Postmortem Distribution of 3‐Beta‐Hydroxybutyrate

The concentrations of 3‐beta‐hydroxybutyrate (3HB) in femoral blood, urine, vitreous humor as well as pericardial and cerebrospinal fluids were retrospectively examined in a series of medico‐legal autopsies, which included cases of diabetic ketoacidosis, hypothermia fatalities without ethanol in blood, bodies presenting mild decompositional changes, and sudden deaths in chronic alcoholics. Similar increases in 3HB concentrations were observed in blood, vitreous, and pericardial fluid, irrespective of the cause of death, suggesting that pericardial fluid and vitreous can both be used as alternatives to blood for postmortem 3HB determination. Urine 3HB levels were higher than blood values in most cases. Cerebrospinal fluid 3HB levels were generally lower than concentrations in blood and proved to be diagnostic of underlying metabolic disturbances only when significant increases occurred.     

Determination of Nitrite in Whole Blood by High‐Performance Liquid Chromatography with Electrochemical Detection and a Case of Nitrite Poisoning

Although nitrite is widely used in meat processing, it is a major toxicity hazard to children and is responsible for the blue‐baby syndrome. A simple and effective method to determine nitrite in whole blood has been devised using ion chromatography with suppressed conductivity detection. The blood sample was deproteinized by adding acetonitrile and purified with mini‐cartridges to remove hydrophobic compounds, chloride ions, and metal ions. An aliquot of the filtrate was injected onto the ion chromatography. The retention time for nitrite was 13.8 min and the detection limit of nitrite in whole blood was 0.4 μmol/L. The calibration curve was linear (r² = 0.9999) over the concentration working range. The blood nitrite concentration of a victim who attempted suicide by ingesting sodium nitrite powder was determined using the present method. The basal levels for nitrite in human blood was determined with 7.1 ± 0.9 μmol/L (n = 12).     

Unexpected Serotonin Syndrome,Epileptic Seizures,and Cerebral Edema Following 2,5‐dimethoxy‐4‐bromophenethylamine Ingestion

4‐bromo‐2,5‐dimethoxyphenethylamine (2C‐B) is a designer drug. In Europe, 2C‐B is easily obtained and used for recreational purposes. It is known for its stimulating effects similar to those of 3,4‐methylenedioxymethamphetamine, although in higher doses it has more hallucinogenic effects. Here, we report a case of 2C‐B ingestion, confirmed by liquid chromatography‐tandem mass spectrometry, in an 18‐year‐old man. The neurological consequences were severe, including the development of serotonin syndrome and severe brain edema. Supportive therapy resulted in a stable condition, although, after several months, the patient still suffered from severe neurological impairment due to the drug‐induced toxicity. This case showed that 2C‐B could not be identified with the drugs of abuse screening routinely used in Dutch hospitals. The use of 2C‐B carries many risks, with potentially profound neurological damage, that both consumers and healthcare physicians are unaware of.     

Assessment of the Role Played by N‐propanol Found in Postmortem Blood in the Discrimination Between Antemortem Consumption and Postmortem Formation of Ethanol Using Rats

This study disproves the reliability of n‐propanol as a biomarker to establish whether the ethanol found in postmortem blood is derived from antemortem ingestion or postmortem putrefactive processes. Two groups of rats were given ethanol or normal saline solution, respectively, and sacrificed 1.5 h later. After putrefaction, blood and, in a few cases, urine samples from the rats were analyzed for ethanol and n‐propanol by head‐space gas chromatography equipped with flame ionization detection. Although the concentration ratios of ethanol/n‐propanol in the postmortem blood collected from the bodies without prior alcohol consumption were expected to be <20 (as per limited case reports and previous in vitro studies), in samples from several rats that were on saline solution, this ratio was found to exceed 20. In conclusion, the concentration ratio of ethanol/n‐propanol in postmortem blood does not allow for the discernment between antemortem ingestion and the postmortem synthesis of ethanol.     

Synthesis and Analysis of Glucuronic Acid‐Conjugated Metabolites of 4‐Bromo‐2,5‐Dimethoxyphenethylamine

In the study reported here, two glucuronic acid‐conjugated metabolites of 4‐bromo‐2,5‐dimethoxyphenethylamine (2C‐B)—a ring‐substituted psychoactive phenethylamine—were chemically synthesized for the first time and a method for analyzing them in urine was developed. β‐D‐Glucuronide of 4‐bromo‐2,5‐dimethoxyphenylethylalcohol was successfully synthesized using methyl 2,3,4‐tri‐Ο‐acetyl‐1‐O‐(trichloroacetimidoyl)‐α‐D‐glucuronate as a glucuronyl donor and boron trifluoride diethylether complex as a Lewis acid catalyst. β‐D‐Glucuronide of 4‐bromo‐2,5‐dimethoxyphenylacetic acid was synthesized by condensing 4‐bromo‐2,5‐dimethoxyphenylacetic acid and benzyl D‐glucuronate followed by benzyl group deprotection based on catalytic hydrogenation. Two glucuronic acid‐conjugated metabolites of 2C‐B in urine were qualitatively and semiquantitatively evaluated via direct liquid chromatography/mass spectrometry (LC/MS) analysis of a diluted urine sample. The simple method proposed is expected to be useful for studying the metabolic fate of 2C‐B.     

Identification of 2‐(ethylamino)‐1‐(4‐methylphenyl)‐1‐pentanone (4‐MEAP), a New “Legal High” Sold by an Internet Vendor as 4‐Methyl Pentedrone

Online vendors are offering a new legal high, 4‐methylpentedrone (4‐MPD). Information for potential users provided by internet vendors of 4‐MPD includes incorrect structures and nonexistent CAS numbers. A sample of 4‐MPD was obtained and analyzed using GC‐MS, NMR, and LC‐EIS. The fragmentation data from the GC‐MS and LC‐EIS produced an M‐1 ion that suggested the molecular mass was 219 amu, rather than 205 amu as calculated for 4‐methylpentedrone. The difference in molecular mass corresponded to the addition of a methyl group. Based on the mass and fragmentation pattern, two standards were synthesized, 2‐(ethylamino)‐1‐(4‐methylphenyl)‐1‐pentanone and 1‐(4‐methylphenyl)‐2‐(propylamino)‐1‐butanone. The synthesis involved bromination of the appropriate ketone followed by the reaction with ethylamine or propylamine. Based on the NMR data and unique fragmentation patterns produced by these molecules, the sample was identified as 2‐(ethylamino)‐1‐(4‐methylphenyl)‐1‐pentanone, not 4‐methylpentedrone.     

Site‐, Technique‐, and Time‐Related Aspects of the Postmortem Redistribution of Diazepam,Methadone, Morphine,and their Metabolites: Interest of Popliteal Vein Blood Sampling

Sampling site, technique, and time influence postmortem drug concentrations. In 57 cases, we studied drug concentration differences as follows: subclavian vein‐dissection/clamping versus blind stick, femoral vein‐dissection/clamping versus blind stick, right cardiac chamber, and popliteal vein‐dissection and clamping only. Cases were distributed in group #1 (all cases with both techniques), group #2 (dissection/clamping), and group #3 (blind stick). Sampled drugs were diazepam, methadone, morphine, and their metabolites. To assess PMR, mean concentrations and ratios were calculated for each group. Time‐dependent variations of blood concentrations and ratios were also assessed. Results indicate that site, method, and time may influence postmortem distribution interpretation in different ways. Popliteal blood seems less subject to PMR. In conclusion, our study is the first to evaluate concurrently three main aspects of PMR and confirms that the popliteal vein may represent a site that is more resistant to the changes seen as a result of PMR.     

Fatal Intoxication with α‐PVP,a Synthetic Cathinone Derivative

This study presents the fatal case of a young man who was admitted to the ICAU due to sudden cardiac arrest. An interview revealed that the patient had taken some unspecified crystals. From the moment of admission, his condition deteriorated dramatically as a result of increasing circulatory insufficiency. After a few hours, sudden cardiac arrest occurred again and the patient was pronounced dead. In the course of a medicolegal autopsy, samples of biological material were preserved for toxicology tests and histopathological examination. The analysis of samples using the LC‐MS/MS technique revealed the presence of α‐PVP in the following concentrations: blood—174 ng/mL, urine—401 ng/mL, brain—292 ng/g, liver—190 ng/g, kidney—122 ng/g, gastric contents—606 ng/g. The study also presents findings from the parallel histopathological examination. Based on these findings, cardiac arrest secondary to intoxication with alpha‐PVP was determined as the direct cause of the patient's death.     

Stability of Morphine,Codeine, and 6‐Acetylmorphine in Blood at Different Sampling and Storage Conditions

The stability of drugs in biological specimens is a major concern during the evaluation of the toxicological results. The stability of morphine, codeine, and 6‐acetyl‐morphine in blood was studied after different sampling conditions: (i) in glass, polypropylene or polystyrene tubes, (ii) with addition of dipotassium ethylene diamine tetraacetic acid (K₂EDTA) or sodium oxalate (Na₂C₂O₄), and (iii) with or without the addition of sodium fluoride (NaF). Spiked blood samples were stored at two different temperatures (4 and ?20^°C), analyzed after different storage times and after three freeze–thaw cycles. Opiate concentrations were decreased in all conditions, but the most unstable was 6‐acetyl‐morphine. The addition of NaF as preservative improved the stability of opiates at all conditions studied, whereas the type of anticoagulant did not affect the stability of opiates. It was concluded that blood samples should be stored at ?20^°C in glass tubes containing oxalate and NaF for maximum stability.     

Determination of Both Fetus’ and Mother's Blood Type from an Autopsy Case Immersed in Formalin for Over 50 Years

A female fetus which had been immersed in formalin for more than 50 years was found in Japan. Because no liquid blood could be obtained, we tried to use immunohistochemistry (IHC) methods to tissue samples obtained at autopsy to identify both the fetal and mother's blood type. We detected B antigens on endothelial cells in paraffin sections of the fetal organs. Furthermore, we observed both anti‐A‐ and anti‐B‐positive red blood cells in the intervillous space, which is indicative of the mother's blood type. To our knowledge, this is the first case report on determining the blood type of both the fetus and the mother from tissue immersed in formalin for such a long time. The results suggest that IHC is valuable for the determination of ABO blood type in circumstances of long postmortem duration and unfavorable storage conditions.     

Analysis of 4‐Bromo‐2,5‐DimethoxyphenethylamineAbuser's Urine: Identification and Quantitation of Urinary Metabolites

The metabolites of 4‐bromo‐2,5‐dimethoxyphenethylamine (2C‐B), a psychoactive drug with hallucinogenic activity, were investigated in a urine sample from a user of 2C‐B. The urine sample was deconjugated enzymatically and the metabolites were recovered by liquid–liquid extraction. The extract was analyzed by gas chromatography/mass spectrometry after derivatization, and the results were used to identify and quantitate the metabolites. 4‐Bromo‐2,5‐dimethoxyphenylacetic acid was the most abundant metabolite of 2C‐B in human urine and accounted for 73% of the total amount of detected metabolites, followed by 4‐bromo‐2‐hydroxy‐5‐methoxyphenylacetic acid (13%) and 4‐bromo‐2,5‐dimethoxyphenylethyl alcohol (4.5%). According to the literature, the main metabolites of 2C‐B in rat urine are N‐(4‐bromo‐2‐methoxy‐5‐hydroxyphenylethyl)acetamide and N‐(4‐bromo‐2‐hydroxy‐5‐methoxyphenylethyl)acetamide. However, these metabolites accounted for only a small proportion of the total amount of detected metabolites in human urine, which indicates that there are significant species‐specific differences in the metabolism of 2C‐B. 4‐Bromo‐2,5‐dimethoxyphenylacetic acid, which was the most abundant metabolite in human urine, is thought to be generated by deamination of 2C‐B by monoamine oxidase (MAO) followed by oxidation by aldehyde dehydrogenase. Our results suggest that MAO plays a crucial role in the metabolism of 2C‐B in humans.     

Temperature‐Dependent Postmortem Changes in Human Cardiac Troponin‐T (cTnT): An Approach in Estimation of Time Since Death

Estimation of time of death is an indispensible requirement of every medico‐legal autopsy, but unfortunately, there is not a single method by which it could be determined accurately. This study focused on the temperature‐dependent postmortem degradation of cardiac troponin‐T and its association with postmortem interval (PMI) in human. The analysis involved extraction of the protein, separation by denaturing gel electrophoresis (SDS‐PAGE), and visualization by Western blot using cTnT‐specific monoclonal antibodies. The area of the bands within a lane was quantified by scanning and digitizing the image using Gel Doc (Universal Hood). The results indicate a characteristic banding pattern among human cadavers (n = 6) and a pseudo‐linear relationship between percentage of cTnT degradation and the log of the time since death (r > 0.95), which can be used to estimate the postmortem interval. The data presented demonstrate that this technique can provide an extended time range during which PMI can be more accurately estimated.     

Distribution of Enantiomers of Methadone and Its Main Metabolite EDDP in Human Tissues and Blood of Postmortem Cases

Knowledge concerning the distribution of methadone in postmortem human tissue and the effect of postmortem redistribution on methadone is today limited making the choice of a suitable substitute for femoral blood difficult when this is not available. Cardiac blood, femoral blood, muscle, and brain tissue concentrations of the enantiomers of methadone and its metabolite 2‐ethyl‐1,5‐dimethyl‐3,3‐diphenylpyrrolinium were recorded for 155 postmortem cases. Brain and muscle tissue concentrations exceeded the femoral blood concentrations with a median fold of 2.3 and 1.6, respectively, but both had a better correlation than cardiac blood to femoral blood concentrations. The Kruskal–Wallis test showed a significant dependency on time and body mass index for some of the matrix ratios over femoral blood. We conclude brain or muscle tissue may constitute a better alternative for measurement of methadone than cardiac blood for situations in which femoral blood is not available, despite concentrations in both matrices being systematically higher.     

The Ratio of 6β‐Hydroxycortisol to Cortisol in Urine as a Measure of Cytochrome P450 3A Activity in Postmortem Cases

Poisoning can occur with chronic accumulation of a drug due to reduced metabolic capacity; conversely, under‐treatment may occur due to an increased metabolic rate. Over half of all drugs are metabolized by the cytochrome P450 3A complex (CYP3A). The activity of CYP3A can be assessed by the urinary ratio of 6β‐hydroxycortisol to cortisol. The aim of this study was to determine the usefulness of this ratio as a postmortem marker for determining whether altered CYP3A enzyme activity occurred prior to death. In a series of 244 postmortem cases, this ratio ranged from 0.014 to 78.6 (median 3.50). The median was significantly higher (5.14) in a subgroup of 28 cases that exhibited the presence of CYP3A‐inducing drugs. In cirrhosis, the median ratio was 1.69. This pointed to a reduced metabolic capacity of CYP3A. Thus, the ratio may constitute a rough indicator of the CYP3A metabolic capacity, which could be of value in special cases.     

Postmortem Blood Concentrations of R‐ and S‐Enantiomers of Methadone and EDDP in Drug Users: Influence of Co‐Medication and P‐glycoprotein Genotype

Abstract: We investigated toxicological and pharmacogenetic factors that could influence methadone toxicity using postmortem samples. R‐ and S‐methadone were measured in femoral blood from 90 postmortem cases, mainly drug users. The R‐enantiomer concentrations significantly exceeded that of the S‐enantiomers (Wilcoxon’s test, p < 0.001). The samples were divided into four groups according to other drugs detected (methadone only, methadone and strong analgesics, methadone and benzodiazepines, or methadone and other drugs). There was no significant difference in any of the R‐methadone/total methadone ratios among the four groups. The median R/S ratio was 1.38, which tends to be higher than that reported for the plasma of living subjects. In addition, we investigated whether small nucleotide polymorphisms in the MDR1 gene that encode the drug transporter P‐glycoprotein were associated with the concentrations of R‐ and S‐methadone and its metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine. No significant association was detected.     

Specificity,Sensitivity, and Operability of RSID™‐Urine for Forensic Identification of Urine: Comparison with ELISA for Tamm‐Horsfall Protein

Abstract: In this study, the specificity, sensitivity, and operability of RSID?‐Urine, a new immunochromatographic test for urine identification, was evaluated and compared with ELISA detection of Tamm‐Horsfall protein (THP). Urine was successfully identified among other body fluids using RSID?‐Urine and ELISA detection of THP. The detection limit of RSID?‐Urine equated to 0.5 μL of urine; although the sensitivity of RSID?‐Urine may be lower than that of ELISA detection of THP, it is thought to be sufficient for application to casework samples. However, results from RSID?‐Urine must be interpreted with caution when the sample may have been contaminated with blood or vaginal fluid, because this might inhibit urine detection. The RSID?‐Urine assay can be performed in just 15 min by dropping the extracted sample onto the test cassette. Therefore, RSID?‐Urine should be an effective tool for the forensic identification of urine, in addition to ELISA detection of THP.     

血尿肝中巴比妥类药物硅藻土提取紫外差示导数光谱测定

目的建立一种操作简单、快速、去除杂质能力强、提取率高的硅藻土提取血、尿、肝中巴比妥类药物的方法。方法尿液不经稀释、血液经稀释过硅藻土柱,乙醚洗脱;肝匀浆用6%高氯酸沉淀蛋白,上清液过硅藻土柱,二氯甲烷洗脱药物。洗脱液挥干用0.45mol/L氢氧化钠溶液溶解,将溶液等分为两份,分别用等量的0.45mol/L氢氧化钠溶液和0.6mol/L硼酸-氯化钾溶液稀释,得到pH10和pH14水溶液,以pH10溶液为参比,测定pH14溶液的紫外二阶导数光谱进行药物检测。结果该法提取率血98.6%~100.3%,尿99.7%~103.2%,肝78.4%~102%,检出限均低于1.0μg/g(m l),变异系数小于2.9%,线性范围0.5~5.0μg/m l。结论硅藻土提取血、尿、肝中巴比妥类药物、紫外差示导数光谱法进行测定,适合作为法医毒物常规检验方法。     

Measurement of Postmortem 1,5‐anhydroglucitol in Vitreous Humor for Forensic Diagnosis

In forensic diagnosis, postmortem blood glucose is known to be susceptible to change after death. However, the 1,5‐anhydroglucitol (1,5‐AG) concentrations in plasma and cerebrospinal fluid (CSF) reflect the mean blood glucose level for a short period of time. In this study, we compared the postmortem 1,5‐AG concentrations in vitreous humor and CSF in 47 subjects to evaluate the utility of this concentration in the vitreous humor for forensic diagnosis. The postmortem 1,5‐AG concentrations in vitreous humor (mean±SD: 20.2 ± 8.7 μg/mL) and CSF (16.8 ± 8.7 μg/mL) did not differ significantly and showed a strong correlation (r² = 0.87, p < 0.01). These results suggest that the vitreous humor 1,5‐AG concentration provides useful information on the antemortem blood glucose level, in addition to the HbA1c value and the CSF 1,5‐AG concentration.     

Methadone‐Related Deaths – Epidemiological,Pathohistological, and Toxicological Traits in 10‐year Retrospective Study in Vojvodina,Serbia

The number of methadone‐related deaths (MRDs) during a 10‐year period (2002–2011) in the region of Vojvodina, Serbia, was increased. The cases were evaluated according to epidemiological parameters, pathohistological findings, and toxicological screening. The majority of victims were men, aged from 20 to 38. Pathohistologically, the signs of acute focal myocardial damage were present in the heart of victims with drug abuse history shorter than 2 years, while both signs of recent and chronic focal myocardial damage were developed among victims with longer drug abuse history (2–5 years). In postmortem blood samples of 54.84% of victims, methadone was detected in combination with diazepam, both in therapeutic range. Alcohol was absent in most cases. Other detected drugs were antipsychotics and antidepressants in therapeutic concentrations. These findings raise the attention to the concomitant use of methadone and benzodiazepines with the need for further studies to clarify the mechanism of death in such cases.     

Postmortem Detection of Hepatitis B,C, and Human Immunodeficiency Virus Genomes in Blood Samples from Drug‐Related Deaths in Denmark*

Abstract: Blood‐borne viral infections are widespread among injecting drug users; however, it is difficult to include these patients in serological surveys. Therefore, we developed a national surveillance program based on postmortem testing of persons whose deaths were drug related. Blood collected at autopsy was tested for anti‐HBc, anti‐HBs, anti‐hepatits C virus (HCV), or anti‐human immunodeficiency virus (HIV) antibodies using commercial kits. Subsets of seropositive samples were screened for viral genomes using sensitive in‐house and commercial polymerase chain reaction (PCR) assays. Hepatitis B virus (HBV) DNA was detected in 20% (3/15) of anti‐HBc‐positive/anti‐HBs‐negative samples, HCV RNA was found in 64% (16/25) of anti‐HCV‐positive samples, and HIV RNA was detected in 40% (6/15) of anti‐HIV‐positive samples. The postmortem and antemortem prevalences of HBV DNA and HCV RNA were similar. Postmortem HIV RNA testing was less sensitive than antemortem testing. Thus, postmortem PCR analysis for HBV and HBC infection is feasible and relevant for demonstrating ongoing infections at death or for transmission analysis during outbreaks.