Poison hemlock determination in postmortem samples.

Poison hemlock (Conium maculatum L.) is a weed that grows rampant in many areas of North America. Forensic toxicology laboratories rarely receive requests to analyze biological specimens for the presence of poison hemlock. This report discusses two postmortem cases that were encountered over a decade apart and describes different analytical approaches that may be used to quantify coniine, a primary poison hemlock alkaloid, in biological specimens. The first case is from 2004 and involves a 27-year-old female that was found deceased in a relatively isolated area of California. Based on the presence of plant material at the scene and signs of its ingestion at autopsy, the possibility of hemlock poisoning was considered. Toxicological testing of the blood and gastric content by quantitative selected-ion monitoring Gas Chromatography/Mass Spectrometry (SIM-GC/MS) revealed the presence of coniine at concentrations of 410 ng/mL and 9300 ng/mL, respectively. The second case is from Pennsylvania and was sent for analysis in the spring of 2019. In this case, a male in his forties was found deceased in the kitchen area of a camper. Green substances, in liquid and residue forms, were observed in the sink. Mixtures of leaf-like material were also found in several bowls and pans. Subclavian blood screened positive for coniine by full-scan Gas Chromatography/Mass Spectrometry (GC/MS). Semi-quantitative confirmation testing was performed by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) and showed the presence of coniine at a concentration of 35 ng/mL. These analytical approaches can be used to substantiate or exclude poison hemlock exposure as a cause of death.

Flualprazolam Blood Concentrations in 197 Forensic Investigation Cases.

Flualprazolam is a designer benzodiazepine and novel psychoactive substance that is increasing in prevalence and appearing in forensic investigations. Flualprazolam was quantitatively confirmed in 197 blood samples from medicolegal death investigations and human performance cases reported between August 2019 and February 2020. Drug screening was performed using liquid chromatography-time-of-flight mass spectrometry and quantitative confirmation was performed using liquid chromatography-tandem mass spectrometry. A three-point standard addition protocol was implemented for quantitation in the absence of an available traditionally validated assay. In postmortem cases with quantitative results (n = 167), the mean (±standard deviation [SD]) flualprazolam concentration was 20 (±63) ng/mL, the median concentration was 8.2 ng/mL and the range of concentrations was 2.0-620 ng/mL. Four additional postmortem cases were reported positive (<2.0 ng/mL). In drug impaired driving cases (n = 22), the mean (±SD) flualprazolam concentration was 22 (±18) ng/mL, the median concentration was 14 ng/mL and the range of concentrations was 4.4 to 68 ng/mL. The four remaining cases were of unknown circumstances. This report details the most extensive dataset of flualprazolam intoxication cases reported to date. There was significant overlap in concentrations of flualprazolam between postmortem and DUID cases. Flualprazolam was commonly (83% of the time) found in combination with opioids (e.g. fentanyl). Toxicologists should consider quantitative flualprazolam results in the context of case history, observations, and/or other toxicological findings. Addition of flualprazolam to the scope of drug testing should be considered by all laboratories.

Eutylone Intoxications-An Emerging Synthetic Stimulant in Forensic Investigations.

Synthetic stimulants are the largest class of novel psychoactive substances identified each year by forensic laboratories internationally. While hundreds of these drugs appear in drug powders, only a few proliferate in use among forensically relevant populations and eventually emerge in postmortem and clinical investigations. Beta-keto-methylenedioxyamphetamines (i.e., novel psychoactive substances with names ending in "ylone") are currently the most popular subclass of synthetic stimulants. Leading up to its federal scheduling in 2018, N-ethyl pentylone was the most encountered synthetic stimulant. The popularity of N-ethyl pentylone declined once it was scheduled, but it was quickly replaced by eutylone (bk-EBDB), a structurally related analog from the same family. In cases encountered between January 2019 and April 2020, eutylone was quantitatively confirmed in 83 forensic investigations, including postmortem cases and driving under the influence of drugs cases. Matrix types included blood, urine and tissue. Eutylone was identified in cases submitted from 13 states, demonstrating proliferation around the United States; Florida accounted for 60% of the positive cases. The mean concentration of eutylone in postmortem blood was 1,020 ng/mL (standard deviation = ±2,242 ng/mL; median = 110 ng/mL, range = 1.2-11,000 ng/mL, n = 67). The mean concentration of eutylone in blood from driving under the influence of drugs cases was 942 ng/mL (standard deviation = ±1,407 ng/mL; median = 140 ng/mL, range = 17-3,600 ng/mL, n = 7). This report includes cause and manner of death data for 22 postmortem cases. Further analysis of authentic human specimens revealed the presence of three eutylone metabolites, including one unique biomarker and one metabolite in common with butylone. Laboratories should be aware that eutylone may be present in cases of suspected Ecstasy, "Molly" and/or methylenedioxymethamphetamine use, causing or contributing to impairment or death.

Quantification of Designer Opioids by Liquid Chromatography-Tandem Mass Spectrometry.

Opioids including heroin and commonly prescribed drugs such as oxycodone and fentanyl are among the most commonly abused drugs. In recent years, the abuse of opioids has spread beyond these commonly encountered analytes and now includes novel psychoactive drugs such as AH-7921 and U47700 and a variety of fentanyl-related compounds such as acetyl fentanyl and furanyl fentanyl. The assay described is for the quantitative determination of 19 designer opioids in serum, plasma, and whole blood. Also included is a discussion on the challenges of keeping an analytical method current as new analytes appear on the illicit drug market.

Rapid and sensitive screening and confirmation of thirty-four aminocarbonyl/carboxamide (NACA) and arylindole synthetic cannabinoid drugs in human whole blood.

We describe the development and validation of a method for the screening and confirmation of a range of chemically diverse synthetic cannabinoid drugs in human whole blood. The method targets the better known arylindole compounds as well as the emerging aminocarbonyl/ carboxamide (NACA) compounds. The approach consists of two separate extraction procedures designed to optimize recovery of each of these two classes, followed by analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The most significant novel compounds added were AB-FUBINACA, ADBICA, 5 F-ADBICA, ADB-PINACA, ADB-FUBINACA, ADB-FUBINACA, 5 F-ADB-PINACA, 5 F-ADB-PINACA, AB-PINACA, AB-CHMINACA, and ADB-CHMINACA. A third procedure is described for the quantitative confirmation of those compounds for which deuterated internal standards permitted quantitative analysis, including JWH-018, JWH-122, JWH-081, JWH-210, AM-2201, XLR-11, and UR-144. The methods were successfully validated according to Scientific Working Group in Forensic Toxicology (SWGTOX) protocol for 34 compounds in common use in the United States in the period of 2014 and 2015, although other substances, unknown at the time may have been introduced to the market over the same time period. The method was determined to be free from carry-over between samples, and no interference was found from other common therapeutic abused or novel psychoactive drugs. The methods were applied to the analysis of 1142 blood samples from forensic investigations, including post-mortem examinations and driving impairment cases. The drugs most frequently detected were AB-CHMINACA (18.6%), ADB-CHMINACA (15%), XLR-11 (5.5%), AB-FUBINACA (4.5%), AB-PINACA (3.9%), and ADB-FUBINACA (2.3%). Copyright © 2016 John Wiley & Sons, Ltd.

Quantitation of amphetamine-type stimulants by LC-MS/MS.

Amphetamines or amphetamine-type stimulants (ATSs) refer to a group of pharmacological and -toxicological agents that have a common phenethylamine structural backbone and typically impart effects that include, but are not limited to, vasoconstriction, anorexia, central nervous system stimulation, and/or hallucinations. While differences in side chain chemistry can impart different pharmacological or toxicological effects, for some compounds, e.g., MDMA (Ecstasy), alterations of the phenyl part of the molecule impart other significant effects. ATSs are used both therapeutically and recreationally, with significant abuse and addiction potential. Therapeutically, these agents are mainly used to treat hyperactivity disorders or aid in weight loss. Toxicological effects include hypertension, arrhythmia, excitability, aggressiveness, psychoses, coma, and death.Traditional analytical methods to analyze amphetamines include gas chromatography-mass spectrometry where derivatization is often required to facilitate analysis. Besides sample preparation issues, it has been demonstrated that injection port chemistry in the GC can lead to misleading results with some members of the amphetamine class. To circumvent these issues, liquid chromatography-mass spectrometry (LC-MS/MS) offers the promise of a simpler sample preparation procedure and fewer analytical concerns. This chapter describes an LC-MS/MS technique for the analysis of 14 ATSs in blood, serum/plasma, and urine. The method is quantitative and has reporting limits in the low ng/mL range. Electrospray ionization is used in the positive ion mode. Two transitions for each compound are monitored along with ion ratios.

Qualitative identification of rodenticide anticoagulants by LC-MS/MS.

Rodenticide anticoagulants are used in the control of rodent populations. In addition to accidental ingestions in humans, such agents have also been used for homicidal and suicidal purposes. There are two major groups of rodenticide anticoagulants - hydroxycoumarins and indanediones. Before the advent of LC-MS/MS, analysis for such agents was relegated to such techniques as TLC and HPLC with nonspecific modes of detection. LC-MS/MS has been used to determine any given number of rodenticide anticoagulants in animal tissues, foods, plasma, etc. Use of this technique allows for the simultaneous identification of individual compounds within both classes of rodenticide anticoagulants. The LC-MS/MS method presented allows for simultaneous qualitative identification of brodifacoum, bromadiolone, chlorphacinone, dicumarol, difenacoum, diphacinone, and warfarin in blood, serum, and plasma using ESI in the negative mode. Two transitions are monitored for each analyte after a simple sample preparation. Chromatographic separation is accomplished using a gradient of ammonium hydroxide in water and ammonium hydroxide in methanol. Chloro-warfarin is used as internal standard.

LC-MS/MS method for the detection of common laxatives.

Laxatives refer to a group of diverse substances used to induce bowel movements. There exist various classes of laxatives, which work through different pharmacological means. Based on the potential medical cause of use, one particular class of laxative may be preferred over another. Additionally, abuse of laxatives in both adults and children occurs. Some of the signs and symptoms of excessive laxative use/abuse can not only mimic various pathological conditions, but cause such conditions. Based on the potential abuse of laxatives, as well as for compliance purposes, a test to identify the use of common laxatives is of significant value. While stool and stool water can be used for such analyses, isolation and identification of analytes can be difficult due to matrix constituents and potential interferences. Ideally, a sensitive urine test for detection of laxative use/abuse with specific detection would be preferable. Described is an LC-MS/MS procedure for the detection of four metabolites related to common laxatives-desacetylbisacodyl, aloe-emodin, emodin, and rhein. Deuterated internal standards for desacetylbisacodyl and emodin are employed while an analog internal standard, biochanin A is used for rhein and aloe-emodin. Sample preparation consists of deconjugation of analytes in urine followed by a simple organic solvent extraction. Analysis is carried out using a pentafluorophenyl column employing a gradient mobile phase of formic acid in water/methanol. Mass spectral ionization conditions employ both positive and negative ESI. Two transitions are monitored for each analyte of interest.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the identification and quantification of JWH-018, JWH-073, JWH-019, and JWH-250 in human whole blood.

A sensitive and specific method for the quantification of JWH-018, JWH-073, and JWH-250 and the qualitative identification of JWH-019 in whole blood was developed and validated. Samples fortified with JWH-018-d9 and JWH-073-d9 underwent liquid-liquid extraction and were analyzed by liquid chromatography-positive ion electrospray ionization-tandem mass spectrometry. Two transitions were monitored for all analytes except JWH-250, for which there was only one available transition. JWH-019 did not meet the stringent requirements for quantitative analysis, and thus this method is only appropriate for the qualitative identification of this compound in whole blood. The linear range was 0.1-20 µg/L for all quantitative analytes. The maximum average within and between-run imprecision was 7.9% and 10.2%, respectively, and all controls quantified within 8.2% of target concentrations. Process efficiency, a measurement that takes into effect extraction efficiency and matrix effect, was ≥ 32.0% for all quantitative analytes; similar results were obtained for the deuterated internal standards. All analytes were stable at room, refrigerated, and frozen temperatures for at least 30 days. The method was used to quantify JWH-018 and JWH-073 in a blood specimen collected from a person known to have used an herbal incense blend containing these substances.

Inhibition of morphine-potentiated HIV-1 replication in peripheral blood mononuclear cells with the nuclease-resistant 2-5A agonist analog, 2-5A(N6B).

Opioids potentiate HIV-1 infection in vitro at least partly by suppressing immunoresponsive processes in human lymphocytes and monocytes. For example, it appears that morphine inhibits the interferon (IFN)-alpha, -beta, and -gamma-mediated natural antiviral defense pathways in human peripheral blood mononuclear cells (PBMC). In this study, we show that restoration of a key component of the antiviral pathway reverses morphine-potentiated HIV-1 infection of human PBMC. The data show that HIV-1 replication is potentiated and RNase L activity is inhibited after morphine administration. Because HIV-1 inhibits the antiviral pathway at the level of 2',5'-oligoadenylate (2-5A) synthetase and p68 kinase, antiviral enzymes that require double-stranded RNA, we overcame this blockade by the addition of the nuclease-resistant, nontoxic 2-5A agonist, 2-5A(N6B), to PBMC in culture. Addition of 2-5A(N6B), but not zidovudine or saquinavir, to morphine-treated PBMC completely reversed the morphine-induced potentiation of HIV-1 infection. Further, 2-5A(N6B) significantly enhanced expression of both IFN-alpha and IFN-gamma. Also, increased expression of IFN-gamma was associated with a significant increase in expression of RANTES and monocyte chemotactic protein (MCP)-1, chemokines that may inhibit HIV-1 infection by blocking viral attachment to CCR2 and CCR5 co-receptors. Our results suggest that reactivation of the antiviral pathway by 2-5A agonists may be useful to inhibit opioid-potentiated HIV-1 replication.