Site-dependent production of gamma-hydroxybutyric acid in the early postmortem period

This study compared endogenous gamma-hydroxybutyric acid (GHB) concentrations in various postmortem fluid samples of 25 autopsy cases. All bodies were stored between 10-20 degrees C until autopsy, and the intervals between death and autopsy were less than 2 days (6-48 h). GHB concentrations were measured by headspace gas chromatography after GHB was converted to gamma-butyrolactone. Endogenous GHB concentrations were significantly higher in femoral venous blood (4.6+/-3.4 microg/ml, n=23) than in cerebrospinal fluid (1.8+/-1.5 microg/ml, n=9), vitreous humor (0.9+/-1.7 microg/ml, n=8), bile (1.0+/-1.1 microg/ml, n=9) and urine (0.6+/-1.2 microg/ml, n=12). GHB concentrations were similar in blood samples taken from different sites. Cut-off limits of 30 and 10 microg/ml are proposed for blood and urine, respectively, to discriminate between exogenous and endogenous GHB in decedents showing no or little putrefaction (postmortem intervals usually 48 h or less). The criterion established for endogenous GHB in postmortem urine may also be applicable to analytical results in cerebrospinal fluid, vitreous humor and bile from deceased persons.     

Quantification of propane in biological materials by head-space GC

Propane was measured in specimens taken from two persons who died from a LPG explosion in an apartment using head-space-GC/FID. Because of the variation of instrument performance and sample injection, the internal standard pentane was used. Calibration standards were prepared by injecting each calculated volume of pure propane gas into capped vials containing 2 ml of blood and 5 microl of internal standard. The calibration curve revealed good linearity from 0.09 microg/ml to at least 90 microg/ml. The method validation data also included repeatability and recovery. The propane quantities in blood, fat and brain tissue were between 0.27 and 71 microg/ml (microg/g) with the highest concentration observed in fat. The confirmation of propane was conducted by the means of solid phase micro-extraction and mass spectrometry.     

Intoxication due to 1,4-butanediol

We report a case of intoxication resulting from the ingestion of a liquid, sold in the illicit market as "liquid ecstasy," which was found to contain 1,4-butanediol, a metabolic precursor of gamma-hydroxybutiric acid (GHB). Identification of the substance in the liquid was performed by gas chromatography-mass spectrometry (GC-MS).The toxicological analysis of blood, urine and gastric content of the victim was performed by immunoassay and gas chromatography with nitrogen-phosphorus detection as screening techniques and by means of GC-MS for confirmation and quantitation of 1,4-butanediol and GHB. The following drug concentrations were found: 82 microg/ml (blood), 401 microg/ml (urine) and 7.4 microg/ml (gastric content) for 1,4-butanediol and 103 microg/ml (blood), 430.0 microg/ml (urine) for GHB. In addition to these, other drugs detected and their blood concentration found in this case were methylenedioxymethylamphetamine (MDMA) 0.23 microg/ml and its metabolite methylenedioxyphenylamphetamine (MDA) 0.10 microg/ml. In the urine, a concentration of 0.10 microg/ml of benzoylecgonine was also found.     

Amphetamine derivative related deaths in northern Greece

Until 1997, only one amphetamine related derivatives (AMPs) fatality had been reported in Greece. Since then, amphetamine (AMP) or AMPs have been found in seven out of 1,500 post-mortem toxicological cases. The cause and manner of death of these seven cases were: 3,4-methylenedioxy-N-methamphetamine (MDMA) and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) poisoning (n = 1), drowning in water (n = 4), cranial injuries caused by a traffic accident (n = 1) and heart failure (n = 1). In the case where the use of AMP or AMPs was considered, the immediate cause of death post-mortem toxicological analysis revealed 2 microg/ml MDMA and 0.7 microg/ml MDEA in blood. MDMA was identified in two cases of drowning (2 microg/ml in blood in the first case and 1.7 microg/g in liver in the second case) and in the traffic accident case (0.4 microg/g in liver). Methamphetamine was detected in two cases of drowning (2.5 microg/ml in blood in the first case and 6 microg/g in liver in the second case). AMP was found in the heart failure case (0.2 microg/g in liver). Alcohol was present, together with AMP or AMPs, in four cases. These findings indicate an increase in the illegal abuse of AMPs in Greece. Because of this, we now routinely screen for AMPs.     

A fentanyl fatality involving midazolam

A case is presented of a 35-year-old black African male anesthesiology resident, found dead in his apartment. At the scene a syringe, butterfly intravenous line and a bottle of Versed (Midazolam) were recovered. A comprehensive screen for common drugs of abuse and therapeutic agents failed to detect any drugs in blood and urine. The blood ethanol concentration was 0.06 g/dl. A GC/MS SIM assay for midazolam was developed. A sub-therapeutic midazolam blood concentration of 7.5 ng/ml was detected and concentrations (ng/ml or ng/g) in bile, urine, and liver were 3.3, 7.5, and 96, respectively. The syringe fluid was then analyzed and found to contain only fentanyl, midazolam was absent. The blood fentanyl concentration was 4.9 ng/ml which is consistent with those reported in fentanyl fatalities. Fentanyl concentrations (ng/ml or ng/g) in bile, urine, and liver were 8.8, 5.0, and 5.9, respectively. The cause of death was ruled to be fentanyl intoxication and the manner of death undetermined.     

Application of solvent microextraction to the analysis of amphetamines and phencyclidine in urine

A fast and simple method to detect some commonly abused illicit drugs, amphetamine, methamphetamine, 3,4-methylendioxy-amphetamine (MDA), 3,4-methylendioxy-methamphetamine (MDMA), 3,4-methylendioxy-N-ethylamphetamine (MDEA) and phencyclidine (PCP) in urine using solvent microextraction (SME) combined with gas chromatography (GC) analysis has been developed. The extraction is conducted by suspending a 2 microl drop of chloroform in a 2 ml urine sample. Following 8 min of extraction, the organic solvent is withdrawn into the syringe and injected into a GC with a pulsed discharge helium ionization detector (PDHID). The effects of different extraction solvents and times, pH and sample preparation were studied. The optimized method was capable of detecting drugs in urine at concentrations below Substance Abuse and Mental Health Services Administration (SAMHSA) established cut-off values for preliminary testing. Good linearity and reproducibility of extraction were obtained. The limits of detection were 0.5 microg/ml for amphetamine, 0.1 microg/ml for methamphetamine and MDA, 0.05 microg/ml for MDMA, 0.025 microg/ml for MDEA and 0.015 microg/ml for PCP. Relative standard deviation (R.S.D.) values ranged between 5 and 20% for the studied drugs.     

Urinary endogenous concentrations of GHB and its isomers in healthy humans and diabetics

Urinary endogenous concentrations of gamma-hydroxybutyric acid (GHB), alpha-hydroxybutyric acid (AHB) and beta-hydroxybutyric acid (BHB) have been investigated for both healthy humans and diabetics by using a newly optimized GC-MS procedure. The endogenous concentrations in healthy volunteers' urine ranged 0.16-2.14 microg/ml for GHB, 0.10-2.68 microg/ml for AHB and 8.51-34.7 microg/ml for BHB. In diabetics, the concentrations ranged 0.17-3.03 microg/ml for GHB, 0.14-124 microg/ml for AHB and 4.94-4520 microg/ml for BHB. Although notably elevated BHB and AHB concentrations were observed for severely uncontrolled diabetics, their GHB concentrations ranged within or near the range seen in healthy humans. The results of this study confirm the previously suggested 10 microg/ml cutoff concentration of urinary GHB to distinguish exogenous GHB, even for uncontrolled diabetic patients suffering severe ketoacidosis.     

Headspace solid phase microextraction for the gas chromatographic analysis of methyl-parathion in post-mortem human samples. Application in a suicide case by intravenous injection

A simple and rapid procedure for the determination of methyl-parathion (m-p) in post-mortem biological samples was developed using headspace solid phase microextraction (SPME) and gas chromatography (GC) with nitrogen-phosphorous detection (NPD). Methyl-parathion was extracted on 85 microm polyacrylate SPME fiber. Salt addition, extraction temperature, and extraction time were optimized to enhance the sensitivity of the method. The linearity (y = 0.0473x - 0.0113, R2 = 0.9992) and the dynamic range (0.1-40 microg/ml) were found very satisfactory. The recoveries of methyl-parathion were found to be 46% in spiked human whole blood, 53% in spiked homogenized liver tissue, and 54% in spiked homogenized kidney tissue compared with samples prepared in water. The coefficients of variations for 2, 4, and 20 microg/ml of methyl-parathion in blood ranged from 0.9 to 5.1%, whereas the detection limit of the method was satisfactory (1 ng/ml in aqueous samples, 50 ng/ml in whole blood). The developed procedure was applied to post-mortem biological samples from a 21-year-old woman fatally poisoned (suicide) by intravenous injection of methyl-parathion. The intact insecticide was found in the post-mortem blood at a concentration of 24 microg/ml. No methyl-parathion was detected in the liver, kidneys, and gastric contents.     

Tissue distribution of nitrazepam and 7-aminonitrazepam in a case of nitrazepam intoxication

We report a case of nitrazepam poisoning in which the distribution of nitrazepam and 7-aminonitrazepam was determined in body fluids and tissues. A 52-year-old woman was found dead in a shallow ditch (approximately 5 cm in depth), in the winter. Ambient temperature was 2-8 degrees C. The postmortem interval was estimated to be approximately 1 day and no putrefaction was observed. The cause of death was thought to be drowning due to nitrazepam overdose and cold exposure. Blood concentrations of nitrazepam and 7-aminonitrazepam were very site dependent (0.400-0.973 microg/ml and 0.418-1.82 microg/ml). In addition, the concentration of the same analytes in the bile were 4.08 and 1.67 microg/ml, respectively, and in the urine: 0.580 and 1.09 microg/ml, respectively. A high accumulation of both substances was observed in various types of brain tissue (2.17-6.22 microg/g and 2.49-5.11 microg/g). Only small amounts of nitrazepam and 7-aminonitrazepam were detected in the liver (0.059 and 0.113 microg/g, respectively). Large differences in the observed concentrations of nitrazepam and 7-aminonitrazepam among arterial and venous blood samples were thought to be mainly due to dilution of arterial blood by water entering the circulation through lungs at the time of death. Bacterial metabolism of nitrazepam may also have contributed to the observed differences.     

Forensic intoxication with clobazam: HPLC/DAD/MSD analysis

Clobazam (Castillium, Urbanil), a benzodiazepine often used as an anxiolytic and in the treatment of epilepsy, is considered a relatively safe drug. The authors present a fatal case with a 49-year-old female, found dead at home. She had been undergoing psychiatric treatment and was a chronic alcoholic. The autopsy findings were unremarkable, except for multivisceral congestion, steatosis and a small piece of a plastic blister pack in the stomach. Bronchopneumonia, bronchitis and bronchiolitis were also diagnosed. Anhigh-performance liquid chromatography (HPLC)/diode array detector (DAD)/mass spectrometry detection (MSD) with electrospray method was developed in order to detect, confirm and quantify clobazam in the post-mortem samples. In the chromatographic separation, a reversed-phase column C18 (2.1 x 150 mm, 3.5 microm) was used with a mobile phase of methanol and water, at a 0.25 ml/min flow rate. Carbonate buffer (pH 10.5) and 20 microl of prazepam (100 microg/ml) as internal standard were added to the samples. A simple and reliable liquid-liquid extraction method for the determination of clobazam in post-mortem samples was described. Calibration curves for clobazam were performed in blood, achieving linearity between 0.01 and 10 microg/ml and a detection limit of 1.0 ng/ml. The clobazam concentration found in post-mortem blood was 3.9 microg/ml, higher than the reported therapeutic concentration (0.1-0.4 microg/ml). The simultaneous acquisition by photodiode array detection and mass spectrometry detection results allowed benzodiazepines to be identified with sufficient certainty. An examination of all the available information suggested that death resulted from respiratory depression due to clobazam toxicity.     

Nutmeg (myristicin) poisoning--report on a fatal case and a series of cases recorded by a poison information centre

In literature, cases of nutmeg abuse have been described repeatedly, but only one fatal case of poisoning was reported [1]. In the present case, myristicin (4 microg/ml) was detected for the first time in the postmortal serum of a 55-year-old woman. Identification was achieved with the aid of UV-VIS spectroscopy and TLC; for quantification, HPLC was used. Because also flunitrazepam (0.072 microg/ml) was found, death had probably been due to the combined toxic effect of both substances. From 1996 to 1998, in a series of cases, seven poisonings with nutmeg were recorded by the Erfurt Poison Information Centre. Even where higher doses (20-80 g of powder) had been ingested, a life-threatening situation was never observed. In one of these cases, a myristicin blood level of 2 microg/ml was measured 8h after ingestion of two to three tablespoonful of nutmeg powder (approx. 14-21 g, or 280-420 mg/kg).     

LC-MS/MS analysis of pholedrine in a fatal intoxication case

Pholedrine (4'-hydroxymethamphetamine) is a cardiovascular agent exerting hypertensive and adrenergic effects. High doses may cause a drop in the peripheral circulation blood flow and increase blood pressure, heart rate and body temperature up to a state of central respiratory paralysis. A 15-year-old girl who suffered from heavy agitation and hallucinations was admitted to the intensive care unit in a comatose state. The clinical findings included a maximum heart rate of 170 bpm and a body temperature of 43.8 degrees C. Resuscitation measures were in vain and abandoned after approximately 2h. A toxicological emergency analysis using GC/MS revealed a considerable amount of pholedrine in blood and urine. A method for determining pholedrine in human body fluids utilizing high-performance liquid chromatography (HPLC)/tandem mass spectrometry (LC-MS/MS) with a turbo ion-spray source was developed, using D11-methamphetamine and D5-methylenedioxymethamphetamine as internal standards. Samples were prepared by SPE extraction using SPEC-C18AR/MP3((R)) columns, which yielded the best extraction recovery (67%). Chromatographic separation was achieved at pH 5 on an RP-18 stationary phase applying gradient elution from 50 to 70% of B (methanol/acetonitrile 3/1 (v/v), 0.02% acetic acid) in A (5mM ammonium acetate/acetonitrile 95/5 (v/v), 0.02% acetic acid). Supra-pure acetic acid was added to the post-column effluent with a flow rate of 0.2 microl/min to optimize ionization. Detection was carried out in the positive ionization, multiple reaction monitoring (MRM) mode. The chromatograms showed no interference from other substances. The limit of detection (LOD, S/N=3) of pholedrine was 0.8 ng/ml and its lower limit of quantification (LLOQ, S/N=10) 3ng/ml. The calibration curve was linear (r=0.999) in the range 1-100 ng/ml. Samples with higher concentrations were diluted to suit the working range. The intra-day R.S.D. between 5 and 80 ng/ml were 3.8-8.7% and the inter-day R.S.D. between 5 and 100 ng/ml were 6.7-10.7%. The pholedrine concentrations in blood and urine collected when the girl was still alive were 16.1 microg/ml (R.S.D. 10.5%) and 1120 microg/ml (R.S.D. 8%), respectively. In post-mortem samples, they were 23.0 microg/ml (R.S.D. 5.1%) in heart blood and 27.3 microg/g (R.S.D. 6.6%) in the liver.     

Concentration of drugs in blood of suspected impaired drivers

Analytical records concerning 440 living drivers suspected of driving under the influence of drug (DUID) were collected and examined during a 2 years period ranging from 2002 to 2003 in canton de Vaud, Valais, Jura and Fribourg (Switzerland). This study included 400 men (91%) and 40 women (9%). The average age of the drivers was 28+/-10 years (minimum 16 and maximum 81). One or more psychoactive drugs were found in 89% of blood samples. Half of cases (223 of 440, 50.7%) involved consumption of mixtures (from 2 to 6) of psychoactive drugs. The most commonly detected drugs in whole blood were cannabinoids (59%), ethanol (46%), benzodiazepines (13%), cocaine (13%), amphetamines (9%), opiates (9%) and methadone (7%). Among these 440 cases, 11-carboxy-THC (THCCOOH) was found in 59% (median 25 ng/ml (1-215 ng/ml)), Delta(9)-tetrahydrocannabinol (THC) in 53% (median 3 ng/ml (1-35 ng/ml)), ethanol in 46% (median 1.19 g/kg (0.14-2.95 g/kg)), benzoylecgonine in 13% (median 250 ng/ml (29-2430 ng/ml)), free morphine in 7% (median 10 ng/ml (1-111 ng/ml)), methadone in 7% (median 110 ng/ml (27-850 ng/ml)), 3,4-methylenedioxymethamphetamine (MDMA) in 6% (median 218 ng/ml (10-2480 ng/ml)), nordiazepam in 5% (median 305 ng/ml (30-1560 ng/ml)), free codeine in 5% (median 5 ng/ml (1-13 ng/ml)), midazolam in 5% (median 44 ng/ml (20-250 ng/ml)), cocaine in 5% (median 50 ng/ml (15-560 ng/ml)), amphetamine in 4% (median 54 ng/ml (10-183 ng/ml)), diazepam in 2% (median 200 ng/ml (80-630 ng/ml)) and oxazepam in 2% (median 230 ng/ml (165-3830 ng/ml)). Other drugs, such as lorazepam, zolpidem, mirtazapine, methaqualone, were found in less than 1% of the cases.     

A case of fatal hemorrhage in the cerebral ventricles following intravenous use of methamphetamine

We describe a case of massive hemorrhage in the cerebral ventricles, probably caused by methamphetamine abuse. A 44-year-old male was found dead in a prone position in a hotel room. Old and new injection marks were observed in his right cubital fossa. Petechiae were observed on the conjunctiva of his right eye, laryngeal mucosa, epicardium and under the capsule of the liver (to a slight or moderate degree). The brain, weighing 1.67 kg, was heavily edematous; the lateral and fourth ventricles were filled with hematomas. Subarachnoid, intracerebral hemorrhages were not observed. Cerebral vascular abnormalities were not evident. There were no remarkable changes in other organs, other than congestion. Gas chromatographic-mass spectrometric analysis of the urine disclosed the presence of methamphetamine and amphetamine. The concentration of methamphetamine within the femoral venous blood and intraventricular hematoma was 0.347 microg/ml and 0.189 microg/g, respectively. Amphetamine was not detected in either sample. Urine contained 3.15 microg/ml methamphetamine and 0.063 microg/ml amphetamine. These results indicate that intraventricular hemorrhage might have occurred shortly after intravenous self-administration of methamphetamine. Cerebral arterial spasm and hypertension resulting from the administration of methamphetamine might have resulted in intraventricular hemorrhage.     

Detection of the calcium antagonist nicardipine and its metabolites by gas chromatography-mass spectrometry

An 89-year-old male patient, hospitalized with Parkinson's syndrome, suddenly died shortly after an intravenous drug injection. The conditions indicated that an overdose of nicardipine (1.3 mg/(mlkg)) may be given to the patient. At the autopsy, no pathological changes were noted. With a capillary gas chromatograph with mass spectrometer (GC-MS), nicardipine (4.97 microg/ml) and its pyridine metabolite (M-5, 5.0 microg/ml) were detected in the heart blood of the deceased. This result indicated that an overdose of intravenous nicardipine caused a sudden death of a patient in a poor condition.     

Suicide by sodium tetraoxoselenate(VI) poisoning

Selenium is one of the most toxic elements necessary for the life of mammals. Only a narrow range separates therapeutic (connected with a protective effect) and toxic doses. Selenium incorporated into animal or human tissues in larger amounts can exceed normal human levels and may be toxic (only elemental selenium and selenium sulphide are poorly absorbed). Acute poisonings with selenium or its compounds, especially fatal ones, occur extremely rarely in humans. Levels of selenium in four fatal cases are reviewed, and the levels in a fatal poisoning with sodium tetraoxoselenate(VI) are evaluated. Postmortem tissue selenium contents in the latter case were the following: brain, 1.45 and 1.60 microg/g; stomach, 6.12 and 6.37 microg/g; small intestine, 4.37 and 4.13 microg/g; large intestine, 4.53 and 4.43 microg/g; liver, 4.20 and 4.35 microg/g; kidney, 3.35 and 3.60 microg/g; lung, 1.80 and 1.60 microg/g; blood, 1.43 and 1.41 microg/ml measured by the use of ETA-AAS and fluorimetric methods, respectively.     

Overdose and death with olanzapine: two case reports

Olanzapine is an antipsychotic drug that has been on the market since 1996. Olanzapine-related deaths are very rare; the literature reports only one. However, in a recent 5-month period one medical examiner's office found two such cases that are reported in this paper. One is a suicide and the other is not. The toxicologic and anatomic findings for each are described. Blood olanzapine concentrations ranged from 0.237 microg/ml for one to 0.675 microg/ml for the other. Gastric content concentrations also exhibited a wide range, varying from 0.197 microg/ml to 17.400 microg/ml for the other. Distribution studies of the liver, kidney, and brain produced nondetectable concentrations for the drug. There were no consistent pathologic anatomic findings for cause of death except for moderate coronary atherosclerosis in the nonsuicide case. Both deaths were attributed to olanzapine toxicity.     

A fatal poisoning with 5-methoxy-N,N-diisopropyltryptamine, Foxy

A fatal overdose involving case by 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is reported. 5-MeO-DIPT and its two metabolites, 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT) and 5-methoxy-N-isopropyltryptamine (5-MeO-NIPT), were identified by LC-MS. The level of 5-MeO-DIPT, 5-OH-DIPT and 5-MeO-NIPT in blood and urine was 0.412, 0.327 and 0.020 microg/ml, and 1.67, 27.0 and 0.32 microg/ml, respectively. These blood and urine levels were higher than published data for such poisoning.     

Postmortem diffusion of drugs from the bladder into femoral venous blood.

We describe significantly elevated drug concentrations in the femoral venous blood due probably to postmortem diffusion from the bladder. A 16-year-old deceased male was found in a shallow ditch in winter. The estimated postmortem interval was 9 days and putrefaction was not advanced. The cardiac chambers contained fluid and coagulated blood and a small amount of buffy coat clots. Diffused hemorrhages were found in the gastric mucosa. The bladder contained approximately 600 ml of clear urine. Gas chromatographic-mass spectrometric analysis of the urine disclosed allylisopropylacetylurea (a fatty acid ureide sedative), diphenhydramine, chlorpheniramine and dihydrocodeine. The cause of death was considered to be drowning due to a drug overdose and cold exposure. The concentrations of diphenhydramine, free dihydrocodeine and total dihydrocodeine in the femoral venous blood (1.89, 3.27 and 3.30 microg/ml, respectively) were much higher than those in blood from the right cardiac chambers (0.294, 0.237 and 0.240 microg/ml, respectively). Urine concentrations of diphenhydramine, free dihydrocodeine and total dihydrocodeine were 22.6, 37.3 and 43.1 microg/ml, respectively. The stomach contained negligible amounts of diphenhydramine, free dihydrocodeine and total dihydrocodeine (0.029, 0.018 and 0.024 mg, respectively); concentrations of these drugs in the femoral muscle were 0.270, 0.246 and 0.314 microg/g, respectively. These results indicate that postmortem diffusion of diphenhydramine and dihydrocodeine from the bladder resulted in the elevated concentrations of these drugs in the femoral venous blood. Not only high urinary drug concentrations but also a large volume of urine in the bladder might accelerate the postmortem diffusion.     

Detection of benzodiazepines in different tissues, including bone, using a quantitative ELISA assay.

Benzodiazepines were analyzed in different tissue samples, including hone, by ELISA. The sensitivity of detection for different benzodiazepines was consistent with the manufacturer's reports of the cross reactivities of the antibodies used, with the greatest sensitivity for midazolam and the least for diazepam; in addition the pharmacokinetics was consistent with the known duration of action of the different benzodiazepines, with midazolam cleared rapidly, and diazepam slowly. Following intramuscular injection of 300 microg of midazolam at 16 h intervals for ten days, the drug was detectable in bone tissue samples obtained from skeletonized remains buried in soil at room temperature for three weeks.