Psychotropic Drug Use in Recreational Scuba Divers and its Effect on Severe Narcosis.

Recreational scuba diving is no longer reserved for young healthy individuals, and as a result, medical drug consumption is on the rise in the diving population. Due to the possible potentiation of nitrogen narcosis by psychotropic drugs, the latter are hence discouraged and are subject to contraindications for practice. However, there are no available experimental data to support this theoretical assumption. The objective of this study is to investigate whether psychotropic drug users are more at risk of severe narcosis. An online survey was sent to the licensed divers from the East of France registered with the French Underwater Federation. Divers were surveyed regarding their consumption of psychotropic drugs, the occurrence of nitrogen narcosis as well as their respective diver's curriculum vitae.1 608 divers responded to the survey of which 15.2% confirmed having used psychotropic drugs and 7.8% since they became divers. Overall, 40.0% and 5.5% experienced severe and critical narcosis. In multivariate analysis, neither severe nor critical narcosis was associated with psychotropic drug use (OR 0.97 [0.59-1.57] and 0.76 [0.29-2.00], respectively).In conclusion, despite the recommendations, a significant proportion of divers use psychotropic drugs but do not seem to be more prone to severe narcosis.

Heroin markers in hair of a narcotic police officer: active or passive exposure?

On March 2007, a police officer (46-year-old man) and a clerk (37-year-old woman) were arrested and subjected to investigation on the charges of drugs of abuse trafficking. The loving couple was exploiting their administrative positions to make money with the resale of seized drugs. The laboratory was requested to analyse their hair for drugs of abuse. Hair of the 2 subjects tested positive for heroin by GC-MS. A few days later, analysis of hair obtained from 11 other police officers of the same unit was requested, in order to compare the results, as external contamination was proposed to account for the positive results. The aim of the investigations was to demonstrate that passive contamination could not occur for persons dealing every day with drugs of abuse with minimal caution and hygiene, and that the measured concentrations in the arrested subjects correspond to personal abuse. All the narcotic team tested negative, irrespective of the compound.

Interpretation of hair findings in children after methadone poisoning.

Methadone is not licensed for use in children though it can be employed for the management of neonatal opiate withdrawal syndrome. During the last 2 years, our laboratory has been asked to test for methadone and EDDP, its major metabolite, in hair from children that were admitted to hospital unconscious and where methadone had already been identified in a body fluid (4 cases) or where the children were deceased and evidence of methadone overdosage having already been established (2 cases). In all of these cases, segmental analysis revealed approximately the same amount of drug along the hair lock. As a consequence, contamination was considered as an issue and interpretation of the results was a challenge that deserves particular attention. After decontamination with dichloromethane and segmentation the hair was cut into small pieces, incubated overnight at 40 degrees C, liquid-liquid extracted and analysed with LC-MS/MS, using 2 transitions per compound. The LOQ for both methadone and EDDP was 10 pg/mg. In the first series involving children admitted to hospital, the following results were obtained: * case 1: 4 x 1 cm section, methadone at 0.05-0.08 ng/mg, no EDDP detected, * case 2: 4 x 1 cm section, methadone at 0.13-0.15 ng/mg, EDDP at 0.02 ng/mg, * case 3: 3 x 1.5 cm section, methadone at 0.07-0.09 ng/mg, EDDP at 0.01-0.03 ng/mg, * case 4: 6 x 2 cm section, methadone at 0.06-0.13 ng/mg, EDDP at 0.02-0.03 ng/mg. The following concentrations were obtained from the children who had died following a methadone overdose: * case 5: 2 x 2 cm section, methadone at 0.53-0.58 ng/mg, no EDDP detected, * case 6: 4 x 1 cm section, methadone at 0.44-0.77 ng/mg, EDDP at 0.04-0.06 ng/mg. The first observation is that all these concentrations are low by comparison with those observed in adults on methadone maintenance therapy. However, the more surprising observation is the relative homogenous concentrations along the hair locks in each specific case. This raises concerns around the possibility that contamination could have occurred prior to sampling and makes it hard to reach a conclusion regarding the possibility of repeated methadone exposure in the months prior the incidents. In these cases it was impossible to conclude that the children were deliberately administered methadone. The results of the analysis of hair could indicate that they were in an environment where methadone was being used and where the drug was not being handled and stored with appropriate care. The homogenous concentrations found on segmental analyses could be indicative of external contamination that may have arisen not only from direct contamination with the drug but also via contamination with body fluids at the post mortem or from sweat produced close to the time of the incident. In view of these results we concluded that a single determination should not be used firmly to discriminate long-term exposure to a drug.

Hair analysis to demonstrate administration of sildenafil to a woman in a case of drug-facilitated sexual assault.

The drug sildenafil (Viagra, Pfizer) and, more recently, tadalafil (Cialis, Lilly-Icos) and vardenafil (Levitra, Bayer), has drawn public attention to aphrodisiacs. The search for such substances dates back millennia. Adverse effects associated with these drugs include hypotension, tachycardia, headache, flushing, blurred vision, dyspepsia, and musculoskeletal pain. Although sildenafil has been marketed for erection of the penis, recent attention has been paid to its application for women, including enhancement of success of in vitro fertilization but also better sexual responses (increased desire, satisfaction, and orgasm) in cases of sexual disorders. Today, there is a debate on internet forums about the potential properties of sildenafil to enhance women's sexual pleasure. This laboratory was asked to analyze a 12-cm length of light brown hair submitted by a British police force following an allegation that a young female had been subjected to sexual assaults over a two-year period. The female was 15-17 years of age at the time. The alleged perpetrator was her stepfather, and there was some suspicion that drugs may have been administered to facilitate the attacks. After decontamination and segmentation (6 x 2-cm section), the specimen was analyzed by liquid chromatography coupled with tandem mass spectrometry after alkaline (pH 9.5) extraction using dichloromethane/ isopropanol/n-heptane (25:10:65, v/v/v). The limit of quantitation was 5 pg/mg. The proximal segment tested positive for sildenafil at 38 pg/mg, and all others proved negative. This was in accord with the victim's claim. In the absence of any controlled studies, it was impossible to put any quantitative interpretation on the measured concentration.

Smoking cessation with varenicline: a suicidal fatality.

The most effective smoking cessation programs involve a combination of pharmacotherapy and behavioral and/or cognitive counseling to improve abstinence rates. Varenicline (Champix in France and the U.K.), the most recently approved agent for tobacco cessation, is the first drug in a new class (alpha4beta2 partial agonist) that binds to the nicotinic receptors to release dopamine and alleviate withdrawal symptoms. As the literature reports psychiatric disorders being linked to varenicline as an issue, we describe the case of a man who committed suicide while receiving therapy with this drug. The deceased (a 39-year-old man) was found dead at his home address with slash wounds to his wrist. The deceased had been prescribed varenicline for several months at a dose of 1 tablet (1 mg) twice daily. The lab received a blood specimen to perform a screening for unknown drugs, including varenicline. Because of its selectivity and sensitivity, liquid chromatography coupled to tandem mass spectrometry was chosen as the best approach to develop a procedure for varenicline. One milliliter of blood was extracted with 5 mL of a mixture of dichloromethane/isopropanol/n-heptane (25:10:65) at pH 9.5 (phosphate buffer) in the presence of diazepam-d(5), which was used as an internal standard (IS). The resultant blood extract was separated on an XTerra MS C18 column using a gradient of acetonitrile and formic acid in water. Drugs were identified by three or two transitions (m/z 212 > 169, 212 > 183, and 212 > 195 and 290 > 154 and 290 > 198 for varenicline and IS, respectively). The limit of quantitation of varenicline was 1 ng/mL. The concentration of varenicline in the blood was determined to be 10 ng/mL. This concentration could not be compared with therapeutic levels, as there are no therapeutic concentrations reported in the literature. Because of its potential effects on behavior, the influence of the drug on the mental functioning of the user should be considered in cases of suicide.

Evaluation of the Cozart DDSV test for cannabis in oral fluid.

Saliva or "oral fluid" has been presented as an alternative matrix to establish drug exposure. The noninvasive collection of an oral fluid sample, which is relatively easy to perform and can be achieved under close supervision, is one of the most important benefits when testing for driving under the influence of drugs. Moreover, the detection of Delta9-tetrahydrocannabinol (THC) in oral fluid is a better indication of recent use than a positive urine test, so there is a higher probability that the subject is experiencing pharmacological effects at the time of sampling. Twenty-five subjects (5 free and 20 addicts from a heroin detoxification center) were included in a study to evaluate the potential application of a new device, the Cozart DDSV (drug detection system visual), to detect cannabis in oral fluid. The time cannabis was last smoked was recorded by the medical staff after interview with each subject. Samples were collected with the Cozart DDS Oral Swab and diluted with the Cozart DDS buffer as proposed by the manufacturer. The Cozart DDSV test was conducted on site at the time of collection, and the remainder of the sample retained for confirmation analysis by gas chromatography with mass spectrometry (GC/MS) after methylation of THC (limit of quantitation 0.5 ng/mL). All 25 samples were analyzed by GC/MS. On-site results were obtained within 10 minutes. The 5 drug-free subjects were negative for cannabis, irrespective of the method. From the 20 subjects declaring that they had smoked cannabis between 30 minutes and 24 hours previously, the DDSV device identified 8 positive subjects (with THC concentrations in the buffer in the range 15-219 ng/mL), whereas 18 subjects tested positive using GC/MS. THC concentrations in the Cozart buffer using GC/MS analysis ranged from 0.7 to 219 ng/mL. These concentrations represent about one third the authentic THC concentrations in oral fluid due to the dilution by the liquid of the device. Given the results, the DDSV device was considered as an acceptable tool to detect cannabis abuse in oral fluid within a period of 2-3 hours after smoking.

Chemical abuse in the elderly: evidence from hair analysis.

The use of a drug to modify a person's behavior for criminal gain is not a recent phenomenon. However, the recent increase in reports of drug-facilitated crimes (sexual assault, robbery) has caused some alarm in the general public. Drugs involved can be pharmaceuticals such as benzodiazepines (flunitrazepam, lorazepam, clonazepam), hypnotics (zopiclone, zolpidem), sedatives (neuroleptics, some antihistamines), or anesthetics (GHB, ketamine); drugs of abuse such as cannabis, ecstasy, or LSD; or, more often, ethanol. Mistreatment of older people, whether it is abuse or neglect, can be classified as physical, psychologic, or financial/material. Several types of mistreatment may occur simultaneously. Very few data are available in the international literature. It seems that mental abuse and neglect are more frequent, but physical abuse such as beating, pushing, kicking, and possibly sexual abuse have also been reported. Drugs used to facilitate sexual assaults can be difficult to detect (active products at low dosages, chemical instability), can possess amnesic properties, and can be rapidly cleared from the body (short half-life). In these situations, blood, or even urine, can be inadequate. This is the reason why some laboratories have developed an original approach based on hair testing. Hair was suggested as a valuable specimen in situations in which, as a result of a delay in reporting the crime, natural processes have eliminated the drug from typical biologic specimens. Hair analysis may be a useful adjunct to conventional drug testing in sexual assault. It should not be considered as an alternative to blood and urine analyses, but as a complement. Mass spectrometry/mass spectrometry technologies appear to be required for analyses in drug-facilitated cases. The experience of the authors is presented in cases involving the elderly and chemical poisoning.

Ethyl glucuronide: unusual distribution between head hair and pubic hair.

Ethyl glucuronide (EtG) is a minor metabolite of ethanol that can be detected in hair. In some specific situations, head hair can be missing, and therefore, alternative anatomical locations of hair are of interest. In this study, paired hair specimens (head hair and pubic hair) from eight social drinkers were analyzed for EtG. Each sample was decontaminated by two dichloromethane bathes (5 ml) for 2 min. After cutting into small pieces, about 50 mg of hair was incubated in 2 ml water in the presence of 10 ng of EtG-d5, used as internal standard and submitted to ultra-sonication for 2 h. The aqueous phase was extracted by SPE using Oasis MAX columns. The hair extract was separated on an ACQUITY BEH HILIC column using a gradient of acetonitrile and formate buffer. Detection was based on two daughter ions: transitions m/z 221-85 and 75 and m/z 226-75 for EtG and the IS, respectively. This laboratory is using a positive cut-off at 50 pg/mg. All eight head hair specimens were negative for EtG at a limit of quantitation fixed at 10 pg/mg. Surprisingly, EtG was identified at high concentrations in pubic hair, in the range 12-1370 pg/mg. It appears, therefore, that it is not possible to document the drinking status of a subject by simply switching from head hair to pubic 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.

Clenbuterol determination in calf hair by UPLC-MS-MS: case report of a fraudulent use for cattle growth.

A method for clenbuterol determination in hair has been developed. Hair specimens collected from two calves were decontaminated using hot water followed by methylene chloride. Hair was cut into small pieces, and 100 mg was incubated in 1 mL 0.1M hydrochloric acid overnight at 45 degrees C in the presence of 1 ng acebutolol used as internal standard. After neutralization with 1 mL 0.1M NaOH, 2 mL of bicarbonate buffer (pH 8.6) were added and the preparation was then purified using solid-phase extraction with an Isolute C18 column. Methanolic eluent was evaporated to dryness and the residue was reconstituted with 50 microL methanol. A 5-microL portion was injected onto an ACQUITY UPLC BEH C18 column (2.1 x 50 mm, 1.7 microm) and separation was achieved using a gradient of acetonitrile and formate buffer delivered at a flow rate of 0.6 mL/min. Detection was done on a Waters Micromass Quattro Micro API triple-quadrupole mass spectrometer. Ionization was achieved using electrospray in positive mode. Clenbuterol was identified by two transitions (m/z 277.1 > 203.2 and m/z 277.1 > 132.1). Quantitation was performed with the most intensive transition (m/z 277.1 > 203.2) versus the internal standard monitored using the transition (m/z 337.3 > 116.1). When compared with gas chromatography methods that are generally used for the determination of beta-adrenergics, the major advantages of this method were the sensitivity, a shorter run time, and the absence of a derivatization step. The analysis of two hair samples from calves suspected of drug administration showed low clenbuterol concentrations at 3.6 and 4.8 pg/mg.

Hair analysis for diphenhydramine after surreptitious administration to a child.

Diphenhydramine is one of the first effective antihistamine agents to have been discovered. The compound is also used for its sedative and antiemetic effects. The first case involving repetitive sedation linked to the use of diphenhydramine as a drug-facilitated crime and subsequent impairment of a 9-year-old female victim is reported. Due to the long delay between the alleged crime and clinical examination, collection of blood or urine was of little value. Hence, the laboratory developed an original approach based on hair testing by LC-MS/MS. A single strand of hair from the victim was sampled about 7 weeks after the last suspected administration and was cut into small segments. After cutting into small pieces, about 20 mg of hair per segment was incubated overnight in a phosphate buffer (pH 8.4). The aqueous phase was extracted with 5 ml of a mixture of methylene chloride/diethyl ether (80/20), in presence of diazepam-d5, used as internal standard (IS). The hair extract was separated on an XTerra MS C18 column using a gradient of acetonitrile and formate buffer. Detection was based on two daughter ions: transitions m/z 256.2-152.1 and 167.1 and m/z 289.9-154.0 for diphenhydramine and the IS, respectively. In the hair of the child, diphenhydramine was detected at concentrations in the range 33-39 pg/mg, depending on the segment.

Testing for atropine and scopolamine in hair by LC-MS-MS after Datura inoxia abuse.

Datura inoxia belongs to the family of Solanaceae. This is a very common plant in New Caledonia that contains two main toxic alkaloids, l-atropine and l-scopolamine. In this study, we report the case of a 20-year-old male admitted to an Emergency Unit after consumption of 6 dried flowers in hot water for hallucinations, mydriasis, and agitation associated with tachycardia and increase of systolic blood pressure to 180. Full recovery was observed after one week. Three weeks later, a lock of about 80 hairs (200 mg) was collected from the subject in vertex posterior with scissors to be tested for both atropine and scopolamine. After decontamination with dichloromethane, a strand of hair was segmented into three parts, cut into small segments (< 1 mm), incubated overnight in 1 mL pH 8.4 phosphate buffer in the presence of 2.5 ng atropine-d(3), the internal standard, then extracted with 5 mL dichloromethane/isopropanol/n-heptane (50:17:33). The residue was reconstituted in 100 microL of methanol, from which 10 microL was injected into an XTerra MS C18 column (100 x 2.1 mm, 3.5 microm) eluted with a gradient of acetonitrile and formate buffer delivered at a flow rate of 0.2 mL/min. A Quattro Micro triple-quadrupole mass spectrometer (MS) was used for analyses. Ionization was achieved using electrospray in the positive ionization mode. For each compound, detection was related to two daughter ions (atropine: m/z 290.2 to 124.0 and 92.9; atropine-d(3): m/z 293.1 to 127.0 and 92.9; scopolamine: m/z 304.1 to 138.0 and 156.0). Although atropine was never detected (limit of detection = 2 pg/mg), scopolamine was identified in the three segments, in the range 14 to 48 pg/mg. The absence of atropine in hair is consistent with its very low dosage in the flower of Datura inoxia. Hair segmentation indicated that the subject was previously exposed on several occasions to the plant. Liquid chromatography-tandem MS appears to be a necessity for testing tropane alkaloids of the Datura group, given the low concentrations to be measured.

Oral fluid testing for cannabis: on-site OraLine IV s.a.t. device versus GC/MS.

Saliva or "oral fluid" has been presented as an alternative matrix to document drug use. The non-invasive collection of a saliva sample, which is relatively easy to perform and can be achieved under close supervision, is one of the most important benefits in a driving under the influence situation. Moreover, the presence of Delta9-tetrahydrocannabinol (THC) in oral fluid is a better indication of recent use than when 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) is detected in urine, so there is a higher probability that the subject is experiencing pharmacological effects at the time of sampling. In the first part of the study, 27 drug addicts were tested for the presence of THC using the OraLine IV s.a.t. device to establish the potential of this new on-site DOA detection technique. In parallel, oral fluid was collected with the Intercept DOA Oral Specimen Collection device and tested for THC by gas chromatography mass spectrometry (GC/MS) after methylation for THC (limit of quantification: 1 ng/mL). The OraLine device correctly identified nine saliva specimens positive for cannabis with THC concentrations ranging from 3 to 265 ng/mL, but remained negative in four other samples where low THC concentrations were detected by GC/MS (1-13 ng/mL). One false positive was noted. Secondly, two male subjects were screened in saliva using the OraLine and Intercept devices after consumption of a single cannabis cigarette containing 25mg of THC. Saliva was first tested with the OraLine device and then collected with the Intercept device for GC/MS confirmation. In one subject, the OraLine on-site test was positive for THC for 2 h following drug intake with THC concentrations decreasing from 196 to 16 ng/mL, while the test remained positive for 1.5 h for the second subject (THC concentrations ranging from 199 to 11 ng/mL). These preliminary results obtained with the OraLine IV s.a.t. device indicate more encouraging data for the detection of THC using on-site tests than previous evaluations.

Determination of trimeprazine-facilitated sedation in children by hair analysis.

Trimeprazine or alimemazine is largely used as an antipruritic agent, but it is also used for insomnia, cough, and oral premedication in pediatric day surgery. The first cases involving repetitive sedation linked to the use of trimeprazine as a drug-facilitated crime and subsequent impairment of two children are reported. Because of the long delay between the alleged crime and clinical examination, collection of blood or urine was of little value. This is the reason why the laboratory developed an original approach based on hair testing by liquid chromatography-tandem mass spectrometry. A strand of hair from each child was sampled about 2 months after the first suspicion of administration and was cut into small segments. After cutting into small pieces, 20 mg of hair was incubated overnight in a phosphate buffer (pH 8.4). The aqueous phase was extracted by 5 mL of a mixture of diethyl ether/methylene chloride (80:20) in presence of diazepam-d(5) used as the internal standard (IS). Hair extract was separated on a XTerra MS C18 column using a gradient of acetonitrile and formate buffer. Detection was based on two daughter ions: transitions m/z 299.3 to 299.0 and 100.0 and m/z 289.9 to 154.0 for trimeprazine and the IS, respectively. In the hair of the two subjects, trimeprazine was detected at concentrations in the range 23 to 339 pg/mg. The stepmother, who was the perpetrator in both cases, did not challenge the use of trimeprazine as a sedative drug.

Hair analysis for drug detection.

Given the limitations of self-reports on drug use, testing for drugs of abuse is important for most clinical and forensic toxicological situations, both for assessing the reality of the intoxication and for evaluation of the level of drug impairment. It is generally accepted that chemical testing of biological fluids is the most objective means of diagnosis of drug use. The presence of a drug analyte in a biological specimen can be used to document exposure. The standard in drug testing is the immunoassay screen, followed by the gas chromatographic-mass spectrometric confirmation conducted on a urine sample. In recent years, remarkable advances in sensitive analytical techniques have enabled the analysis of drugs in unconventional biological specimens such as hair. The advantages of this sample over traditional media, like urine and blood, are obvious: collection is noninvasive, relatively easy to perform, and in forensic situations it may be achieved under close supervision of law enforcement officers to prevent adulteration or substitution. The window of drug detection is dramatically extended to weeks, months or even years when testing hair. It seems that the value of alternative specimen analysis for the identification of drug users is steadily gaining recognition. This can be seen from its growing use in preemployment screening, in forensic sciences, in clinical applications and for doping control. Hair analysis may be a useful adjunct to conventional drug testing in urine. Methods for evading urinalysis do not affect hair analysis. The aim of this review is to document toxicological applications of hair analysis in drug detection.

Hair to document exposure to glibenclamide.

Among the drugs that are used to incapacitate victims such as kids or elderly for sedation or for criminal gain such as sexual offences or robberies, glibenclamide, an antidiabetic was never mentioned. To document the interest of hair testing in such forensic situations, we have developed an original method to test for glibenclamide. A 30-year-old man was admitted to the Emergency Unit for coma and seizures after a party with some members of his family. Blood glucose was 0.40 g/l. A hair specimen was collected several weeks after the event and divided into two segments of 2 cm. Twenty milligrams of each segment cut into small pieces were incubated overnight in a phosphate buffer (pH 5.5), in presence of gliclazide used as internal standard (IS). A liquid/liquid extraction was realized with a mixture of diethyl ether/methylene chloride, and hair extract was separated on a XTerra MS C18 column using a gradient of acetonitrile and formate buffer. Detection of glibenclamide was achieved using two transitions: m/z 493.9 to 168.9 and 493.9 to 368.8. Linearity was observed from 5 to 1000 pg/mg (r2 = 0.956) with a limit of quantification at 5 pg/mg and a clean-up recovery of about 61%. Within-batch precision and bias were 9.0 and 9.5%, respectively. Ion suppression tested on drug-free hair was about 50%. Glibenclamide tested positive in the two consecutive segments (root to 2 cm: 23 pg/mg and 2-4 cm: 31 pg/mg). These findings were in accordance with a repetitive exposure to the drug. The concentrations were compared with those obtained after a single and a daily dose administration. In the hair of a subject receiving a single 5mg dose and collected 4 weeks later, glibenclamide was detected in the proximal segment at 5 pg/mg. After a 20 mg/day dose, the hair concentration of a subject under glibenclamide therapy was 650 pg/mg.

Doping control for metandienone using hair analyzed by gas chromatography-tandem mass spectrometry.

A sensitive, specific and reproducible method for the quantitative determination of the anabolic metandienone in human hair has been developed. The preparation involved a decontamination step with methylene chloride. The hair sample (about 50 mg) was solubilised in 1 ml 1 M NaOH, 10 min at 95 degrees C, in presence of 2 ng of nandrolone-d(3) used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase extraction (Isolute C(18) eluted with methanol) and a liquid-liquid extraction with pentane. The residue was derivatized by adding 5 microl MSTFA/NH(4)I/2-mercaptoethanol (250 microl; 5 mg; 15 microl) and 45 microl MSTFA, then incubated for 20 min at 60 degrees C. A 1 microl aliquot of derivatized extract was injected into the column (HP5-MS capillary column, 5% phenyl-95% methylsiloxane, 30 m x 0.32 mm i.d., 0.25 microm film thickness) of a Hewlett Packard (Palo Alto, CA, USA) gas chromatograph (6890 Series). Metandienone was identified using three transitions (its daughter ions at m/z 339 and 206 for the parent 444 and 191 for 206) using a Waters Quattro Micro MS-MS system. The transition m/z 444 to 206 has been used as quantification transition and the others as identification transitions. The assay was capable of detecting 2 pg/mg of metandienone when approximately 50 mg of hair material was processed. Linearity was observed for metandienone concentrations ranging from 2 to 500 pg/mg with a correlation coefficient of 0.9997. Intra-day and between-day precisions at 50 pg/mg were 13.4-16.5% and 22.0%, respectively, with an extraction recovery of 48%. The analysis of hair, cut into four segments, obtained from an athlete, revealed the presence of metandienone at the concentrations of 78, 7, 10 and 108 pg/mg in each segment of hair (0-1, 1-2, 2-3 and 3 cm to the tip).

Windows of detection of tetrazepam in urine, oral fluid, beard, and hair, with a special focus on drug-facilitated crimes.

Reducing the capacity of a victim to react against sexual assault, coupled with a possible abrupt unconsciousness-inducing effect and ease of administration in spiked drinks, have resulted in the use of sedative agents in cases of drug-facilitated offence. Among these compounds, tetrazepam may impair an individual rapidly. The chances of detecting this substance increase if the most sensitive methods are used and if the biologic matrix that allows the longest possible detection time is available. To document the window of detection of tetrazepam, 50 mg of the drug was administered orally to 2 volunteers, and the following samples were collected: oral fluid (n = 1) over 515 minutes, urine (n = 2) over 236-240 hours, hair (n = 2) 4 weeks after exposure, and beard (n = 1) over 34 days. Tetrazepam was analyzed by LC-MS/MS (Micromass Quattro Micro) after alkalinization and extraction by dichloromethane/diethyl ether in the presence of diazepam-d5, used as internal standard. Reversed-phase separation on an XTerra MS C18 column was achieved in 12 minutes, under gradient conditions. Pseudo-molecular ions selected were m/z 289.2 and 290.2 for tetrazepam and the internal standard (IS), respectively, and the corresponding daughter ions selected were m/z 225.2 and 253.2 for tetrazepam and m/z 154.1 and 198.3 for the IS. Urine tested positive for tetrazepam over 236-240 hours (14-13 ng/mL). Oral fluid tested positive for tetrazepam over 515 minutes (2.5 ng/mL). Tetrazepam was detected in beard over 27 days (6.5 pg/mg). A single tetrazepam dose was detected in hair 4 weeks after intake (123-175 pg/mg). Tetrazepam tested positive over the studied time intervals but would be expected to be detectable for a considerably longer time. Therefore, in cases of drug-facilitated crimes in which tetrazepam is involved, hair and beard analyses can be an important complement to urine analyses to document exposure, particularly if LC-MS/MS is used.

Unusually high concentrations in a fatal GHB case.

The first case in France involving a fatal overdose resulting from the ingestion of gamma-hydroxybutyrate (GHB) is presented. GHB was tested by gas chromatography-mass spectrometry (GC-MS) after precipitation. Briefly, 20 microL of body fluids (blood, bile, urine, gastric contents, or vitreous humor) was pipetted in a glass tube, followed by 20 microL GHB-d6 and 45 microL acetonitrile. After vortex mixing and centrifuging, the supernatant was collected and evaporated to dryness. The residue was derivatized with BSTFA with 1% TMCS for 20 min at 70 degrees C. After injection on a 30-m HP5 MS capillary column, GHB (m/z 233, 204, and 147) and GHB-d6 (m/z 239) were identified by MS. GHB was also tested in pubic hair after incubation in 0.01 N NaOH, neutralization, acidification, extraction in ethyl acetate and derivatization with MTBSTFA, using GC-MS-MS. GHB was positive in all the tested specimens, with the following concentrations 2937, 33,727, 1800, and 2856 mg/L in femoral blood, urine, bile, and vitreous humor, respectively. This seems to be the highest blood concentration ever observed. Postmortem redistribution appears weak, as the concentration in cardiac blood was 3385 mg/L (cardiac blood/femoral blood ratio of 1.15). Oral route was suggested with GHB at 7.08 g in 100 mL of gastric contents. Pubic hair analysis clearly indicated chronic GHB abuse, with concentrations along the shaft in the range 19.4 to 25.0 ng/mg (in comparison with physiological concentrations < 2 ng/mg). Methylenedioxymethamphetamine was present in femoral blood at 144 ng/mL. These results are consistent with an acute fatal overdose of GHB.

Screening method for benzodiazepines and hypnotics in hair at pg/mg level by liquid chromatography-mass spectrometry/mass spectrometry.

A procedure is presented for the screening of 16 benzodiazepines and hypnotics in human hair by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon and zolpidem). The method involves decontamination of hair with methylene chloride, hair cut into small pieces, incubation of 20 mg in phosphate buffer (pH 8.4) in the presence of 1 ng diazepam-d5 used as internal standard, liquid-liquid extraction with diethyl ether/methylene chloride (10/90) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.5 to 5 pg/mg using a 20-mg hair sample. Linearity is observed from the limit of quantification of each compound to 200 pg/mg (r2 > 0.99). Coefficients of variation measured on six points and at two concentrations (10 and 50 pg/mg) range from 5 to 20% for all drugs but one. Extraction recovery, measured at the two same concentrations range from 32 to 76%. These results were found suitable to screen for 16 benzodiazepines in hair and detect them at very low concentrations, making this method suitable to monitor single dose.