Hair testing in postmortem diagnosis of substance abuse: An unusual case of slow-release oral morphine abuse in an adolescent.

Morphine sulfate misuse is essentially observed among regular heroin injectors. To our knowledge, primary addiction to morphine sulfate is exceptional, especially among young adolescents. A 13-year-old girl, with no history of addiction, was found dead with three empty blisters of Skenan(®) LP 30 mg at her side. Opiates were detected in biological fluids and hair by chromatographic methods. Blood analyses confirmed morphine overdose (free morphine: 428 ng/mL; total morphine: 584 ng/mL) and segmental hair analysis confirmed regular exposure over several months (maximum morphine concentration 250 pg/mg). Suspecting the victim's mother of recreational use of Skenan(®), the magistrate ordered analysis of her hair, with negative results. From an epidemiological viewpoint, this case of oral morphine sulfate abuse in an adolescent with no previous history suggests the emergence of a new trend of morphine sulfate consumption. From a toxicological viewpoint, it demonstrates the value of hair testing, which documented the victim's regular exposure and made an important contribution to the police investigation.

A tendency for re-offending in drug-facilitated crime.

The authors present 3 cases that demonstrate a return to DFC following periods of inactivity. The offences occurred in Paris and its suburbs and in each of the cases there were two distinct periods of activity by the offenders with 2, 8 and 22 victims attributed to each of the perpetrators. To 20mg of decontaminated and cut hair, 100 pg/mg of clonazepam-d4 was added as internal standard. Hair specimens were extracted with CH(2)Cl(2)/ether after incubation overnight at 56 degrees C in pH 7.6 buffer. Extractions were performed on blood and urine using Toxi-tube A with 5 ng/mL of clonazepam-d4. The residues were analyzed by LC-ESI-MS/MS. Calibration curves in blood and urine (0.5-500 ng/mL) were prepared by spiking aliquots of blank fluids (r(2)>0.9816 for all drugs). LOD in body fluids ranged 0.5-10 ng/mL. Calibration curves in hair (0.5-100 pg/mg) were prepared by spiking aliquots of blank hair (r(2)>0.9877 for all drugs). LOD in hair ranged 0.5-5 pg/mg. Case #1: Two young women were raped with an interval of approximately 1 year between the incidents. Lorazepam (present, <2 pg/mg) was detected in hair obtained from the first victim, and zolpidem (19 pg/mg) in hair of the second one. The offender was in jail between the two offences. Case #2: The offender approached a total of 8 men and women who were aged over 50 years. The offender was in jail between the two series of respectively 3 and 5 victims. Zopiclone was detected in victims' hair (n=7) at concentrations 13-42 pg/mg. Case #3: The offender stole thousands of Euros using credit cards obtained from 22 different wealthy victims. He employed a cocktail of up to 6 drugs made up of: flunitrazepam, clonazepam, doxylamine, cyamemazine, zolpidem and lorazepam. Drugs were detected in all victims' hair (n=18) at concentrations in the range 1-81 pg/mg for all drugs. Between the two series (of respectively 4 and 16 victims) the offender spent 6 months in jail, and then police spent 6 months looking for him while he was under judiciary control prior to his judgment. Segmental hair analysis permits retrospective information on drug exposure and should be considered in the investigation of drug-facilitated crimes not only to prove single exposure but also when there has been any appreciable delay in samples being obtained for analysis. Indeed, in 56% cases reported in this paper, due to the long time that elapsed between offences and the opportunity to obtain samples for analysis hair analysis was considered the only viable matrix to investigate the possibility of drug involvement in the crimes. Our experience demonstrates that the incidence of re-offending in DFC after a period of inactivity (often due to imprisonment) may be of concern, notably in big cities.

The role of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to test blood and urine samples for the toxicological investigation of drug-facilitated crimes.

The authors present an overview of the drug-facilitated crime (DFC) phenomenon, especially in France. Recently, there has been an increase in reports of incidents (mainly sexual assaults and robbery) as well as in scientific publications and congress presentations on the topic. From enquiries conducted nationally, a list of drugs reportedly associated with DFC was established and includes benzodiazepines and benzodiazepine-like drugs (zolpidem, zopiclone), minor tranquilizers and neuroleptics, barbiturates, narcotics, hallucinogens, and anaesthetics. Some of these molecules are specific to France in DFC cases. A study using healthy volunteers who had taken benzodiazepines (lorazepam, bromazepam, flunitrazepam, clonazepam), zolpidem and zopiclone, showed that the only way to increase the duration of detection of these drugs is to use liquid chromatography-tandem mass spectrometry (LC-MS/MS) to test blood and urine samples. The very high sensitivity of this method appears to be an essential condition to document the cases, because the drugs tested were still detectable in urine at least 6 days after the ingestion of one therapeutic dose. Limits of detection were always lower than 0.5 ng/mL in urine. The actual list of molecules and metabolites the authors screened for in urine and blood by LC-MS/MS, in every DFC, is given in detail: 25 benzodiazepines and benzodiazepine-like drugs, 11 minor tranquilizers and neuroleptics, 2 barbiturates, 12 narcotics, 4 hallucinogens, and 1 anaesthetic. However, the distinction between continual therapeutic use of a psychotropic drug or illegal narcotic and a single ingestion has to be documented by sequential analysis of hair, again with LC-MS/MS.

Determination of ibogaine and noribogaine in biological fluids and hair by LC-MS/MS after Tabernanthe iboga abuse Iboga alkaloids distribution in a drowning death case.

Tabernanthe iboga belongs to the Apocynaceae family. In this study, we report the case of a 37-year-old black male working as a security agent in Paris and found dead naked on the beach in Gabon after consumption of iboga. Autopsy revealed a drowning fatality and a myocardial abnormality (myocardial bridging). Samples of blood, urine, bile, gastric content, liver, lungs, vitreous, spleen and hair were taken. Biological fluids were liquid-liquid extracted with saturated NH4Cl pH 9.5 and methylene chloride/isopropanol (95/5, v/v) in presence of clonazepam-d(4), used as internal standard. After decontamination with dichloromethane, hair was cut into small pieces then sonicated for 2h in saturated NH4Cl pH 9.5 before extraction by methylene chloride/isopropanol (95/5, v/v). After evaporation the residues were reconstituted in methanol/ACN/formate buffer pH 3, from which 10 microL were injected into an ODB Uptisphere C(18) column (150 mm x 2.1mm, 5 microm) and eluted with a gradient of acetonitrile and formate buffer delivered at a flow rate of 200 microL/min. A Quantum Ultra triple-quadrupole mass spectrometer was used for analyses. Ionization was achieved using electrospray in the positive ionization mode (ESI). For each compound, detection was related to three daughter ions (ibogaine: m/z 311.4-->122.1, 174.1 and 188.1; noribogaine: m/z 297.4-->122.1, 159.1 and 160.1; clonazepam-d(4): m/z 319.9-->218.1, 245.1 and 274.1). Ibogaine and noribogaine were detected in all autopsy samples. Hair segmentation was not possible as hair was very short and frizzy. Concentrations of 1.2 and 2.5 ng/mg, respectively were detected. Neither other licit or illicit drugs nor alcohol were found. The presence of ibogaine and noribogaine in all autopsy samples was consistent with the recent absorption of Tabernanthe iboga, which was assumed to be responsible of the drowning fatality. The history of exposure, regarding hair analysis, is discussed. LC-MS/MS appears to be the best method for analyzing complex and poorly volatile alkaloids in autopsy samples and particularly in hair, due to the presence of a nitrogen ring and the relatively low concentrations to be measured.

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

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

Liquid chromatography-tandem mass spectrometry for the determination of colchicine in postmortem body fluids. Case report of two fatalities and review of the literature.

Poisoning by colchicine may occur following ingestion of this alkaloid used for the treatment of acute gouty arthritis. The authors report two fatalities and describe a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) triple-quadrupole method for the determination of colchicine in autopsy samples. One milliliter of heart blood, femoral blood, urine, bile, gastric, and vitreous each were extracted with saturated NH4Cl at pH 9.6 and dichloromethane/5% isopropanol. Separation was achieved on a C18-Xterra column with a mobile phase consisting of 2 mM ammonium formate buffer (pH 3)/acetonitrile in a gradient mode. Four product ions of the protonated molecule were monitored. The method was fully validated in whole blood (1 mL) and was linear in the range of 0.5-50 ng/mL (r2>0.99). The limit of detection was 0.1 ng/mL (50 times S/N), and the limit of quantitation was 0.5 ng/mL with RSDs<11.8% intraday (n=6), <18.7% interday (n=18), and accuracy<3% (n=18). Case #1: a 33-year-old nurse committed suicide by the ingestion of 80 colchicine 1-mg tablets. She died 61 h later after resuscitation procedures. Colchicine was found in heart blood at 5.2 ng/mL, femoral blood at 17.4 ng/mL, urine at 19.4 ng/mL, bile at 42.8 ng/mL, gastric at 348 ng/mL, and vitreous at 3 ng/mL. Case #2: a 57-year-old man with gout was found dead at home. Colchicine was found in heart blood at 22.8 ng/mL, femoral blood at 21.9 ng/mL, lung blood at 45.2 ng/mL, urine at 148.5 ng/mL, bile at 1818.5 ng/mL, gastric at 219.8 ng/mL, and vitreous at 0.5 ng/mL. These results were consistent with death. Because of its good sensitivity, this LC-ESI-MS-MS triple-quadrupole method is suitable for the determination of colchicine not only in fatalities but also for pharmacokinetic studies.

A case in south-eastern France: a review of drug facilitated sexual assault in European and English-speaking countries.

Drug facilitated sexual assaults (DFSA) have been increasingly reported in the medical literature since the 1980s but their legal recognition is more recent, at least in Europe. From a case treated in south-eastern France, whose judicial consequences were known, it seemed of interest to carry out an international study of jurisprudence concerning this type of rape. While from the medical viewpoint the drugs used are well-known and their presence can be clinically verified, the legal consequences of their use in subsequent criminal prosecution is less clear-cut. Some European countries have no jurisprudence in this area, while others consider the use of drugs as an aggravating circumstance. In France, it was only in 2003 that the first case of DFSA was truly punished by the judicial system, with considerable media attention. By contrast, in English-speaking countries, particularly the United States, the use of drugs to facilitate sexual assault has frequently been recognized in legislation and in criminal prosecutions. Prevention is fundamental and is recognised as demonstrated by campaigns in various countries.

Hair analysis by liquid chromatography-tandem mass spectrometry in toxicological investigation of drug-facilitated crimes: report of 128 cases over the period June 2003-May 2004 in metropolitan Paris.

In recent years, reports of drug-facilitated crimes (DFC) have been increasing. The drugs involved are sometimes difficult to detect, because of their low dosages and the long time ellapsed between alleged DFC and blood and urine sampling. In order to detect benzodiazepines and benzodiazepine-like hypnotics, we developed an approach for hair analysis by liquid chromatography-tandem mass spectrometry using a triple stage quadrupole with an electrospray ionization (LC-ESI-MS/MS). Separation was performed on an Uptisphere ODB C18 column using a gradient of 2mM formate buffer and acetonitrile. For the 23 compounds studied, detection limits are lower than 2 pg/mg, but a specific extraction procedure is needed for 7-amino metabolites. Over a 1-year period within the city limits of Paris and three suburbs, we tested blood and urine from victims of sexual assaults, robbery and battery in which psychoactive substances were suspected of being involved. Hair was collected 4-8 weeks after the alleged DFC. Over the 128 cases studied, results of simultaneous analysis of blood, urine, and hair allowed us to conclude that 23 cases were real DFC. In 18 cases, no conclusion was possible since no hair was sampled and/or results were negative. In 56 cases, victims were previously using narcotics, cannabis, and/or a prescribed drug, according to the compounds detected in hair strands. Thirty-one cases were not DFC cases. This study indicates that the prevalence of zolpidem and clonazepam is high, followed by bromazepam, nordazepam, and midazolam. Others benzodiazepines and analogs are rare. LC-ESI-MS/MS is a good tool for toxicological investigations of DFC. Testing blood, urine, and hair by this technique may reveal drug presence, even if it was administered at a single therapeutic dose. That may be helpful to prosecute perpretators or to exclude a drug-facilitated crime.

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.

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.

Determination of endogenous levels of GHB in human hair. Are there possibilities for the identification of GHB administration through hair analysis in cases of drug-facilitated sexual assault?

We have developed a GC-MS-MS assay for GHB in human hair. Five milligrams of washed hair were hydrolyzed by 1M or 0.01M NaOH before a liquid-liquid extraction with ethyl acetate under acidic conditions. GHB-d(6) was used as the internal standard. TMS derivatives were formed before injection. TBDMS derivatives were used in cases of strong chromatographic interferences or in a confirmatory procedure. Analysis of basal levels of GHB in 61 drug-free donors gave the following results: the mean measured concentration for blond hair was 0.60 ng/mg (n = 12), SD = 0.19 ng/mg, and extreme figures were in the range 0.35-0.95 ng/mg. For brown hair, the mean measured concentration was 0.90 ng/mg (n = 30), SD = 0.42 ng/mg, and extreme figures 0.41-1.86 ng/mg. For black hair, the mean measured concentration was 0.90 ng/mg (n = 19), SD = 0.37 ng/mg, and extreme figures 0.32-1.54 ng/mg, showing no significant differences depending on hair color. Analysis of basal levels of GHB of 12 or more specimens in segmented hair showed a mean concentration of 1.22 ng/mg (0.31-8.4 ng/mg) and a relative standard deviation for each individual ranging from 6.75% to 37.98%. GHB was administered to a healthy 53-year-old white male (light brown hair) at oral dosages of 30, 45, and 60 mg/kg. Beard hair was collected just before administration and 24 h after (and each day for one week for the last dose), and a 7.5-cm scalp hair lock was collected 7 days after the last dose. A rise in GHB concentration was observed in beard hair for the 45 and 60 mg/kg dosages with a maximum at 24 h, whereas no change was observed for the 30 mg/kg dosage. Scalp hair was segmented into 3-mm long segments. The three proximal last segments showed significantly (0.0005 < p < 0.005) different concentrations of GHB (1.22, 1.27, and 1.66 ng/mg, respectively) when compared with the basal physiological level of GHB in this same person (mean = 0.62 ng/mg, SD = 0.15 ng/mg). A case of daily GHB abuse during bodybuilding allowed us to determine a concentration of GHB of 14 ng/mg, in a 2-cm long segment (black hair). A case of rape under the influence of GHB was documented through hair analysis (black hair) and positive analysis of the glass she used. Sampled 7 days after the sexual assault, the three last 3-mm long proximal segments tested for GHB exhibited concentrations of 3.1-5.3 and 4.3 ng/mg, respectively, whereas the mean physiological level determined in this woman was 0.71 ng/mg, SD = 0.17 ng/mg. The authors advise a two-step hair sampling as evidence of GHB consumption: the first sample at the time of exposure to show the contamination by sweat of the proximal segment in case of recent administration with a significant rise of hair level at the root, and the second after at least 3 or 4 weeks to avoid this contamination and determine the levels incorporated in the hair matrix before, during, and after the exposure.

An unusual case of death: suffocation caused by leaves of common ivy (Hedera helix). Detection of hederacoside C, alpha-hederin, and hederagenin by LC-EI/MS-MS.

We report one fatal case of asphyxia caused by leaves of common ivy. Macroscopic examination of the corpse during the autopsy disclosed an incredible quantity of leaves of Hedera helix in the mouth and throat of the decedent. In order to rule out the possibility of poisoning by the toxic saponins contained in the plant, we have developed an efficient LC-EI/MS-MS assay of hederacoside C, alppha-hederin, and hederagenin in biological fluids and plant material. Sample cleanup involved solid-phase extraction of the toxins on C18 cartridges followed by LC analysis under reversed-phase conditions in the gradient elution mode. Solute identification was performed using full scan MS-MS spectrum of the analytes. Oleandrine was used as internal standard. Under these conditions, saponins in powdered dried leaves of Hedera helix were measured at a concentration of 21.83 mg/g for hederacoside C, 0.41 mg/g for alpha-hederin and 0.02 mg/g for hederagenin. No toxin was detected in cardiac blood, femoral blood, or urine of the deceased, but hederacoside C was quantitated at 857 ng/mL in the gastric juice. These findings led us to conclude that the man committed suicide and that the death was caused by suffocation by leaves of common ivy.