A fatal doxepin poisoning associated with a defective CYP2D6 genotype

It has been suggested that the polymorphism of the CYP2D6 gene can contribute to occurrence of fatal adverse effects. We therefore investigated postmortem toxicology cases of fatal drug poisonings related to CYP2D6 substrates, with the manner of death denoted as accidental or undetermined. CYP2D6 genotypes were determined in 11 consecutive cases with samples available for DNA analysis. A case of fatal doxepin poisoning with an undetermined manner of death was found to coincide with a completely nonfunctional CYP2D6 genotype (*3/*4), indicating a total absence of CYP2D6 enzyme and suggesting a poor metabolizer phenotype. The doxepin concentration was 2.4 mg/L, the concentration of nordoxepin 2.9 mg/L, and the doxepin/nordoxepin ratio 0.83, the lowest found among the 35 nordoxepin-positive postmortem cases analyzed during the same year. No alcohols or other drugs were detected in the case. The CYP2C19 genotype was determined as that of an extensive metabolizer. The high N-desmethylmetabolite concentration is not consistent with acute intoxication. It is therefore probable that the defective genotype has contributed to the death, possibly involving repeated high dosage of doxepin. Our case strongly emphasizes that a pharmacogenetic analysis in postmortem forensic setting may reveal new insight to the cause or manner of death.     

Sudden Death by Spontaneous Epiglottic Hematoma Secondary to High Blood Levels of Warfarin

A 67‐year‐old man was found dead, at his home. On external examination, we found a voluminous purplish black ecchymosis of the anterior neck area. On internal examination, we found a voluminous epiglottis hematoma completely obstructing the upper airway. It was associated with other sites of intra‐abdominal hemorrhage. Toxicological studies revealed the presence of warfarin at a concentration of 8.4 mg/L in peripheral blood, which supposes an INR well above 4.5. To conclude, we supposed death was due to asphyxia secondary to a spontaneous epiglottic hematoma caused by a high blood concentration of warfarin. Hemorrhage in the epiglottis is very rare. To our knowledge, our patient is the only case of “sudden death” reported with spontaneous epiglottic hematoma due to high blood concentration of warfarin. In forensic practice, an anterior neck ecchymosis, without trauma, may suggest hemorrhage into soft airway tissues. Pathology findings make it possible to exclude exogenous trauma.     

A fatal dichlorvos poisoning: concentrations in biological specimens

Abstract:  A 54-year-old man was found dead with a bottle containing a brownish fluid near him. A toxicological screening was carried out in blood, urine, and stomach content. Only dichlorvos (2,2 dichlorovinyl O-O dimethylphosphate or DDVP) was found. A simple and rapid method, using DDVP-D₆ as an internal standard, was developed for the determination of DDVP by gas chromatography/mass spectrometry (GC/MS). The method was linear from 1 to 10 mg/L. Intraday and interday precisions were all <15%. DDVP concentration in cardiac blood was approximately four times higher than in peripheral blood. A high concentration was found in the heart showing a cardiac tropism of DDVP, kidney and lung concentrations being much lower. No DDVP was found in liver. DDVP stomach content was 38 g. The amount presumed ingested was 82 g, c . 1000 mg/kg of body. The oral LD₅₀ for DDVP ranges between 20 and 1090 mg/kg in animals but is not known for humans.     

Genotyping of five short tandem repeat loci via triplex and duplex PCR

We have developed a triplex PCR method for D3S1359, HumTH01 and HumTPO tetranucleotide loci and a duplex PCR method for HumFES/FPS and HumvWA31A tetranucleotide loci using high resolution polyacrylamide gel electrophoresis and silver staining. The methods were evaluated for paternity testing and individual identification and allele frequencies at these loci are reported for 189–3387 unrelated individuals in the Finnish population. The D3S1359 locus, especially, was found to be a highly informative locus. Seventeen alleles were found in the D3S1359 locus with a highest observed allele frequency of 0.199, a high exclusion power (PE) in paternity testing (0.78) and a high observed heterozygosity (0.89). The combined PE for these five loci was 0.99.     

CYP2D6 and CYP2C19 genotypes and amitriptyline metabolite ratios in a series of medicolegal autopsies

In a series of 202 postmortem toxicology cases, the CYP2D6 and CYP2C19 genes were genotyped, and the concentrations of amitriptyline (AT) and six metabolites were analyzed. The polymorphic CYP2D6 and CYP2C19 genes encode enzymes participating in the metabolism of several potentially toxic drugs, and mutations in these genes may lead to adverse drug reactions, possibly even intoxications. AT was chosen as the substrate of interest because it is mainly metabolized by these enzymes, is considered relatively toxic, and ranks among the major causes of fatal drug poisoning in Finland. Our objective was to evaluate genetically determined interindividual variation in conjunction with metabolite ratios of drugs found in toxicological analysis in a series of medicolegal autopsies. Positive correlations were found between the proportion of trans-hydroxylated metabolites and the number of functional copies of CYP2D6 and between the proportion of demethylated metabolites and the number of functional copies of CYP2C19. None of the accidental or undetermined AT poisonings coincided with the CYP2D6 or CYP2C19 genotype which predicts a poor metabolizer phenotype. However, an unusually high femoral blood concentration of AT, 60mg/l, was found in one suicide case with no functional CYP2D6 genes. Our study shows a concordance of AT metabolite patterns with CYP2D6 and CYP2C19 genotypes in the presence of confounding factors typical for postmortem material. This result demonstrates the feasibility of postmortem pharmacogenetic analysis and supports the dominant role of genes in drug metabolism.     

Coexistence and concentrations of ethanol and diazepam in postmortem blood specimens: risk for enhanced toxicity?

Both ethanol and diazepam are classified as depressants of the central nervous system and exert their effects via the GABAA receptor complex. We report the coexistence and concentrations of ethanol, diazepam, and its primary metabolite nordiazepam in a case series of 234 forensic autopsies collected over a ten-year period. Diazepam, nordiazepam, and ethanol were determined in femoral venous blood by highly selective gas chromatographic methods. The mean (median) femoral blood concentrations were ethanol 0.24 g/100 mL (0.25 g/100 mL), diazepam (D) 0.23 microg/g (0.10 microg/g), nordiazepam (ND) 0.24 micro/g (0.20 microg/g), sum (D + ND) 0.43 microg/g (0.30 microg/g), and the ratio D/ND was 1.19 (1.0). When cause of death was attributed to alcohol and/or drug intoxication (N = 50), the mean and median blood-ethanol concentration was higher, being 0.36 g/100 mL and 0.38 g/100 mL, respectively, whereas the mean (median) and range of blood-diazepam concentrations were about the same, 0.23 microg/g (0.10 microg/g) and 0.05 to 1.2 microg/g. The femoral-blood concentrations of diazepam and nordiazepam were highly correlated (r = 0.73), but there was no correlation between the concentrations of ethanol and diazepam (r = -0.15). In another 114 fatalities (all causes of death) with diazepam and/or nordiazepam as the only drugs present, the mean (median) and range of blood-diazepam concentrations were 0.22 microg/g (0.10 microg/g) and 0.03 to 3.5 microg/g. The pathologists report showed that none of these deaths were classed as drug intoxications. The impression gleaned from this study of ethanol-diazepam deaths is that high blood-ethanol concentration is the major causative factor. We found no evidence that concurrent use of diazepam enhanced the acute toxicity of ethanol, although interpretation is complicated by the high blood-ethanol concentration (median 0.38 g/100 mL), making it difficult to discern an added effect of diazepam.     

Acute intoxication caused by overdose of flunitrazepam and triazolam: high concentration of metabolites detected at autopsy examination

ABSTRACT: A 52-year-old woman was found dead on the floor of the living room on the first floor of a house, which belonged to the man with whom she shared the house. On visiting the site, her clothes were found to be undisturbed. Packages of flunitrazepam (Silece, 2 mg/tablet) and triazolam (Halcion, 0.25 mg/tablet) were found strewn around the victim. Toxicological analysis was performed, and the concentrations of flunitrazepam, triazolam, and their metabolites in the victim's blood and urine were measured by high-performance liquid chromatography coupled with photodiode array and mass spectrometry. A high blood concentration of 7-aminoflunitrazepam was detected (1,270 ng/g), and further metabolites such as 7-acetamidoflunitrazepam, 7-acetamidodesmethylflunitrazepam, and 7-aminodesmethylflunitrazepam were detected in the blood and urine samples. In addition, 4-hydroxytriazolam and α-hydroxytriazolam were detected in her urine at a concentration of 950 and 12,100 ng/mL, respectively.On the basis of the autopsy findings and toxicology results of high concentrations of both flunitrazepam and triazolam derivatives, the cause of death was determined to be acute intoxication from flunitrazepam and triazolam.     

The time-dependant post-mortem redistribution of antipsychotic drugs

The post mortem redistribution of ten commonly prescribed antipsychotic drugs (APs) was investigated. Femoral blood was collected from 273 cases at admission to mortuary (AD) and at post-mortem (PM). The PM samples were collected at various times up to nine days after admission and the sample pairs analysed using LC-MS/MS. The drugs included in this study were 9OH-risperidone (paliperidone), amisulpride, chlorpromazine, clozapine, haloperidol, olanzapine, promethazine, quetiapine, risperidone, and zuclopenthixol. Haloperidol, quetiapine and risperidone showed minimal changes between AD and PM specimens, whereas the majority of drugs showed significant changes between the sample pairs collected at different time points post mortem (p<0.01) in addition to an average concentration change greater than the uncertainty of measurement of the applied method. Average increases in blood concentrations after admission to the mortuary ranged up to 112% (chlorpromazine and olanzapine) but also decreases up to -43% (9OH-risperidone) were seen. There were large standard deviations between sample pairs and substantial day-to-day unpredictable changes that highlight the difficulty in the interpretation of drug concentrations post-mortem. Based on the presented data, we recommend that specimens for toxicological analysis should to be taken as soon as possible after admission of a deceased person to the mortuary in order to minimise the effects of the PM interval on the drug concentration in blood.     

Fatal Intoxication with Methoxetamine

Methoxetamine (MXE) is a new synthetic drug of abuse structurally related to ketamine and phencyclidine. A case of a 29-year-old male with acute toxicity related to the analytically confirmed use of MXE is reported. The man was found dead at his residence. Biological material was analyzed using liquid chromatography–tandem mass spectrometry. The concentration of MXE in urine of the deceased was 85 μg/mL. Despite the vial containing the blood sample being destroyed during transportation and the blood leaking out into the cardboard packaging, the blood level of MXE was estimated. After determination of the cardboard grammage (approx. 400 g/m³) and the mean mass of the blood obtained after drying (0.1785 ± 0.0173 g per 1 mL), the estimated blood concentration of MXE was found to be 5.8 μg/mL. The high concentration of MXE in blood and urine and the circumstances of the case indicate an unintentional, fatal intoxication with this substance.     

Cocaine fatality: an unexplained blood concentration in a fatal overdose

A case is presented of a 26-year-old female who died as a result of cocaine intoxication. A blood cocaine concentration of 330 mg/l, about 1.5 times greater than the highest concentration previously reported, was found. Blood benzoylecgonine and ecgonine methyl ester concentrations were 50 and 18 mg/l, respectively. The unusually high blood concentrations of cocaine and the metabolites are suggestive of a massive administration, however, the history suggests a series of recreational uses. The manner of death was undetermined.     

Determination of ethchlorvynol in body tissues and fluids after embalmment

A 54 year-old female expired at her residence. Her husband, a physician, signed a certificate stating that her death was due to cerebrovascular accident (CVA) and released her body to a funeral home, where she was embalmed. Since the deceased had a long history of medical problems and drug abuse, an autopsy was performed and no evidence of CVA was found. Toxicological analyses of body fluids and tissues revealed the presence of ethchlorvynol in high concentration in the bile (112 mg/l). The bloody fluid collected from the heart contained a concentration of ethchlorvynol below the limit for quantitation. Other findings included phenobarbital (32.8 mg/l) in heart bloody fluid and methanol (an ingredient of embalming fluid). The significance of the findings is discussed in relation to embalmment prior to autopsy and toxicological analyses. Ethchlorvynol concentration in the bile is compared to other fatal cases due to ethchlorvynol overdose.     

New alleles and mutational events in D12S391 and D8S1132: sequence data from an eastern German population.

To investigate the DNA mutation rate and pattern in the hypervariable short tandem repeat (STR) locus D12S391 and in the locus D8S1132, samples from an eastern German population (Dresden area) were analysed. A duplex PCR was applied, using short amplification products for D12S391 (129-177bp) and a modified reverse primer for D8S1132 (127-182bp). The sequences of some rare and new variant alleles are described. At the locus D12S391, 13 regular and six incomplete alleles with different lengths were found, exhibiting several sequence structures. Two isolated father/child mismatches were observed in a total of 648 meioses. Novel alleles 13.1, 14.1 and 27 were discovered at the locus D8S1132. Three parent/child mismatches were found in a total of 672 meioses.     

Relationship between the concentration of ethanol and methanol in blood samples from Swedish drinking drivers

Headspace gas chromatography was used to determine the concentration of ethanol and methanol in blood samples from 519 individuals suspected of drinking and driving in Sweden where the legal alcohol limit is 0.50 mg/g in whole blood (11 mmol/l). The concentration of ethanol in blood ranged from 0.01 to 3.52 mg/g with a mean of 1.83 +/- 0.82 mg/g (+/- S.D.). The frequency distribution was symmetrical about the mean but deviated from normality. A plot of the same data on normal probability paper indicated that it might be composed of two subpopulations (bimodal). The concentration of methanol in the same blood specimens ranged from 1 to 23 mg/l with a mean of 7.3 +/- 3.6 mg/l (+/- S.D.) and this distribution was markedly skew (+). The concentration of ethanol (x) and methanol (y) were positively correlated (r = 0.47, P less than 0.001) and implies that 22% (r2) of the variance in blood-methanol can be attributed to its linear regression on blood-ethanol. The regression equation was y = 3.6 + 2.1 x and the standard error estimate was 0.32 mg/l. This large scatter precludes making reliable estimates of blood-methanol concentration from measurements of blood-ethanol concentration and the regression equation. But higher blood-methanol concentrations are definitely associated with higher blood-ethanol in this sample of Swedish drinking drivers. Frequent exposure to methanol and its toxic products of metabolism, formaldehyde and formic acid, might constitute an additional health risk associated with heavy drinking in predisposed individuals. The determination of methanol in blood of drinking drivers in addition to ethanol could indicate long-standing ethanol intoxication and therefore potential problem drinkers or alcoholics.     

Determination of bromadiolone in whole blood by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A rapid, sensitive and selective high-performance liquid chromatography tandem mass spectrometric method (HPLC/MS-MS) has been developed and validated for the determination of bromadiolone in whole blood using warfarin as an internal standard (IS). Bromadiolone was extracted from the whole blood samples by liquid-liquid extraction with ethyl acetate. Multiple-reaction monitoring (MRM) was used to detect bromadiolone and IS, using precursor --> product ion combinations at m/z 527 --> 465 and 307 --> 161, respectively. The calibration curve was linear (r2=0.998) in the concentration range of 0.5-100.0 ng/mL with a lower limit of quantification of 0.5 ng/mL in whole blood. Intra- and inter-day relative standard deviations (R.S.D.s) were less than 7.5 and 11.9%, respectively. Recoveries of bromadiolone ranged from 82.1 to 85.2%. This method is found to be determined trace bromadiolone in whole blood and can be used in the diagnosis of the poisoned human beings.     

Identification of a D8S1179 primer binding site mutation and the validation of a primer designed to recover null alleles

A population study of Chamorros and Filipinos using short tandem repeat (STR) loci amplified with the AmpFlSTR Profiler Plus PCR amplification kit demonstrated an excess of observed homozygosity at the D8S1179 locus. Use of a different set of D8S1179 primers to type the same samples did not demonstrate an excess of homozygosity and showed discordant genotypes at the D8S1179 locus. A single point mutation, G-to-A transition, 16 nucleotides from the 3' end of the reverse primer, was identified to cause allele dropout when using the AmpFlSTR Profiler Plus primer set. An additional D8S1179 reverse primer specific for the variant was constructed resulting in the recovery of the null allele. The primer was included in the newly developed AmpFlSTR Identifiler PCR amplification kit. No deleterious effects or non-specific peaks were observed in validation experiments evaluating primer concentration, Mg2+ concentration, annealing temperature and population samples.     

Evaluation of breath-alcohol instruments. III. Controlled field trial with Alcolmeter pocket model

A breath-alcohol screening device, Alcolmeter pocket model, was evaluated in a controlled field trial with policeman operating the instruments. The results of tests made with subjects before they drank alcohol were always zero. The standard deviation (S.D.) of breath alcohol determinations increased with increase in the concentration of alcohol in the sample, being 0.036 mg/ml at a mean blood-ethanol concentration of 0.53 mg/ml. The S.D. varied among subjects tested (from 0.022 to 0.053 mg/ml) as well as among the instruments used (from 0.023 to 0.054 mg/ml). The breath test results were on average less than the actual blood-ethanol concentrations when a 2100: 1 blood/breath ratio was used to calibrate the Alcolmeter device. Blood ethanol (x) and Alcolmeter readings (y) were highly correlated (r = 0.95 +/- 0.018) and the regression equation was y = -0.017 + 0.95x. At a mean blood-ethanol concentration of 0.50 mg/ml, the Alcolmeter instrument will indicate 0.46 mg/ml on average. The standard error estimate was 0.085 mg/ml, being 17% of the mean Alcolmeter reading and this corresponds to 95% confidence limits of +/- 0.17 mg/ml. The results of this study show that Alcolmeter pocket-model is a useful device for breath-alcohol screening purposes at a blood-alcohol level of 0.50 mg/ml. A blood/breath ratio of 2300 should be used to calibrate the Alcolmeter device.     

Screening for cannabinoids in blood using EMIT: concentrations of delta-9-tetrahydrocannabinol in relation to EMIT results.

The EMIT d.a.u. cannabinoid assay of methanolic extracts of blood was found to be usable as a screening method in cases of suspected impairment by cannabis, when delta-9-tetrahydrocannabinol (THC) was analysed in the subsequent assay. A prerequisite is that the blood sample is taken some time after cannabis smoking. When a cut-off limit corresponding to 50 nM delta-9-tetrahydrocannabinol carboxylic acid (17 ng/ml) was used, 86% of the EMIT positive blood samples contained THC concentrations above the cut-off limit of 1 nM (0.3 ng/ml). A high EMIT result gave a high probability of finding a high THC concentration in the subsequent confirmation analysis.     

Determination of dimethoate in blood and hemoperfusion cartridge following ingestion of formothion: a case study

A 57-year-old male who had ingested not more than 22 g of formothion was semicomatose on admission to hospital, approximately 1.5 h after ingestion. Dimethoate, a hydrolyzed formothion, was found in blood samples collected from the patient and in the charcoal column in the direct hemoperfusion cartridge which was used 6 to 7.5 h after ingestion. It was extracted and purified by Extrelut column extraction. A gas chromatograph, equipped with a flame photometric detector and a gas chromatograph-mass spectrometer, were used to detect and confirm the presence of dimethoate. The blood dimethoate concentrations which were taken approximately 1.5 and 6 h after ingestion were 21.4 and 12.7 micrograms/g, respectively. A blood dimethoate concentration of 21.4 micrograms/g would appear to indicate a high level of formothion intoxication. The total amount of dimethoate found in the charcoal column used was 15 mg.     

Toxicological characteristic of neuroleptics--substituted benzamides

At present in the medical practice the group of neuroleptics, substituted benzamides is widely used. According to literature data they cause acute intoxications under particular conditions. The group of substituted benzamides includes drugs such as amisulpride, tiapride, sulpiride and sultoprid. Substituted benzamides intake causes toxic actions: psychotropic and neurotoxic. The neuroleptic intoxication is an effect of overdosage, abuse and hypersensitization. Severe intoxications with drugs of this group may cause lethal outcome.     

The rapid analysis of heroin drug seizures using micellar electrokinetic chromatography with short-end injection

A simple and rapid method for the analysis of heroin seizures by micellar electrokinetic chromatography with short-end injection is described. Separations were performed using an uncoated fused silica capillary, 50 cm x 50 microm I.D. x 360 microm O.D. with an effective separation length of 8 cm. The system was run at 25 degrees C with an applied negative voltage of -25 kilovolts. Injection of each sample was for 2 s at -50 mbar. UV detection was employed with the wavelength set at 210 nm. The background electrolyte consisted of 85:15 (water:acetonitrile, v/v) containing final concentrations of 25 mM SDS and 15 mM sodium borate, pH 9.5. Samples and standards were prepared in 0.1% v/v acetic acid and diluted in the run buffer containing 1 mg/ml of N,N-dimethyl-5-methoxytryptamine as an internal standard. Under these conditions a text mixture containing caffeine, paracetamol, morphine, codeine, heroin, and acetylcodeine was resolved within 1.5 min. The method was used to determine the concentration of heroin in heroin seizure samples, and the results were in good agreement with those obtained by a validated gas chromatographic method.