Detection of lisdexamfetamine and its metabolite d-amphetamine in urine and gastric contents collected from a cadaver at forensic autopsy.

PURPOSE: Lisdexamfetamine (LDX), which is used for the treatment of attention-deficit/hyperactivity disorder and narcolepsy, is composed of L-lysine attached to dextroamphetamine (d-amphetamine). In this article, we report a forensic autopsy case in which prescription drugs were unknown at autopsy. While amphetamine was detected, methamphetamine could not be detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in any of samples collected. Thus, we aimed to quantify LDX concentrations in autopsy samples and to prove that the amphetamine detected in this case was due to metabolized LDX. METHODS: Femoral vein blood, cardiac whole blood, urine, and gastric content samples were taken at autopsy for toxicological analysis. Qualitative and quantitative analyses were performed using LC-MS/MS. In addition, optical isomer separation for the amphetamine detected was conducted. The stability of LDX in whole blood and urine was also examined at three different temperatures. RESULTS: The concentrations of LDX were < 4.00, 30.9, and 4.42 ng/mL in whole blood, urine, and gastric content samples, respectively. The concentrations of amphetamine were 329, 510, 2970, and 915 ng/mL in femoral vein blood, heart whole blood, urine, and gastric contents, respectively. The amphetamine detected in this case was identified to be only d-amphetamine by optical isomer separation. The d-amphetamine detected was considered to be derived from LDX. Stability experiments revealed that LDX in whole blood decreased at ambient temperature. CONCLUSIONS: The results in the present case report may be useful in interpreting whether or not the amphetamine detected in a cadaver is a metabolite of LDX.

Ropinirole involved in a fatal case: blood and urinary concentrations.

PURPOSE: Ropinirole is an antiparkinsonian  drug and has recently been suggested to be effective in amyotrophic lateral sclerosis. It is expected that ropinirole prescriptions will increase in the near future. However, the fatal concentration in blood is unclear at this time. Therefore, we report a fatal case involving ropinirole intoxication and discuss the fatal concentrations with reference to several autopsy cases involving ropinirole. METHODS: Ropinirole was quantified in femoral vein blood, cardiac blood, and urine from five autopsy cases in which ropinirole was detected by drug screening in our laboratory. One is a ropinirole intoxication case (this report) and the others  were non-intoxication cases. Their ropinirole concentrations were compared and discussed. RESULTS: The ropinirole concentration in this case was 100 ng/mL in femoral blood, 160 ng/mL in cardiac blood, and 1840 ng/mL in urine. The ropinirole concentrations in the four non-ropinirole poisoning cases were 7-35 ng/mL (mean: 24 ng/mL) in femoral blood, 13-100 ng/mL (mean: 60 ng/mL) in cardiac blood, and 140-1090 ng/mL (mean: 640 ng/mL) in urine. Cardiac/peripheral ratios were in the range of 1.6-2.1 (mean 1.8). CONCLUSIONS: There were no obvious signs of overdose, and the high cardiac/peripheral blood ratio suggested that postmortem redistribution may have occurred, but the  peripheral blood ropinirole concentration (100 ng/mL) was obviously higher than that reported in the previous fatal case of ropinirole poisoning (64 ng/mL). Based on these results, the cause of death in this case was considered to be shock and fatal arrhythmia due to ropinirole poisoning. This case provides important data on postmortem blood and urinary levels of ropinirole poisoning.

Simple and simultaneous quantification of cyanide, ethanol, and 1-propanol in blood by headspace GC-MS/NPD with Deans switch dual detector system.

Cyanide is a powerful and rapidly acting poison. In Japan, cyanide poisoning is rare, and regular cyanide testing can be costly and time consuming. In contrast, alcohol analysis is routinely performed in most forensic laboratories. In this study, we attempted to develop a method for the simultaneous quantification of cyanide and alcohols in blood using headspace gas chromatography (HS-GC). As nitrogen-phosphorus detection (NPD) is more sensitive to hydrogen cyanide than mass spectrometry (MS), a Deans switch was used to switch the detectors during a single run. The separation provided by three analytical columns, PoraBOND Q, CP-Sil 5 CB, and HP-INNOWax, was investigated, and PoraBOND Q was selected. The use of HS-GC-MS/NPD with a Deans switch enabled the simple and simultaneous quantification of cyanide, ethanol, and 1-propanol. Eighteen other volatile compounds were detected in the SIM/scan mode of the MS.

Association between sexual activity-related death and non-prescription use of phosphodiesterase type 5 inhibitors.

In recent years, there has been an increase in the use of phosphodiesterase type 5 inhibitors (PDE5i) that are purchased from abroad without a doctor's diagnosis via the Internet or other means. We report six cases in which nonprescription use of PDE5i may have led to death. Among the four deceased individuals who were believed to have experienced sudden cardiac death, three (cases 1-3) had a history of cardiovascular disease, which is a contraindication, and the remaining case (case 4) involved combined use of multiple PDE5i. Sildenafil (0.063 µg/mL, 0.087 µg/mL) was detected in two of the four cases of sudden cardiac death. Tadalafil (0.096 µg/mL) was detected in one of the remaining two cases, and tadalafil (0.197 µg/mL) and vardenafil (0.011 µg/mL) were detected in the other case. Sildenafil (0.032 µg/mL), tadalafil (0.062 µg/mL), and ethanol were detected in a traffic accident case with a history of contraindications. In a case of asphyxiation by vomit aspiration, autopsy showed 90% stenosis in the anterior descending branch of the coronary artery, and sildenafil (0.063 µg/mL) was detected. To the best of our knowledge, this is the first report of postmortem blood levels of tadalafil and vardenafil likely contributing to the cause of death. Despite all the warnings about the dangers of using PDE5 inhibitors, cases of PDE5i contributing to death are still identified during autopsies. Therefore, raising public awareness of the risks of the risks associated with the imported drug use by individuals is necessary.

A Fatal Case of Poisoning with Fentanyl Transdermal Patches in Japan.

Fentanyl transdermal patches have been used to treat cancer- and noncancer-related chronic pain. However, its inappropriate or illegal application may cause fatal poisoning. We herein present the case of a Japanese woman in her 40s who was found dead with seven 25-µg/h fentanyl transdermal patches on her body. We established a detailed toxicological analysis procedure to quantify fentanyl, and its metabolite norfentanyl, and other drugs (acetaminophen, allylisopropylacetylurea, celecoxib, estazolam, promethazine, and sertraline) in human whole blood by ultra-high-performance liquid chromatography-tandem mass spectrometry. The measured fentanyl and norfentanyl concentrations in the femoral and cardiac blood were 0.051 and 0.072 µg/mL and 0.033 and 0.076 µg/mL, respectively. The decedent's fentanyl concentrations were consistent with previously reported postmortem blood levels for fatal cases of poisoning by fentanyl transdermal patches. Based on the decedent's case history, autopsy findings, and toxicological analyses, the cause of death was identified as intoxication with transdermal fentanyl.

Forensic analysis using ultra-high-performance liquid chromatography-tandem mass spectrometry with solid-phase extraction of α-solanine and α-chaconine in whole blood.

PURPOSE: The potato glycoalkaloids (PGAs), α-solanine and α-chaconine can exert adverse effects on human health when consumed in excess. This study aimed to investigate the optimal extraction method for the quantitative analysis of PGAs in whole blood by using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and to apply this validated method to postmortem blood. METHODS: A total of 200 µL of human whole blood was prepared for PGA extraction. For validation, a solid-phase extraction (SPE) using Oasis® PRiME HLB, in which extraction could be performed in three simple steps (sample loading, washing, and elution) was used, with no need for both conditioning and equilibration of columns for sample preparation. RESULTS: In this method, the limit of detection and the lower limit of quantification (LLOQ) of both α-solanine and α-chaconine were 1 and 2 µg/L, respectively. The calibration curves of the two compounds were obtained with good linearity in the range of 2-100 µg/L. The recovery rates at the LLOQ of α-solanine and α-chaconine were ≥ 91.8% and ≥ 85.9%, respectively. The validation data (intra- and inter-day combined) for accuracy ranged from 93.5 to 106.6% for α-solanine and from 93.9 to 107.7% for α-chaconine. This validated method was successfully applied to one forensic autopsy case, and the concentrations of α-solanine and α-chaconine in the postmortem cardiac blood were 45.1 and 35.5 µg/L, respectively. CONCLUSIONS: This validated UHPLC-MS/MS with SPE for quantitative analysis of PGAs could be useful in forensic toxicology.

Olanzapine-Induced Fatal Ketoacidosis with Pneumomediastinum and Subcutaneous Emphysema.

We report a case of fatal olanzapine-induced ketoacidosis in which pneumomediastinum (PM) and subcutaneous emphysema (SE) were detected on postmortem computed tomographic (CT) images. A man in his forties was found in a state of cardiopulmonary arrest with profuse perspiration, and 50 empty capsules of olanzapine (10 mg) and flunitrazepam (1 mg) were found in his room. The major findings of postmortem CT prior to autopsy were PM and SE from the lower half of the face to the height of the first rib. The results of autopsy, biochemical tests, and toxicological analyses indicated the cause of death to be fatal ketoacidosis induced by olanzapine intoxication. No injuries, medical interventions, or particular diseases were evident, suggesting that PM and SE were caused by ketoacidosis. Our findings indicated that toxicological analyses should be performed when PM and SE are detected on CT images.

High-Throughput Functional Evaluation of Variants of Unknown Significance in ERBB2.

Purpose: The advent of next-generation sequencing technologies has enabled the identification of several activating mutations of Erb-B2 receptor tyrosine kinase 2 (ERBB2) among various cancers. However, the significance of infrequent mutations has not been fully investigated. Herein, we comprehensively assessed the functional significance of the ERBB2 mutations in a high-throughput manner.Experimental Design: We evaluated the transforming activities and drug sensitivities of 55 nonsynonymous ERBB2 mutations using the mixed-all-nominated-in-one (MANO) method.Results: G776V, G778_S779insG, and L841V were newly revealed to be activating mutations. Although afatinib, neratinib, and osimertinib were shown to be effective against most of the ERBB2 mutations, only osimertinib demonstrated good efficacy against L755P and L755S mutations, the most common mutations in breast cancer. In contrast, afatinib and neratinib were predicted to be more effective than other inhibitors for the A775_776insYVMA mutation, the most frequent ERBB2 mutation in lung cancer. We surveyed the prevalence of concurrent ERBB2 mutation with gene amplification and found that approximately 30% of ERBB2-amplified urothelial carcinomas simultaneously carried ERBB2 mutations, altering their sensitivity to trastuzumab, an mAb against ERBB2. Furthermore, the MANO method was applied to evaluate the functional significance of 17 compound mutations within ERBB2 reported in the COSMIC database, revealing that compound mutations involving L755S were sensitive to osimertinib but insensitive to afatinib and neratinib.Conclusions: Several ERBB2 mutations showed varying sensitivities to ERBB2-targeted inhibitors. Our comprehensive assessment of ERBB2 mutations offers a fundamental database to help customize therapy for ERBB2-driven cancers.We identified several ERBB2 mutations as activating mutations related to tumorigenesis. In addition, our comprehensive evaluation revealed that several ERBB2 mutations showed varying sensitivities to ERBB2-targeted inhibitors, and thus, the functional significance of each variant should be interpreted precisely to design the best treatment for each patient. Clin Cancer Res; 24(20); 5112-22. ©2018 AACR.

Fatal Poisoning with Both Dichlorvos and Phenthoate.

Organophosphates are widely used as pesticides. However, organophosphates are occasionally orally ingested to commit suicide. In this case, a man in his late 80s committed suicide by ingesting both dichlorvos and phenthoate. Autopsy findings revealed a characteristic volatile odor from his mouth, stomach, lungs, liver, and kidneys. The esophageal mucosa was denatured and had lost elasticity. Serum cholinesterase activity was 9 IU/L. Toxicological analyses performed using high-performance liquid chromatography-tandem mass spectrometry revealed that dichlorvos concentrations in the left and right cardiac blood samples were 11.6 and 4.6 µg/mL, respectively. Phenthoate concentrations in the left and right cardiac blood samples were 5.8 and 0.51 µg/mL, respectively. The total amounts of dichlorvos and phenthoate in the stomach were 7.35 and 4.55 g, respectively. The case history, autopsy findings, and toxicological analyses indicated that the cause of death was acute fatal poisoning after oral ingestion of both dichlorvos and phenthoate.

Experimental Study on the Postmortem Redistribution of the Substituted Phenethylamine, 25B-NBOMe.

2-(4-Bromo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25B-NBOMe) is a substituted phenethylamine, which has become highly prevalent worldwide since 2014. Recently, in an autopsy case involving fatal 25B-NBOMe intoxication, we found the postmortem increase of 25B-NBOMe concentration in the cardiac blood approximately 2 days after death. The aim of this study was to investigate the distribution of 25B-NBOMe and reproduce the postmortem redistribution using a rat model. Sprague-Dawley rats were killed 30 min after intraperitoneal injection of 25B-NBOMe (0.5 mg/kg) and left for 0, 3, 6, 9, 15, or 24 h (six rats at each time point). Postmortem 25B-NBOMe concentrations in the cardiac blood increased by more than 10-fold at 6-h postmortem. 25B-NBOMe accumulated primarily in the lung. Moreover, this postmortem redistribution occurred even in rats that had died 1 week following the 25B-NBOMe administration. These findings indicate that attention should be paid to sample collection and data interpretation in the toxicological analysis of 25B-NBOMe.

MDMA induces cardiac contractile dysfunction through autophagy upregulation and lysosome destabilization in rats.

The underlying mechanisms of cardiotoxicity of 3,4-methylenedioxymethylamphetamine (MDMA, "ecstasy") abuse are unclear. Autophagy exerts either adaptive or maladaptive effects on cardiac function in various pathological settings, but nothing is known on the role of autophagy in the MDMA cardiotoxicity. Here, we investigated the mechanism through which autophagy may be involved in MDMA-induced cardiac contractile dysfunction. Rats were injected intraperitoneally with MDMA (20mg/kg) or saline. Left ventricular (LV) echocardiography and LV pressure measurement demonstrated reduction of LV systolic contractility 24h after MDMA administration. Western blot analysis showed a time-dependent increase in the levels of microtubule-associated protein light chain 3-II (LC3-II) and cathepsin-D after MDMA administration. Electron microscopy showed the presence of autophagic vacuoles in cardiomyocytes. MDMA upregulated phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) at Thr172, mammalian target of rapamycin (mTOR) at Thr2446, Raptor at Ser792, and Unc51-like kinase (ULK1) at Ser555, suggesting activation of autophagy through the AMPK-mTOR pathway. The effects of autophagic inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) on LC3-II levels indicated that MDMA enhanced autophagosome formation, but attenuated autophagosome clearance. MDMA also induced release of cathepsins into cytosol, and western blotting and electron microscopy showed cardiac troponin I (cTnI) degradation and myofibril damage, respectively. 3-MA, CQ, and a lysosomal inhibitor, E64c, inhibited cTnI proteolysis and improved contractile dysfunction after MDMA administration. In conclusion, MDMA causes lysosome destabilization following activation of the autophagy-lysosomal pathway, through which released lysosomal proteases damage myofibrils and induce LV systolic dysfunction in rat heart.

Relationship between the matrix effect and the physicochemical properties of analytes in gas chromatography.

The phenomenon "matrix-induced chromatographic response enhancement" (matrix effect) causes quantitative errors in gas chromatography (GC) analyses. This effect varies according to the analyte nature, matrix type and concentration, and GC-system parameters. By focusing on the physicochemical properties of analytes, a predictive model was developed for the matrix effect using quantitative structure-property relationships. Experimental values of the matrix effect were determined for 58 compounds in a serum extract obtained from solid-phase extraction as the matrix. Eight molecular descriptors were selected, and the matrix-effect model was developed by multiple linear regression. The developed model predicted values for the matrix effect without any further experimental measurements. It also indicated that the molecular polarity (particularly H-bond donors) and volume of the analyte increase the matrix effect, while hydrophobicity and increasing number of nonpolar carbon atoms in the analyte decrease the matrix effect. The model was applied to the analysis of barbiturates. The predicted values indicated that N-methylation decreases the matrix effect, and the relative predicted values were effective for the selection of an internal standard. The obtained insight into the matrix effect and the prediction data will be helpful for developing quantitative analysis strategies.

Acute fatal poisoning with Tolfenpyrad.

The authors present a fatal case of poisoning with Tolfenpyrad (TFP), a pesticide first approved in Japan in 2002. A man in his fifties was found dead in the supine position at his son's home and the small towel with a smell of naphthalene was found nearby. Forensic autopsy was unremarkable, except for a very small amount of light pink fluid in the stomach, with naphthalene odour. The toxicological analyses revealed the presence of TFP and its major metabolite PTCA (4-[4-[(4-chloro-3-ethyl-1-methylpyrazol-5-yl)carbonylaminomethyl]phenoxy]benzoic acid), together with naphthalene and methyl naphthalenes in the post-mortem sample, with liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) respectively. The plasma concentrations of each substance were quantified as 1.97 µg/ml (TFP), 2.88 µg/ml (PTCA), 1.70 µg/ml (naphthalene), 0.67 µg/ml (1-methyl naphthalene) and 1.44 µg/ml (2-methyl naphthalene). According to these results together with autopsy findings, the cause of his death was determined to be acute Tolfenpyrad poisoning. This is the first case report of fatal poisoning attributable to an intake of TFP product.

Analysis of tolfenpyrad and its metabolites in plasma in a tolfenpyrad poisoning case.

Tolfenpyrad (TFP) is a pesticide that was first approved in 2002 in Japan under the trade name of Hachi-hachi. Analyses of TFP and its major metabolite, 4-[4-[(4-chloro-3-ethyl-1-methylpyrazol-5-yl)carbonylaminomethyl]phenoxy]benzoic acid (PTCA), in plasma obtained from a cadaver suspected to have died of TFP poisoning, were conducted by liquid chromatography-mass spectrometry. The existence of TFP and PTCA was confirmed by scan mode and quantitative analysis was performed by selected ion monitoring mode. Calibration curves showed good linearity over the range of 0.1-4 and 0.25-4 µg/mL, and concentrations were estimated to be 1.97 ± 0.02 and 2.88 ± 0.04 µg/mL for TFP and PTCA, respectively. The plasma extract was further examined to find other metabolites using quadrupole time-of-flight MS, and the results revealed three more metabolites, which were suggested to be hydroxy-TFP, dehydro-TFP and hydroxy-PTCA. Plausible metabolic pathways of TFP in humans are: (i) oxidation of the methyl group on the benzene ring, and (ii) hydroxylation followed by dehydration at the ethyl group on the pyrazole ring.

Identification of active ingredients in dietary supplements using non-destructive mass spectrometry and liquid chromatography-mass spectrometry.

A mid-forties woman purchased seven different dietary supplements from Thailand on the internet and subsequently died after taking these supplements. Since there were no ingredient labels on the supplements, we identified the active ingredients using direct analysis in real time-mass spectrometry (DART-MS), direct exposure probe-MS (DEP-MS), and liquid chromatography-MS (LC-MS). DART-MS gives exact molecular weights and DEP-MS shows the fragmentation of a molecule by electron ionization. Analyses using these two instruments are rapid and do not require extraction of the sample. The compounds predicted by DART-MS and DEP-MS were confirmed by LC-MS and the active ingredients of the seven dietary supplements were identified.

Autopsy report on pseudo-Bartter syndrome with renal calcification induced by diuretics and diet pills.

A woman in her mid-forties had repeated vomiting and diarrhoea accompanied by muscle weakness soon after she started taking seven different diet pills imported from Thailand. After she had taken the pills for 8 days, respiratory depression progressed rapidly to arrest. Blood tests at the Emergency Department showed severe hypokalaemia with metabolic alkalosis. We diagnosed that she had developed pseudo-Bartter syndrome from the findings based on ionic abnormalities and high renin and aldosterone levels, and hyperplasia of the juxtaglomerular apparatus. A postmortem blood analysis indicated subtherapeutic levels of furosemide. We concluded that the patient died from pseudo-Bartter syndrome, which was triggered by chronic self-administration of furosemide and aggravated by the diet pills. This is the first pseudo-Bartter syndrome autopsy report to show histological localisation of calcification in the kidneys.

Biochemical blood markers and sampling sites in forensic autopsy.

Forensic pathologists often hesitate to use biochemical blood markers due to the risk of large postmortem changes and deviations from healthy subjects. Biochemical analyses of postmortem blood, if possible, may help to evaluate pathological status and determining the cause of death in forensic diagnosis, for example, in sudden unexpected death without obvious cause, or young adults with no apparent cause of death or antemortem information. Even commercially available biochemical markers were re-evaluated in the blood samples of 164 forensic autopsy cases. Biochemical markers examined were HbA1c, fructosamine, blood nitrogen urea (BUN), creatinine, total protein, total bilirubin, gamma-glutamyl transpeptidase (gamma-GTP), triglyceride, total cholesterol, C-reactive protein (CRP) and pseudocholine esterase (pChE). We collected cardiac blood (left cardiac blood and right cardiac blood) and peripheral blood (femoral vein blood) to clarify the differences in measured values by sampling site. The measured values were analyzed in relation to postmortem interval, etiology of death and sampling sites. Of all eleven markers, HbA1c is the most useful and reliable because of its negligible postmortem changes and small deviation from healthy subjects. Total bilirubin, BUN, CRP and total cholesterol can be useful if we set appropriate limit ranges and pay attention to the interpretation. For the evaluation of changes due to postmortem intervals, none of the markers except for triglyceride showed significant changes up to three days postmortem. As for sampling sites, femoral vein blood is generally recommended considering postmortem changes, but left cardiac blood was suitable for creatinine, pChE, and total cholesterol. For clinical forensic diagnosis of biochemical blood markers, we must determine the "forensic abnormal value" after collecting more cases by known causes with more information about the population.