Postmortem Blood Specimens Research Articles

The interest of using vitreous humor for γ-hydroxybutyrate (GHB) quantification in related fatalities: Stability evaluation, case report and literature review

Analysis and interpretation of the findings for γ-hydroxybutyrate (GHB) in related fatalities remains problematic. Indeed, GHB is a naturally occurring compound present in both the mammalian central nervous system and peripheral tissue. Moreover, a postmortem increase in endogenous GHB concentration has been observed, especially in blood. Facing this issue, the use of an alternative matrix such as vitreous humor (VH) can thus be particularly interesting for GHB testing and quantification. VH is considered to be less prone to postmortem redistribution, is easy to collect, and has relatively few interfering compounds for the analytical process. In this context, the authors report the case of a GHB-related fatality involving 22-year-old male. In this case, GHB femoral blood (FB) (790 mg/L) and vitreous (750 mg/L) concentrations appeared similar with a FB to VH (FB/VH) ratio of 1.05. In addition, other similar cases with both GHB blood and vitreous concentrations were reviewed. Five cases were identified. The blood to VH ratios ranging from 0.13 to 2.58. Finally, GHB stability was documented in postmortem blood and VH, in order to address the reliability of VH as an alternative matrix for GHB quantitation at postmortem. GHB appeared relatively stable in postmortem blood specimens (at 50 mg/L) over a period of 28 days when stored at +4 °C or −20 °C. The same results were observed in VH specimens.

Distribution of donepezil in postmortem casework

Donepezil is one of the primary treatments options for patients suffering from Alzheimer's Disease. In a review of more than 2200 postmortem donepezil positive blood specimens, 76% of concentrations were higher than the proposed therapeutic range. Means and medians were similar between central blood specimens and peripheral specimens, indicating minimal postmortem redistribution.Postmortem concentrations may not reflect those circulating antemortem. Mean and median postmortem blood concentrations were approximately 3-fold higher than those in antemortem blood specimens. Additionally, in cases where antemortem blood was available for testing, large increases in donepezil concentrations were reported between antemortem and postmortem specimens without documented administration by medical personnel. Elevated blood donepezil concentrations have been reported in multiple postmortem cases where cause of death was unrelated. The blood concentrations reported in cases where donepezil did not contribute to death overlapped with those in suspected drug overdose cases where other drugs may have been present. In 4 out of 5 suspected donepezil overdose cases, blood concentrations greater than 1000 ng/mL were reported, whereas less than 1% of all postmortem blood samples reviewed achieved these concentrations. Blood concentrations greater than 1000 ng/mL should be considered contributory when a drug overdose is suspected. Postmortem donepezil concentrations should be interpreted with caution in the context of a comprehensive case history.

Evaluating the Reliability of Dipstick Drug Screens on Vitreous and Postmortem Blood as a Triage Modality in Forensic Pathology.

Dipstick drug screens are cheap, easy to use, and quick presumptive tests to detect common drugs of abuse. Dipsticks are designed for drug detection in urine. There is no literature regarding their potential use on fluids different from urine. The study aimed to determine the performance of dipstick screening tests on postmortem vitreous and blood specimens compared to urine dipsticks and final confirmatory toxicology analyses on blood. The study population included cases in which a complete toxicology analysis was performed. Each subject was screened for three substances: cocaine, fentanyl, and opiates. Dipstick results were checked by visual inspection. Results were compared with urine screening tests and quantitative, confirmatory toxicological analyses by gas chromatography/mass spectrometry on postmortem blood samples as the gold standards for screening and confirmatory analysis, respectively. There was a high number of false-negative results for opiates. Cocaine dipsticks in blood showed the highest reliability. Fentanyl dipsticks in vitreous showed a high number of false-negative results. Both vitreous and blood dipstick screening tests for all substances performed well on negative cases. When both blood and vitreous screening tests are negative, the chance that the confirmatory toxicology analysis will be positive is very low.

“Spice”-related deaths in and around Munich, Germany: A retrospective look at the role of synthetic cannabinoid receptor agonists in our post-mortem cases over a seven-year period (2014–2020)

Synthetic cannabinoid receptor agonists (SCRAs, “Spice”) are a diverse group of recreational drugs, with their structural and pharmacological variability still evolving. Forensic toxicologists often rely on previous reports to assess their role in intoxication cases. This work provides detailed information on the “Spice”-related fatalities around Munich, Germany, from 2014 to 2020. All cases underwent an autopsy. Pharmaceutical and illicit drugs were detected and quantified in post-mortem peripheral blood or liver by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Based on circumstantial evidence, only those cases for which a prior consumption was suspected underwent additional analyses for SCRAs and other new psychoactive substances in post-mortem blood, liver or antemortem specimens. Drug concentrations, pathological findings at autopsy and case histories were considered to assess and rank the SCRAs’ involvement in each death. Concentration ranges for the individual substances in blood were defined and their distribution patterns over the investigated period were determined and correlated with their legal status and local police seizures. We identified 41 different SCRAs among 98 fatalities. 91.8% were male, at a median age of 36 years. SCRAs played a causative role in 51%, contributory role in 26%, and an insignificant role in 23% of cases. In correlation with local police seizures and legal status, 5F-ADB was the most prevalent in our cases, followed by 5F-MDMB-PICA and AB-CHMINACA. Cumyl-CBMICA and 5F-MDMB-P7AICA were among the least frequently detected SCRAs. “Spice”-related fatalities and SCRAs’ causative role have significantly decreased among our cases since the German New Psychoactive Substances Act.

Post‐translational modification and phenotype

Post‐translational modification and phenotype

Microbial degradation products of lurasidone and their significance in postmortem toxicology.

Recent research reported that lurasidone degrades in unpreserved ante-mortem human whole blood inoculated with microorganisms known to dominate postmortem blood specimens. In vitro degradation occurred at a similar rate to risperidone, known to degrade in authentic postmortem specimens until below analytical detection limits. To identify the lurasidone degradation products formed, an Agilent 6520 liquid chromatograph quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS) operating in auto-MS/MS mode was used. Numerous degradation products not previously reported in prior in vitro or in vivo pharmacokinetic studies or forced degradation studies were detected. Accurate mass data, mass fragmentation data, acetylation experiments, and a proposed mechanism of degradation analogous to risperidone supports initial identification of the major degradation product as N-debenzisothiazole-lurasidone (calculated m/z [M + H]+ = 360.2646). A standard was unavailable to conclusively confirm this identification. Retrospective data analysis of postmortem cases involving lurasidone identified the presence of the major degradation product in four of six cases where lurasidone was also detected. This finding is significant for toxicology laboratories screening for this drug in postmortem casework. The major postmortem lurasidone degradation product has consequently been added to the LC-QTOF-MS drug screen at Forensic Science SA (FSSA) to indicate postmortem lurasidone degradation in authentic postmortem blood specimens and as a marker of lurasidone administration in the event lurasidone is degraded to concentrations below detection limits.

Simultaneous Quantitation of Seven Phenethylamine-Type Drugs in Forensic Blood and Urine Samples by UHPLC-MS-MS.

Abuse of new psychoactive substances (NPS) has become a health and social issue of global concern. p-Methoxyamphetamine (PMA)/p-methoxymethamphetamine (PMMA) with fluoro- or chloro-derivatives of amphetamine and methamphetamine were among the most common drugs found in specimens from fatal cases in Taiwan during the January 2011 to December 2018 period. A liquid-liquid extraction sample preparation protocol with highly sensitive ultra-high performance liquid chromatography-tandem mass spectrometry approach was developed for the simultaneous analysis of seven phenethylamine-type drugs-PMA, PMMA, p-methoxyethylamphetamine, 4-fluoroamphetamine (4-FA), 4-fluoromethamphetamine (4-FMA), 4-chloroamphetamine (4-CA) and 4-chloromethamphetamine (4-CMA)-in postmortem blood and urine specimens. Separation by liquid chromatography was performed by Agilent Zorbax SB-Aq column. Tandem mass spectrometry was operated in Agilent Jet Stream Technology electrospray ionization in positive-ion multiple reaction monitoring mode. An analytical methodology was evaluated using drug-free blood and urine after fortification with 100-2,000 ng/mL of the seven target analytes. Average extraction recoveries were >80%; slightly higher ion suppression was observed for PMA and 4-CA; intra-/inter-day precision (% coefficient of variation) and accuracy were in the ranges of 0.52-12.3% and 85-110%, respectively. Limit of detection and lower limit of quantitation for these seven analytes were both in the 0.5-5 ng/mL range. Interference and carryover were not significant. This relatively simple methodology was found effective and reliable for routine identification and quantitation of these seven analytes in postmortem and antemortem blood and urine specimens received in 2018. Analytical data obtained from these actual cases indicated the following: (i) compared to findings reported during the 2007-2011 period, the use of substituted phenethylamine-type drugs decreased in 2018; (ii) ketamine and 7-aminonimetazepam (the main metabolite of nimetazepam) were the most common co-ingested substances in specimens containing PMA/PMMA, 4-FA/4-FMA, or 4-CA/4-CMA; and (iii) in drug fatalities, the concentration of PMA was significantly higher than the concentration of PMMA in both urine and blood, while the reverse was true in urine specimens from antemortem cases.

Method Validation of Seven Synthetic Cathinones by LC-MS/MS Analysis and the Prevalence of N-Ethylpentylone in Postmortem Casework.

N-ethylpentylone (NEP, ephylone, bk-EBDP) was the most prevalent synthetic cathinone detected by the Miami-Dade Medical Examiner Toxicology Laboratory from 2016-2018. There is limited information regarding the toxicity of NEP, however the few published reports suggest that NEP can cause serious toxic effects and sudden death. The purpose of this publication is to describe a validated LC-MS/MS method for seven synthetic cathinones (methylone, ethylone, butylone, dibutylone, α-PVP, pentylone, and NEP) and to present a detailed summary regarding the presence of NEP in postmortem casework at the Miami-Dade Medical Examiner Department. Post-mortem iliac blood, serum, liver, and brain specimens were prepared by solid-phase extraction with analysis by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). Analyte linearity was established from 0.01 to 0.5 mg/L on a six-point calibration curve. A total of 101 NEP quantitations were performed using this method. Concentrations in postmortem case samples ranged from 0.01 mg/L to 2.7 mg/L. Iliac blood concentrations averaged 0.312 mg/L with a median of 0.137 mg/L (n=72) across all causes and manners of death. Approximately half of the cases were homicides in which the decedent was the victim of gunshot wounds or stabbing. Two of the three highest concentration cases of NEP (2.7 mg/L and 1.7 mg/L) involved 38-year old white males who were tasered by police prior to death. The psychostimulant effect of NEP may result in an excited delirium and/or hallucinogenic state. The concentration of NEP detected in accidental intoxication and polydrug cases overlapped with those attributed to other causes, including homicides and police involved deaths.

RECiQ: A Rapid and Easy Method for Determining Cyanide Intoxication by Cyanide and 2-Aminothiazoline-4-carboxylic Acid Quantification in the Human Blood Using Probe Electrospray Ionization Tandem Mass Spectrometry.

In this study, we developed a rapid and easy method to determine cyanide (CN) intoxication by quantification of CN and 2-aminothiazoline-4-carboxylic acid (ATCA), which is a new and reliable indicator of CN exposure, in the human blood using probe electrospray ionization tandem mass spectrometry (PESI/MS/MS) named RECiQ. For CN, we applied the previously reported one-pot derivatization method using 2,3-naphthalenedialdehyde and taurine, which can directly derivatize CN in the blood. The analytical conditions of the CN derivatization were optimized as a 10 min reaction time at room temperature. In contrast, ATCA could be directly detected in the blood by PESI/MS/MS. We developed quantitative methods for the derivatized CN and ATCA using an internal standard method and validated them using quality control samples, demonstrating that the linearities of each calibration curve were greater than 0.995, and intra- and interday precisions and accuracies were 5.1–15 and 1.1–14%, respectively. Moreover, the lower limit of detections for CN and ATCA were 42 and 43 ng/mL, respectively. Finally, we applied RECiQ to three postmortem blood specimens obtained from victims of fire incidents, which resulted in the successful quantification of CN and ATCA in all samples. As PESI/MS/MS can be completed within 0.5 min, and the sample volume requirement of RECiQ is only 2 μL of blood, these methods are useful not only for the rapid determination of CN exposure but also for the estimation of the CN intoxication levels during an autopsy.

The role of ethanol in fatalities in Jeddah, Saudi Arabia

In Saudi Arabia, alcohol consumption is prohibited by law, but interpreting postmortem ethanol can be complicated by its postmortem production. This study developed and validated a method using headspace gas chromatography with flame ionization detection and liquid chromatography tandem mass spectroscopy to detect ethanol and its polar metabolites (ethyl glucuronide [EtG] and ethyl sulfate [EtS]) in postmortem blood and urine specimens, respectively. All calibration curves were linear with coefficients of determination greater than 0.999. The limits of detection ranged 4.5–5.0mg/dL for ethanol and 0.05–0.06mg/L for EtG and EtS. The limits of quantification were 10.0mg/dL for ethanol and 0.075mg/L for EtG and EtS. Within-run precision was less than 11% for all analytes of interest. Matrix effects for EtG and EtS ranged 3–47%. After excluding matrix effects, analytical recoveries ranged 72–100%. This validated method was then used for routine postmortem forensic toxicology analyses in 592 routine postmortem cases to distinguish between antemortem ethanol consumption and its postmortem microbial formation. Among them, 98 blood samples (17%) were positive for ethanol or its polar metabolites. Thirty-two of these cases (33%) were positive for EtG and EtS and therefore due to antemortem ethanol consumption. The remaining 66 (67%) cases were negative for both EtG and EtS and therefore due to postmortem ethanol synthesis. Because this is the first study to report the problem of alcohol consumption in Saudi Arabia, further studies are essential for validating these findings.

High Throughput Detection of 327 Drugs in Blood by LC-MS-MS with Automated Data Processing.

The described procedure provides a rapid technique for the detection and semi-quantitation of a large number of drugs in blood. This procedure uses a minimal sample volume and employs a one-step liquid extraction and automated data processing to yield rapid turnaround times. A total of 327 of the most commonly used medicinal and illicit drugs in Australia were selected including various amphetamines, anesthetics, antidepressants, antipsychotics, anticonvulsants, benzodiazepines, beta blockers, opioid and nonopioid analgesics, stimulants, THC and a large number of synthetic cannabinoids and other novel psychoactive substances. The extracts were subject to 5-minute chromatography using a Kinetex C18 50×4.6mm 2.6μm solid-core analytical column and analyzed using a Sciex 3200 Q-TRAP MS-MS (+ ESI, MRM mode, two transitions per analyte). The method was fully validated in accordance with international guidelines. Matrix effects and extraction efficiencies were acceptable with most analytes showing > 80% response and low variation (within 25%RSD). Cannabinoids were most affected by the matrix and yielded poorest recovery values but were still detectable. Precision, accuracy, repeatability and multipoint linearity were assessed for all analytes. The method has been used in routine practice in the forensic toxicology service at the Victorian Institute of Forensic Medicine in over 6000 coronial investigations using both postmortem and clinical blood specimens. This technique has greatly increased throughput, reduced turnaround times and allowed for rapid same-day analysis of results when needed. The method is routinely used in routine overnight testing with results reported to pathologists within 4h of data acquisition. This rapid toxicological technique is used in conjunction with other investigative processes such as full-body CT imaging, review of case circumstances and medical histories to provide an efficient death investigation process.

Identification of five mitragyna alkaloids in blood and tissues using liquid chromatography-quadrupole/time-of-flight mass spectrometry

Kratom is a botanical drug with psychoactive properties that is increasingly being used recreationally and “therapeutically” in a non-medically supervised setting. Analytical methods for the detection of kratom use in biological matrices are limited in scope. Prevalence of these alkaloids and their metabolites in forensic specimens is not well understood. The purpose of this study was to develop and validate a procedure to identify five Mitragyna alkaloids in blood and tissues using liquid chromatography quadrupole time–of–flight mass spectrometry (LC–Q/TOF–MS). Mitragynine (MG), speciociliatine (SC), paynantheine (PY), speciogynine (SG) and 7-hydroxymitragynine (7-MG-OH) were identified in postmortem blood (n = 40) and liver specimens (n = 20). Mitragyna alkaloids were determined quantitatively using targeted acquisition and metabolites were identified qualitatively using full scan (untargeted) acquisition. The analytical procedure was validated in accordance with published recommendations. Limits of quantitation were 0.5–2 ng/mL for the five targeted alkaloids. Precision, bias, and matrix effects were all within acceptable thresholds. Concentrations of MG in central and peripheral blood were 1–422 ng/mL and 1–412 ng/mL. Liver concentrations of MG ranged from 1450 ng/g. Metabolites of MG (7-MG-OH, 9-O-demethylmitragynine and 16-carboxymitragynine) were also identified in postmortem blood with 7-MG-OH being identified in at least 95% of cases. Interestingly, SC concentrations were frequently identified in excess of MG concentrations. A validated LC–Q/TOF–MS method for the analysis of five Mitragyna alkaloids is described. In addition, minor Mitragyna alkaloids and metabolites can serve as biomarkers of kratom use in blood and tissues.

Fast increase of postmortem fentanyl blood concentrations after transdermal application: A call to careful interpretation

BackgroundInterpretation of postmortem fentanyl concentrations after transdermal application remains a challenge. There are indications that fentanyl shows relevant postmortem redistribution. The aim of this study was to investigate the time course of these changes and to develop recommendations for toxicological case work. Material and methodBlood specimens were collected from palliative care patients who were treated with fentanyl transdermal patches. Antemortem reference samples (ethylenediaminetetraacetic acid (EDTA) and serum specimens) were collected at stable dose rates. Postmortem femoral venous blood specimens were collected at four postmortem time-points: 2hpm (hours postmortem), 6–8hpm, 11–16hpm and approximately 24hpm. Liquid chromatography tandem mass spectrometry was applied to quantify fentanyl and norfentanyl. ResultsTen patients were included in the study (8 men, 2 women). Fentanyl patches with delivery rates of 12–150μg/h were applied. Antemortem fentanyl levels in EDTA samples varied between 0.19 and 4.64μg/L. At 6 to 8hpm, blood concentrations of fentanyl were already significantly (p=0.05) higher in postmortem samples compared to the paired antemortem reference. On average, the antemortem concentration (range: 0.19–4.64μg/L) increased 3-fold within 6–8hpm (range: 0.4–14.9μg/L), and 5.5-fold within 24hpm (range: 0.39–21.88μg/L). Norfentanyl concentrations increased significantly (p=0.01) within 6–8hpm, too. In half of the patients, norfentanyl concentrations were below fentanyl concentrations, antemortem as well as postmortem. ConclusionPostmortem fentanyl concentrations increased quickly. As early as 6–8h after death, postmortem concentrations differ significantly from antemortem ones.Our results strongly indicate that postmortem blood concentrations of fentanyl after transdermal application should be interpreted carefully.

Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Assay for Quantifying Fentanyl and 22 Analogs and Metabolites in Whole Blood, Urine, and Hair.

Recently, synthetic opioid-related overdose fatalities, led by illicitly manufactured fentanyl and analogs, increased at an alarming rate, posing a global public health threat. New synthetic fentanyl analogs have been constantly emerging onto the drug marked for the last few years, to circumvent the laws and avoid analytical detection. Analytical methods need to be regularly updated to keep up with the new trends. In this study, we aimed to develop a new method for detecting the newest fentanyl analogs with a high sensitivity, in whole blood, urine, and hair. The method is intended to provide to clinical and forensic toxicologists a tool for documenting consumption. We developed a comprehensive ultra-high-performance liquid chromatography-tandem mass spectrometry method for quantifying fentanyl and 22 analogs and metabolites. Urine samples were simply diluted before injection; a liquid-liquid extraction was performed for blood testing; and a solid phase extraction was performed in hair. The chromatographic separation was short (8 min). The method was validated with a high sensitivity; limits of quantifications ranged from 2 to 6 ng/L in blood and urine, and from 11 to 21 pg/g in hair. The suitability of the method was tested with 42 postmortem blood, urine, or hair specimens from 27 fatalities in which fentanyl analogs were involved. Average blood concentrations (±SD) were 7.84 ± 7.21 and 30.0 ± 18.0 μg/L for cyclopropylfentanyl and cyclopropyl norfentanyl, respectively (n = 8), 4.08 ± 2.30 μg/L for methoxyacetylfentanyl, (n = 4), 40.2 ± 38.6 and 44.5 ± 21.1 μg/L for acetylfentanyl and acetyl norfentanyl, respectively (n = 3), 33.7 and 7.17 μg/L for fentanyl and norfentanyl, respectively (n = 1), 3.60 and 0.90 μg/L for furanylfentanyl and furanyl norfentanyl, respectively (n = 1), 0.67 μg/L for sufentanil (n = 1), and 3.13 ± 2.37 μg/L for 4-ANPP (n = 9). Average urine concentrations were 47.7 ± 39.3 and 417 ± 296 μg/L for cyclopropylfentanyl and cyclopropyl norfentanyl, respectively (n = 11), 995 ± 908 μg/L for methoxyacetylfentanyl, (n = 3), 1,874 ± 1,710 and 6,582 ± 3,252 μg/L for acetylfentanyl and acetyl norfentanyl, respectively (n = 5), 146 ± 318 and 300 ± 710 μg/L for fentanyl (n = 5) and norfentanyl (n = 6), respectively, 84.0 and 23.0 μg/L for furanylfentanyl and furanyl norfentanyl, respectively (n = 1), and 50.5 ± 50.9 μg/L for 4-ANPP (n = 10). Average hair concentrations were 2,670 ± 184 and 82.1 ± 94.7 ng/g for fentanyl and norfentanyl, respectively (n = 2), and 10.8 ± 0.57 ng/g for 4-ANPP (n = 2).

Quantification of seven novel synthetic opioids in blood using LC–MS/MS

The objective of this study was to develop, optimize, and validate a method for the simultaneous quantification of U-47700, AH-7921, U-49900, U-50488, MT-45, W-18, and W-15 in whole blood using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Blood samples (500 µL) were fortified with mixed calibrator or quality control (25 µL) and internal standard (10 µL) solutions. Analytes were isolated via a solid-phase extraction procedure. Analysis was performed using an Agilent 1290 Infinity II liquid chromatograph coupled to an Agilent 6470 triple quadrupole mass spectrometer. The method was validated in accordance with Scientific Working Group for Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology and applied to the analysis of postmortem blood specimens. Lower limits of quantification were 0.25–1 ng/mL and the upper limits of quantification were 100 ng/mL. The coefficients of determination (R2) for the calibration curves were > 0.99. Analytical bias, within-run imprecision, and between-run imprecision were within ± 15%, ≤ 16%, and ≤ 17%, respectively. All analytes were found to be stable at room temperature for 24 h, refrigerated (4 °C) for 72 h, and in the autosampler (4 °C) for 72 h. Authentic blood samples (n = 30) were analyzed using the validated method. Mean (range) U-47700 concentrations were 214 (3.2–1448) ng/mL in 15 cases. A quantification method for seven synthetic opioids (U-47700, AH-7921, U-49900, U-50488, MT-45, W-18, and W-15) in whole blood was developed, optimized, and validated in accordance with SWGTOX standard practices for method validation in forensic toxicology. This sensitive method was successfully applied to postmortem casework.

Dibutylone (bk-DMBDB): Intoxications, Quantitative Confirmations and Metabolism in Authentic Biological Specimens

The number of emerging novel stimulants modified based on beta-keto variations of amphetamine-like substances continues to rise. Dibutylone reports described in the medical and toxicological literature are limited, therefore little information is available in terms of quantitative confirmation or metabolism. During this study, authentic human specimens, including blood, urine, vitreous humor, oral fluid and liver were quantitatively and qualitatively analyzed for the presence of dibutylone and butylone, with paired case history and demographic information. Dibutylone concentrations were variable across all specimen types, specifically ranging from 10 to 1,400 ng/mL in postmortem blood specimens. The metabolic profile of dibutylone was mapped by in vitro incubation with human liver microsomes (HLM). Samples were analyzed using a SCIEX TripleTOF® 5600+ quadrupole time-of-flight mass spectrometer. Data processing was conducted using MetabolitePilot™. Authentic human specimens, including blood, urine, vitreous humor, oral fluid and liver, were utilized for in vivo verification of five HLM-generated metabolites in analytically confirmed cases of dibutylone use. Butylone was confirmed as a metabolite of dibutylone, but issues involving co-ingestion of these two novel stimulants or potential co-existence from synthesis lead to ineffectiveness as a true biomarker. Hydrogenation of the beta-ketone of dibutylone resulted in the most prominent metabolite found in human specimens, and its uniqueness to dibutylone over other stimulants leads to its classification as an appropriate biomarker for dibutylone ingestion. This is the first study to map the metabolic profile of dibutylone, including verification in authentic specimens, confirming metabolic conversion to butylone and identifying biomarkers more useful in forensic toxicological drug testing.

Detection of human immunodeficiency virus, hepatitis C virus, and hepatitis B virus in postmortem blood specimens using infectious disease assays licensed for cadaveric donor screening

Evaluation of assay performance on postmortem blood specimens (obtained after cessation of the heartbeat) presents unique scientific and regulatory challenges. In the United States, assay performance is evaluated in part by spiking postmortem specimens. Fifty-four specimens obtained from decedents known to be infected with human immunodeficiency virus (HIV), hepatitis C virus (HCV), or hepatitis B virus (HBV), including some coinfections, were tested for each virus using Food and Drug Administration (FDA)-licensed donor screening tests for nucleic acid, antibody, and antigen. For each disease, >95% of subjects who were reported to have an infection at the time of death had a positive test result on at least one of the donor screening assays for that infection. Licensed donor screening tests were positive on postmortem specimens obtained within 24hours of death from individuals dying with HIV, HCV, and/or HBV, and were able to detect presence of the virus. The use of multiple tests (including antibody and direct viral detection methods) is necessary to adequately evaluate donors.

Simultaneous Quantitation of Methamphetamine, Ketamine, Opiates and their Metabolites in Urine by SPE and LC-MS-MS.

Heroin, methamphetamine and ketamine have been the most commonly abused drugs in Taiwan. The presence of these drugs and their metabolites in postmortem specimens has been routinely monitored in our laboratory mostly by gas chromatographic-mass spectrometric methods. This study aimed to evaluate a more effective approach to simultaneously quantify these analytes (i.e., amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), morphine, codeine, 6-acetylmorphine, 6-acetylcodeine, ketamine and norketamine) in postmortem urine and blood specimens by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Samples (1 mL) were extracted via solid-phase extraction, evaporated and reconstituted in the mobile phase for injection into the LC-MS-MS system. Respective deuterated analogs of these analytes were used as internal standards. Chromatographic separation was achieved by an Agilent Zorbax SB-Aq analytical column at 50°C. Mass spectrometric analysis was performed by electrospray ionization in positive-ion dynamic multiple reaction monitoring mode with optimized collision energy for respective precursor ion selected for each analyte, and the monitoring of two transition ions. Performance characteristics were assessed using drug-free samples that were fortified with 50-1,000 ng/mL of the 10 analytes. Analytical parameters evaluated and resulting data are as follows: (i) average extraction recoveries (n= 3) were better than 80%, except for MDMA (71%) and morphine (74%); (ii) inter-day and intra-day precision ranges (%CV) were 1.59-8.80% and 0.57-3.89%, respectively; (iii) calibration linearity (r2), detection limit and quantitation limit for all analytes were >0.999, 1 and 5 ng/mL, respectively; (iv) matrix effects (ion suppression) were observed for three analytes, but were satisfactorily compensated for by the deuterated internal standards adopted in the analytical protocol. This method was successfully applied to the analysis of specimens collected from unknown death cases from various district prosecutors' offices in Taiwan, and was also found helpful to understanding whether the detected opiates were derived from heroin or legal morphine/codeine-containing medications.

Detection of Carfentanil by LC-MS-MS and Reports of Associated Fatalities in the USA.

Carfentanil is a mu (μ) opioid receptor agonist and is estimated to be ~10,000 times more potent than morphine in animal (non-human) models. It is not approved for human use and is only used to immobilize large exotic animals in veterinary medicine. In mid-2016, carfentanil emerged as a contaminant in street heroin in the USA and was central to a large number of emergency department visits and deaths. We describe an analytical method for the detection and quantification of carfentanil in whole blood specimens via a protein precipitation extraction with acetonitrile and liquid chromatography with triple quadrupole mass spectrometry. From 1 September 2016 to 1 January 2017, carfentanil was identified in 262 postmortem blood specimens. Blood concentrations ranged from 10.2 to 2,000 ng/L, with a mean concentration equal to 193 ng/L and a median concentration equal to 98.4 ng/L. We describe 13 fatalities from the Midwest region (Indiana, Kentucky, Michigan and Ohio) of the USA in which our laboratory performed comprehensive toxicology and in which carfentanil was detected and associated with cause of death. We recommend that any analytical method applied to the detection of this substance in human whole blood specimens be sufficiently sensitive to detect sub-100 ng/L concentrations and preferably utilize a 10-50 ng/L reporting limit.

Gabapentin concentrations and postmortem distribution

Gabapentin is a widely prescribed medication used primarily for the treatment of epilepsy and neuropathic pain. Gabapentin has a favorable adverse effect profile in therapeutic dosing with the most common reported effects being dizziness, fatigue, drowsiness, weight gain, and peripheral edema. Even with intentional self-poisonings, serious effects are generally rare. In this report, gabapentin analyses were performed on 30 postmortem cases that had peripheral blood, central blood and liver tissue. Overall the central to peripheral blood (C/P) ratio mean was 0.90±0.24 (mean±standard deviation), and a median of 0.97. The liver to peripheral blood (L/P) ratio mean was 0.68±0.26L/kg (mean±standard deviation), and a median of 0.65L/kg. An additional case, where both antemortem blood and postmortem peripheral blood specimens were available, revealed the same gabapentin concentration in both specimens. Taken together, the data presented suggests that gabapentin is unlikely to show postmortem redistribution.