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.

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.

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.

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.

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).

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.

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.

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.

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.

Screening and confirmatory method for benzodiazepines and hypnotics in oral fluid by LC-MS/MS.

A procedure is presented for the screening of 17 benzodiazepines and hypnotics in oral fluid after collection with the Intercept(R) device by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon, zopiclone and zolpidem). The method involves extraction of 0.5 mL of oral fluid (previously stored in the Intercept blue buffer) treated with 0.5 mL of phosphate buffer (pH 8.4) in the presence of 5 ng diazepam-d(5) used as internal standard, with 3 mL of diethyl ether/methylene chloride (50/50) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.1 to 0.2 ng/mL. Linearity is observed from the limit of quantification of each compound to 20 ng/mL (r(2)>0.99). Coefficients of variation at 2 ng/mL, measured on 6 points range from 4 to 8% for all drugs, except zopiclone (34%). Extraction recovery, measured at the same concentration was higher than 90%. Ion suppression was evaluated for each compound and was lower than 10% for all drugs except zopiclone (93%). These results were found suitable to screen for 17 benzodiazepines in oral fluid and detect them at very low concentrations, making this method suitable for monitoring subjects under the influence.

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.

Detection of cannabis use in drivers with the drugwipe device and by GC-MS after Intercept device collection.

Saliva or "oral fluid" has been presented as an alternative matrix in the establishment of drug exposure. The noninvasive 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 the drug is detected in urine, so there is a higher probability that the subject is experiencing pharmacological effects at the time of sampling. At 3 check points organized by the Swiss police in Bern, 61 drivers were tested for the presence of drugs of abuse using the Drugwipe 5 device. In parallel, oral fluid was collected with the Intercept DOA Oral Specimen Collection device and tested by gas chromatography-mass spectrometry (GC-MS) after methylation of THC (limit of quantitation 1 ng/mL). The Drugwipe device identified 1 exposed driver, but with GC-MS, 18 drivers tested positive. THC concentrations in the Intercept buffer ranged from 2.1 to 205.1 ng/mL. These concentrations represent about 1/2 to 1/3 the authentic THC concentrations in oral fluid because of the dilution by the blue liquid of the device. Two main limitations of oral fluid were 1. the amount of matrix collected is smaller when compared to urine and 2. the levels of drugs in urine are higher than in oral fluid. A current limitation of the use of this specimen for roadside testing is the absence of a suitable immunoassay that detects the parent compound in sufficiently low concentrations.

Drug-facilitated sexual assault and analytical toxicology: the role of LC-MS/MS A case involving zolpidem.

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 alarm in the general public. Drugs involved can be pharmaceuticals, such as benzodiazepines (flunitrazepam, lorazepam, etc.), hypnotics (zopiclone, zolpidem), sedatives (neuroleptics, some histamine H1-antagonists) or anaesthetics (gamma-hydroxybutyrate, ketamine), drugs of abuse, such as cannabis, ecstasy or lysergide, or more often ethanol. Drugs said to be used to facilitate sexual assaults can be difficult to detect (active products at low dosages, chemical instability), possess amnesic properties and can be rapidly cleared from the body (short half-life). We present here a case involving a 23-year old girl that declared a sexual assault 6 days after the event was said to have occurred. To the Police, the victim claimed a total amnesia of the offense associated with intense sedation. Toxicological analyses for unknown sedative drugs achieved by LC-MS/MS revealed the presence of zolpidem (Stilnox), a non-benzodiazepine hypnotic. Concentrations after 6 days were 16 and 32 pg/mL in blood and urine, respectively. The drug tested also positive in the corresponding hair segment at 0.75 pg/mg. The requested extraordinary sensitivity of LC-MS/MS appears as a pre-requisite to document any case involving drug-facilitated sexual assault.

Testing for zolpidem in oral fluid by liquid chromatography-tandem mass spectrometry.

The purported enhancement of sexuality, coupled with a possible abrupt coma-inducing effect and ease of administration in spiked drinks have resulted in the use of hypnotics to facilitated a sex offence. Among these compounds, zolpidem possesses amnesic properties, is short-acting and can rapidly impair an individual. In order to document zolpidem exposure, we have developed an original analytical method in oral fluid. 500 microl of oral fluid was added to 500 microl pH 7.6 Soerensen buffer was extracted by 2 ml dichloromethane in presence of 5 ng diazepam-d5, used as internal standard. An aliquot of the extract was injected into a 5 microm Novapak C18 column (150 mm x 2.1 mm). Reversed-phase separation was achieved in 6 min, under isocratic conditions (90% acetonitrile, 10% 2 mM ammonium formate pH 3.6) at a flow rate of 150 microl/min. Detection was achieved by tandem mass spectrometry. Molecular ions (m/z 308 and 290 for zolpidem and the IS, respectively) were selected in Q1 and the corresponding daughter ions (m/z 235 and 263 for zolpidem and m/z 154 and 198 for the IS) were detected in Q3 after collision with argon. Linearity was observed for zolpidem concentrations ranging from 0.2 to 100 ng/ml, and the assay was capable of detecting 0.05 ng/ml. Oral fluid was collected for 480 min after oral zolpidem administration of 10 mg to 2 subjects. In both cases, zolpidem was detectable (0.4 ng/ml) after 15 min intake. Peak zolpidem concentrations were obtained at 150 min (53.5 ng/ml) and 180 min (75.7 ng/ml), respectively. Oral fluid tested positive for zolpidem for over 8 h (9-15 ng/ml).

Ultra-rapid procedure to test for gamma-hydroxybutyric acid in blood and urine by gas chromatography-mass spectrometry.

Gamma-hydroxybutyric acid (GHB) is a substance naturally present within mammal species. Properties of a neurotransmitter or neuromodulator are generally suggested for this substance. GHB is therapeutically used as an anaesthetic, but can be used for criminal offences (date-rape drug). It appears that the window of detection of GHB is very short in both blood and urine, and therefore its presence is very difficult to prove after a rape case. Twenty microl of blood or urine were pipetted into a glass tube, followed by 20 microl GHB-d(6) and 45 microl acetonitrile. After vortexing and efficient centrifugation, the supernatant was collected and evaporated to dryness. The residue was derivatized with BSTFA+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-d(6) (m/z 239) were identified by mass spectrometry. The procedure was linear from 1 to 200 mg/l for both blood and urine. Precisions were in the range 4 to 11%. The method appears simple, specific and rapid as an accurate result can be obtained within 1 h.

Determination of bromazepam, clonazepam and metabolites after a single intake in urine and hair by LC-MS/MS. Application to forensic cases of drug facilitated crimes.

The number of reports on drug facilitated crimes is increasing these last years. Apart from ethanol and cannabis, benzodiazepines (BZD) and analogs are the most common drugs reported to be used probably due to their amnesic and sedative properties. We have developed a rapid and sensitive method using LC-MS/MS triple stage quadrupole (TSQ) for the determination of single exposure to bromazepam (Lexomil, 6 mg) and clonazepam (Rivotril, 2 mg) in urine and hair of healthy volunteers. Chromatography was carried out on a Uptisphere ODB 5 microm, 2.1 mm x 150 mm column (Interchim) with a gradient of acetonitrile and formate 2 mM buffer, pH 3. Urine was extracted with Toxitube A (Varian) and allowed the detection of bromazepam, 3-hydroxy-bromazepam, clonazepam and 7-Aminoclonazepam for more than 6 days. Head hair, collected 1 month after the exposure, was treated by incubation with Soerensen buffer pH 7.6, followed by liquid-liquid extraction with dichloromethane for common BZD. A specific pre-treatment for amino-BZD, with an incubation of 15 min at 95 degrees C in 0.1 N NaOH before liquid-liquid extraction with dichloromethane, gave better recoveries and repeatability. After single exposure, bromazepam was present in powdered hair at 28 pg/mg and 7-Aminoclonazepam at 22 pg/mg in the first 1-cm segment, while no clonazepam was detectable. This method was applied in two forensic cases. It allowed us to determine bromazepam in urine 3 days after the alleged offense and in cut head hair at a concentration of 6.7 pg/mg only in the 2-cm proximal segment. The other case showed the presence of clonazepam and 7-Aminoclonazepam in urine a few hours after the offense and the presence of 7-Aminoclonazepam at about 3.2 pg/mg in axillary hair 4 months later.

GHB in postmortem toxicology. Discrimination between endogenous production from exposure using multiple specimens.

Since gamma-hydroxybutyrate (GHB) is present in both blood and urine of the general population as an endogenous compound, toxicologists must be able to discriminate between these endogenous levels and a concentration resulting from exogenous exposure. The implementation of a cut-off concentration must be done cautiously, due to the wide distribution of endogenous concentrations. To verify the accuracy of a proposed 50 mg/l postmortem blood cut-off, we tested 71 autopsy cases of subjects where the cause of death could exclude GHB exposure. The delay between death and autopsy ranged from 12 to 72 h. GHB was tested by gas chromatography-mass spectrometry (GC-MS) after precipitation. Briefly, 20 microl of blood, bile, or vitreous humor was pipetted in a glass tube, followed by 20 microl of GHB-d6 and 45 microl of acetonitrile. After vortexing and centrifugation, the supernatant was collected and evaporated to dryness. The residue was derivatized with bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (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 tested positive in all the 71 whole blood (cardiac) specimens, with concentrations in the range 0.4-409 mg/l, with a major distribution in the range 10-40 mg/l. A concentration >50 mg/l was observed in 14 cases. As there was no data to support GHB exposure, this was considered as postmortem formation. In order to discriminate this contamination, when available, femoral blood, bile or/and, vitreous humor were tested. The following results were obtained: cardiac blood (55-409 mg/l) versus bile (6.1-238 mg/l) in seven cases; cardiac blood (51-409 mg/l) versus femoral blood (17-44 mg/l) in five cases, and cardiac blood (51-409 mg/l) versus vitreous humor (3.9-2 mg/l) in six cases. It is obvious that bile does not fit the requirements for discrimination and that femoral blood and mostly vitreous humor can be of particular interest. These results demonstrate that a positive (>50 mg/l) postmortem blood GHB concentration cannot support alone drug exposure and that it is essential to document the case with other specimens, including peripheral blood and vitreous humor.

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

The purported lowering of sex opposition, coupled with a possible abrupt unconsciousness-inducing effect and ease of administration in spiked drinks have resulted in the use of hypnotics in cases of drug-facilitated offense. Among these compounds, lorazepam possesses amnesic properties and can impair an individual rapidly. The chances to detect this substance increase if the most sensitive methods are used and if the biological fluid which allows the longest possible detection time is available. In order to document the window of detection of lorazepam, we have orally administered 2.5 mg of the drug to three volunteers and collected oral fluid (n = l) over 8 h, urine (n = 2) over 144 h and hair (n = 3) 4 weeks after exposure. Lorazepam was analyzed by LC-MS/MS after alkalinisation (to pH 8.4 with phosphate buffer) and extraction by dichloromethane/diethyl ether in presence of diazepam-d5, used as internal standard. Reversed-phase separation on a XTerra C18 column was achieved in 12 min, under gradient conditions. Molecular ions (m/z 321 and 290 for lorazepam and the IS, respectively) were selected in Ql and the corresponding daughter ions (m/z 303 and 275 for lorazepam and m/z 154 and 198 for the IS) were detected in Q3 after collision with argon. Urine tested positive for lorazepam over 144 h (2-4 ng/ml), with a peak detected after 24 h exposure (411-880 ng/ml). Oral fluid tested positive for lorazepam over 8 h (0.7 ng/ml). Despite a limit of quantitation at 1 pg/mg, we were unable to detect a single lorazepam dose in hair, contrarily to most other benzodiazepines that are detectable. Therefore, in case of drug-facilitated crimes involving lorazepam, urine appears as the best specimen to document exposure, particularly if LC-MS/MS is used.

Hair to document drug-facilitated crimes: four cases involving bromazepam.

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, so-called DFSA, robbery) has caused alarm in the general public. Drugs used can be difficult to detect (active products at low dosages, chemical instability), possess amnesic properties, and can be quickly cleared from body fluids. In case of long delay between the alleged crime and clinical examination, collection of blood or even urine can be of little value. This is the reason why this laboratory developed an original approach based on hair testing by liquid chromatography-tandem mass spectrometry. To explore the detectability of a single absorption of bromazepam in hair, two volunteers (male and female) received a 6-mg dose. A strand of hair was sampled about one month after exposure and was cut into three segments of 2-cm long. After pulverization, 20 mg of hair was incubated overnight in a phosphate buffer (pH 8.4). The aqueous phase was extracted with 5 mL of a mixture of diethyl ether/methylene chloride (80:20) in the presence of diazepam-d5, which was used as 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 316.0 to 182.2 and 209.3 and m/z 290.1 to 154.1 and 198.2 for bromazepam and the IS, respectively. In the hair of the two subjects, bromazepam was detected in the proximal segment at 0.8 and 4.7 pg/mg, respectively. Hair analysis was applied to four authentic criminal cases. In the two first cases, bromazepam tested positive in the corresponding hair segment at 5.7, and 10.3 pg/mg. In another case, head hair was sampled 19 weeks after the alleged offense, and its length (< 4 cm) did not allow analysis of the corresponding period. However, 4.1 pg/mg of bromazepam was quantified in the victim's pubic hair. In these three cases, concentrations were consistent with a single exposure to bromazepam. In the last case, bromazepam was detected at 15 pg/mg in the segment corresponding to the period of the alleged offence but also in the range 2 to 7 pg/mg in the four other consecutive segments, making a single exposure statement difficult.