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.     

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.     

Improved gas chromatography-negative ion chemical ionization tandem mass spectrometric method for determination of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in hair using mechanical pulverization and bead-assisted liquid-liquid extraction

A gas chromatography-negative ion chemical ionization tandem mass spectrometric (GC-NCI-MS/MS) method was developed and validated for the determination of 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in human hair. After decontamination, hair samples were weighed (25mg), mechanically pulverized with a bead mill, and incubated in 0.7 mL of 1.0M sodium hydroxide at 95 °C for 30 min. Bead-assisted liquid-liquid extraction was performed with n-hexane:ethyl acetate (9:1, v/v), a method developed in our laboratory. The extract was evaporated to dryness, derivatized with pentafluoropropanol and pentafluoropropionic anhydride, and analyzed by GC-MS/MS in the negative ion chemical ionization mode using methane as the reagent gas. The linear ranges were 0.05-10.0 pg/mg for THC-COOH with the coefficient of determination (r(2) = 0.9976). The intra-day and inter-day precisions were within 1.7 and 13.8%, respectively. The intra-day and inter-day accuracies were -4.8 to 10.0% and -3.9 to 3.8%, respectively. The limit of detection and quantification were 0.015 and 0.05 pg/mg, respectively. The recoveries were in the range of 79.4-87.1%. The results indicate that the proposed method is simple, rapid, accurate, and precise for determination of THC-COOH in hair. The method identified THC-COOH in hair specimens from suspected marijuana abusers.     

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.     

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.     

Determination of ethyl glucuronide in human hair by SPE and LC-MS/MS

A method for the sensitive and selective determination of ethyl glucuronide (EtG) in hair has been developed using solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Washed and cut hair segments were extracted by ultrasonication (3h, 50 degrees C) and the extracts were cleaned-up with aminopropyl SPE columns. LC-MS/MS analysis was performed using a polar-endcapped phenyl-hexyl-RP-phase with negative mode electrospray ionisation (ESI) using a triple quadrupole mass spectrometer (Sciex API 365) with a turboionspray source and post-column addition of acetonitrile for enhanced sensitivity. The MS/MS transitions monitored were m/z 221 -->75 for EtG and 226 -->75 for D(5)-EtG as an internal standard. The method was selective and sensitive, with a detection limit of 51 pg/mg hair at a signal-to-noise ratio of 3:1. The mean recovery was 96%, with an intra- and inter-day precision of less than 11.7% at a concentration of 200 pg/mg. The linearity was assessed in the range of 25-2000 pg/mg hair, with a correlation coefficient of 0.997. The method was successfully applied to 97 human hair samples which were taken at autopsies from persons with known alcoholism or were obtained from alcoholics who were hospitalized for ethanol withdrawal, from social drinkers and from children having not consumed any alcohol. Although, approximately two-third of the alcoholics showed EtG concentrations in hair of higher than 51 pg/mg (up to >4000 pg/mg), in one-third the EtG concentration was below the detection limit. However, only in one of five hair samples of "social drinkers", the EtG concentration was above the detection limit (51 pg/mg). No EtG has been detected in the hair of children. These investigations demonstrate that heavy alcohol consumption may be but not necessarily has to be detectable by EtG analysis in hair.     

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 degrees C. A 100 microL aliquot was collected and evaporated to dryness in presence of 100 microL of methanol/hydrochloric acid (99:1, v/v) to avoid amphetamines loss. The dried extract was dissolved in 100 microL 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.     

Development and validation of a liquid chromatography-tandem mass spectrometry assay for hair analysis of atomoxetine and its metabolites: Application in clinical practice

Atomoxetine (ATX) is a potent inhibitor of the noradrenaline reuptake transporter approved since 2002 for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children, adolescents, and adults as alternative treatment to methylphenidate. A procedure based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of ATX and its main metabolites (4-hydroxyatomoxetine - 4 hydroxyATX - and N-desmethylatomoxetine - des-methylATX) in hair of one treated child and five treated adolescents. Since hair samples can be easily collected without the need for specials skills and exposing a patient to discomfort, hair testing of ATX and eventually of its metabolites should be useful, especially in case of pediatric patients, to check compliance in a wider time-window. After addition of duloxetine as internal standard, hair samples were overnight digested with 2ml 1M NaOH at 45°C. Then, analytes were extracted from alkaline solution with two different 2ml aliquots of tert-butyl methyl ether. Chromatographic separation was achieved at ambient temperature using a reverse-phase column and a mobile phase of 40% of water-60% 5mM ammonium acetate, 50mM formic acid, 4mM trifluoroacetic acid in acetonitrile-water (85:15, v/v). The mass spectrometer was operated in positive ion mode using multiple reaction monitoring. The method was linear over the concentration range 0.2-50ng/mg hair for the all analytes under investigation, with an intra- and inter-assay imprecision and inaccuracy always less than 20% and an analytical recovery between 33.1% and 76.1%, depending on the considered analyte. Only ATX and 4-hydroxyATX were detected in hair samples with concentrations varying from 0.2 to 2.0ng/mg hair and from 0.3 to 1.0ng/mg, respectively. Notwithstanding the absence of any dose-hair concentration relationship, hair monitoring of ATX and concomitant medications commonly administrated in ADHD children and adolescents can be crucial in verifying long-term compliance to prescribed medication in individuals displaying a non negligible tendency to refuse drugs and to lie on the adherence to therapy as a specific symptom of the disease.     

Ethyl glucuronide in human hair after daily consumption of 16 or 32 g of ethanol for 3 months

The overall objectives of the study were to develop a sensitive method for ethyl glucuronide (EtG) determination in hair and then investigate if a low or moderate intake of ethanol could be differentiated from total abstinence. Forty-four subjects were included in the study, 12 males (7 drinkers and 5 abstinent) and 32 females (14 drinkers and 18 abstinent). The study lasted 3 months and the female drinkers consumed one glass (16 g of ethanol) and the males consumed two glasses (32 g of ethanol) of wine (13.5-14%) daily. Hair samples were collected as close as possible above the skin and the proximal 2 cm were analyzed for EtG. Hair was cut into pieces of about 0.5 cm length and washed before incubation overnight in water and then extracted on Clean Screen EtG Carbon columns. The LC/MS/MS system consisted of a Waters ACQUITY UPLC connected to an API 4000 triple quadrupole instrument. Two transitions for EtG and one for the internal standard EtG-D(5) were measured. The method was linear from 60 to 10,000 pg/sample. Imprecision studies were performed at three levels as well as with an authentic sample. Total imprecision was 16% at 200 pg/sample, 8% at 1000 pg/sample, 6% at 8000 pg/sample and 13% at 29 pg/mg in the authentic sample. Of those who drank two glasses of wine every day, four had measurable amounts of EtG in their hair (5-11 pg/mg), and in only one of the females drinking one glass of wine EtG was quantified (3 pg/mg). Among the 23 abstinent subjects two had traces of EtG in the hair. We conclude that persons who ingested 16 or 32 g of ethanol daily for 3 months presented with low concentrations of EtG in hair, well below the proposed threshold for overconsumption set at 30 pg/mg. In addition, none of those who ingested 16 g/day had concentrations over the proposed abstinence threshold of 7 pg/mg.     

Doping control for beta-adrenergic compounds through hair analysis

An original procedure was developed to simultaneously test beta2-agonists (salbutamol and clenbuterol) and beta-blockers (atenolol, acebutolol, pindolol, betaxolol, propranolol, timolol, sotalol, metoprolol, tertatolol, bisoprolol, labetalol and oxprenolol) in both human and animal hair. After decontamination with methylene chloride (2 times, 2 min), a 200 mg hair strand is pulverized in a ball mill. Then, a 100 mg portion is incubated overnight in 2 mL 0.1 N HCl, at 56 degrees C, in the presence of carteolol, which was used as an internal standard. After neutralization of the acid phase with 0.1 N NaOH, a 2 mL bicarbonate buffer (pH 8.6) is added to the preparation, which is then purified by solid-phase extraction with Isolute C18 columns. Drugs are derivatized using a mixture of trimethylboroxine-ethyl acetate for 15 min at 80 degrees C to form methaneboronate derivatives. Drugs are detected using GC/MS on an HP 6890-5973 system. A 4 microL portion of the derivatized extract is injected using a pulsed mode in a 30 m HP5 MS capillary column. Linearity was observed for all compounds in the range 25 pg/mg to 10 ng/mg. Limits of detection were in the range 2 to 10 pg/mg. At 1 ng/mg, recoveries were in the range from 37 to 100%, with a within-run precision of 5.9 to 14.1% (n = 8). The application of the method can be documented by the following examples: (1) Hair from asthmatic patients (n - 11), including two cases of asthma deaths, tested positive for salbutamol in the range of 27 to 210 pg/mg. (2) A 24-year-old swimmer who tested positive in urine for salbutamnol denied the results. Hair analysis confirmed salbutamol exposure, with a concentration of 71 pg/mg. (3) A shooting specialist was assumed to chronically use metoprolol (100 mg/daily during some periods). Hair concentration of metoprolol was 8.41 ng/mg. (4) An archery specialist was assumed to chronically use sotalol (80 mg/daily, during some periods). Hair concentration of sotalol was 261 pg/mg. (5) Hair from two calves revealed chronic exposure to clenbuterol, which was used to increase the mass of the animals at a concentration of 30 and 48 pg/mg.     

Determination of benzodiazepines in human hair by on-line high-performance liquid chromatography using a restricted access extraction column.

A method is described for the identification of five frequently prescribed benzodiazepines (BZD) (clonazepam, diazepam, flunitrazepam, midazolam and oxazepam) in human hair samples by reversed phase HPLC, following on-line simple enrichment and clean-up on a restricted access extraction column. 50mg of powdered hair were incubated (2h at 45 degrees C) after sonication (1h) in 1 ml of the following solution (methanol:ammonia, 97.5/2.5, v/v). The aliquot was centrifuged and the methanolic phase transferred to a conical tube and evaporated under a gentle stream of nitrogen. The residue was reconstituted by adding 100 microl of a mixture of phosphate buffer (20mM, pH=2.2) and acetonitrile (94/6, v/v). A total of 80 microl were injected into the system with the column switching technique. The pre-column or clean-up column was washed with phosphate buffer pH=7.2. The drugs retained on the pre-column were then eluted in the back-flush mode and separated on a C(8) semi micro column, Lichrospher select B, 125 mm x 3 mm. The BZD were determined by a photodiode-array detector at 254 nm, using reference data (retention time and UV spectra) stored in a personal library. The method showed excellent linearity between 0.5 and 20 ng/mg of hair for clonazepam, flunitrazepam and midazolam and between 0.5 and 100 ng/mg of hair for diazepam and oxazepam. Finally, the present method has been applied to a number of forensic cases in our laboratory.     

Determination of cathinone,cathine and norephedrine in hair of Yemenite khat chewers

A sensitive and reproducible method for the quantitative determination of cathinone (CTN), norpseudoephedrine (NPE, cathine) and norephedrine (NE) from hair was developed. The compounds were extracted for 4 hours with phosphate buffer pH 2.0, followed by a standard solid phase extraction procedure on a mixed phase column, derivatization with heptafluorobutyric anhydride (HFBA) and GC-MS separation and quantification using D(3)-ephedrine (D(3)-E) and alpha-aminoacetophenone (AAP) as the internal standards. The diastereomers NPE and NE were satisfactorily separated. In the validation, the limits of detection and of quantification were determined at 0.03-0.08 ng/mg and 0.10-0.24 ng/mg, respectively and the interday standard deviation was between 10 and 15%. The method was applied to hair samples of 24 Yemenite khat chewers. All three compounds were detected in 23 of these cases. The concentrations ranged from 0.57 to 23.9 ng/mg for NPE, 0.19-25.0 ng/mg for NE and 0.11-22.7ng/mg for CTN. A highly significant correlation was found between the self-reported data about the khat consumption habits of the volunteers (4-56h chewing per week) and the concentrations of norephedrine and norpseudoephedrine in hair.     

固体进样杆-GC/MS-SIM技术检验头发中6-单乙酰吗啡

目的建立固体进样杆-GC/MS-SIM技术,检测人头发中毒品海洛因的主要代谢产物6-单乙酰吗啡的方法。方法样品头发洗净剪碎后,经MSTFA衍生化,采用GC/MS-SIM定性定量分析。结果 6-单乙酰吗啡在浓度0.1ng/mg～18.0ng/mg范围内线性相关系数为0.9972,方法检出限为0.1ng/mg。结论本方法头发使用量小,准确度、精密度良好、灵敏度高,是高效、快速、实用的测定头发中6-单乙酰吗啡的方法。     

头发中5-MeO-DiPT等12种新型色胺类致幻剂的UPLC-MS/MS分析方法研究

目的建立同时分析头发中5-MeO-DiPT等12种新型色胺类致幻剂的超高效液相色谱-串联质谱(UPLC-MS/MS)方法。方法以赛洛西宾-d4和赛洛新-d10为内标,20 mg头发样品加入1 mL提取液(含内标2 ng/mL的0.1%甲酸水溶液)后冷冻研磨,离心取上清液,经Waters Acquity^TM UPLC HSS T3色谱柱分离,采用多反应监测模式同时测定12种新型色胺类致幻剂。结果头发样品中12种色胺类新精神活性物质的检出限为1~10pg/mg,定量限为3~50pg/mg。在相应浓度范围内,12种色胺类新精神活性物质均具有良好的线性关系,相关系数大于0.99。本方法准确度为91.3%~113.5%,日间和日内精密度(RSD)均小于15%,回收率大于80%,无明显基质效应。结论该方法前处理简单、灵敏度高、选择性好,适用于法医毒物分析中新型色胺类致幻剂的鉴定。     

Detection of THCCOOH in hair by MSD-NCI after HPLC clean-up

Regular consumption of cannabis can easily be detected by examination of hair for tetrahydrocannabinol, cannabinol, and cannabidiol. Although several studies have demonstrated that after contamination with smoke or treatment with THC containing shampoos THC is not detectable, or only in small traces, the detection of 11-nor-9-carboxy-delta 9-tetrahydrocannabinol (THCCOOH) should be offered to prove the consumption and metabolisation of THC. Up to now this confirmation was only available using tandem MS techniques combined with negative chemical ionisation. A new method using a normal quadrupole GC/MS is described. The lack of expensive instruments has to be paid for by a costly and time consuming extraction and clean-up. After the sample has been digested by 2 M NaOH at 95 degrees C and the neutralised liquid has been extracted with a mixture of n-hexane and ethyl acetate the dried residue is reconstituted in acetonitrile-methanol-0.01 M sulfuric acid (49:21:30, v/v/v) and the cannabinoids separated by HPLC. Each fraction is collected over 1 min. Another extraction with n-hexane-ethyl acetate is followed by evaporation, derivatisation, and GC/MS determination. The calibration with THCCOOH spiked hair led to a LOD of 0.3 pg/mg and a LOQ of 1.1 pg/mg.     

Use of solid-phase microextraction (SPME) for the determination of methadone and EDDP in human hair by GC-MS

Solid-phase microextraction (SPME) is a new extraction technique with many advantages: small sample volume, simplicity, quickness and solvent-free. It is mainly applied to environmental analysis, but is also useful for the extraction of drugs from biological samples. In this paper the use of SPME is proposed for the determination of methadone and its main metabolite EDDP in hair by GC-MS. The hair samples were washed, cut into 1-mm segments, and incubated with Pronase E for 12 h. A 100-micron polydimethylsiloxane (PDMS) film fibre was submerged for 30 min in a diluted solution of the hydrolysis liquid (1:4 with borax buffer) containing methadone-d3 and EDDP-d3 as internal standards. Once the microextraction was concluded the fibre was directly inserted into the CG injection port. Linearity was found for methadone and EDDP in the range studied, 1.0-50 ng/mg hair, with correlation coefficients higher than 0.99. Interassay relative standard deviation (R.S.D) was determined to be less than 13.30% for methadone and less than 8.94% for EDDP, at 3.0 and 30.0 ng/mg. Analytical recoveries were close to 100% for both compounds on spiked samples. The method was applied to the analysis of real hair samples from eight patients of a methadone maintenance programme. The concentration of methadone in hair ranged from 2.45 to 78.10 ng/mg, and for EDDP from 0.98 to 7.76 ng/mg of hair.     

Evidence of mephedrone chronic abuse through hair analysis using GC/MS

Mephedrone is a synthetic derivative of cathinone which is becoming more common on the recreational drug market. Several intoxications following mephedrone abuse have been reported though published papers have focused essentially on analytical approaches for biological fluids and only one has involved a hair sample. After the development and validation of a new method, the first series of positive results for mephedrone in hair specimens is reported here. After decontamination of the hair strand in methylene chloride, hair segments were cut into small pieces with scissors, weighed and incubated overnight in Soerensen buffer pH 7.0 in the presence of deuterated 3,4-methylenedioxymethamphetamine (MDMA) at 40°C. The incubation medium was extracted using ethyl acetate after alkalinisation with 1N sodium hydroxide (NaOH). Before injection, the dry extract was derivatized using a mixture of heptafluorobutyric anhydride/ethyl acetate (100:50, v/v), evaporated and dissolved in ethyl acetate (25μl). After introduction of 1μl of the extract onto a splitless injector, chromatographic separation was achieved on a HP 6890 gas chromatograph equipped with a 5-MS capillary column. Detection was achieved in single ion monitoring mode (m/z 254-119-210 for mephedrone, m/z 258-213 for MDMA-d5) using a 5973 MSD operating in electron impact mode. Sixty-seven hair specimens were tested for mephedrone. Thirteen of them were found positive for mephedrone with concentrations ranging from 0.2 to 313.2ng/mg with a mean concentration of 26.8ng/mg. It was difficult to compare our findings due to a lack of reference data, nevertheless mephedrone seems well incorporated into hair (concentrations in the ng/mg range) like other stimulant drugs such as amphetamines or cocaine. The aim of this work was to develop a specific and accurate method for mephedrone analysis in hair specimens and its application to a large number of samples (n=67). The developed analytical method appears sensitive enough to reveal occasional to regular use of mephedrone.     

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.     

Testing for zopiclone in hair application to drug-facilitated crimes

The use of a drug to modify a person's behaviour for criminal gain is not a recent phenomenon. However, the recent increase in reports of drug-facilitated crimes (sexual assault, robbery) has caused alarm in the general public. The drugs involved can be difficult to detect due to low dosages or chemical instability. They possess amnesic properties and can be quickly cleared from the body fluids. In these situations, blood or even urine can be of poor interest. This is the reason why this laboratory developed an original approach based on hair testing by LC-MS/MS. Zopiclone (Imovane), due to its short half-life associated with rapid hypnotic activity, is considered as a compound of choice to sedate victims. To document the detection of zopiclone in hair, we first tested specimens obtained from two volunteers who had ingested a single 7.5 mg Imovane tablet, and from repetitive consumers of zopiclone. After pH 8.4 buffer incubation and extraction with methylene chloride/diethyl ether (80/20 (v/v)), hair extracts were separated on a Xterra MS C18 column using a gradient of acetonitrile and formate buffer. Zopiclone and diazepam-d5, used as internal standard, were detected by tandem mass spectrometry. A single exposure to zopiclone was detectable in the first hair segment of two volunteers at concentration of 5.4 and 9.0 pg/mg, respectively. Hair from repetitive consumers tested positive for zopiclone at concentrations of 37 and 66 pg/mg. Hair analysis was applied to two authentic criminal cases. In the first one, zopiclone tested positive in the corresponding hair segment at 4.2 pg/mg, in accordance with a single exposure to the drug. In the other expertise, zopiclone was detected in the two segments analyzed, at 21.3 and 21.5 pg/mg, making unlikely the hypothetical single exposure to zopiclone.     

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.