Hair analysis and detectability of single dose administration of androgenic steroid esters

Detection of anabolic steroids in hair samples has been possible only in fatal cases or in cases of high-continuous dosages. In order to verify the possibility of detecting an acute administration, a sensitive and specific assay has been developed for the simultaneous determination of testosterone, nandrolone and some of their esters in hair. The analytes were extracted from finely cut hair with methanol-trifluoroacetic acid overnight. After the incubation, the mixture was evaporated to dryness, redissolved and extracted with hexane. The dried organic layer was silanised and analysed by GC-MS and GC-MS-MS. A sensitivity of at least 20 pg injected was obtained for all the analytes. In guinea pigs treated with a single intramuscular dose of 10 mg/kg nandrolone decanoate, neither nandrolone decanoate nor nandrolone were found in hair collected after 13 days, while both compounds were clearly detectable after four repeated doses (each dose every 3-4 days) of 20 mg/kg nandrolone decanoate. Neither nandrolone decanoate nor nandrolone could be detected in hair from a male healthy volunteer 1 month after treatment with 50 mg nandrolone decanoate, while his urine still tested highly positive for the main nandrolone metabolite (> 100 ng/ml). Testosterone esters could not be detected in hair of healthy subjects collected respectively 3, 2 and 1 month after a single intramuscular administration of 250 mg testosterone enanthate (five subjects), a single intramuscular coadministration of 25 mg testosterone propionate plus 110 mg testosterone enanthate (one subject), or a single oral administration of 120 mg testosterone undecanoate (three subjects). Otherwise, hair analysis revealed an increase of testosterone concentration corresponding to the period of treatment. Analysis of blood and urine samples confirmed the absorption of those compounds. At the sensitivity achieved by the present method, no detection of nandrolone, nandrolone decanoate nor testosteron esters in hair seems to be obvious after a single dose administration.     

Testing for anabolic steroids in hair from two bodybuilders.

Two male bodybuilders were recently arrested by the French customs in Strasbourg (France) in possession of 2050 tablets and 251 ampoules of various anabolic steroids. It was claimed that the steroids were for personal use and not for trafficing as suggested by the police. Urine and hair specimens were collected from both suspects to clarify the claims. Nandrolone, stanozolol, testosterone and their corresponding metabolites were identified in the urine of both subjects. After decontamination, the hair was hydrolyzed by sodium hydroxide in presence of deuterated internal standards. After extraction with ethyl acetate and silylation, the drugs were identified by GC-MS in the electron impact mode. Hair from both males were positive for nandrolone (196 and 260 pg/mg), testosterone (46 and 71 pg/mg) and stanozolol (135 and 156 pg/mg), clearly indicating steroids abuse. Although not yet recognized by the International Olympic Committee, hair analysis may be a useful adjunct to conventional drug testing in urine from athletes.     

Hair analysis of seven bodybuilders for anabolic steroids,ephedrine, and clenbuterol

Several bodybuilders, all winners of international competitions, were arrested for trafficking of a number of doping agents including anabolic steroids, ephedrine, beta-adrenergics, human chorionic gonadotropin, antidepressants, and diuretics. In accordance with the recent French law against doping, the judge asked to test seven bodybuilders to identify doping practices. Hair and urine specimens were collected for analysis. After decontamination, a 100 mg hair strand was pulverized in a ball mill, hydrolyzed, extracted, and derivatized to be tested by GC/MS for anabolic steroids, beta-adrenergic compounds, ephedrine, and other doping agents. Urine was analyzed for anabolic steroids and metabolites, beta-adrenergic compounds, ephedrine, and human chorionic gonadotropin, in addition to a broad spectrum screening with GC/MS. The following compounds were detected in urine: ephedrine (29 and 36 ng/mL, n = 2), clenbuterol (0.2 to 0.3 ng/mL, n = 3), norandrosterone (4.7 to 100.7 ng/mL, n = 7), norethiocholanolone (0.9 to 161.8 ng/mL, n = 6), stanozolol (1 to 25.8 ng/mL, n = 4), methenolone (2.5 to 29.7 ng/mL, n = 4), testosterone (3 to 59.6 ng/mL, n = 7), epitestosterone (1 to 20.4 ng/mL, n = 7) and ratio testosterone/epitestosterone >6 for four subjects (18.5 to 59.6). The following drugs were detected in hair: ephedrine (0.67 and 10.70 ng/mg, n = 2), salbutamol (15 to 31 pg/mg, n = 3), clenbuterol (15 to 122 pg/mg, n = 6), nandrolone (1 to 7.5 pg/mg, n = 3), stanozolol (2 to 84 pg/mg, n = 4), methenolone (17 and 34 ng/ml, n = 2), testosterone enanthate (0.6 to 18.8 ng/mg, n = 5), and testosterone cypionate (3.3 to 4.8 ng/mg, n = 2). These results document the doping practice and demonstrate repetitive exposure to anabolic compounds and confirm the value of hair analysis as a complement to urinalysis in the control of doping practice.     

Quantitative Analysis of Gamma‐Hydroxybutyrate at Endogenous Concentrations in Hair using Liquid Chromatography Tandem Mass Spectrometry

Abstract: A method capable of quantifying endogenous concentrations of gamma‐hydroxybutyrate (GHB) in human head hair was developed and validated using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Hair was digested under alkaline conditions, and GHB was isolated using liquid–liquid extraction. LC/MS/MS was performed using atmospheric pressure chemical ionization in the negative mode, multiple reaction monitoring, and deuterated internal standard (GHB‐D₆). Linearity was observed between 0.1 and 100 ng/mg GHB (R² = 1.000). The limits of detection and quantitation in human hair were 0.2 and 0.4 ng/mg, respectively. Accuracy at 2 ng/mg and 10 ng/mg was determined to be 97% and 94%, and intra‐assay CVs at these concentrations were 5.2% and 7.4% (n = 4). Beta‐hydroxybutyrate (BHB), alpha‐hydroxybutyrate, gamma‐butyrolactone, and 1,4‐butanediol did not produce an interference, and there was negligible ion suppression or enhancement from the matrix.     

GC/MS、GC/NPD法检测毛发中的氯胺酮

目的采用液-液萃取、衍生化和GC/MS、GC/NPD方法,进行毛发中氯胺酮定性定量分析。方法选择4-苯基丁胺为内标,毛发样本用NaOH、HCl及芳基硫酸酯酶/β-葡萄糖醛酸酶等3种方式进行水解,再进行衍生化后,采用GC/MS和GC/NPD方法定性定量分析。对不同水解和衍生化条件以及提取溶剂进行比较优化,并考察方法精密度、稳定性和检出限。结果方法的提取回收率大于95%,精密度和样品稳定性良好,日内和日间标准偏差小于6%;采用GC/NPD和GC/MS直接分析毛发中的氯胺酮,检出限为0.2ng/mg和2.0ng/mg,线性范围为10.0～250.0ng/mg,相关系数均大于0.99;采用酰化衍生化后分析,GC/NPD和GC/MS检出限分别提高至0.1ng/mg和0.2ng/mg。结论该方法回收率高、检测限低,可以用于毛发中氯胺酮的定性定量分析检验。     

Detection of thiopental and pentobarbital in head and pubic hair in a case of drug-facilitated sexual assault

The quali-quantitative determination of two barbiturates, thiopental and its metabolite pentobarbital, in head and pubic hair samples of a woman who had been sexually assaulted during hospitalisation, is reported. Hair was analysed by means of solid-phase microextraction (SPME) and gas chromatography-multiple mass spectrometry (GC-MS-MS), in chemical ionisation conditions. Thiopental and pentobarbital were found in three proximal head hair segments (sample 1A: 0.30 and 0.40 ng/mg; sample 1B: 0.20 and 0.20 ng/mg; sample 3: 0.15 and 0.20 ng/mg) and pubic hair sample. Two distal head hair segments were negative for both barbiturates. Despite the lack of collection and toxicological analysis of blood or urine samples within the hospital setting, analytical findings from hair revealed the use of the anaesthetic agent thiopental to sedate the victim quickly and deeply and commit sexual assault.     

Solid-phase microextraction for the determination of cocaine and cocaethylene in human hair by gas chromatography-mass spectrometry

A method for the simultaneous determination of cocaine (COC) and cocaethylene (CE) in human hair was developed, using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) as analytical technique to identify and quantify the drugs. Selected ion monitoring (SIM) mode was used to obtain higher sensitivity. The deuterated-labeled analogues were used as internal standards. The detector response was linear for the drugs studied over the range 0.4-15 ng/mg, with correlation coefficients higher than 0.995. The coefficients of variation oscillated between 0.65% and 14.18% and the accuracy was in the range from 0.73% to 11.20%. The limits of quantitation and detection were found to be acceptable. Finally, this method was applied to 15 hair samples from cocaine users, obtaining positive results in all cases. The mean concentrations were 5.39 ng/mg (range: 0.43-8.98 ng/mg) for cocaine and 1.11 ng/mg (range: 0.42-2.23 ng/mg) for cocaethylene.     

Simultaneous analysis of six amphetamines and analogues in hair, blood and urine by LC-ESI-MS/MS. Application to the determination of MDMA after low ecstasy intake

A rapid and sensitive method using LC-MS/MS triple stage quadrupole for the determination of traces of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"), 3,4-methylenedioxyethamphetamine (MDEA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair, blood and urine has been developed and validated. Chromatography was carried out on an Uptisphere ODB C(18) 5 microm, 2.1 mm x 150 mm column (Interchim, France) with a gradient of acetonitrile and formate 2 mM pH 3.0 buffer. Urine and blood were extracted with Toxitube A (Varian, France). Segmented scalp hair was treated by incubation 15 min at 80 degrees C in NaOH 1M before liquid-liquid extraction with hexane/ethyl acetate (2/1, v/v). The limits of quantification (LOQ) in blood and urine were at 0.1 ng/mL for all analytes. In hair, LOQ was <5 pg/mg for MA, MDMA, MDEA and MBDB, at 14.7 pg/mg for AP and 15.7 pg/mg for MDA. Calibration curves were linear in the range 0.1-50 ng/mL in blood and urine; in the range 5-500 pg/mg for MA, MDMA, MDEA and MBDB, and 20-500 pg/mg for AP and MDA. Inter-day precisions were <13% for all analytes in all matrices. Accuracy was <20% in blood and urine at 1 and 50 ng/mL and <10% in hair at 20 and 250 pg/mg. This method was applied to the determination of MDMA in a forensic case of single administration of ecstasy to a 16-year-old female without her knowledge during a party. She suffered from hyperactivity, sweating and agitation. A first sample of urine was collected a few hours after (T+12h) and tested positive to amphetamines by immunoassay by a clinical laboratory. Blood and urine were sampled for forensic purposes at day 8 (D+8) and scalp hair at day 60 (D+60). No MDMA was detected in blood, but urine and hair were tested positive, respectively at 0.42 ng/mL and at 22 pg/mg in hair only in the segment corresponding to the period of the offence, while no MDA was detectable. This method allows the detection of MDMA up to 8 days in urine after single intake.     

Determination of opiates and cocaine in hair using automated enzyme immunoassay screening methodologies followed by gas chromatographic-mass spectrometric (GC-MS) confirmation

The objective of this study was to develop a two-step strategy for analysis of opiates and cocaine in hair samples involving an immunological screening procedure followed by confirmation of results using gas chromatography-mass spectrometry (GC-MS). A semi-quantitative automated competitive enzyme-linked immunosorbent assay (ELISA) methodology using Oral Fluid Micro-Plate Enzyme Immunoassays (Orasure Technologies, Inc.) was developed and validated. Applicability was proven by analysis of authentic head hair samples from drug users (n=103) and from opiate associated fatalities (n=21). The optimum cutoff values for the ELISA tests were 0.1 ng cocaine-equivalents/mg hair and 0.05 ng morphine-equivalents/mg hair using a 50 mg hair sample. Both ELISA tests had a sensitivity of 100%, the specificity was 66% for cocaine-equivalents and 42% for morphine-equivalents. The intraassay precision was 11% for the cocaine and 3% for the opiates ELISA, while interassay precision was 12% for the cocaine and 4% for the opiates ELISA test. The actual analyte concentrations in the hair samples were determined using GC-MS and were between 0.04 and 5.20 ng/mg for heroin (HER), between 0.04 and 30.01 ng/mg for 6-monoacetylmorphine (MAM), between 0.03 and 11.87 ng/mg for morphine (MOR), between 0.02 and 1.84 ng/mg for codeine (COD), between 0.02 and 2.48 ng/mg for acetylcodeine (AC), between 0.01 and 21.37 ng/mg for cocaine (COC), between 0.03 and 10.51 ng/mg for benzoylecgonine (BE) and between 0.05 and 1.26 ng/mg for cocaethylene (CE). The automated ELISA tests were proven to be valid screening procedures for the detection of cocaine and opiates in hair as confirmed by GC-MS. Screening methods provide rapid and inexpensive automated pre-test procedures to detect drugs in hair or other matrices. For forensic purposes screening therefore represents an ideal complement to routinely applied GC-MS procedures.     

Detection of nandrolone metabolites in urine after a football game in professional and amateur players: a Bayesian comparison.

Nandrolone (19-nortestosterone) is a widely used anabolic steroid in sports where strength plays an essential role. Once nandrolone has been metabolised, two major metabolites are excreted in urine, 19-norandrosterone (NA) and 19-noretiocholanolone (NE). In 1997, in France, quite a few sportsmen had concentrations of 19-norandrosterone very close to the IOC cut off limit (2ng/ml). At that time, a debate took place about the capability of the human male body to produce by itself these metabolites without any intake of nandrolone or related compounds. The International Football Federation (FIFA) was very concerned with this problematic, especially because the World Cup was about to start in France. In this respect, a statistical study was held with all football players from the first and second divisions of the Swiss Football National League. All players gave a urine sample after effort and around 6% of them showed traces of 19-norandrosterone. These results were compared with amateur football players (control group) and around 6% of them had very small amounts of 19-norandrosterone and/or 19-noretiocholanolone in urine after effort, whereas none of them had detectable traces of one or the other metabolite before effort. The origin of these compounds in urine after a strenuous physical activity is still unknown, but three hypotheses can be put forward. First, an endogenous production of nandrolone metabolites takes place. Second, nandrolone metabolites are released from the fatty tissues after an intake of nandrolone, some related compounds or some contaminated nutritive supplements. Finally, the sportsmen may have taken something during or just before the football game.     

基质辅助激光解吸飞行时间质谱分析生物样品中甲基苯丙胺

目的建立尿样和头发中甲基苯丙胺的基质辅助激光解吸飞行时间质谱（matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS）分析方法。方法尿样采用液液提取,头发经0.1mol/L盐酸水解后采用液液提取,以碳纳米管为基质应用MALDI-TOF-MS法检测。结果尿样中甲基苯丙胺的最低检测限（LOD）为0.5μg/mL,线线范围为线性范围为0.5～100μg/mL（R2=0.9970）;毛发中甲基苯丙胺的最低检测限（LOD）为0.4ng/mg,线性范围为0.4～60ng/mg（R2=0.9976）,对送检案例中尿样和头发检材进行检测,效果良好。结论本方法适用于尿样和头发中甲基苯丙胺的分析,与传统气相色谱质谱联用和液相色谱-质谱联用相比,分析速度更快,适合大批量样品同时分析。     

Hair as a specimen to document tetramethylene disulfotetramine exposure

Tetramethylene disulfotetramine (tetramine) is a rodenticide that has been banned for many years in China. Since 2005, inhabitants of a village in the Henan Province have been suffering from grand mal seizures. To investigate the possibility of tetramine as the cause, we developed a method to determine tetramine in human hair. Sample preparation involved external decontamination, frozen pulverization, and ultrasonication in 2 mL ethyl acetate in the presence of cocaine-d3 as an internal standard. The method exhibited good linearity; calibration curve was linear over a range of 0.1-20 ng/mg hair. The limit of detection for the assay was 0.05 ng/mg hair. Except for one subject (No. 4), all head and pubic hair samples were positive for tetramine. The concentrations of tetramine in pubic hair were significantly higher than those in the same subjects' head hair samples. Because of a long retention in body, segmental head hair analysis cannot provide an accurate exposure history of tetramine in the body.     

Simultaneous analysis of six amphetamines and analogues in hair, blood and urine by LC-ESI-MS/MS: Application to the determination of MDMA after low Ecstasy intake

A rapid and sensitive method using LC-MS/MS triple stage quadrupole for the determination of traces of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”), 3,4-methylenedioxyethamphetamine (MDEA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair, blood and urine has been developed and validated. Chromatography was carried out on an Uptisphere ODB C₁₈ 5 μm, 2.1 mm × 150 mm column (Interchim, France) with a gradient of acetonitrile and formate 2 mM pH 3.0 buffer. Urine and blood were extracted with Toxitube A^® (Varian, France). Segmented scalp hair was treated by incubation 15 min at 80 °C in NaOH 1 M before liquid–liquid extraction with hexane/ethyl acetate (2/1, v/v). The limits of quantification (LOQ) in blood and urine were at 0.1 ng/mL for all analytes. In hair, LOQ was <5 pg/mg for MA, MDMA, MDEA and MBDB, at 14.7 pg/mg for AP and 15.7 pg/mg for MDA. Calibration curves were linear in the range 0.1–50 ng/mL in blood and urine; in the range 5–500 pg/mg for MA, MDMA, MDEA and MBDB, and 20–500 pg/mg for AP and MDA. Inter-day precisions were <13% for all analytes in all matrices. Accuracy was <20% in blood and urine at 1 and 50 ng/mL and <10% in hair at 20 and 250 pg/mg. This method was applied to the determination of MDMA in a forensic case of single administration of ecstasy to a 16-year-old female without her knowledge during a party. She suffered from hyperactivity, sweating and agitation. A first sample of urine was collected a few hours after (T + 12 h) and tested positive to amphetamines by immunoassay by a clinical laboratory. Blood and urine were sampled for forensic purposes at day 8 (D + 8) and scalp hair at day 60 (D + 60). No MDMA was detected in blood, but urine and hair were tested positive, respectively at 0.42 ng/mL and at 22 pg/mg in hair only in the segment corresponding to the period of the offence, while no MDA was detectable. This method allows the detection of MDMA up to 8 days in urine after single intake.     

Evaluation of the One-Step ELISA kit for the detection of buprenorphine in urine, blood, and hair specimens

A solid-phase enzyme immunoassay involving microtiter plates was recently proposed by International Diagnostic Systems corporation (IDS) to screen for buprenorphine in human serum. The performance of the kit led us to investigate its applicability in other biological matrices such as urine or blood, and also hair specimens. Low concentrations of buprenorphine were detected with the ELISA test and confirmed by HPLC/MS (buprenorphine concentrations measured by HPLC/MS: 0.3 ng/mL in urine, 0.2 ng/mL in blood, and 40 pg/mg in hair). The intra-assay precision values were 8.7% at 1 ng/mL of urine (n = 8), 11.5% at 2 ng/mL in serum (n = 8), and 11.5% at 250 pg/mg of hair (n = 8), respectively. The immunoassay had no cross-reactivity with dihydrocodeine, ethylmorphine, 6-monoacetylmorphine, pholcodine, propoxyphene, dextromoramide, dextrometorphan at 1 and 10 mg/L, or codeine, morphine, methadone, and its metabolite EDDP. A 1% cross-reactivity was measured for a norbuprenorphine concentration of 50 ng/mL. Finally, the immunoassay was validated by comparing authentic specimens results with those of a validated HPLC/MS method. From the 136 urine samples tested, 93 were positive (68.4%) after the ELISA screening test (cutoff: 0.5 ng/mL) and confirmed by HPLC/MS (buprenorphine concentrations: 0.3-2036 ng/mL). From the 108 blood or serum samples screened, 27 were positive (25%) after the ELISA test with a cutoff value of 0.5 ng/mL (buprenorphine concentrations: 0.2-13.3 ng/mL). Eighteen hair specimens were positive (72%) after the screening (cutoff: 10 pg/mg) and confirmed by LC/MS (buprenorphine concentrations: 40-360 pg/mg). The ELISA method produced false positive results in less than 21% of the cases, but no false negative results were observed with the immunological test. Four potential adulterants (hypochloride 50 mL/L, sodium nitrite 50 g/L, liquid soap 50 mL/L, and sodium chloride 50 g/L) that were added to 10 positive urine specimens (buprenorphine concentrations in the range 5.3-15.6 ng/mL), did not cause a false negative response by the immunoassay.     

生物检材中苯丙胺类兴奋剂和氯胺酮的LC-MS/MS分析

目的建立生物检材中苯丙胺类兴奋剂和氯胺酮的液相色谱-串联质谱(LC-MS/MS)分析方法。方法生物检材包括血液、尿液和毛发,采用稀释法和液液提取的前处理方法,应用两个不同的液相柱,优化LC-MS/MS分析方法,并考察了血液和尿液基质的离子抑制作用。结果同时分析苯丙胺和MDA,液相1在3m in内完成,液相2可用于确认分析或复杂基质分离。尿液稀释法检材用量少,前处理简便快速。毛发中苯丙胺类兴奋剂和氯胺酮的最低检测限(LOD)为0.005~0.05ng/mg。对送检案例检材产妇头发和胎毛进行苯丙胺类兴奋剂和氯胺酮的分析。结论本方法可用于生物检材中苯丙胺类兴奋剂和氯胺酮的同时分析,血、尿等生物检材的离子抑制作用是影响本方法灵敏度的主要原因。     

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。     

Diagnosis of chronic alcohol consumption. Hair analysis for ethyl-glucuronide

This paper describes a procedure for the detection and quantification of ethyl-glucuronide (EtG) in hair samples. During method development the efficacy of extraction of EtG from hair was compared in four extraction methods: (a) methanol; (b) methanol:water (1:1); (c) water; and (d) water:trifluoroacetic acid (9:1). In addition, three derivatizing agents were compared as well: N,O-bistrimethylsilyl-trifluoroacetamide (BSTFA): trimethylchlorosilane (TMCS) (99:1), pentafluoropropionic anhydride (PFPA) and heptafluorobutyric anhydride (HFBA). Water was found to be the best extracting solvent and PFPA the best derivatizing agent. Both provided the highest recoveries, with cleaner extracts and more stable derivatives. The final method is as follows: about 100mg of hair are sequentially washed with water and acetone. The decontaminated sample is finely cut with scissors, then the deuterated internal standard (EtG-d5) and 2 mL of water are added. After sonication for 2 h, the sample is maintained at room temperature overnight. Derivatization is performed with PFPA. Derivatives are injected into a GC-MS system in the electronic impact mode. The method shows linearity over the range of concentrations from 0.050 to 5 ng/mg. Detection and quantification limits are 0.025 and 0.050 ng/mg, respectively. Mean recoveries for the three studied concentrations (low, medium and high) are higher than 87%. The coefficients of variation in intra- and inter-assay precision are always lower than 7%. The method is being routinely applied in our lab for the diagnosis of chronic alcohol consumption.     

Simultaneous hair testing for opiates, cocaine, and metabolites by GC-MS: a survey of applicants for driving licenses with a history of drug use

A sensitive GC-MS method for the simultaneous determination of opiates, cocaine, and metabolites in hair at a cut-off level of 0.1 ng/mg was adopted to assess past exposure to these drugs in applicants for driving licenses with a history of drug use. The sampling protocol consisted of collection of one hair (sample A, 5-cm length) and one urine sample. When hair and urine (EMIT Syva, cut-off levels: 0.3 mg/l for opiates, 0.15 mg/l for cocaine, GC-MS confirmation of positives) were both positive or negative the protocol was concluded. In the other cases, the assessment of 'current exposure' to drugs was carried out, in order to avoid seriated random urinalysis, by collecting a second hair sample (sample B) 6 weeks later and analysing the proximal 1-cm segment. Out of the 214 'A' hair samples analyzed, 14 (6.5%) tested positive for morphine and/or 6-acetylmorphine (6AM), and 26 (12%) for cocaine and/or benzoylecgonine (BE), whereas none of the samples tested positive for both drugs. Levels between 0.1 and 1 ng/mg of the single analytes were found in eight out of the 14 morphine-6AM positives (57%) and in 18 out of the 26 cocaine-BE positives (69%). The time course of positive cases showed a progressive decrease of morphine-6AM positives and a corresponding increase of cocaine-BE positives within the study period September 1995-February 1999. No cases with positive urine and negative hair were observed. Among the 40 positive cases, seven (four and three for opiates and cocaine, respectively) were found to be 'currently exposed to drug', four by urinalysis (three and one) and three by analysis of the hair sample B (1 and 2).     

Analysis of ketamine and norketamine in urine by automatic solid-phase extraction (SPE) and positive ion chemical ionization-gas chromatography-mass spectrometry (PCI-GC-MS)

Ketamine (KT) is widely abused for hallucination and also misused as a "date-rape" drug in recent years. An analytical method using positive ion chemical ionization-gas chromatography-mass spectrometry (PCI-GC-MS) with an automatic solid-phase extraction (SPE) apparatus was studied for the determination of KT and its major metabolite, norketamine (NK), in urine. Six ketamine suspected urine samples were provided by the police. For the research of KT metabolism, KT was administered to SD rats by i.p. at a single dose of 5, 10 and 20mg/kg, respectively, and urine samples were collected 24, 48 and 72 h after administration. For the detection of KT and NK, urine samples were extracted on an automatic SPE apparatus (RapidTrace, Zymark) with mixed mode type cartridge, Drug-Clean (200 mg, Alltech). The identification of KT and NK was by PCI-GC-MS. m/z238 (M+1), 220 for KT, m/z 224 (M+1), 207 for NK and m/z307 (M+1) for Cocaine-D(3) as internal standard were extracted from the full-scan mass spectrum and the underlined ions were used for quantitation. Extracted calibration curves were linear from 50 to 1000 ng/mL for KT and NK with correlation coefficients exceeding 0.99. The limit of detection (LOD) was 25 ng/mL for KT and NK. The limit of quantitation (LOQ) was 50 ng/mL for KT and NK. The recoveries of KT and NK at three different concentrations (86, 430 and 860 ng/mL) were 53.1 to 79.7% and 45.7 to 83.0%, respectively. The intra- and inter-day run precisions (CV) for KT and NK were less than 15.0%, and the accuracies (bias) for KT and NK were also less than 15% at the three different concentration levels (86, 430 and 860 ng/mL). The analytical method was also applied to real six KT suspected urine specimens and KT administered rat urines, and the concentrations of KT and NK were determined. Dehydronorketamine (DHNK) was also confirmed in these urine samples, however the concentration of DHNK was not calculated. SPE is simple, and needs less organic solvent than liquid-liquid extraction (LLE), and PCI-GC-MS can offer both qualitative and quantitative information for urinalysis of KT in forensic analysis.     

Use of headspace solid-phase microextraction (HS-SPME) in hair analysis for organic compounds

Headspace solid phase microextraction (HS-SPME) has advantages of high purity of the extract, avoidance of organic solvents and simple technical manipulation and can be used in combination with gas chromatography-mass spectrometry (GC-MS) in the hair analysis of a number of drugs. HS-SPME coupled with the hydrolysis of the hair matrix by 4% sodium hydroxide in the presence of excess sodium sulphate and of a suitable internal standard proved to be a convenient one-step method for the measurement of many lipophilic basic drugs such as nicotine, amphetamine derivatives, local anaesthetics, phencyclidine, ketamine, methadone, diphenhydramine, tramadol, tricyclic antidepressants and phenothiazines. Detection limits were between 0.05 and 1.0 ng/mg. From spiked 10-mg hair samples absolute recoveries between 0.04 and 5.7% were found. These recoveries decreased considerably if larger sample amounts were used, perhaps due to increased drug solubility in the aqueous phase or to elevated viscosity in the presence of dissolved hair proteins. Because of the phenolic hydroxyl group a change of pH after alkaline hair digestion (by adding excess orthophosphoric acid) was necessary for the detection of delta 9-tetrahydrocannabinol (delta 9-THC), cannabinol (CBN) and cannabidiol (CBD) by HS-SPME. Nevertheless, the detection limits were such that only CBN could be detected in hair of a consumer. Clomethiazole, a compound hydrolysed in alkali, was measured by HS-SPME after extraction with aqueous buffer. The detection limit was 0.5 ng/mg. Cocaine could not be detected by HS-SPME. The application of HS-SPME to hair samples from several forensic and clinical cases is described.