A fatal diazinon poisoning

A case of fatal suicidal ingestion of diazinon insecticide is presented. Diazinon concentrations in post-mortem body fluids and tissues were determined using electron capture and flame ionization gas-liquid chromatography. The highest concentrations of diazinon were found in blood, stomach contents, bile and adipose tissue.     

Massive acute arsenic poisonings

Arsenic poisonings are still important in the field of toxicology, though they are not as frequent as about 20-30 years ago. In this paper, the arsenic concentrations in ante- and post-mortem materials, and also forensic and anatomo-pathological aspects in three cases of massive acute poisoning with arsenic(III) oxide (two of them with unexplained criminalistic background, in which arsenic was taken for amphetamine and one suicide), are presented. Ante-mortem blood and urine arsenic concentrations ranged from 2.3 to 6.7 microg/ml, respectively. Post-mortem tissue total arsenic concentrations were also detected in large concentrations. In case 3, the contents of the duodenum contained as much as 30.1% arsenic(III) oxide. The high concentrations of arsenic detected in blood and tissues in all presented cases are particularly noteworthy in that they are very rarely detected at these concentrations in fatal arsenic poisonings.     

Olanzapine-induced hyperglycemic ketoacidosis and corresponding acetone concentrations post-mortem: a forensic interpretation

Olanzapine has been shown to cause or have a contributory role in the development of hyperglycemia and diabetes mellitus. Without careful monitoring for the development of these conditions and control of the resulting adverse effects, patients receiving olanzapine may be at risk of developing fatal ketoacidosis. A review of post-mortem toxicological reports has revealed an increase in the incidence of post-mortem findings of acetone in decedents who were taking olanzapine over the past decade. A review of the current literature and a comprehensive review of case histories and toxicological findings were conducted at the Centre of Forensic Sciences (Toronto, Ontario). Olanzapine concentrations ranging from <62.5 to 858 ng/mL and acetone concentrations as high as 95 mg/dL were detected concurrently. Due to the unstable nature of olanzapine, in several instances quantitation was not possible despite elevated responses during qualitative screening procedures. Five cases suggesting olanzapine-induced ketoacidosis were identified based on the case history and toxicological findings. These data have been compiled and examined with respect to acetone concentrations following olanzapine use and the forensic relevance of post-mortem olanzapine and acetone concentrations are discussed.     

The possible influence of micro-organisms and putrefaction in the production of GHB in post-mortem biological fluid

In recent years, the post-mortem production of the drug of abuse gamma-hydroxybutyric acid (GHB) in biological fluids (e.g. blood and urine) has caused various interpretative problems for toxicologists. Previously, other researchers have shown certain microbial species (Pseudomonas spp. and Clostridium aminobutyricum) possess the necessary enzymes to convert GABA to GHB. A preliminary investigation involving putrefied post-mortem blood indicated there was no observed relationship between "endogenous" GHB concentrations and concentrations of common putrefactive markers (tryptamine and phenyl-2-ethylamine). Microbiological analysis identified the presence of various micro-organisms: Clostridia spp., Escherichia coli, Proteus vulgaris, Enterococcus faecalis and Aeromonoas spp. Equine plasma, human blood and urine samples were inoculated with these and an additional micro-organism (Pseudomonas aeruginosa) and incubated at 22 degrees C for 1 month. Following comparison with control samples and pre-inoculation concentrations, the data indicated an apparent production of GHB in unpreserved P. aeruginosa inoculated blood (2.3 mg/l). All other fluoride-preserved and unpreserved samples (including controls) had GHB concentrations <1mg/l. Although this concentration is lower than is typically associated with "endogenous" post-mortem GHB concentrations, this paper proposes a potential microbial production of GHB with time.     

An unusual case of post-mortem redistribution of ethanol

We report an unusual case of post-mortem redistribution of ethanol in a woman diver who died by drowning in seawater. The ethanol concentrations were right heart blood 0.60 g/l, left heart blood 2.08 g/l, femoral venous blood 0.63 g/l, gastric contents 5.87 g/l, bile 0.83 g/l. The mechanisms of post-mortem redistribution of ethanol described in the literature, that is, early redistribution from the stomach or the lung parenchyma in the case of inhalation of gastric contents, are inadequate to account for the degree of variation observed between the measurements. We believe that this difference in concentration is explained by the presence of seawater in the pulmonary alveoli at the time of death.     

Identification and quantitation by 1H-NMR of metabolites in animal organs and tissues. An application of NMR spectroscopy in forensic science

1H-nuclear magnetic resonance (NMR) was applied to the study of metabolites in rat organs and tissues. From 1H-NMR spectra of D2O extracts of various organs and tissues, lactate, pyruvate, and some other substances were simultaneously identified and quantitated. On the basis of the results, the use of 1H-NMR for estimating post-mortem time was suggested.     

Post-mortem toxico-kinetics of trazodone.

Trazodone is a structurally unique bicyclic anti-depressant, said to be significantly less toxic than other anti-depressants following an acute overdose. We studied the tissue distribution and post-mortem redistribution of trazodone in two fatalities, one of which allowed comparison with trimipramine, a typical tricyclic anti-depressant. Case 1, a 53-year-old female weighing 72 kg, had femoral vein concentrations of trimipramine 5.5 micrograms/ml, trazodone 14.4 micrograms/ml and alcohol 107 mg%. Case 2, a 48-year-old female of 70 kg, had a femoral vein trazodone of 15.5 micrograms/ml and alcohol 34 mg%, with no other drugs detected. For case 1 and case 2 respectively, trazodone tissue concentrations were: skeletal muscle 7.3 and 9.0 micrograms/g; left and right lungs 13.3, 12.9 and 35.3, 40.1; myocardium, 30.9 and 28.9; kidneys 34.7 and 39.6; liver 73.7 and 82.4; fat 18.5 and 16.5; brain 48.6 and 20.9. For case 1 and 2, respectively, blood trazodone concentrations in 10 initial autopsy samples ranged from 13.7-17.3 and 14.4-16.9 micrograms/ml. Twenty-four and forty-eight hours later the respective ranges were 12.8-18.0 and 12.4-19.9 for case 1, 12.5-20.1 and 12.7-27.0 for case 2. By contrast, for trimipramine, blood concentrations at 0 time, 24 h and 48 hours ranged from 5.5-11.4, 5.2-14.3, and 4.2-18.2, respectively. We conclude that trazodone shows little preferential concentration in solid organs and consequently has relatively stable post-mortem blood concentrations with little drug redistribution artefact. Both the clinical pharmacokinetics and post-mortem toxicokinetics of trazodone differ significantly from the tricyclic anti-depressants.     

Post-mortem changes in calmodulin binding proteins in muscle and lung

Estimation of post-mortem interval (PMI) remains an elusive issue in forensic investigations. In this study, we examined the possible use of calmodulin (CaM) binding proteins (CaMBPs) as indicators of PMI. Whole CaMBP populations from homogenized rat lung and rat skeletal muscle removed at 0, 24, 48 and 96 h post-mortem at 21 degrees C were detected by the calmodulin binding overlay technique (CaMBOT) using 35S-VU1-CaM and visualized by autoradiography. CaMBOT showed that, in both tissues, the CaMBP population remained relatively stable for up to 96 h post-mortem with the exception of a single approximately 200 kDa CaMBP that increased in 24 h post-mortem samples then showed decreasing amounts at subsequent times. Immunoblot analysis of the specific CaMBPs, Ca(2+)/CaM-dependent kinase II (CaMKII), calcineurin A (CNA), myristoylated alanine-rich C-kinase substrate (MARCKS) and inducible nitric oxide synthase (iNOS) were done on lung tissue samples. CaMKII levels did not change appreciably over the 96 h PMI examined. In contrast to iNOS levels, which varied from sample to sample, CNA and MARCKS showed predictable patterns of change: the level of MARCKS decreased steadily in the 0-96 h post-mortem lung samples while CNA underwent a shift in mobility on SDS-PAGE by 24 h post-mortem before slowly decreasing in amount. The stability of CaMKII levels over 96 h was also seen in skeletal muscle tissue while CNA showed variable levels at 0, 48 and 96 h with the presence of the rapidly migrating band at 24 h. These patterns of change in CaMBPs provide some insight into the post-mortem changes in calmodulin-mediated signaling components in lung and skeletal muscle and support the further study of CNA and CaMKII as potential markers for estimating short- and long-term PMIs.     

Rapid determination of carboxyhemoglobin in blood by Oximeter

Different methods to determine carboxyhemoglobin (COHb) in blood are described in the literature. In our laboratory three methods to analyze COHb in post-mortem blood samples were compared: the spectrophotometric method of Maehly, a gas chromatographic method with a thermal conductivity detector (GC-TCD) and the Oximeter. Several COHb containing blood samples of deceased persons were analyzed. Results of all three methods were comparable for low concentrations (ca. 10% COHb) as well as for high concentrations (ca. 80% COHb) regardless of the viscosity of the blood samples. The advantages of the Oximeter when compared to Maehly's method and GC-TCD are extreme short time of analysis (<1min), very small blood volume required (<0.1ml) and easy handling. In our opinion application of the Oximeter is not limited to analyses of blood samples from living persons (e.g. in clinical toxicology); it can as well be used for the determination of COHb in post-mortem blood samples. Hence it is a useful and time saving tool in forensic toxicology.     

Two fatal intoxication cases with imidacloprid: LC/MS analysis

Imidacloprid [1-(6-chloro-3pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] is a new and potent nitromethylene insecticide with high insecticidal activity at very low application rates. It is the first highly effective insecticide that, like nicotine, acts on the nervous system, causing blockage of postsynaptic nicotinergic acetylcholine receptors. Two fatal cases with this insecticide in two male individuals, of 33 and 66 years old, are presented. An LC/MS with electrospray method for measuring imidacloprid and its metabolites in post-mortem samples is described. In the chromatographic separation, a reverse-phase column XTerra MS C18 (2.1mm i.d.x 150 mm, 5 microm) was used and the mobile phase composed with acetonitrile and 0.1% formic acid (15:85), at a 0.25 mL/min flow rate. Samples were prepared with a liquid-liquid extraction procedure with dichloromethane. Calibration curves for imidacloprid in blood and urine samples were linear from 0.2 to 15 microg/mL. The mean recovery was 86% with a coefficient of variation of +/-5.9%. The detection limit was 0.002 microg/mL. Quantitative results were obtained for all post-mortem matrices available of the two fatal cases: blood, urine, stomach contents, lung, liver and kidney. The imidacloprid blood concentrations found in two-cases were 12.5 and 2.05 microg/mL. The authors validated a method to detect and quantify imidacloprid in post-mortem samples, and to our knowledge for the first time a post-mortem tissue distribution was performed on various samples for this insecticide.     

A preliminary study of changes in scalp impedance during the early post-mortem period in rats.

An accurate and reproducible technique was used for estimation of scalp impedance (1 kHz) in each of eight rat cadavers, maintained at 9.0 +/- 0.7 degrees C, during the 1 to 144 hour post-mortem period. Scalp impedance increased non-linearly, and in a bimodal manner, between 24 and 144 hours post-mortem. The relationship between scalp impedance and post-mortem interval, and the degree of individual variation in the time course of impedance change, were such that scalp impedance is unlikely to be a suitable tool for estimation of time since death.     

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.     

Post-mortem biochemical investigations of vitreous humor

For several decades vitreous humor has been used for post-mortem biochemical investigations with the objective of a post-mortem diagnosis of pre-existing diseases and the clarification of forensic issues, in particular the determination of the post-mortem interval. For the determination of measured concentrations in vitreous humor pre-analytic factors as well as analytical and instrumental variations have to be taken into consideration. The aim of this study was a methodical investigation of two methods of sample pre-treatment as influencing variables. The compared methods were centrifugation and treatment in the ultrasonic bath. The determined parameters were sodium, potassium, chloride, calcium, lactate, urea, glucose and creatinine. Analyses were performed photometrically or by an ion-selective electrode. For some of the analytes a dilution was necessary before analysing. Regarding to the two pre-treatment methods, significant differences in the measured concentrations were not found. The precision proved to be mostly unsatisfying and was clearly better in diluted samples than in undiluted aliquots. A comparison of the vitreous humor of the two ocular bulbs did not lead to significant differences.     

Suspected clozapine poisoning in the UK/Eire, 1992-2003

OBJECTIVE: Toxicological analyses are often performed to investigate suspected poisoning, but the interpretation of results may not be straightforward. We studied suspected poisoning cases 1992-2003 where blood clozapine and N-desmethylclozapine (norclozapine) were measured in order to assess the relationship of these parameters to outcome. METHODS: Samples were referred from clinicians, pathologists/coroners, or via the Clozaril Patient Monitoring Service (CPMS, Novartis). Information was gathered from clinical, post-mortem, or coroners' reports. RESULTS: There were seven fatal [five male, two female; median (range) age 28 (24-41) year] and five non-fatal [four male, one female; median age 35 (26-41) year] clozapine overdoses. The median post-mortem blood clozapine and norclozapine concentrations were 8.2 (3.7-12) and 1.9 (1.4-2.4)mg/L, respectively [median clozapine:norclozapine ratio 4.4 (2.9-5.1)]. The median plasma clozapine and norclozapine concentrations (first or only sample) were 3.9 (1.7-7.0) and 0.40 (0.30-0.70)mg/L, respectively [median clozapine:norclozapine ratio 7.6 (5.3-18)] in the remainder. These overdoses were in patients who were poorly or non-adherent to clozapine, or who had taken tablets prescribed for someone else. In 54 further people who died whilst receiving clozapine [38 male, 16 female; median age 41 (22-70) year], the median post-mortem blood clozapine and norclozapine concentrations were 1.9 (0-7.7, n = 43) and 1.4 (0-6.0, n = 39)mg/L, respectively [median clozapine:norclozapine ratio 1.5 (0.4-7.6, n = 38)]. The median post-mortem increase in blood clozapine and norclozapine as compared to the most recent ante-mortem measurement was 489 (98-5,350)% and 371 (139-831)%, respectively [median sample time before death 14 (0-30, n = 21) days]. CONCLUSION: Clozapine poisoning cannot be diagnosed on the basis of blood clozapine and norclozapine concentrations alone. The analysis of ante-mortem blood specimens collected originally for white cell count monitoring and the blood clozapine:norclozapine ratio may provide additional interpretative information.     

The redistribution of selected psychiatric drugs in post-mortem cases

The post-mortem redistribution of a number of psychiatric drugs was investigated. A portion of liver, the gastric contents and blood collected from heart and femoral sites was obtained from 13 cases and analyzed by liquid chromatography-mass spectrometry. Drugs detected included five selective serotonin reuptake inhibitors; venlafaxine, a serotonin/noradrenaline reuptake inhibitor; and risperidone, an atypical antipsychotic. Heart blood concentrations were significantly higher (3.4-fold on average) than those measured in femoral blood when results from all drugs were included together. The range for parent drug concentrations in these two blood specimens was 0.5-6.2. There was no significant correlation of the post-mortem interval, the liver concentration and content of drugs in the gastric contents to the heart:femoral blood concentration ratio. These data serve to demonstrate that variable increases in blood concentration occur post-mortem and limit the interpretative value of such toxicological data.     

Biochemical reconstruction of three cases of death — Results of international cooperation

Biochemical serotonin and histamine determinations were applied to the reconstruction of three suspected homicide cases. To distinguish between ante-mortem and post-mortem wounds and to time the ante-mortem injuries the concentrations of free histamine and serotonin in the wound samples and in the control samples from neighbouring intact skin were examined. The results of these biochemical determinations allowed a reconstruction of the events and one of the three cases was shown to be suicide instead of homicide.These methods can be used at least during the first 4 – 5 days after death and some-times even longer. This allows for international cooperation when investigating and reconstructing complicated cases of death.     

Postmortem toxicology of drugs of abuse

Conducting toxicology on post-mortem specimens provides a number of very significant challenges to the scientist. The range of additional specimens include tissues such as decomposing blood and other tissues, hair, muscle, fat, lung, and even larvae feeding on the host require special techniques to isolate a foreign substance and allow detection without interference from the matrix. A number of drugs of abuse are unstable in the post-mortem environment that requires careful consideration when trying to interpret their significance. Heroin, morphine glucuronides, cocaine and the benzodiazepines are particularly prone to degradation. Moreover, redistributive process can significantly alter the concentration of drugs, particularly those with a higher tissue concentration than the surrounding blood. The designer amphetamines, methadone and other potent opioids will increase their concentration in blood post-mortem. These processes together with the development of tolerance means that no concentration of a drug of abuse can be interpreted in isolation without a thorough examination of the relevant circumstances and after the conduct of a post-mortem to eliminate or corroborate relevant factors that could impact on the drug concentration and the possible effect of a substance on the body. This article reviews particular toxicological issues associated with the more common drugs of abuse such as the amphetamines, cannabinoids, cocaine, opioids and the benzodiazepines.     

Interpretation of a 3,4-methylenedioxymethamphetamine (MDMA) blood level: discussion by means of a distribution study in two fatalities

The amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy" is a currently used or abused designer drug and fatalities are frequently encountered in forensic practice. However, the question remains open whether an MDMA blood level can be toxic or even potentially lethal. In order to provide insight in the interpretation of a detected MDMA concentration, the distribution of MDMA and its metabolite 3,4-methylenedioxyamphetamine (MDA) in various body fluids and tissues was studied and discussed in two different fatalities. Apart from peripheral blood samples (such as femoral and subclavian blood), various blood samples obtained centrally in the human body and several body fluids (such as vitreous humour) were examined. In addition, various tissues such as cardiac muscle, lungs, liver, kidneys, and brain lobes were analysed. In contrast to the peripheral blood levels, high MDMA and MDA levels were found in cardiac blood and the majority of the organs, except for the abdominal adipose tissue. The high concentrations observed in all lung lobes, the liver and stomach contents indicate that post-mortem redistribution of MDMA and MDA into cardiac blood can occur and, as a result, blood sampled centrally in the body should be avoided. Therefore, our data confirm that peripheral blood sampling remains "the golden standard". In addition, a distinct difference in peripheral blood MDMA concentrations in our two overdose cases was established (namely 0.271 and 13.508 microg/ml, respectively). Furthermore, our results suggest that, if a peripheral blood sample is not available and when putrefaction is not too pronounced, vitreous humour and iliopsoas muscle can be valuable specimens for toxicological analysis. Finally, referring to the various mechanisms of death following amphetamine intake, which can result in different survival times (e.g. cardiopulmonary complications versus hyperthermia), the anatomo-pathological findings and the toxicological results should be considered as a whole in arriving at a conclusion.     

UPLC-MS/MS法检测佐匹克隆及其在大鼠体内动态分布研究

目的建立安眠镇静药佐匹克隆的检测方法及其在大鼠体内动态分布模型。方法实验组大鼠用佐匹克隆橄榄油溶液(47.25mg/kg)灌胃给药,空白对照组大鼠采用橄榄油灌胃,分别于0.5、1、1.5、2.5、5、8、12h后采集心血后处死大鼠,分别取心、肝、肺、脾、肾、胃、大脑组织,采用超高效液相色谱-串联质谱法(UPLC-MS/MS)检测各组织中佐匹克隆的质量浓度。结果佐匹克隆与内标物SKF525A出峰时间分别为1.43、1.6min。各组织中佐匹克隆在5~5000ng/mL(g)线性关系良好。佐匹克隆在10、100、1000ng/mL三个浓度下日间、日内精密度良好,在各组织中平均萃取回收率高。灌胃给药后大鼠各组织中佐匹克隆含量在0.5~1h内呈上升趋势,在1h时达到峰值,在各时间点,佐匹克隆在胃壁组织中含量较其他组织高,心血和大脑组织中相对较少。结论本课题建立的UPLCMS/MS法动态检测大鼠各组织中佐匹克隆的含量具有高效性、可靠性的特点,这对今后法医学案件中涉及到佐匹克隆定性定量检验有一定的参考价值。     

利用尸食性蚤蝇推断死亡时间的法医学研究进展

死亡时间的准确推断一直是法医学的难题。昆虫学方法已被认为是死亡时间推断的有效方法。尸食性蝇类发育生物学在死亡时间推断中有着重要地位。蚤蝇体形微小,在相对密闭的空间,可成为尸体上主要的甚至是唯一的昆虫证据。本文从尸食性蚤蝇的作用、种属鉴定以及日龄推断方面,综述了利用尸食性蚤蝇推断死亡时间的法医学研究进展。