidic hair matrix. When using hair analysis as a matrix during investigative analysis, e.g. workplace drug testing, doping, driving under the influence, drug-facilitated crime, the question of importance is to know whether the analytical procedure was sensitive enough to identify traces of drugs; this is particularly important when the urine sample(s) of the subject was positive and the hair sample(s) was negative. It has been accepted in the forensic community that a negative hair result cannot exclude the administration of a particular drug, or one of its precursors and the negative findings should not overrule a positive urine result. Nevertheless, the negative hair findings can, on occasion, cast doubt on the positive urine analysis, resulting in substantial legal debate and various consequences for the subject. The concept of minimal detectable dosage in hair is of interest to document the negative findings, but limited data is currently available in the scientific literature. Such data includes cocaine, codeine, ketamine, some benzodiazepines and some unusual compounds. Until laboratories will have sensitive enough methodologies to detect a single use of drug, care should be taken to compare urine and hair findings.     

Quantitative analysis of methamphetamine in hair of children removed from clandestine laboratories--evidence of passive exposure?

In New Zealand many children have been removed from clandestine laboratories following Police intervention. In the last few years it has become standard procedure that these children have hair samples taken and these samples are submitted to the laboratory for analysis. There are various mechanisms for the incorporation of drugs into hair. The hair follicle has a rich blood supply, so any drug that may be circulating in the blood can be incorporated into the growing hair. Another mechanism is via external contamination, such as spilling a drug on the hair or through exposure to fumes or vapours. Hair samples were analysed for methamphetamine and amphetamine. From the 52 cases analysed 38 (73%) were positive for methamphetamine (>0.1 ng/mg) and amphetamine was detected in 34 of these cases. In no case was amphetamine detected without methamphetamine. The hair washes (prior to extraction) were also analysed (quantified in 30 of the positive cases) and only 3 had a wash to hair ratio of >0.1 (all were <0.5), which may be indicative of a low level of external contamination. This low level of evidence of external contamination suggests that the children are exposed to methamphetamine and are incorporating it into the hair through the blood stream.