Opiate levels in hair

By means of radioimmunoassay-technique, hair samples of users, drug related fatalities, carcinoma patients receiving morphine and of experimental guinea pigs receiving codeine were investigated for opiates. The RIA-investigations require a minimum of material; our routine procedures need only 50 mg of hair. No correlation existed between administered doses of opiates and their concentrations in hair of both human and experimental animals. By sectioning the hair, the approximate period of drug use in man could be detected. However, these findings could not be confirmed by the animal experiments. The growth rate of the hair, diffusion and adhesion processes may influence the transport of drugs along the hair. External contaminations and washing procedures were shown to increase or diminish the drug concentration of the samples.     

Interlaboratory comparison of quantitative determinations of drug in hair samples

Testing human hair for drugs of abuse is a relatively new technique which requires control before being fully accepted in Justice applications. A consensus procedure was recently proposed to the four French Laboratories performing hair analysis for opiates and cocaine. Results of two independent controls have shown that the laboratories have performed very well quantitatively, using the recommended method. In order to compare these results with those obtained by other procedures, one sample was sent to 15 laboratories concerned with the analysis of human hair for drugs of abuse in Germany, Italy, Spain, and United States. Results from this study have indicated that the French recommended method is in accordance with the general procedures.     

HAIRVEQ 2006: evolution of laboratories' performance after different educational actions

HAIRVEQ is a proficiency testing program for hair analysis of illicit drugs organized by the Istituto Superiore di Sanità (Rome, Italy) and the Institut Municipal d'Investigació Mèdica (Barcelona, Spain). The aim of the three exercises performed in 2006 was the evaluation of 32 laboratories' performance when analyzing the same hair sample containing opiates, cocaine and methadone, after carrying out some specific educational interventions. In the first round, the sample was sent to be analyzed following laboratory routine methodology. In the second round, standard operating procedures (SOP) for hair testing including sample preparation, method validation and qualitative and quantitative data evaluation, and an open hair sample for SOP training were also sent together with other hair samples including the one used for performance evaluation. After the second round, a workshop was held with participant laboratories to discuss methodological issues and interpretation of obtained results. An additional amount of open samples was distributed to the laboratories for implementing the SOPs. In the third round, the same unknown sample containing opiates, cocaine and methadone was resent for the final evaluation of laboratory performance. In the first round, 11 incorrect qualitative results (10 false negative and 1 false positive) were reported by seven laboratories (22%), in the second round, a reduction in the number of incorrect results was observed (4 false negatives and 1 false positive were reported by four laboratories, 13%) and in the third round, 5 false positives and 5 false negatives were reported by seven laboratories (22%). Concerning quantitative results, the scatter was similar between the three rounds and similar to the ones reported by other proficiency tests in hair analysis. More educational actions should be addressed to a group of laboratories, which did not yet show satisfying qualitative and quantitative results.     

生物检材中吗啡类生物碱的LC-MS／MS分析

目的针对滥用药物分析鉴定实践中亟待解决的问题,开展LC-MS/MS分析生物检材中吗啡类生物碱的应用研究。方法满足不同的鉴定需要,分别建立血液、尿液、唾液和头发等生物检材的样品前处理方法,确定同时分析海洛因、单乙酰吗啡、吗啡、可待因、乙酰可待因、二氢可待因酮和氢吗啡酮等吗啡类生物碱的LC-MS/MS方法。将方法应用于实际案例。结果所建立的方法对吗啡类生物碱分离良好。尿液稀释法、尿液提取法和头发中吗啡的最低检测限(LOD)分别为10ng/mL、0.01ng/mL和0.01ng/mg。结论所建立的方法简便、快速、特异性强、灵敏度高。目标物中加入二氢可待因酮和氢吗啡酮扩大了方法的实用范围。     

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.     

Simultaneous quantification of opiates, cocaine and cannabinoids in hair

The present paper describes a sensitive method developed in our laboratory for the simultaneous analysis of opiates (morphine, codeine and monoacetylmorphine), cocainics (cocaine and benzoylecgonine) and cannabinoids (Δ⁹-tetrahydrocannabinol and 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid) in hair samples. After decontaminating the sample with dichloromethane, two consecutive hydrolyses were performed in order to achieve the best conditions for extracting the three kinds of drugs from the protein matrix. First the opiate and cocainic compounds were extracted by means of a soft acidic hydrolysis with 0.1 N HCl at 50 °C overnight and organic solvent extraction at pH 9.2. The cannabinoids need a stronger basic hydrolysis with 11.8 N KOH for 10 min at laboratory temperature. After adding maleic acid, the cannabinoids were extracted with an organic solvent. The derivatization was carried out with heptafluorobutyric anhydride and hexafluoropropanol. Calibration curves were linear between 0.5–100 ng/mg of hair. Recovery and reproducibility were assured. The quantification limits ranged between 0.04–0.26 ng/mg of hair. Seventy hair samples from known drug abusers were cut into 1-cm segments and analyzed by this method. The ranges of measured concentrations (ng/mg) were 0.31–89 for cocaine, 0.1–5.76 for benzoylecgonine, 0.34–45.79 for morphine, 0.45–39.59 for codeine, 0.09–48.18 for monoacetylmorphine, 0.06–7.63 for THC and 0.06–3.87 for THC---COOH. The results of sectional analyses agreed with the self reported drug histories. The usefulness of this method is in assessing earlier drug consumption, and also at the same time obtaining a chronological profile of the consumption of these three types of drugs.     

Decontamination procedures for drugs of abuse in hair: are they sufficient?

This paper reviews the methods for decontaminating hair exposed to external solutions of drugs of abuse. Exposure of hair to cocaine at 1 μg/ml for 5 min is sufficient to contaminate hair, yet decontamination is a very slow process. Using externally contaminated hair, a number of decontamination procedures were attempted, and none removed all the contamination. The percentage of external contamination removed depended on the hair type, with thick black hair being the most resistant to decontamination. Hair treated by dying incorporated externally applied drugs differently, depending on the hair type. Thick black hair became more absorbent whereas thin brown hair became less absorbent. Kinetic wash criteria are evaluated for their ability/inability to determine if hair has been contaminated from external sources. A theoretical framework for the incorporation and removal of drugs from hair is discussed, and the hypothesis that inaccessible domains exist in hair which trap drugs is critically examined. The results presented in this paper strongly suggest that much more information on the decontamination of hair and the differentiation of exogenously and endogenously incorporated drugs is needed before hair analysis can be employed in most forensic applications. We propose that the radioactive tracer methods discussed herein are well suited for evaluating any new decontamination or extraction technique.     

Analysis of pain management drugs, specifically fentanyl, in hair: application to forensic specimens

This article discusses the immunoassay screening of pain management drugs, and the mass spectrometric confirmation of fentanyl in human hair. Hair specimens were screened for fentanyl, opiates (including oxycodone), tramadol, propoxyphene, carisoprodol, methadone, and benzodiazepines and any positive results were confirmed using gas chromatography or liquid chromatography with mass spectral detection. The specific focus of the work was the determination of fentanyl in hair, since autopsy specimens were also available for comparison with hair concentrations. Using two-dimensional gas chromatography with electron impact mass spectrometric detection, fentanyl was confirmed in four of nine hair specimens collected at autopsy. The accuracy of the assay at 10 pg/mg was 95.17% and the inter-day and intra-day precision was 5.04 and 13.24%, respectively (n=5). The assay was linear over the range 5-200 pg/mg with a correlation of r(2)>0.99. The equation of the calibration curve forced through the origin was y=0.0053x and the limit of quantitation of the assay was 5 pg/mg. The fentanyl concentrations detected were 12, 17, 490, and 1930 pg/mg and the results were compared with toxicology from routine post-mortem analysis. The screening of pain management drugs in hair is useful in cases where other matrices may not be available, and in routine testing of hair for abused drugs.     

Evaluation of the IDS One-Step™ ELISA kits for the detection of illicit drugs in hair

This work presents the validation of a new immunological assay, the One-Step™ enzyme-linked immunosorbent assay (ELISA) tests from International Diagnostic Systems Corp. for the screening of drugs of abuse (cannabis, amphetamines, opiates, and cocaine) in human hair, with subsequent GC–MS confirmation. After decontamination and segmentation into small pieces, 50 mg of hair sample were incubated in 1 ml of methanol during 16 h at 40 °C. A 100 μL aliquot was collected and evaporated to dryness in presence of 100 μL of methanol/hydrochloric acid (99:1, v/v) to avoid amphetamines loss. The dried extract was dissolved in 100 μL of the “sample and standard diluent” solution included in the kit. This solution was submitted to analysis according to the recommended instructions of the manufacturer. During the validation phase, GC–MS confirmations were conducted according to our fully validated and published methods for opiates, cocaine, cannabis, and amphetamines determinations in hair. In a last development step, these procedures were slightly modified to directly confirm ELISA results by GC–MS using the methanolic extract. Ninety-three specimens were simultaneously screened by the ELISA tests (103 for tetrahydrocannabinol (THC)) and confirmed by GC–MS. Twenty were found positive for cannabis (THC: 0.10–6.50 ng/mg), 21 for cocaine (0.50–55.20 ng/mg), 24 for opiates (6-acetylmorphine (6-AM): 0.20–11.60 ng/mg, MOR: 0.20–8.90 ng/mg, codeine (COD): 0.20–5.90 ng/mg), and 13 for amphetamines (AP: 0.20 and 0.27 ng/mg, methamphetamine (MAP): 0.30 and 1.10 ng/mg, methylenedioxymethamphetamine (MDMA): 0.22–17.80 ng/mg). No false negative results were observed according to the Society of Hair Testing's (SoHT) cutoffs (0.5 ng/mg for cocaine, 0.2 ng/mg for opiates and amphetamines, and 0.1 ng/mg for THC). The One-Step™ ELISA kits appear suitable due to their sensitivity and specificity for drug of abuse screening in hair. This technology should find interest in workplace drug testing or driving license regranting, especially when many samples have to be tested with a high rate of negative samples, as ELISA is an easy and high-throughput method.     

The detection of sedatives in hair and nail samples using tandem LC-MS-MS

Drug screening methods were developed to detect alprazolam, clobazam, clonazepam, diazepam, midazolam, oxazepam, temazepam, triazolam, zopiclone, and selected metabolites in human hair and nail samples employing liquid-liquid extraction and tandem liquid chromatography-mass spectrometry (LC-MS-MS). Hair and nail samples were obtained from patients who had recently discontinued or were currently prescribed one or more of the targeted drugs. Prazepam was used as the internal standard for all compounds. Some components in the hair matrix gave the same transitions as some of the analytes but did not compromise the analyses because their retention times differed from those for the target compounds. The analytical run time was 8-10min. Results of the hair analysis of a DFSA victim are also presented.     

Comparison of molecularly imprinted solid-phase extraction (MISPE) with classical solid-phase extraction (SPE) for the detection of benzodiazepines in post-mortem hair samples

This preliminary study compares the benzodiazepine results for 10 post-mortem scalp hair samples using a classical solid-phase extraction (SPE) and a molecularly imprinted solid-phase extraction (MISPE) system. The hair samples selected for testing were from drug-related deaths where a positive benzodiazepine blood result was obtained. Samples were decontaminated with 0.1% sodium dodecyl sulfate, distilled water and dichloromethane, incubated overnight in methanol/25% aqueous ammonium hydroxide (20:1), extracted by SPE or MISPE and subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Both extraction methods detected diazepam, nordiazepam, oxazepam, temazepam and nitrazepam in the samples. Diazepam was detected in a greater number of samples using MISPE due to both its lower limit of detection (LOD) and higher extraction recovery as a result of excellent molecular recognition of the template (diazepam) imparted by the imprinting process. The selective recognition of two diazepam analogues, nordiazepam and oxazepam, was demonstrated using MISPE since they were also detected in a greater number of samples. In contrast, another diazepam analogue, temazepam, was detected in a greater number of samples using SPE since the LOD using this extraction was lower than with MISPE. Nitrazepam was detected in one sample using both extraction methods. Overall the MISPE and SPE hair results were in good qualitative agreement. For the samples, where both extraction methods detected nordiazepam, temazepam and oxazepam, the concentrations were always higher for SPE. This is probably due to the MIP procedure producing extracts with fewer matrix interferences than the extracts produced using the classical SPE method. MISPE could be used as a complementary method to classical SPE for the analysis of benzodiazepine positive hair samples collected from chronic users.     

Analysis of drugs of abuse in hair: evaluation of the immunochemical method VMA-T vs. LC-MS/MS or GC-MS

Hair analysis is an elaborate and time-consuming multi-step process. The immunometric test VMA-T from Comedical has been evaluated as screening assay for hair analysis. From routine work, authentic samples were selected that were positive for opiates, cocaine, MDMA-type drugs, amphetamines, methadone or THC. These hair samples were investigated by LC-MS or GC-MS and the VMA-T procedure, respectively. Using the cut-off values recommended by the Society of Hair Testing, the VMA-T method discriminates with good sensitivity between negative and positive hair samples and is an expedient screening method for drugs in keratinized matrices such as hair. In order to save time and resources, the residue of the VMA-T extraction solution can be reused for confirmation analysis by LC-MS except for cocaine.     

Segmental analysis for cocaine and metabolites by HPLC in hair of suspected drug overdose cases

Hair samples of eight postmortem cases were analyzed in segments of 1 to 3 cm for cocaine, benzoylecgonine and cocaethylene. Samples were prepared for analysis by digestion in 0.1 M HCl and subsequent extraction with mixed-mode solid-phase extraction columns. Measurement was made by reversed-phase, narrow-bore HPLC and fluorescence detection using two laboratory-made internal standards. The concentrations were in the region of 0.29–316 ng/mg of hair for cocaine, 0.43–141 ng/mg of hair for benzoylecgonine and 0.93–1.83 ng/mg of hair for cocaethylene. All eight investigated cases had cocaine-positive segments. In six of the cases, all segments were positive, suggesting regular cocaine use and two showed in-between negative segments indicating an interruption or a change of the abuse intensity. The results showed a second, remarkable observation, i.e. enormous concentration differences (factor >150) for both cocaine and benzoylecgonine between the different subjects. Furthermore, interindividual cocaine/benzoylecgonine ratios ranged from 0.02 to 8.43. We believe these observations could in part be attributed to both some of the still existing limitations in the analytical approach(es), especially the mandatory hair washing steps, and in our still too limited knowledge of the hair incorporation processes. Nevertheless, in some cases, segmental analysis proved to be an important tool to distinguish, together with postmortem examination, deadly chronic abuse from single acute drug overdosage.     

Tandem mass spectrometry: a helpful tool in hair analysis for the forensic expert

The Bavarian State Bureau of Investigation in Munich has the exclusive responsibility for investigation of criminal acts. One considerable expertise is that of hair analysis. According to the legal system in Germany, there is a special interest when some clients' hair tested positive for illicit drugs. An accused with a lot of drugs in his hair will be treated as a supposed addict and will be guaranteed extenuating circumstances. The instrumentation used for hair analysis is a powerful analytical tool: a Varian 3400 gas chromatograph linked to a Finnigan Tandem-MS (TSQ 700). The methanol extraction method is used for the detection of illegal drugs and metabolites: amphetamine, methamphetamine, MDA, MDMA (ecstasy), MDE, MBDB, methadone, THC, EDDP (metabolite of methadone), cocaine, benzoylecgonine, cocaethylene, opiates (dihydrocodeine, codeine, heroin, 6-monoacetylmorphine, morphine, acetylcodeine). For the detection of 9-carboxy-THC by negative chemical ionization the hair sample is hydrolyzed under alkaline conditions. Solid-phase extraction is used for clean-up. The LOQ for the determination of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic-acid is 0.16 pg/mg hair. An unsurpassed combination for rendering an expert opinion based on hair analysis may be: a forensic expert using diligence and experience, coupled with the performance of a sophisticated analytical instrument.     

Influence of bleaching on the enantiomeric disposition of amphetamine-type stimulants in hair

The aim of this work was to study the influence of hair bleaching on the enantiomeric ratios of amphetamine-type stimulants (ATS), including amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyethylamphetamine. Hair specimens from 14 STA users were treated with a commercial bleaching product during 40 min. After alkaline digestion and solid-phase extraction of bleached and non-bleached hair, the STA enantiomers were derivatised with an in-house synthesised chiral reagent, the (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride. The diastereoisomers were quantified by GC/MS-NCI. The results showed that the concentrations of all enantiomers decreased in bleached hair in comparison with the non-treated hair (median values between 20 and 39%). The enantiomeric ratios of the STA in bleached hair were not significantly different from those determined in non-treated hair. Our findings pointed out that bleaching treatments decrease concentrations of STA in hair without influencing their enantiomeric ratios.     

Optimized conditions for simultaneous determination of opiates, cocaine and benzoylecgonine in hair samples by GC--MS

The present paper describes a qualitative and quantitative method for the simultaneous detection of opiates, cocaine and benzoylecgonine from human hair samples. Every step of the analytical procedure was studied to find the optimized conditions. Nine different incubation systems were examined. The influence of different pH values of samples on the isolation of analytes from the incubation media by Bond Elut cartridges and the stability of the compounds of interest in the different incubation media and conditions were investigated. The extracting power of different incubation media was studied as well. The phosphate buffer 0.1 N at pH 5 was chosen as the extraction medium in an optimized procedure for simultaneous determination of opiates, cocaine and benzoylecgonine in hair samples. The method developed was validated. Recoveries were 90% for morphine (M), 81% for 6-monoacetylmorphine (6-AM), 90% for codeine (CD), 86% for cocaine (C) and 90% for benzoylecgonine (BE). Relative standard deviation for inter-day precision was better than 12%. The limits of detection resulted as 0.05 ng/mg for M and C, as 0.08 for 6-AM and as 0.2 ng/mg for BE. Forty hair samples collected from drug abusers admitted to centers for detoxification treatment were analyzed obtaining 23 positive results for opiates and/or cocaine. Twelve hair specimens longer than 10 cm were analyzed following a sectional approach. In the six positive cases, it was interesting to find that the 6-AM/M ratio generally decreased for each sample from the proximal segment to the distal segments. Moreover, the 6-AM/M ratio was generally lower than 1 in the intermediate and distal segments.     

Segmental hair analysis in order to evaluate driving performance

On the 31st of July 2002 the Lombardy local government issued a memorandum, C.R. 35/SAN, providing "guidelines to investigate drugs of abuse addiction in order to judge driving performance". About hair samples, this memorandum advises that the proximal lock of 6 cm-length would be analysed for opiates, cocaine, cannabinoids, amphetamine and derivatives, divided into two segments of 3 cm each. The Local Medical Driving Licence Commissions (CML) can decide whether or not to enforce these instructions; from our survey it resulted that most CMLs do not abide by the memorandum, not requiring segmental analysis. The purpose of our study was to verify whether this procedural discordance could affect analytical results and, consequently, the evaluation of the subject's driving performance. We analysed hair samples taken from subjects who were requesting the renewal of their driving licence in our Laboratory during the period from 1 August 2002 to 31 December 2006. We divided samples into two groups: (1) samples previously analysed in one single segment which resulted positive for at least one analyte, but under the cut-off (0.5 ng/mg), were re-analysed in accordance with the guidelines; (2) samples previously processed following guidelines which resulted positive in one of the segments were newly analysed in a single segment. Comparing the new results with the original ones, an increase of positive results emerged in the first group. The second set of results fully supported the first ones. These results underscore the importance of the 35/SAN memorandum, so if the guidelines had been followed there would have been a larger amount of driving licence renewal denied.     

Hair analysis to document a clinical case of TCDD over-exposure

Dioxins and related compounds (furans) are persistent environmental contaminants that cause adverse biological effects. Their influence on humans is still unclear, except for accidental high-dose exposure. Chronic exposure to these compounds seems to be involved in cancer, endocrine disruption and neurobehavioral effects. For several years, a large concern about the potential health risks of dioxins is emerging in Europe and United States. The case of a 50-year-old man victim of an acute over-exposure to tetrachloro dibenzo-p-dioxin (TCDD or Seveso dioxin) is reported here with particular focus on hair investigations. The developed method involved the decontamination of the hair strand using picograde level methylene chloride, the homogenization of hair segments with scissors and their extraction in presence of 13C12-marked dioxin congeners under reflux of toluene using a Soxhlet, 8h at 130 degrees C. After reduction of the toluene fraction to 1 ml and addition of purification marker (37Cl4-2,3,7,8-TCDD), dioxins purification was achieved using three successive columns: silica, alumina/sodium sulfate and carbon/Celite columns. Finally, the toluene eluent was evaporated and the extract injected in the analytical system. After chromatographic separation, detection was achieved in single ion monitoring mode using a high resolution mass spectrometer operating in electron impact ionization mode (40 eV, minimal resolution of 10,000). The analysis of the first hair segment (0-6 cm) revealed the presence of 2,3,7,8-TCDD at 65 fg/mg when the distal one remained negative (LOQ=0.3 fg/mg). All other congeners (n=16) were in the range of those determined in the general population (0.62 and 0.89 fg/mg in the two hair segments, respectively). The extremely low dioxin levels generally found in hair specimens (low fg/mg range) lead us to analyze them using the very sensitive and specific gas chromatography-high resolution mass spectrometry apparatus. From the best of our knowledge, this is the first case of over-exposure to Seveso dioxin through hair analysis reported in the literature.     

新型微流体液相色谱(micro-LC)串联四极杆质谱检测头发中5种苯二氮卓类药物

目的建立了新型微流体液相色谱(micro-LC)串联四级杆质谱检测头发中5种苯二氮卓类药物(三唑仑,α-羟基咪达唑仑,咪达唑仑,α-羟基阿普唑仑,普拉西泮)。方法采用液-液萃取法(LLE)提取毛发样品,微流体液相色谱串联四级杆质谱检测。结果在回归系数大于0.99的情况下,目标分析物的良好线性。精密度和萃取效率分别在2.7%~17.1%和66.5%~120.5%之间。每个分析物的检出限和定量限分别为0.001~0.08 pg·mg^-1和0.0125~0.25pg·mg^-1。结论本方法与传统的液相色谱串联质谱法(LC/MS-MS)相比灵敏度出高2-10倍。本研究展示了新型微流体分离系统串联质谱方法的实用性,并为法医毒理学领域关于头发样本中痕量药物检测提供了一种新的检测手段。     

萃取方式对海洛因滥用者毛发中代谢物分析的影晌

目的比较液相萃取和同相萃取对毛发中海洛因毒品代谢物分析的影响。方法对海洛因吸食者毛发和空白添加标准品毛发经甲醇超声后的提取液分别进行液相萃取、同相萃取,然后进行衍生化和GC／MS—SIM检测。结果利用同相萃取法对添加6-单乙酰吗啡的毛发进行萃取和测试,6-单乙酰吗啡的回收率为32．O％,相对标准偏差（RSD）为2．4％;而液相萃取回收率为52．6％,相对标准偏差（RSD）为4．6％。结论固相萃取较之液液萃取,有更好的重复性,更少的杂质干扰和有机溶剂消耗等优势,但甲醇超声液需要挥干后才能进行同相萃取,而且6-单乙酰吗啡的水解率高。