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.     

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.     

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.     

Effect of murine retroviral infection on hair and serum levels of cocaine and morphine.

LP-BM5 retrovirally infected female C57BL/6J mice were administered cocaine, morphine or both by daily intraperitoneal injection for 9 weeks. Drug concentrations were measured by radioimmunoassay in serum and in hair extracts. Hair samples obtained from all drug-treated mice were positive for the drug injected, while none of the saline-treated mice had detectable drug levels in hair or serum. The average morphine concentration obtained from non-infected mice was 11 ng/mg hair whereas the amount found in the LP-BM5-infected mice was significantly higher (20 ng/mg hair). Mice injected with both morphine and cocaine were given 50% of the regular dose of each drug and drug levels in the hair of these animals were approximately half that of mice injected with the full dose of the single drug. Non-infected mice treated with both drugs had a mean value of 7 ng morphine/mg hair and 374 ng cocaine/mg hair while retrovirus-infected mice had significantly higher concentrations, 10 ng morphine/mg hair and 1160 ng cocaine/mg hair (P less than 0.001). Serum concentrations of cocaine and morphine were significantly higher (P less than 0.01) in the retrovirus-infected animals from 40 min to 1.5 h. The increased concentrations of cocaine and morphine in serum during retrovirus infection are accompanied by a significant increase in the amount of drug incorporated into the hair matrix. This change indicates that retroviral infection may influence the disposition of these drugs in the systemic circulation.     

A Fatality Following Ingestion of the Designer Drug Meta‐Chlorophenylpiperazine (mCPP) in an Asthmatic—HPLC‐MS/MS Detection in Biofluids and Hair

A 20‐year‐old man, a cocaine addict and regular ecstasy user, with a medical history of allergic asthma died after ingesting half a tablet earlier the same day. The white tablet, stamped with a “smiling sun” logo looked very much like an ecstasy tablet and was sold as such. He experienced a severe asthma attack just after ingesting the half tablet and it evolved over the next few hours into fatal cardiorespiratory arrest. Biological samples, taken after embalming, were analyzed by high‐performance liquid chromatography tandem mass spectrometry (HPLC‐MS/MS). Analysis revealed meta‐chlorophenylpiperazine (mCPP) in concentrations of 45.8 mg in a similar tablet obtained later from the drug dealer, 5.1 ng/mL in the bile, 0.3 ng/g in the liver, 15.0 ng/mL in the urine, and its absence in a hair sample (<0.02 ng/mg), which indicated he was not a regular user (whereas strong concentrations of MDMA and cocaine were found in the hair). Interrogated by the police after his arrest, the dealer said that he had sold the victim and for the very first time two tablets with the same “smiling sun” logo. The tablet used for analysis was from the same brand as the one ingested by the victim. The autopsy excluded other causes of death, while the histological analyses showed a large number of polynuclear eosinophils in the bronchial walls, confirming the asthmatic pathology. None of the other organs examined (larynx, liver, heart, adrenal glands, and kidneys) showed any distinctive signs, and in particular no inflammatory infiltrate. The death was the result of an asthma attack in an asthmatic person, violently decompensated following ingestion of approximately 20 mg of mCPP.     

Gas chromatographic detection of cocaine and cocaethylene in hair of mice chronically injected with cocaine or cocaethylene and fed ethanol.

GC and GC/MS analysis was used to detect cocaine and cocaethylene in hair extracts of mice injected with 20 mg/kg cocaine hydrochloride or an equivalent dose of cocaethylene fumarate twice daily for 3 weeks. Some mice were fed liquid Lieber-DeCarli diets containing ethanol (26% of total calories) and injected twice daily with the same doses of cocaine or cocaethylene or combination of cocaine and morphine (5 mg/kg). The average concentrations of cocaine in different experimental groups were in the range of 0.9-2.4 ng/mg of hair and for cocaethylene, 2.4-2.8 ng/mg of hair. There were no significant differences in hair concentrations of cocaine among groups receiving cocaine treatment, nor were there significant difference in cocaethylene concentration in hair in the two groups administered cocaethylene. In hair extracts of mice treated with cocaine and ethanol, levels of cocaethylene were below the limit of detection.     

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.     

Methamphetamine body packer: acute poisoning death due to massive leaking of methamphetamine

We encountered three methamphetamine (MA) body packers presenting simultaneously, one of whom died. Three Nigerian men (39, 35, and 37 years old) who attempted to smuggle were found to contain 35 (498 g), 21 (292 g), and 5 packages (73 g) of methamphetamine hydrochloride (MA-HCl) in their stomachs, respectively. Packages were wrapped with plastic film and Scotch tape. The 39-year-old man died with acute poisoning from c. 20 g of MA-HCl that had leaked from the packages into the stomach. His plasma MA concentration was 8.6 microg/mL when he was hospitalized (17 h before his death). Autopsy findings showed extreme pulmonary congestion and edema as well as moderate hepatic edema and several petechiae. Quantitative analysis was performed by gas chromatography/mass spectrometry. Extremely high concentrations of MA and its metabolite amphetamine (AP) were found in cardiac blood (63.5 microg/mL and 1.2 microg/mL), urine (4,518 microg/mL and 72.4 microg/mL), gastric contents (8,490 microg/mL and 16.9 microg/mL), and in all other autopsy samples. These high concentrations confirmed that the cause of death was acute MA poisoning. Furthermore, impurity-profiling analysis of the seized MA revealed that the MA smuggled by the three suspects originated from the same batch.     

Simultaneous determination of opiates,cocaine and major metabolites of cocaine in human hair by gas chromotography/mass spectrometry (GC/MS)

A procedure is presented for the simultaneous identification and quantification of morphine (MOR), codeine (COD), ethylmorphine (EM), 6-monoacetylmorphine (6-MAM), cocaine (COC), benzoylecgonine (BZE), ecgonine methylester (EME) and cocaethylene (CE), contained in the hair of opiates and cocaine addicts. The method involves decontamination in dichloromethane, pulverization in a ball mill, heat-acid hydrolysis, addition of deuterated internal standards, liquid-liquid extraction and gas chromatography/mass spectrometry (GC/MS) after silylation. The limit of detection (LOD) was ~0.1–0.8 ng/mg for each drug, using a 30-mg hair sample. The method is reproductible, with a coefficient of variation (CV) of ~8–17%. Cocaine and 6-monoacetylmorphine were the major compounds detected in cases of cocaine (14 cases) and heroin (68 cases) intake. Concentrations were in the range 0.4–78.4 ng/mg (COC), 0.0–36.3 ng/mg (BZE), 0.0–1.6 ng/mg (EME), 0.0–2.1 ng/mg (CE), 0.0–84.3 ng/mg (6-MAM), 0.2–27.1 ng/mg (MOR) and 0.1–19.6 ng/mg (COD). An application in forensic sciences, involving multi-sectional analysis, is given.     

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).     

Hair analysis for opiates,cocaine and metabolites. Evaluation of a method by interlaboratory comparison

The aim of this study was to evaluate the performance of a technique for the simultaneous testing of opiates, cocaine and metabolites in hair by interlaboratory comparison. Sixteen forensic and clinical laboratories with different degrees of experience in hair analysis participated voluntarily in the study (no selection criteria were applied). The suggested analytical procedure, the one routinely used in our laboratory, consisted of incubation in HCl 0.1N (45 degrees C, overnight), solid phase extraction (with Bond Elut Certify) cartridges), derivatisation (trimethylsilyl (TMS) derivatives) and GC-MS analysis. Three different mixtures of finely cut (1 mm or less) hair were prepared using drug-users' and drug-free hair: one 'negative' sample (<0.1 ng/mg for morphine, 6-acetylmorphine (6AM), cocaine and benzoylecgonine (BE)), one 'low concentration' sample (between 0.5 and 2 ng/mg) and one 'high concentration' sample (>3 ng/mg). Accuracy and precision (CV% lower than 5.1, 9.9, 5.2, 3.8, 7.3 and 8.3% for morphine, 6AM, codeine, cocaine, BE, and methylecgonine (ME), respectively; range 0.5-5 ng/mg) of the method and homogeneity of the mixtures were evaluated in our laboratory by intraday (CV% lower than 12% for all analytes) and interday analyses (CV% lower than 17% for all analytes except 6AM, 25%). Participants in the study were grouped into: (1) laboratories (n = 6) obtaining the best qualitative and quantitative values, corresponding to those with long experience in hair analysis; (2) laboratories (n = 5) with no reported false positive and/or false negatives; (3) laboratories (n = 5) with one or more reported false positives/false negatives. The results obtained by the labs of the first group were used as reference values. The scatter of data was similar to those obtained in other published studies.     

Evaluation of cocaine, amphetamines and cannabis use in university students through hair analysis: preliminary results

The evaluation of drug abuse in a defined population was performed through toxicological hair analysis. Hair samples from university students ranging from 18 to 25 years of age were anonymously collected and screened for cocaine, amphetamines and cannabinoids by radioimmunoassay (RIA). Positive results (cut-off values adopted were 2 ng/mg for cocaine and amphetamines and 0.5 ng/mg for cannabinoids) were confirmed by GC/MS. Preliminary results showed 19% of positive results for cocaine on 200 samples analysed. No confirmed positive results were obtained for amphetamine analysis. RIA technique demonstrated its unsuitability for cannabinoids preliminary screening on hair, giving a high percent of false positive results.     

Analysis of hair after contamination with blood containing 6-acetylmorphine and blood containing morphine

The study was carried out to investigate external contamination of hair by blood in heroin-related post-mortem cases. Solutions were prepared containing 0.05, 0.1, 0.2, 0.5 and 3.0μg/mL of 6-monoacetylmorphine (6-AM) only or morphine only in human blood. Samples of approximately 3.2g of drug-free hair were contaminated by soaking in the blood solutions for 5min. They were then removed and left at room temperature. Approximately 0.5g of hair was collected from each of the blood soaked hair samples at 6h, 1, 2, 4 and 7 days after contamination. As each hair sample was collected it was shampoo-washed to prevent further drug absorption. Hair samples were analysed in triplicate using a fully validated method described previously. 6-AM broke down to morphine in all samples. In hair contaminated with blood containing 0.05, 0.1 and 0.2μg/mL 6-AM or morphine drug was either not detected or was detected below the limit of quantitation (0.2ng/mg hair) at all contamination times. In hair contaminated with blood spiked with 0.5μg/mL morphine, the concentration in hair ranged from 0.54 to 0.91ng/mg and in hair contaminated with blood spiked with 3.0μg/mL, from 3.25 to 5.77ng/mg. The concentrations of 6-AM ranged from 0.65 to 1.11ng/mg and morphine from 0.34 to 0.80ng/mg in hair contaminated with 0.5μg/mL 6-AM in blood. 6-AM ranged from 2.12 to 3.67ng/mg and morphine from 0.84 to 2.05ng/mg in hair contaminated with 3μg/mL 6-AM in blood. For 6-AM and morphine ANOVA statistical evaluation showed no significant difference among the concentrations over time.     

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.     

Atropine Eye Drops: An Unusual Homicidal Poisoning

In March 2009, the body of a 51‐year‐old man was found in the boot of his car. The body had been frozen before being dismembered at the abdomen. The autopsy failed to determine the cause of death. Systematic toxicological analyses of the victim's peripheral blood and urine showed the presence of atropine, a powerful anticholinergic. Atropine was therefore specifically detected and quantified throughout the victim's biologic samples by HPLC‐MS² in the biologic fluids and UHPLC‐MS² in the hair. The atropine concentrations were 887 ng/mL in the cardiac blood, 489 ng/mL in the peripheral blood, 6693 ng/mL in the gastric contents (1.1 μg), 6753 ng/mL in the urine, and 2290 pg/mg in the hair. The blood concentrations measured in the decedent were consistent with an overdose of atropine, which was determined as the cause of death. The manner of death was a homicide with criminal intent.     

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.     

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.     

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.     

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.     

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.