Testing for zopiclone in hair application to drug-facilitated crimes.

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

Windows of detection of zolpidem in urine and hair: application to two drug facilitated sexual assaults.

A LC-MS/MS method for the detection of zolpidem in hair was developed to detect this drug after a single dose in possible drug facilitated sexual assaults. To determine the window of detection of zolpidem in both urine and hair, three volunteers received a 10 mg dose. Urine specimens were collected each 12 h for 144 h. Hair was sampled 3-5 weeks after exposure. Hair and urine extracts were separated on a Xterra MS C18 column using a gradient of acetonitrile and formate buffer. For each compound, detection was related to two daughter ions. Zolpidem was detected for up to 60 h in urine with peak concentrations obtained at 12 h. A single exposure to zolpidem was detected in hair at concentrations ranging from 1.8 to 9.8 pg/mg. Hair analysis was applied to two possible criminal cases. In the first case, zolpidem tested positive in the corresponding hair segment at 4.4 pg/mg. In the other case, zolpidem was detected in all the segments analyzed, demonstrating likely previous drug use in addition to recent exposure associated with a positive blood result.

Entomotoxicology for the forensic toxicologist: much ado about nothing?

We present a series of 29 necropsies in which organic compounds (including benzodiazepines, barbiturates, antidepressants, phenothiazine, opiates, cannabinoids, meprobamate, digoxin and nefopam) were detected in arthropod larvae sampled on human corpses. No correlation was observed between drug concentrations in the larvae versus human samples. When tested, inter-larvae and inter-site variations of drug concentrations (i.e., within larvae when analyzed separately, and within anatomic sites when larvae were grouped according to their site of sampling) were enormous and not reproducible from one case to another, confirming that arthropod larvae are unreliable for quantitative toxicological analysis. Since drugs identified in maggots are always detectable in the cadaver too, we conclude that larvae analysis is of almost no interest for practical forensic casework.

Testing for kavain in human hair using gas chromatography-tandem mass spectrometry.

A sensitive, specific and reproducible method for the quantitative determination of kavain in human hair has been developed. The sample preparation involved a decontamination step of the hair with methylene chloride. The hair sample (about 50 mg) was incubated in 1 ml of methanol for 1 h, in an ultrasonic bath, in presence of 20 ng of methaqualone-d7 used as internal standard. The methanolic solution was evaporated to dryness, and the residue reconstituted by adding 30 microl of methanol. A 2 microl aliquot of the extract was injected onto the column (Optima5-MS capillary column, 5% phenyl-95% methylsiloxane, 30 m x 0.25 mm i.d. x 0.25 mm film thickness) of a Hewlett-Packard (Palo Alto, CA) gas chromatograph (5890). Kavain was detected by its parent ion at m/z 230 and daughter ions at m/z 111 and 202 through a Finnigan TSQ 700 MS/MS system. The assay was capable of detecting 30 pg/mg of kavain (limit of detection (LOD)). Linearity was observed for kavain concentrations ranging from 100 to 2000 pg/mg with a correlation coefficient of 0.998. Intra-day precision at 400 pg/mg was 13.7%. The analysis of a segment of hair, obtained from an occasional consumer, revealed the presence of kavain at the concentration of 418 pg/mg. A higher concentration (1708 pg/mg) was detected in the corresponding pubic hair.

HPLC/MS findings in a fatality involving sustained-release verapamil.

A fatality involving verapamil, a calcium channel blocker agent, is presented. A 51-year old male ingested 7200 mg of sustained-release (SR) verapamil at T0 and died 40 hours later of refractory, mixed shock and multiorgan failure. The symptoms displayed during hospitalization were quite typical and involved altered consciousness, hypotension, bradycardia, atrioventricular block, metabolic acidosis and renal failure. Verapamil and its primary metabolite, norverapamil, were assayed on eight plasma and two urine samples, successively taken between the admission to the ICU (T0 + 4 hours) and time of death, using an original high-performance liquid chromatography/mass spectrometry (HPLC/MS) procedure with verapamil-d3 as internal standard. Plasma verapamil and norverapamil levels on admission were 0.94 and 1.36 microg/mL, respectively, then verapamil remained practically unchanged throughout the hospitalization (0.85 microg/mL at T0 + 40 hours). The discussion focuses on the detrimental role of SR formulations in overdose, with special emphasis on the risk of pharma-cobezoar development already reported with SR-verapamil. To our knowledge, this is the first report of a verapamil fatality documented by repeated plasma measurements of the drug during the antemortem period.

HPLC-MS for the determination of sildenafil citrate (Viagra) in biological fluids. Application to the salivary excretion of sildenafil after oral intake.

An original high-performance liquid chromatography-mass spectrometry (HPLC-MS) procedure was developed for the determination of sildenafil in biological fluids. Liquid-liquid extraction was performed by chloroform/2-propanol/n-heptane (25:10:65, v/v) at pH 9.5 with 300 ng of buprenorphine-d4 as the internal standard (IS). After agitation (10 min) and centrifugation (3500 x g, 10 min), the organic phase was evaporated and the dry extract resuspended in 25 microL methanol, from which 2 microL was injected onto a NovaPak C18 (Waters) HPLC column. Separation was carried out by a gradient of (acetonitrile + 10 microg/mL trimethylamine) in 2mM NH4COOH pH 3.0 buffer (35-70% in 9 min). Detection was done by a PerkinElmer Sciex API-100 single-quadrupole mass analyzer with an ionspray interface operated in positive-ion mode. MS data were collected as either TIC or SIM at m/z (475 + 534) or (475 + 283) for sildenafil, depending on the potential applied at the ion sampling orifice (0 V or + 100 V). The retention times of sildenafil and the IS were 4.20 and 5.07 min, respectively. Extraction recoveries were always > 87%. LOD and LOQ were 0.2 and 0.5 ng/mL whatever the biological fluid tested. The method appears specific, extremely sensitive, and relatively simple in both equipment and sample preparation. As an example, we present the results of a preliminary study on the salivary excretion of sildenafil following the oral intake (T0) of 25 mg Viagra in a 38-year-old volunteer. Sildenafil was detectable in oral fluid at T0 + 0.5 h (1.2 ng/mL) and peaked at T0 + 1.5 h (8.3 ng/mL), whereas at the same time its plasma concentration was 72.4 ng/mL. Salivary concentrations then rapidly decreased, and the last detectable value (0.9 ng/mL) was at T0 + 5.5 h. It is suggested that the salivary excretion pattern of sildenafil resembles that of benzodiazepines (high plasma protein binding, low saliva-to-plasma ratio).

Fatal overdosage with nefopam (Acupan).

This paper presents a fatality due to massive, intravenous self-administration of nefopam (Acupan), a non-opiate central analgesic, in a 37-year-old female. Nefopam was measured in various postmortem samples by means of high-pressure liquid chromatography coupled to mass spectrometry via an ionspray interface. Heart blood concentration was 4.38 microg/mL and exceeded by approximately 30 times the highest therapeutic levels with the usual reservations concerning possible postmortem redistribution. This is only the third case of death following nefopam overdose reported in the literature.

Determination of blood cyanide by HPLC-MS.

An original high-performance liquid chromatographic-mass spectrometric (HPLC-MS) procedure was developed for the determination of cyanide (CN) in whole blood. After the addition of K13C15N as internal standard, blood was placed in a microdiffusion device, the inner well of which was filled with a mixture of taurine (50mM in water)/naphthalene-2,3-dicarboxaldehyde (NDA, 10mM in methanol)/methanol/ concentrated (approximately 20%) ammonia solution (25:25:45:5, v/v). Concentrated H2SO4 was added to the blood sample, and the microdiffusion chamber was sealed. After 30 min of gentle agitation, 2 microL of the contents of the inner vial were pipetted and directly injected onto a NovaPak C18 HPLC column. Separation was performed by a gradient of acetonitrile in 2mM NH4COOH, pH 3.0 buffer (35-80% in 10 min). Detection was done with a Perkin-Elmer Sciex API-100 mass analyzer with an ionspray interface, operated in the negative ionization mode. MS data were collected as either TIC or SIM at m/z (299 + 191) and (301 + 193) for the derivatives formed with CN and 13C15N, respectively. Inspired by previous works dealing with the complexation of CN by NDA + taurine to form a 1-cyano [f] benzoisoindole derivative analyzed by HPLC-fluorimetry, this method appears simple, rapid, and extremely specific. Limits of detection and quantitation for blood CN are 5 and 15 ng/mL, respectively. The use of 13C15N as internal standard allows the quantitation of CN with elegance and accuracy in comparison with previously reported methods.

Fatal overdosage with sildenafil citrate (Viagra): first report and review of the literature.

A fatal case of sildenafil citrate (Viagra) overdosage is presented. The deceased was a 56-year old male found dead at home, with a past history of diabetes mellitus, hypertension, chronic alcoholism, anxio-depressive disorders, and erectile dysfunction. The main autopsy findings were cardiomegaly (650 g) with dilated cardiomyopathy, diffuse coronary atherosclerosis with no sign of acute ischaemic disease, and extensive fibrosis of the myocardium, especially affecting the cardiac conducting tissue. As measured by HPLC/MS, sildenafil concentration in postrmortem blood (6.27 microg/mL) exceeded at least four times the highest therapeutic levels previously reported. The results are discussed in the light of the literature about the cardiovascular side effects of sildenafil, with special emphasis on the recently evidenced arrhythmogenic potential of the drug. This is the first report of a fatality caused by sildenafil overdosage.

Repeated measurements of aldicarb in blood and urine in a case of nonfatal poisoning.

A nonfatal case of poisoning involving aldicarb, an extremely toxic carbamate pesticide, is presented. A 39-year-old female ingested an unknown amount of aldicarb, together with alprazolam and sertraline. On admission to ICU (T0), she displayed marked cholinergic symptoms and a deep coma. The patient was given pralidoxime and atropine. Her condition gradually improved on days 2 and 3 and she was discharged at T0+80 h. Aldicarb was assayed by high-performance liquid chromatography on 21 blood and 8 urine samples successively taken during hospitalization. At the same time, serum pseudocholinesterase activity was followed on 21 successive samples. Blood aldicarb level was 3.11 microg/mL at T0 and peaked at T0+3.5 h (3.22 microg/mL), then followed a two-slope decay with a terminal half-life of ca. 20 h. Aldicarb was detected in all urine samples (peak level: 6.95 microg/mL at T0+31.5 h) and was still present at the time of discharge. Serum pseudo-cholinesterase activity remained low (< or = 10% of normal) until the 30th hour then rapidly increased and returned to normal after the 60th hour. The patient's clinical picture closely followed blood aldicarb levels and serum pseudo-cholinesterase activities. To our knowledge, this is the first report of an aldicarb poisoning documented by repeated measurements of the drug in the intoxicated person.

Fatal blunt pancreatic trauma secondary to assault and battery: a case report.

The authors report on an fatal case of closed trauma of the pancreas in a context of violence. A 55-year-old man was found unconscious on the sidewalk and died a short time after being taken to the hospital. He had been hit with several punches to the face and abdomen 6 h before. The post-mortem examination showed numerous bruises over the whole body, a haemoperitoneum, a fissuration of the spleen and a massive peripancreatic haemorrhage associated with a complete dilaceration of the pancreas head. Histological examination of the pancreas revealed a massive necrosis associated with a subtotal disappearance of the acini, numerous sites of cytosteatonecrosis and a large haemorrhagic suffusion of the peripancreatic tissue. This case illustrates the possibilities of pancreatic injuries induced by blunt force aimed at the abdomen in a context of violence. Even if this occurs as an isolated injury it can result in rapid death because of the particular type of pancreatic fracture which is frequently involved. In postmortem situations, the pancreas should be systematically checked at necropsy and a histological examination should be carried out at the slightest doubt of a pancreatic lesion or suspicion of blunt force abdominal injuries.

The distribution of laudanosine in tissues after death from atracurium injection.

A case is presented involving an acute fatality resulting from self-administration of atracurium, a muscle relaxant by a 45-year-old nurse. In the body, atracurium undergoes a spontaneous non-enzymatic degradation to laudanosine and an acrylate moiety. Laudanosine was quantified using gas chromatography coupled to mass spectrometry after extraction with chloroform-isopropanol-n-heptane (50:17:33 v/v) at pH 9.5 and separation on a HP5-MS capillary column. Laudanosine was subject to postmortem redistribution due to release from drug-rich tissues such as the lung and heart. The heart blood (917 ng/ml) to peripheral blood (390 ng/ml) ratio was 2.4. No other drugs, including ethanol were detected.

Aldicarb poisoning.

Aldicarb (2-methyl-2(methylthio) propanal o-[(methylamino)-carbonyl] oxime) is a pesticide manufactured since 1965. This carbamate ester is sold under the tradename, Temik, and is used as insecticide and nematicide. The Environmental Protection Agency has classified aldicarb in the highest toxicity category and has defined a strict control for its delivery and use. In Brazil and the Caribbean island, aldicarb is illegally used as a household rodenticide with a widespread risk of poisoning. Our study presents the first review of aldicarb poisoning circumstances associated with clinical and analytical findings. Moreover, the oxime treatment is discussed. Eighteen patients with cholinergic symptoms admitted to the Emergency Unit and two deceased with a history of aldicarb poisoning were included in the study. As agricultural workers, only two of them could legally use Temik. Seventy percent of the patients was managed by the Emergency Mobil Unit. Serum cholinesterase activity was always lower than 30% of the normal range and aldicarb was identified by UV spectra and retention time after liquid chromatography separation. The most common muscarinic effect was diarrhea, the main nicotinic sign fasciculation and almost half of the poisoned patients had central nervous system (CNS) depression (Glasgow Coma Score lower than 8). Four patients had serious conduction abnormalities and two of them died. These results suggest that aldicarb intoxication is always severe. Oxime treatment did not produce side effects and should be recommended whenever the pesticide involved is unknown. Effective measures should be implemented to stamp out the illicit use of aldicarb.

Diatom analysis in victim's tissues as an indicator of the site of drowning.

The diagnosis of drowning is one of the most difficult in forensic pathology and previously we proposed criteria for a positive tissue analysis according to the qualitative and quantitative diatom investigations. In the positive cases, we studied the reliability of determining the site of drowning by comparing the diatom taxa found in the lung samples with those of the water samples or in the absence of these samples with the results of the water diatom monitoring programme set up in our region. In this study, we present two series of cases, the first is one of 20 corpses who died from accidental or suicidal drowning with known drowning site, and the second of 20 corpses for which the drowning site was unknown. The results showed that a concordance of the abundance of the diatom taxa in tissues compared to the site of drowning and their distribution relative to one another was 65% in the group where the site of drowning was known and 35% in the other group. The concordance of the individual distribution in the lungs of water diatom taxa may be an interesting method to guide the investigations for determining the site of drowning. The two limiting factors are the concentration of diatoms in the lungs and the development of a river monitoring programme in the district of the study.

[Acute poisoning during substitution therapy based on high-dosage buprenorphine. 29 clinical cases--20 fatal cases]. / Intoxications aiguës par traitement substitutif à base de buprénorphine haut dosage. 29 observations cliniques--20 cas mortels.

OBJECTIVES: Buprenorphine has been an important advance in care for drug abusers, but the toxic risk may be fatal. We report here two original series of buprenorphine poisoning in opiate abusers on substitution therapy. PATIENTS: The first series included 20 males and 9 females, aged 20-35 years (mean = 27.5) with non-fatal poisoning. The second series included 20 subjects (19 males, 1 female) aged 14-48 years (mean = 26.6) with a fatal outcome. All subjects were opiate addicts taking high-dosage sublingual buprenorphine formulation as substitution therapy. RESULTS: Blood concentrations of buprenorphine were found in all cases to remain at a low level (1.0-2.3 ng/ml, m = 1.4 ng/ml, and 1.1-29.0 ng/ml, m = 8.4 ng/ml in non-fatal and fatal cases respectively). Almost all cases involved concomitant intake of psychotropic medications, especially benzodiazepines (18 non-fatal and 17 fatal cases). DISCUSSION: These observations confirm previously reported data on the danger of buprenorphine-benzodiazepine combinations. Intravenous injection of crushed tablets also appears to be a risk factor (8 deaths and 10 non-fatal poisonings). This series highlights the need for improvement in the recently developed French program for substitution therapy with high-dosage buprenorphine in heroin addicts.

Buprenorphine-related deaths among drug addicts in France: a report on 20 fatalities.

This paper reports a series of 20 fatalities involving a high-dose, sublingual buprenorphine (BUP) formulation recently marketed in France for the substitutive therapy of opiate addicts. The files were recorded over a 16-month period from five different urban areas in France. All subjects but one were male, aged 14-48 (mean 26.6). BUP and its primary metabolite norbuprenorphine (norBUP) were assayed in postmortem fluids and viscerae by HPLC-MS. Blood levels for BUP and norBUP ranged from 1.1 to 29.0 ng/mL (mean 8.4 ng/mL) and 0.2 to 12.6 ng/mL (mean 2.6 ng/mL), respectively, that is, within or slightly over the therapeutic range. BUP exhibited extensive tissue distribution, with average postmortem concentrations of 6.0, 35.0, 45.5, and 80.0 ng/g in the myocardium, kidney, brain, and liver, respectively. In blood, as in viscerae, norBUP levels were generally lower than BUP. The highest concentrations were found in the bile for both BUP (range 575-72,650 ng/mL) and norBUP (range 41-30,000 ng/mL). Therefore, bile may represent a sample of choice for postmortem screening. BUP was identified in 9 of the 11 hair samples assayed at concentrations ranging from 6 to 597 ng/g (mean 137 ng/g), whereas norBUP was never detected. Intravenous injection of crushed tablets, a concomitant intake of psychotropics (especially benzodiazepines), and the high dosage of the BUP formulation available in France appear to be the major risk factors for such fatalities.

Car crash after massive ingestion of digoxin and midazolam.

In a case where a 32-year-old man lost control of his vehicle, urine and blood samples were taken 6 h after the crash for toxicological investigations. In the hospital, the driver admitted consumption of some drugs, in particular digoxin and midazolam just before the crash which corresponded to the results of blood analyses. Toxicological findings indicated the presence of digoxin at 12.9 ng/ml and midazolam at 7 ng/ml in the blood. These results suggested that at the moment of the crash digoxin and midazolam blood levels were in the range of toxic and therapeutic concentrations, respectively. Therefore the respective roles of the drugs in the impairment of the ability to drive at the moment of the crash is discussed.

Enantioselective analysis of methadone in sweat as monitored by liquid chromatography/ion spray-mass spectrometry.

In recent years, remarkable advances in sensitive analytical techniques have enabled the analysis of drugs in unconventional samples, such as sweat. In a study conducted during a methadone maintenance program, PharmChek sweat patches were applied to 20 subjects. The subjects were orally administered methadone in 1 dosage/day, and doses ranged from 80 to 100 mg. The sweat patch was applied 10 minutes before administration and removed 72 hours later just before a new administration of methadone. The absorbent pad was stored at -20 degrees C until analysis in plastic tubes. Methadone was extracted in 5 ml methanol in presence of 200 ng of racemic methadone-d3, used as internal standard. After a 30-minute agitation, the methanol solution was evaporated to dryness. Enantioselective separation of methadone was obtained using an alpha-1-acid glycoprotein column (100 x 4 mm ID) and liquid chromatography/ion spray-mass spectrometry. In all 20 specimens obtained from subjects under racemic methadone treatment, R- (the active form) and S-enantiomers of methadone were identified with the following concentrations: 26 to 1118 ng/patch for R-methadone and 28 to 1114 ng/patch for S-methadone. The ratio between R- and S-methadone was in the range of 0.72 to 2.66 and was higher than 1.00 in 15 samples. No correlation between the doses of methadone administered and the concentrations of methadone in sweat was observed.