Determination of bromadiolone and brodifacoum in human blood using LC-ESI/MS/MS and its application in four superwarfarin poisoning cases

Superwarfarin poisoning is a growing health problem. A sensitive and reproducible LC-ESI/MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) method was developed and validated for the determination of bromadiolone and brodifacoum, the most commonly used superwarfarins, in human blood using warfarin-D5 as an internal standard. Bromadiolone and brodifacoum were extracted from whole blood samples by liquid-liquid extraction with ethyl acetate. Multiple-reaction monitoring (MRM) was used to detect bromadiolone and brodifacoum using precursor→product ion combinations of m/z 525→250 and 521→135, respectively. The calibration curves were linear (r(2)=0.9999) in the concentration range of 0.5-100.0ng/mL for bromadiolone and brodifacoum, with a lower limit of detection of 0.1 and 0.2ng/mL, respectively, in whole blood. This method detected trace levels of bromadiolone and brodifacoum in whole blood samples and can be used in the diagnosis of poisoned human beings.     

曲马多在家兔体内的死后分布研究

目的研究曲马多在中毒家兔体内死后分布规律,为曲马多中毒检材采取提供实验依据。方法家兔经口给予10倍LD50曲马多,待家兔死亡后迅速解剖取样,气相色谱／质谱联用和气相色谱-FTD法测定其体液、脏器、大脑及右上肢和右下肢肌肉中曲马多的含量,比较其变化规律。结果血液和肝脏中曲马多的最低检出限分别为0．05μg/mL和0．05μg/g,提取回收率为97．60％±0．65％～103．10％±1．24％。曲马多在家兔体内的死后分布为：肾〉胃〉肝〉脾〉肺〉脑〉心〉上肢肌肉〉下肢肌肉〉〉体液（尿〉胆汁、心血〉玻璃体液）。结论大剂量曲马多中毒致死后在体内分布不均匀,组织中曲马多含量明显高于心血、胆汁等体液。     

Ethyl sulphate and ethyl glucuronide in vitreous humor as postmortem evidence marker for ethanol consumption prior to death

To clarify the circumstances of death, the degree of inebriation is of importance in many cases, but for several reasons the determination of the ethanol concentration in post-mortem samples can be challenging and the synopsis of ethanol and the direct consumption markers ethyl glucuronide (EtG) and ethyl sulphate (EtS) has proved to be useful. The use of a rather stable matrix like vitreous humor offers further advantages. The aim of this study was to determine the concentrations of ethanol and the biomarkers in the robust matrix of vitreous humor and to compare them with the respective levels in peripheral venous blood and urine. Samples of urine, blood from the femoral vein and vitreous humor were taken from 26 deceased with suspected ethanol consumption prior to death and analyzed for ethanol, EtS and EtG. In the urine samples creatinine was also determined. The personal data, the circumstances of death, the post-mortem interval and the information about ethanol consumption prior to death were recorded. EtG and EtS analysis in urine was performed by LC-ESI-MS/MS, creatinine concentration was determined using the Jaffé reaction and ethanol was detected by HS-GC-FID and by an ADH-based method. In general, the highest concentrations of the analytes were found in urine and showed statistical significance. The mean concentrations of EtG were 62.8mg/L (EtG100 206.5mg/L) in urine, 4.3mg/L in blood and 2.1mg/L in vitreous humor. EtS was found in the following mean concentrations: 54.6mg/L in urine (EtS100 123.1mg/L), 1.8mg/L in blood and 0.9mg/L in vitreous humor. Ethanol was detected in more vitreous humor samples (mean concentration 2.0g/kg) than in blood and urine (mean concentration 1.6g/kg and 2.1g/kg respectively). There was no correlation between the ethanol and the marker concentrations and no statistical conclusions could be drawn between the markers and matrices.     

Colchicine poisoning: case report of two suicides

Colchicine overdose is uncommon but potentially life threatening because of the high toxicity of the drug. Poisoning by colchicine may occur following ingestion of medication used in acute attacks of gout and inflammatory diseases. We describe two cases involving suicide by the ingestion of medications marketed in France. In case 1, only heart blood was taken after body external examination. In case 2 an autopsy was performed and heart blood, urine, gastric contents and bile were taken for toxicological analysis. Colchicine was assayed in biological specimens by an HPLC-DAD method, after extraction by dichloromethane at pH 8, adding prazepam as internal standard (IS). Analyses were performed on a Symetry C-8 column. Mobile phase was a gradient of acetonitrile/pH 3.8 phosphate buffer. Colchicine is eluted at 13.1 min and the method is linear for blood, urine and bile over the range 4-1000 ng/mL. LOQ is 4 ng/mL. The concentrations of colchicine detected are: case 1: heart blood 13 ng/mL; case 2: heart blood 66 ng/mL, urine 500 ng/mL, gastric content 12 ng/mL, bile 5632 ng/mL. Our findings are in the range of lethal concentrations previously described, but there is no correlation with the amount of ingested drug. Even after massive overdose, it could be impossible to detect colchicine in blood, and as there is a widespread enterohepatic recirculation before excretion in bile and feces, bile is the target sample to analyse. We conclude in both cases that the cause of death was suicide with colchicine. It appears very important to perform an autopsy in order to obtain bile, urine, heart blood and femoral blood.     

A death involving probenecid.

A death following deliberate ingestion of approximately 75 g of probenecid in a 36-year-old man is described. Tissue concentrations of probenecid were highest in serum (710 mg/L) and liver (550 mg/kg). Probenecid was also detected in vitreous and bile. Ethanol was also detected in blood at 0.13 g/100 mL.     

甲胺磷在犬体内的死后分布

目的建立甲胺磷的犬灌胃染毒致死模型,观察甲胺磷在犬体内的死后分布规律。方法犬经8倍LD50（7.4mg/kg）剂量甲胺磷灌胃后,观察其中毒症状,死亡后即刻解剖,分别取心、肝、脾、肺、肾、脑、右上肢肌、右下肢肌、胸肌、胃组织、心血、胆汁、玻璃体液和尿液,GC/MS和GC法检测其中甲胺磷含量。结果犬8倍LD50甲胺磷灌胃染毒后20min内出现中毒症状（,53.3±14.1）min死亡。各组织脏器及体液中甲胺磷含量由高到低分别为胃（99.84±0.87）μg/g、脾（46.87±28.67）μg/g、肝（43.82±22.74）μg/g、肾（43.79±29.04）μg/g、心血（35.36±13.98）μg/mL、肺（35.25±18.59）μg/g、尿34.81μg/mL、胸肌（19.23±17.18）μg/g、右上肢（16.92±8.98）μg/g、心（15.09±6.11）μg/g、右下肢（12.83±7.63）μg/g、脑（10.91±4.13）μg/g、胆汁（6.75±1.45）μg/mL、玻璃体液（6.22±4.97）μg/mL。结论甲胺磷在犬体内死后分布不均,胃、脾、肝、肾、心血、肺、尿检材中含量较高,可作为疑似甲胺磷中毒毒物分析的检材。     

A fatal poisoning caused by methomyl and nicotine

A 35-year-old male was found lying in a prone position in his room. He was in cardiopulmonary arrest on arrival to hospital and was pronounced dead. There was no attempt at resuscitation. No miosis was observed on admission. At post-mortem his stomach contained 170 g greenish liquid with a small amount of shredded tobacco leaves. The serum cholinesterase activities were 47-90 IU (normal range for male: 200-440 IU). GC and GC-MS analyses showed nicotine (21.8 mg), methomyl (304 mg), and triazolam (1.69 mg) in his stomach. He had consumed tobacco leaves, Lannate containing water soluble methomyl (45%), and Halcion tablets containing 0.25 mg triazolam. Methomyl concentrations in blood were 3-8 ng/ml. Substantial amounts of methomyl (2260-2680 ng/ml) were detected in cerebrospinal fluid and vitreous humor. Nicotine concentrations in blood ranged from 222 to 733 ng/ml. A small amount of triazolam was detected only in bile (176 ng/ml) and liver (23 ng/g). The cause of death was respiratory paralysis produced by the additive effects of methomyl and nicotine shortly after consumption.     

Accidental death from hydromorphone ingestion

Abstract: A 15‐year‐old male orally consumed an unknown but fatal amount of sustained release hydromorphone. He was naïve to opioid use. No other drugs or alcohol were involved. The cause of death was acute aspiration‐related bronchopneumonia, secondary to hydromorphone ingestion; the manner of death was accidental. Hydromorphone and hydromorphone‐3‐glucuronide were quantified in postmortem fluids by tandem liquid chromatography–mass spectrometry. The hydromorphone concentrations in the peripheral blood, urine, and vitreous humor were 57, 4460, and 31 ng/mL, respectively. The hydromorphone‐3‐glucuronide concentrations in the corresponding three fluids were 459, 36,400, and 40 ng/mL. Hydromorphone‐3‐glucuronide accumulation probably did not contribute significantly to the opiate toxicity. The proposed minimum lethal hydromorphone blood concentration in the nontolerant user is in the vicinity of 60 ng/mL.     

Heroin fatality due to penile injection

Death due to heroin overdose and/or rapid injection of heroin is a frequent occurrence among opioid addicts. We present an unusual case of heroin fatality due to the injection of the drug in the penis. Blood, urine, bile, and vitreous humor concentrations of morphine were 0.68, 0.49, 0.32 and 0.062 microg/ml, respectively. Ethanol was detected at concentrations of 104, 124, 106, and 94 mg/dl in the blood, urine, bile, and vitreous humor, respectively. The cause of death was determined to be due to heroin and ethanol intoxication.     

Postmortem determination of the biological distribution of sufentanil and midazolam after an acute intoxication

A case is presented of a death caused by self-injection of sufentanil and midazolam. Biological fluids and tissues were analyzed for midazolam by high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) and for sufentanil by GC/MS. Midazolam was extracted from basified fluids or tissues homogenated with n-butyl chloride and analyzed by HPLC by using a phosphate buffer: acetonitrile (60:40) mobile phase on a mu-Bondapak C18 column at 240 nm. Sufentanil was extracted from basified fluids and tissue homogenates with hexane:ethanol (19:1). GC/MS methodology for both compounds consisted of chromatographic separation on a 15-m by 0.25-mm inside diameter (ID) DB-5 (1.0-micron-thick film) bonded phase fused silica capillary column with helium carrier (29 cm/s) splitless injection at 260 degrees C; column 200 degrees C (0.8 min) 10 degrees C/min to 270 degrees C; and electron ionization and multiple ion detection for midazolam (m/z 310), methaqualone (IS, m/z 235), sufentanil (m/z 289), and fentanyl (IS, m/z 245). Sufentanil concentrations were: blood 1.1 ng/mL, urine 1.3 ng/mL, vitreous humor 1.2 ng/mL, liver 1.75 ng/g, and kidney 5.5 ng/g. Midazolam concentrations were: blood 50 ng/mL, urine 300 ng/mL, liver 930 ng/g, and kidney 290 ng/g. Cause of death was attributed to an acute sufentanil/midazolam intoxication and manner of death a suicide.     

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.     

A mixed-drug intoxication involving venlafaxine and verapamil

This case report describes the suicide of a 52-year-old woman whose cause of death was attributed to a mixed-drug intoxication involving venlafaxine and verapamil. Venlafaxine is prescribed for the treatment of depression and should be used with caution in patients with cardiovascular disease. Verapamil is a calcium channel blocker primarily used for treatment of cardiovascular disorders. The following drug concentrations were determined in postmortem fluids: verapamil--3.5 mg/L (femoral blood), 9.4 mg/L (subclavian blood), and 1.0 mg/L (vitreous fluid); norverapamil--1.0 mg/L (femoral blood), 2.1 mg/L (subclavian blood), and 0.20 mg/L (vitreous fluid); verapamil and norverapamil could not be detected in bile or urine due to the high levels of erythromycin present; venlafaxine--6.2 mg/L (femoral blood), 8.6 mg/L (subclavian blood), 5.3 mg/L (vitreous fluid), 54.0 mg/L (bile), and 72.3 mg/L (urine); and O-desmethylvenlafaxine--5.4 mg/L (femoral blood), 8.3 mg/L (subclavian blood), positive (vitreous fluid), 29.2 mg/L (bile), and 9.5 mg/L (urine). The cause of death was determined to be a mixed-drug intoxication resulting from an overdose of verapamil and venlafaxine. The manner of death was determined to be suicide.     

A multiple drug fatality involving MK-801 (dizocilpine), a mimic of phencyclidine

MK-801 (dizocilpine) is a non-competitive antagonist at the N-methyl-D-aspartate (NMDA) family of glutamate receptors in the central nervous system. It is an anticonvulsant and also shares several pharmacological properties with phencyclidine and ketamine. It is not observed routinely as a substance of abuse. The deceased, a 45-year-old white male, obtained MK-801 surreptitiously in an attempt to treat a self-diagnosed depression. He was discovered the next morning, unresponsive on the bathroom floor. An empty bottle, labeled to contain 25mg of MK-801, was found near the body.The autopsy was performed at the Joseph A Jachimczyk Forensic Center, Houston, TX. Body weight at autopsy was 88kg. Lungs were edematous and congested (right: 775g; left 700g). The heart had proportionate chambers and was otherwise unremarkable. The kidneys (right: 220g; left 225g) were smooth surfaced. The brain (1550g) was congested and without trauma. Microscopic evaluation of the heart, kidneys and lungs showed normal histology and confirmed pulmonary congestion and edema. Samples of heart blood, liver, bile, vitreous humor, stomach contents and urine were collected at autopsy. There were 550ml of stomach contents.Drugs in blood were screened by EMIT II Plus immunoassay procedures and by gas chromatography/mass spectrometry (GC/MS) of an organic solvent extract of basified blood. Alcohol was determined by gas chromatography with headspace injection. MK-801, benzodiazepines and alcohol were detected in blood.Amounts of MK-801 present in blood, bile, liver, vitreous humor and urine were 0.15, 0.29, 0.92, less than 0.1 and 0.36 mg/l (kg), respectively.The cause of death was benzodiazepine, dizocilpine and ethanol toxicity and the manner accidental.     

Development and validation of a LC–MS/MS method for analysis of vortioxetine in postmortem specimens. First data from an authentic case

Vortioxetine is an antidepressant recently licensed in USA and EU for the treatment of major depressive disorder. Neither fatal case due to overdose nor data about postmortem concentrations on blood or other specimens have been reported. The aims of this study were the development and validation of a method for vortioxetine analysis by Liquid Chromatography Tandem Mass Spectrometry (LC–MS/MS) in postmortem samples and its application in an authentic case. The method was validated and applied on blood, vitreous humor, bile, brain, liver, kidney, and gastric content. After protein precipitation, the supernatant was directly injected into LC–MS/MS. Analysis was carried out by Multiple Reaction Monitoring (MRM) mode. The authentic case concerned a 38 years-old woman, affected by depression, who was found hanged at home. The method determined an acceptable sensitivity, selectivity, linearity, precision, and accuracy for all matrices. No interference was shown for all matrices. The matrices do not significantly reduce the peak intensity of vortioxetine. No carryover was shown. Toxicological analysis of the authentic case showed vortioxetine in blood (234 ng/ml), vitreous humor (10.5 ng/ml), brain (490 ng/g), lung (479 ng/g), liver (3751 ng/g), kidney (798 ng/g), bile (2267 ng/ml) and gastric content (253 ng/ml). Our case suggests that even at blood concentrations of vortioxetine equal to 234 ng/ml, the subject was able to stage and carry out the hanging. Vortioxetine concentrations found in the other cadaveric samples (biological fluids, organs, and gastric content) may be helpful to evaluate further similar comparable cases.     

Bupropion Overdose Resulted in a Pharmacobezoar in a Fatal Bupropion (Wellbutrin®) Sustained‐release Overdose: Postmortem Distribution of Bupropion and its Major Metabolites

Bupropion (BUP) overdose commonly causes generalized seizures and central nervous system depression. The case of a 28‐year‐old woman who died from a massive lethal overdose with sustained‐release bupropion (Wellbutrin^® 300 mg) is herein presented. The autopsy revealed the presence of a pharmacobezoar consisting of at least 40 tablets in the stomach. Determination of bupropion and its active metabolites (hydroxybupropion, threobupropion, erythrobupropion) was achieved by a liquid chromatographic mass spectrometry (LC‐MS/MS) method. Postmortem concentrations for bupropion, hydroxybupropion, threobupropion, and erythrobupropion were obtained in intracranial blood, urine, bile, liver, kidney, and vitreous humor. In this case, intracranial blood level of the parent drug was 1.9 mg/L. Threobupropion was the most abundant metabolite in both blood and urine, 59.3 and 890.6 mg/L. Tissue distribution showed the highest concentration in the liver, 12.3 mg/kg. The 0.8 bupropion concentration ratio vitreous/blood suggested that vitreous could be a valuable specimen for toxicological analysis should postmortem blood be unavailable.     

家兔急性有机磷中毒死后玻璃体液化学成分的变化趋势

目的 探讨家兔急性有机磷中毒死后不同时间眼玻璃体液化学成分的变化。方法用5％敌百虫制作家兔急性有机磷中毒动物模型,在死后0～72h内不同时间点,采用留置微量重复取样法吸取眼玻璃体液,以全自动生化分析仪测量眼玻璃体液中钾、镁、磷、钙、钠、氯浓度的变化,统计数据进行二项式回归方程拟合分析。结果家兔急性有机磷中毒死后72h眼玻璃体液中钾、镁、磷随PMI延长而逐渐升高,舻分别为0．9818、0．9700、0．9458,与PMI相关性好,其中30h内镁的相关性更好;钙、钠随PMI延长呈降低趋势,砰分别为0．7124、0．7355,相关性较差;氯表现不稳定掣仅为0．0869。结论家兔急性有机磷中毒死后玻璃体液中钾、镁、磷、钙、钠等离子浓度随PMI延长而改变,72h内总体衡量,钾与PMI最具有相关关系。     

A fentanyl fatality involving midazolam

A case is presented of a 35-year-old black African male anesthesiology resident, found dead in his apartment. At the scene a syringe, butterfly intravenous line and a bottle of Versed (Midazolam) were recovered. A comprehensive screen for common drugs of abuse and therapeutic agents failed to detect any drugs in blood and urine. The blood ethanol concentration was 0.06 g/dl. A GC/MS SIM assay for midazolam was developed. A sub-therapeutic midazolam blood concentration of 7.5 ng/ml was detected and concentrations (ng/ml or ng/g) in bile, urine, and liver were 3.3, 7.5, and 96, respectively. The syringe fluid was then analyzed and found to contain only fentanyl, midazolam was absent. The blood fentanyl concentration was 4.9 ng/ml which is consistent with those reported in fentanyl fatalities. Fentanyl concentrations (ng/ml or ng/g) in bile, urine, and liver were 8.8, 5.0, and 5.9, respectively. The cause of death was ruled to be fentanyl intoxication and the manner of death undetermined.     

Applicability of succinylmonocholine as a marker for succinylcholine administration--comparative analysis of samples from a fatal succinylcholine-intoxication versus postmortem control specimens

Doubts concerning the applicability of succinylmonocholine (SMC) as a succinylcholine (SUX) marker have been issued. A comparative analysis of previously discussed tissues, i.e. brain, liver and kidney, was conducted to further elucidate this question by searching for diagnostically useful differences in analyte content in samples of SUX- versus non-SUX-associated fatalities. Furthermore, possible advantages of vitreous humor as a novel and promising target matrix for SUX analytics were assessed. Sample material of SUX-negative controls as well as the fatal SUX-intoxication was derived from frozen archive material and current autopsies. Samples were analyzed according to a modified protocol of a previously published and validated method employing ion-pairing solid-phase extraction and subsequent HPLC-MS/MS analysis. Standard addition was employed for quantification as well as an estimation of the analytical limits of the method. In all tested matrices, the method was proven to be sufficiently sensitive for the intended application. No indication of native SMC was found in controls of fresh tissues, nor in fresh or frozen vitreous humor. However, most of the samples were found to be positive for a previously reported interference with SMC's main ion transition, thereby falsely suggesting an SMC content of up to 139 ng/g, 126 ng/g, 165 ng/g and 93 ng/ml in brain, liver, kidney and vitreous humor, respectively. Contrasting the results for fresh sample material, SMC was detectable in some of the initially non-putrefied liver samples after long-term storage, as well as in massively decomposed SUX-negative control bodies. In this context, a microbial origin of the analyte may be assumed. All tissues as well as the vitreous humor of the fatal SUX-intoxication were negative for SUX and SMC. Just like serum, tissue and vitreous humor samples therefore do not allow a reliable diagnosis of a SUX-intoxication: in tissues this is due to the pronounced instability of both target analytes in these esterase-containing matrices, for vitreous humor an additional reason could be their insufficient incorporation into this medium.     

溴敌隆及其代谢物-苄叉丙酮在犬体内的死后分布

目的研究溴敌隆及其代谢物-苄叉丙酮在中毒致死犬体内死后分布规律,为溴敌隆中毒检材的采取提供实验依据。方法分别经口给予犬2倍和4倍LD_(50)溴敌隆,待其死亡后迅速解剖取材,气相色谱-质谱联用法测定心血、外周血、尿、胆汁、心、肝、脾、肺、肾、脑、左下肢肌、膀胱、胃、胃内容、胰等脏器和体液中溴敌隆和代谢物-苄叉丙酮的含量。结果犬经2倍和4倍LD_(50)溴敌隆灌胃染毒后3d开始出现出血症状,(178.40±20.94)h后死亡。溴敌隆和代谢物-苄叉丙酮在各组织脏器及体液中的死后分布为:溴敌隆2LD_(50)组溴敌隆:胆汁尿、肝、心、肾心血、外周血、脾、肺等;苄叉丙酮:胆汁、尿、心血、外周血、肺、胃内容中含量高于其他脏器。溴敌隆4LD_(50)组溴敌隆:胆汁、尿肝、外周血心血、胃内容物等脏器。苄叉丙酮:胆汁、尿、肺浓度高于其他脏器。结论溴敌隆及其代谢物-苄叉丙酮在中毒致死犬体内死后分布不均匀,溴敌隆在胆汁、尿、肝脏、心血和外周血含量较高,代谢物-苄叉丙酮在胆汁、尿、肺较高。胆汁、尿、肝脏、心血、外周血可作为疑似溴敌隆中毒毒物分析的检材。