A quantitative LC-MS/MS analysis of Xylazine, p-Fluorofentanyl, Fentanyl and Fentanyl-Related compounds in postmortem blood.

The purpose of this study was to develop and validate a method to quantitate the veterinary sedative xylazine as well as 4-anilino-N-phenethylpiperidine (4-ANPP), acetyl fentanyl, fentanyl, norfentanyl, and p-fluorofentanyl in blood utilizing liquid chromatography tandem mass spectrometry. This method also qualitatively monitors for the presence of o-fluorofentanyl and m-fluorofentanyl isomers. UCT Clean Screen® DAU extraction columns were utilized to isolate the analytes in postmortem blood samples. The extracts were eluted, evaporated, reconstituted, and then analyzed using a Waters Acquity™ UPLC coupled a triple quadrupole mass spectrometer. The lower limit of quantitation was determined to be 0.1 ng/mL for all analytes, except for xylazine (0.2 ng/mL). The upper limit of quantitation for all analytes was 100 ng/mL. No interferences from matrix, internal standard, or common drug analytes were observed. Bias (-13.1-4.6 %) and precision (-13.14-10.3 %) fell within the acceptable ± 20 % criteria range. Dilution integrity at x2, x10, and x100 was evaluated and all results were within ± 20 % of the target concentration. Processed extract stability was evaluated after 72 h and all results were within ± 20 % of the analyte initial concentration. Matrix effects were the most prominent with xylazine, but deemed acceptable as the deuterated internal standard also observed comparable enhancement. Analysis of 89 postmortem blood samples by this method resulted in positive results for fentanyl (0.27-66 ng/mL, n = 82), xylazine (0.24-958 ng/mL, n = 21), 4-ANPP (0.10-38 ng/mL, n = 72), acetyl fentanyl (0.18-1.5 ng/mL, n = 3), p-fluorofentanyl (0.11-33 ng/mL, n = 30), and norfentanyl (0.10-98 ng/mL, n = 73).

Presumptive identification of nitrite by Griess reagent test strips-Case reports of fatal poisoning with sodium nitrite.

The intentional ingestion of sodium nitrite causes toxicity by inducing methemoglobinemia, which can lead to cyanosis, hypotension and death. The number of reported suicide cases has significantly increased in the past 10 years as sodium nitrite is readily available online. The traditional tests for nitrite and nitrate require specialized detection methods which are not typically available in a postmortem toxicology laboratory. This rise in sodium nitrite overdose cases indicates the need for a simple, quick test for suspected nitrite toxicity. In this study, a common Griess reagent color test (MQuant™ Nitrite Test Strips) was used as a presumptive method in cases where the ingestion of sodium nitrite was suspected. The test results were consistent between specimens in all cases, and vitreous humor was identified as a reliable matrix to be used in the cases of suspected sodium nitrite poisonings. Case reports of five patients who died of suicide by sodium nitrite in a 6-month span are presented.

Fatal intoxication involving 2-methyl AP-237.

Novel synthetic opioids contribute considerably to the opioid epidemic, especially with the frequent emergence of structurally similar compounds. This case report describes a fatal intoxication involving 2-methyl AP-237. A 54-year-old Caucasian male was found deceased from an apparent drug overdose. A plastic container labeled "2MAP" and a cut straw were found in the decedent's backpack at the scene. A white substance found in the container tested positive for fentanyl by field testing. According to his medical history, the decedent was treated for a drug overdose 3 years prior to his death. With no diagnostic findings at autopsy, the case was submitted for toxicological analysis. An unknown substance was detected in peripheral blood and urine using gas chromatography with nitrogen phosphorous detection (GC-NPD). Further testing was conducted using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) which confirmed the presence of 2-methyl AP-237 and potential metabolites in blood and urine. Quantitation by GC-NPD revealed concentrations of 2-methyl AP-237 in blood and urine at 480 ng/mL and 4200 ng/mL, respectively. The toxicological analysis also identified and quantitated alprazolam in the blood at 55 ng/mL. Additionally, the metabolism of 2-methyl AP-237 was investigated and three hydroxylated metabolites were identified in peripheral blood and urine. Limited literature is available for the detection and quantitation of 2-methyl AP-237 in postmortem specimens. Given the toxicological findings with unremarkable autopsy findings, this case is an example of a fatal intoxication involving 2-methyl AP-237.

Identification of 11-nor-∆8-Tetrahydrocannabinol-9-Carboxylic Acid in Postmortem Urine.

Laws concerning the growing, selling and consuming of cannabis and its related products have been changing considerably over the last few years. The legalization of hemp in 2018 sparked an interest in ∆9-tetrahydrocannabinol (∆9-THC) isomers and analogs that are derived from hemp and sold with minimal oversight. One example is ∆8-tetrahydrocannabinol (∆8-THC). Although less potent than ∆9-THC, ∆8-THC is gaining popularity and can easily be found where cannabis-related products are sold. The Forensic Toxicology Laboratory at the University of Florida routinely tested decedents for 11-nor-∆9-tetrahydrocannabinol-9-carboxylic acid (∆9-THC-acid), the primary metabolite of ∆9-THC. Urine samples from ∼900 decedents were received by the laboratory between mid-November 2021 and mid-March 2022 and subjected to CEDIA™ immunoassay testing. Subsequent confirmation of 194 presumptive positive samples was performed by gas chromatography--mass spectrometry. A peak eluting immediately after ∆9-THC-acid was identified as 11-nor-∆8-tetrahydrocannabinol-9-carboxylic acid (∆8-THC-acid), a metabolite of ∆8-THC, in 26 of those samples (13%). Six of the samples were positive for ∆8-THC-acid only. Other toxicological findings were consistent with poly-drug use including fentanyl/fentanyl analogs, ethanol, cocaine and methamphetamine. There has been an emergence of ∆8-THC use as indicated by the presence of ∆8-THC-acid in 26 of 194 presumptive positive cases during a four-month period. The majority of individuals were White males with a history of drug and/or alcohol use. ∆9-THC-acid, as well as other drugs, was often present. Given the psychoactive potential and availability of ∆8-THC, monitoring ∆8-THC-acid in decedents is important to characterize the risk and prevalence of ∆8-THC use.

Toxicological Analysis of Fluorofentanyl Isomers in Postmortem Blood.

The opioid epidemic continues to evolve in the USA, with fentanyl the most prevalent synthetic opioid in fatal drug overdoses. Following the scheduling of fentanyl's core structure in 2018, there was a notable decline in the prevalence of fentanyl analogs in decedents; however, fluorofentanyl began being reported in casework in the winter of 2020. Fluorofentanyl has three positional isomers (para-fluorofentanyl (p-FF), ortho-fluorofentanyl (o-FF) and meta-fluorofentanyl (m-FF)), with the most predominant isomer that has recently emerged in the USA being p-FF. The goal of this study was to identify p-FF in postmortem cases between October 2020 and April 2021. Urine and blood were extracted using UCT Clean Screen® extraction columns and then screened using an Agilent 1290 Infinity liquid chromatograph (LC) coupled to an Agilent 6545 accurate mass time-of-flight mass spectrometer (TOF-MS) and quantified using an Agilent 6890N GC system coupled with an Agilent 5973 MS. The limit of quantitation (LOQ) for fentanyl, acetyl fentanyl, butyryl fentanyl, p-FF, o-FF and m-FF was 2.5 ng/mL. The screening method could not differentiate the three positional isomers of fluorofentanyl. Suspected overdose cases (n = 370) received from October 2020 through April 2021 from four Medical Examiner Districts in the state of Florida were analyzed for the presence of fluorofentanyl. The LC-QTOF-MS screen yielded 27 decedents positive for fluorofentanyl, with a majority being Caucasian (93%) and male (70%) with ages ranging from 27 to 63 years. Analysis of the blood and urine by GC-MS yielded 14 decedents positive for p-FF, nine of which were positive in the blood. The blood concentrations (n = 9) for p-FF ranged from

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.

Ethylone-Related Deaths: Toxicological Findings.

Synthetic cathinones are an emerging class of designer drugs, frequently with deceptive labels and a multitude of analogs to circumvent drug control regulations. Research regarding the pharmacological effects and toxicity of these amphetamine derivatives is scarce, heightening the risk to the public health and safety. The composition of synthetic cathinone products continually changes and laboratories began to notice ethylone-positive products in late 2011. This report presents nine postmortem cases in whom ethylone was identified. Ethylone was isolated using solid-phase extraction and detected by gas chromatography-mass spectrometry. Seven of the cases had measurable concentrations of ethylone in blood, ranging from 38 to 2,572 ng/mL; ethylone was detected in the blood sample of one case with a concentration below the assay limit of quantification (25 ng/mL), and one case did not have detectable ethylone in blood. Besides ethylone, all but one case were also positive for 11-nor-9-carboxy-Δ9-tetrahydrocannabinol; seven cases had other drugs quantified in blood, including ethanol, alprazolam, benzoylecgonine, diphenhydramine, morphine and tramadol. In five cases where ethylone was present at blood concentrations >400 ng/mL, no other drugs excluding ethanol, cannabis metabolite and doxylamine (one case) were found. The assay also tested for mephedrone, methylone and three dimethoxyamphetamine analogs; no case was positive for these analytes. The present report documents postmortem blood concentrations of ethylone, a novel synthetic cathinone, along with other concurrently identified substances. The findings provide valuable information for developing analytical assays and evaluating a toxic concentration range of ethylone.