Pharmacokinetics and tissue distribution of methcathinone in rabbits

Methcathinone is one of the most commonly abused designer narcotics. The pharmacokinetics and tissue distribution of methcathinone is not well understood. In this study, methcathinone was intravenously or intragastrically administered to rabbits in order to investigate the pharmacokinetics and tissue distribution of methcathinone. The plasma concentrations of methcathinone and its metabolite cathinone at various timepoints post-methcathinone administration as well as the distribution of methcathinone and cathinone in various tissues were determined and quantified using a liquid chromatography-tandem mass spectrometry (LC-MS/MS). According to our results, the elimination of methcathinone and cathinone was faster after intravenous administration than that after intragastric administration. The methcathinone or cathinone concentration in the plasma dramatically dropped at 16–18 h post-methcathinone administration followed by a rebound. Gastric content and stomach tissue could be better samples for the identification of methcathinone abuse by oral administration while bile and stomach tissue could be ideal samples for the identification of methcathinone abuse in intravenous injection cases. The pharmacokinetic characteristics and tissue distribution pattern of methcathinone and its metabolite cathinone described in this study could benefit future study on identification and control of methcathinone abuse in forensic toxicological analysis.     

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.     

Development of an LC–MS/MS method for quantification of 3-chloro-L-tyrosine as a candidate marker of chlorine poisoning

A simple and sensitive liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI–MS/MS) method for the determination of 3-chloro-L-tyrosine (Cl-Tyr) was developed and validated. For sample preparation, 50 μL of the body fluids or tissue extracts were processed by protein precipitation followed by the derivatization with dansyl chloride. The calibration curve was linear over the concentration range of 2.0–200 ng/mL blood or 4.0–400 ng/g tissue. Our method allowed the reproducible and accurate quantification. That is, the intra- and inter-assay coefficients of variation were below 7.73 and 6.94%, respectively in both the blood and lung. We applied the developed method to the analysis of Cl-Tyr in the human autopsy samples, which were suspected of chlorine poisoning, and detected 55.2 ng/mL and 206.6 ng/g Cl-Tyr in left heart blood and lung, respectively. Furthermore, in more than 20 autopsy samples, which were obtained from other causes of death including burn, drowning, hanging, internal disease, trauma and drug poisoning, Cl-Tyr was almost not detected in their both body fluids and organ tissues. In conclusion, the data here reported demonstrate that the LC/ESI–MS/MS method allows the Cl-Tyr in the autopsy samples and that chlorine exposure strongly affects its level, providing a basis for novel identification tool of chlorine poisoning.     

Development for the measurement of serum thiosulfate using LC–MS/MS in forensic diagnosis of H2S poisoning

Thiosulfate measurement is crucial to diagnosis of hydrogen sulfide (H₂S) poisoning in forensic toxicology. Although GC–MS method is currently regarded as a standard thiosulfate measurement, it requires complicated sample preparation prior to analysis. This study presents a simple, rapid, and highly sensitive method for the quantitative analysis of serum thiosulfate by using liquid chromatography–tandem mass spectrometry (LC–MS/MS). This method is based on selected reaction monitoring and has high sensitivity with a lower quantification limit of 0.5 μM. Precision and accuracy of this method meet the basic requirements for quantitative analysis (intra- and inter-day tests have a relative standard deviation of ⩽10.4%; range of analytical recovery is 94.3–102.6%). On the measurements of serum thiosulfate by our developed method, a thiosulfate concentration as 57.5 μM was detected clearly in the H₂S poisoning case comparing to the non poisoning case in which only a trace amount of thiosulfate was observed.     

A simple method for detecting fenitrothion,its metabolite 3-methyl-4-nitrophenol,and other organophosphorus pesticides in human urine by LC-MS

A simple liquid chromatography-mass spectrometry (LC-MS) method was developed and carefully validated for quantitation of fenitrothion, its metabolite 3-methyl-4-nitrophenol, and five other organophosphorus pesticides (acephate, cyanophos, methidathion, ethyl p-nitrophenyl phenylphosphonothioate, and malathion) in human urine samples. Following deproteinization by acetonitrile, the extracts were injected into an LC system and quantitated by MS in the negative ionization mode. The limits of detection were in the range of 0.25–0.75 μg/ml for each analyte, and the method was validated on its accuracy and precision over the concentration range of 1–15 μg/ml. The method was successfully applied to an actual poisoning case and should prove useful in clinical and forensic toxicology.     

Disputed case of homicide by smothering due to severe amitriptyline intoxication of the victim

We report a fatal case of a female for whom the forensic autopsy revealed injuries to the external respiratory orifices indicating smothering. Subsequent postmortem toxicological analysis confirmed heavy amitriptyline acute intoxication. The victim had serious psychological problems, was under long-term treatment with antidepressants and was a systematic alcohol abuser. Forensic autopsy determined damage to the external airways, along with multiple formal petechial hemorrhages (Tardieu) in various parts of the body. The presence of amitriptyline, nortriptyline and 10-hydroxynortriptyline was confirmed by GC–MS and quantified by HPLC in blood (7.0 μg/ml amitriptyline and 7.4 μg/ml nortriptyline). The cause of death was disputed between severe intoxication (poisoning or suicide attempt) and smothering due to controversial evidence.     

5-HT_（1A）受体激动和5-HT重摄取抑制双靶标新药YL-0919在小鼠体内的组织分布

目的研究5-HT1A受体激动和5-HT重摄取抑制双靶标新药YL-0919在小鼠体内的组织分布特征。方法 15只雄性昆明小鼠随机分为3组,单剂量YL-0919（6 mg/kg）灌胃后,分别于2,5,60 min摘眼球取血,处死后解剖取组织。采用液相色谱串联质谱法测定YL-0919浓度。结果小鼠YL-0919灌胃后2 min即在小肠、胃和肝达到最高值,其他组织在给药后5 min浓度达到最高值,浓度由高到低依次为：小肠〉胃〉肝脏〉脂肪〉肾脏〉肺脏〉心脏〉脾脏〉血〉脑〉肌肉〉睾丸,60 min后各组织的药物浓度均降至较低水平。结论 YL-0919在小鼠体内的吸收和分布较快,药物入血后很快分布到体内各个组织中;药物在组织中的消除也较快,没有组织蓄积现象。     

5-HT1A受体激动和5-HT重摄取抑制双靶标新药YL-0919在小鼠体内的组织分布

目的研究5-HT1A受体激动和5-HT重摄取抑制双靶标新药YL-0919在小鼠体内的组织分布特征。方法 15只雄性昆明小鼠随机分为3组,单剂量YL-0919(6 mg/kg)灌胃后,分别于2,5,60 min摘眼球取血,处死后解剖取组织。采用液相色谱串联质谱法测定YL-0919浓度。结果小鼠YL-0919灌胃后2 min即在小肠、胃和肝达到最高值,其他组织在给药后5 min浓度达到最高值,浓度由高到低依次为:小肠>胃>肝脏>脂肪>肾脏>肺脏>心脏>脾脏>血>脑>肌肉>睾丸,60 min后各组织的药物浓度均降至较低水平。结论 YL-0919在小鼠体内的吸收和分布较快,药物入血后很快分布到体内各个组织中;药物在组织中的消除也较快,没有组织蓄积现象。     

An UPLC-MS/MS method for the determination of valproic acid in blood of a fatal intoxication case

Valproic acid (VPA) has been used as an anticonvulsant for the treatment of epilepsy. The authors present a fatal case involving a 45-year-old female, found dead lying in bed with empty tablets of Diplexil^® next to her. She was a chronic alcoholic and epileptic who had been under psychiatric treatment, having repeatedly demonstrated intent to commit suicide.A rapid method was developed and validated to determine VPA in blood by ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) with electrospray ionization source in negative ion mode.The method involved sample treatment with phosphoric acid followed by solid-phase extraction. Chromatographic separation was achieved using an Acquity UPLC^® BEH (2.1 × 50 mm id, 1.7 μm) column and a mobile phase containing ammonium acetate and acetonitrile, at a 0.5 mL/min flow rate. Detection and quantification of VPA was achieved using multiple reaction monitoring (MRM). The MS/MS transitions used for monitoring were m/z 143.1–143.1 for valproic acid and m/z 296.1–205.0 for hydrochlorothiazide used as an internal standard (IS).The limit of quantification (LOQ) was 0.5 μg/mL and the method was linear in the concentration range of 0.5–100 μg/mL. The coefficients of variation obtained for accuracy and precision were less than 10% and the mean recovery was 95% for the three concentrations levels studied (5 μg/mL, 10 μg/mL and 50 μg/mL). Toxicological results showed high concentration of VPA (556 μg/mL) and therapeutic concentrations of tiapride, mirtazapine, oxazepam and nordiazepam. Blood sample analysis also revealed the presence of ethanol at a concentration of 1.34 g/L.A specific, selective and sensitive method for the determination of VPA in blood was developed and can be used in routine forensic investigation. Toxicological results led the pathologist to rule that death was due to an intoxication caused by the simultaneous ingestion of high VPA concentrations and alcohol, with a suicidal legal-medical etiology.     

Ethyl sulfate in blood shows the potential to distinguish alcoholic death and postmortem alcohol instillation

Alcohol is often found in the blood of the deceased. To cover up the true cause of victim’s death, postmortem instillation of alcohol occurs in some criminal cases. Explaining the finding of alcohol is extremely vital in forensic practice. This study aims to evaluate whether ethyl glucuronide (EtG) and ethyl sulfate (EtS) in blood and vitreous humor (VH) can be used to distinguish alcoholic death and postmortem alcohol instillation. Saline or 12.6 g/kg ethanol (antemortem alcohol poisoning group) was introduced into rabbits’ stomachs 2 h before sacrificed. Same amount of ethanol was introduced into rabbits’ stomachs at 0 h, 0.5 h, 1 h and 2 h after death in four subgroups of postmortem alcohol instillation group, respectively. Cardiac blood and VH were collected at 10 min, 4 h, 10 h and 24 h after death in blank and antemortem alcohol poisoning group, and after instillation of alcohol in postmortem alcohol instillation group. Blood was also collected at 34 h. Ethanol and EtG levels in blood and VH and EtS in VH in antemortem alcohol poisoning group were overlapped with those in postmortem alcohol instillation group. The contents of EtG and EtS in blood in antemortem alcohol poisoning group (mean ≥ 7.833 μg/mL for EtG and ≥ 19.990 μg/mL for EtS) were much higher than those in postmortem alcohol instillation group (mean ≤ 0.118 μg/mL for EtG and ≤ 0.091 μg/mL for EtS), but apparent decomposition was observed in EtG, which might lead to misinterpretation. Blood EtS showed better stability and could be used to distinguish alcoholic death and postmortem alcohol instillation.     

Postmortem diffusion of n-butane and i-butane used for anticontagious plugging spray

Blood and tissue samples from a forensic autopsy of a man in his late 60s, who developed dementia and died of multiple head traumas due to a fall from a moving vehicle, contained certain amounts of n-butane and i-butane. The concentration of n-butane was in the range of 0.48–70.5 μL/g, which would be considered as toxic or lethal levels. We had to distinguish whether the cause of his unexplained behavior was due to his pre-existing condition (dementia), or from a confused state induced by butane abuse. No traces of butane use were found at the scene. Police investigation revealed that a propellant used in an anticontagious plugging spray had been administered to him during a postmortem treatment in the emergency hospital. In order to prove the postmortem butane diffusion had resulted from the spray administration and to estimate the diffused concentration, experimental simulation was conducted by using rats. As a result of postmortem treatment with the spray, n-butane at concentrations of 0.54–15.5 μL/mL or g were found in the rat blood and tissues. In this case, we provided further evidence that the postmortem butane diffusion, caused by using the anticontagious plugging spray containing butane gas as a propellant administered to a cadaver during a postmortem procedure prior to forensic autopsy, should be distinguished from cases of actual butane poisoning.     

Suicidal poisoning with mercaptodimethur–morphological findings and toxicological analysis

In the western countries, the number of fatal intoxications with plant protecting agents has decreased to some extent due to laws restricting the use of highly toxic pesticides like halogenated hydrocarbons. Nevertheless, in consideration of the easy availability of most plant protectants, the small fraction of such fatalities among suicides and intoxications is astonishing. An 80-year-old woman died of an intoxication with methiocarb (mercaptodimethur), a carbamate type pesticide and as such a reversible inhibitor of the acetylcholinesterase. The case is presented because it is the first explicit report on a fatal poisoning of a human with methiocarb. The methiocarb concentrations detected were 6,100 μg/g in stomach content, 4.0 μg/ml in heart blood, 11 μg/g in kidney, 1.9 μg/ml in urine, 25 μg/g in liver, 2 μg/g in bile and 2.5 μg/g in brain tissue.     

LC-MS determination of Taxus alkaloids in biological specimens

A semi-quantitative LC-MS method was developed for the detection of the pseudo alkaloids of Taxus baccata (yew) from human body fluids and tissue samples. This method was used to examine the cause of death of a 43-year-old man who died several hours after he drank a decoction of taxus leaves. Autopsy and histology demonstrated early signs of myocardial hypoxia. Since investigation of the stomach content did not yield evidence of taxus ingestion, the taxus alkaloids were determined in blood, stomach content and tissue samples of the deceased by LC-MS. The samples were prepared by solid phase extraction on RP-18 columns. Chromatographic separation was achieved by HPLC on a RP-8 column, coupled to an ion trap mass spectrometer (Finnigan LCQ). An atmospheric pressure electrospray ionisation was performed. Spectra of the alkaloids were recorded in the single MS mode and in the MS-MS mode and compared with reference spectra obtained from an extract of yew leaves. In the stomach content, the kidneys, the liver and a heart blood sample of the deceased, alkaloids of Taxus baccata, predominantly taxine B and iso-taxine B, were identified. The semi-quantitative evaluation of the heart blood revealed a taxine concentration of 11 µg taxine/g. As far as we know this is the first case in which a semi-quantitative analysis of taxine alkaloids has been performed.     

Preliminary screening method for the determination of inorganic arsenic in urine

A simple and rapid method was developed for the routine determination and classification of inorganic arsenic based on its clinical and forensic properties. Inorganic arsenic was isolated from urine by using copper granules, which was then made to react with ammonium molybdate in order to detect its presence with the naked eye. Based on studies of extraction and reaction conditions, e.g., reaction temperature and time, a colorimetric screening method was established. The reaction mixture was measured by a spectrophotometer, and there was linearity from 0.05 to 2.0 μg/ml and the correlation coefficients of the calibration curves were greater than 0.99. The coefficients of intra-day variation at 0.2 and 2.0 μg/ml of inorganic arsenic in urine were 9.6 and 4.2%, respectively (n = 5). The minimum detectable level in urine is 0.03 μg/ml, and it is possible to detect the lowest level of poisoning according to the published reports. The proposed method was applied to a poisoning case wherein the patient ingested NEOARSEN BLACK® with alcohol, which contained 45% of arsenic trioxide. This method produced positive results in all the urine samples tested, and this method is useful for the screening of inorganic arsenic based on its clinical properties because it enables the detection of inorganic arsenic in urine without expensive equipment.     

Experimental penetration of fragment simulating projectiles into porcine tissues compared with simulants

IntroductionBallistic gelatin is well validated in its ability to simulate the retardation of bullets into homogenous muscle. However the relationship is less clear for fragmentation projectiles and non-homogenous tissues as would truly be found in a human.Method0.16 g, 1.10 g and 2.84 g NATO standardised cylindrical Fragment Simulating Projectiles (FSPs) were fired at a range of velocities (112–1652 m s^?1) into four body areas (thigh, abdomen, thorax or neck) of six pig cadavers as well as 20% gelatin. Cadavers were imaged by Computed Tomography (CT) scanning and FSP Depth of Penetration (DoP) ascertained through radiology followed by dissection by a forensic pathologist.Results106/149 (71%) FSPs were retained in tissues enabling DoP measurements and 43/149 (29%) exited the subjects. There was significantly less retardation of FSPs in the thorax and abdomen compared to gelatin but no difference in retardation in leg and neck tissue compared to gelatin. Although the gradient appeared identical for the 2.84 g FSP as well, there were insufficient FSPs retained in the neck and leg for meaningful analysis to be undertaken.DiscussionPorcine leg and neck muscle was demonstrated to be comparable to 20% ballistic gelatin in terms of retardation, validating the use of projectile penetration algorithms derived from this tissue simulant. The effect of pig skin was significant for the 0.16 g FSP, especially at lower velocities, and we would therefore suggest that specific algorithms for any future numerical injury models be based directly from animal data or validated skin simulants for this smaller sized FSP. Reproducing the retardation effects of FSPs in the thorax and abdomen using tissue simulants alone will be problematic due to the anatomical complexity as well as multiple tissue–air interfaces and we would recommend further research in this area.     

Time-dependent postmortem redistribution of morphine and its metabolites in blood and alternative matrices—application of CT-guided biopsy sampling

Interpretation of postmortem morphine concentrations in forensic toxicology provides several pitfalls such as missing information on tolerance, analyte stability, or postmortem redistribution (PMR). Recently, it had been shown that computed tomography (CT)-guided collection of biopsies using a robotic arm (virtobot) provides a valuable strategy for systematic studies on time-dependent PMR. Using this technique, time-dependent PMR of morphine and its metabolites was investigated in 12 cases. At admission to the institute (t1), femoral and heart blood (right ventricle) as well as biopsies from the right lung, the right kidney, liver, spleen, and muscle tissue were collected. At autopsy approximately 24 h later (t2), samples from the same body regions were collected again. Additionally, gastric contents, urine, brain tissue, and heart blood from the left ventricle was collected. Morphine, normorphine, hydromorphone, morphine-3-glucuronide, morphine-6-glucuronide, and morphine-sulfate were quantified with LC-MS/MS. In femoral blood, significant increase of morphine concentrations was observed, although ultimately not relevant for forensic interpretation. In the alternative matrices, increases as well as decreases were observed without a clear trend. The morphine metabolites did not exhibit relevant concentration changes. Investigation of underlying redistribution mechanisms indicated that concentration change (i.e., increase) of morphine in femoral blood rather resulted from diffusion processes than from release of morphine from its conjugates. Concentration changes in heart blood might have been caused by redistribution from lung tissue or gastric content. This study also proved that CT-guided collection of biopsies using a virtobot arm is an invaluable tool for future studies on PMR redistribution of other substance groups.

     

Determination of 3-chloro-l-tyrosine as a novel indicator of chlorine poisoning utilizing gas chromatography-mass spectrometric analysis

Chlorine gas exposure occurs in chemical warfare, industrial and household accidents. In forensic science, the generation of chlorine gas by mixing sodium hypochlorite detergent and strong acid detergent cannot be overlooked because of the possibility of suicide method (NaClO + 2HCl → NaCl + H₂O + Cl₂). Though typical autopsy findings are obtained in chlorine exposure, such as pulmonary edema, useful biomarkers don’t exist. In this research, we developed an analytical method of 3-chloro-l-tyrosine (Cl-Tyr) in blood as a novel marker of chlorine poisoning utilizing gas chromatography–mass spectrometry (GC–MS). Cl-Tyr was purified using protein precipitation and cation-exchange solid phase extraction, derivatized by the silylation agent and subjected to GC–MS. The quantification range was 10–200 ng/mL and good reproducibility was obtained. We applied the developed method to analyze Cl-Tyr in autopsy sample, which is suspected of chlorine poisoning, and detected 59.7 ng/mL Cl-Tyr in left heart blood. To our knowledge, this is the first report of determination of the chlorinated biomolecule in the human autopsy sample from chlorine poisoning.     

Diagnostic approach to drug-screening tests for fatal diabetic ketoacidosis: Forensic autopsy of a methamphetamine abuser

To diagnose the cause of death in autopsy cases, systematic examinations, such as macroscopic, pathological, biochemical, and toxicological are important. In this case report, drug examinations also gave very useful information to diagnose the cause of death, fatal diabetic ketoacidosis (DKA). A female methamphetamine abuser in her forties was found dead lying on a hotel bed. Diagnosing her cause of death was difficult only from the macroscopic findings because there was no fatal and/or serious injury or disease. On toxicological examination, acetone was detected at a high concentration (682 μg/mL in blood, 887 μg/mL in urine) using gas chromatography (GC). Using gas chromatography–mass spectrometry (GC–MS), methamphetamine was detected in the blood, urine, hair, and visceral organs; however, these concentrations were low. At the same time, GC–MS examination revealed a high glucose peak. From the results of the biochemical examination of urine, acetoacetic acid was 1940 μmol/L, β-hydroxybutyric acid was 14,720 μmol/L, and glucose was 4620 mg/dL. Histologically, Langerhans’ islets in the pancreas were fibrotic and atrophic, and no insulin-immunoreactive cells were observed. The subsequent police investigation also revealed that she had contracted diabetes mellitus type 1; therefore, we concluded that her cause of death was DKA, due to a lack of insulin injection.     

Histological microaggregates as a sign of systemic dissemination due to oral ingestion of povidone-iodine (Betadine®)

Povidone-iodine (PVP-I) in the formulation of Betadine® is widely used in several medical fields, whereas its suicidal ingestion is an unusual occurrence. Therefore, the biological signs of this fatal poisoning remain unclear and elusive, similarly to the histological lesions induced by PVP-I. In fact, there are no forensic articles concerning the histological signs of PVP-I acute poisoning. Specifically, this short communication reports the unique case of a man who ingested 125 ml of Betadine® 10% to commit suicide, for which he died three days later. The autopsy examination showed either local or systemic signs of caustic ingestion; moreover, the histological analysis showed both intra- and extra-cytoplasmic amorphous and brownish microaggregates in almost all the organs. Histological and histochemical techniques resulted to be negative for the detection of physiological and known pigments (e.g., formalin, lipofuscin, hemosiderin). Likewise, such pigments were not associated neither with the man’s diseases nor with his medications. Therefore, the authors supposed that they were povidone-iodine microaggregates, which have been deposited in the organs through the blood circulation. In conclusion, the complete postmortem histological examination could allow the identification and the characterization of PVP-I microaggregates as evidence of systemic toxicity from Betadine®, when dealing with a forensic case of ingestion of povidone-iodine.     

Assisted suicide by fentanyl intoxication due to excessive transdermal application

Herein, we report a case of an assisted suicide committed by application of 34 matrix-based fentanyl-containing transdermal therapeutic systems (TTS) with different release rates. The TTS were supplied by the husband but administered by the deceased herself. Besides routine systematic toxicological analysis (STA), the concentrations of fentanyl and norfentanyl were determined in the blood (femoral and heart), urine, stomach content, brain, lung tissue, musculus iliopsoas, liver, kidney, bile and in some of the used TTS by LC-MS/MS. Blood levels of fentanyl were 60.6 μg/L in femoral blood and 94.1 μg/L in heart blood. These concentrations are in good concordance with levels described in cases with accidental or lethal suicidal fentanyl patch application. The organ distribution indicates an influence of post-mortem redistribution. The levels of residual fentanyl in the TTS were also determined. STA furthermore revealed supratherapeutic levels of bromazepam. Thus, the cause of death was a combination of fentanyl and bromazepam intoxication. However, considering the determined levels of fentanyl and norfentanyl in the entire set of specimens and the high toxicity in comparison to bromazepam, fentanyl was the leading toxic noxa.