Authentic Human Specimens Research Articles

Quantification of Major Metabolites of AB-FUBINACA in Solid Tissues Obtained from an Abuser.

AB-FUBINACA M3 was reported to be a major metabolite of the drug, but its in vivo concentration in authentic human solid tissues has not been quantified yet. Another metabolite AB-FUBINACA M4 did not receive much attention previously and also has not been quantified yet in any authentic human specimens. The aims of this study are to establish a sensitive method for quantification of M3 and M4 in solid tissues and to compare the metabolite profile of AB-FUBINACA in authentic human specimens in vivo with that produced by human hepatocytes in vitro. The quantification was performed by liquid chromatography (LC)-quadrupole-ion trap-tandem mass spectrometry (MS-MS), and the characterization by LC-quadrupole Orbitrap MS-MS The limits of quantification of M3 were 10 pg/mL and 60 pg/g, and those of M4 were 100 pg/mL and 600 pg/g in urine and tissues, respectively. In the present work, M3 and M4 were identified and quantified in human lung, liver and kidney obtained from a cadaver for the first time; the concentrations of M3 were 226, 255, 202 and 155 pg/mL or g, and those of M4 14,400, 768, 637 and 1,390 pg/mL or g in urine, lung, liver and kidney, respectively. The peak intensity profiles of seven metabolites in these specimens were compared with that produced by human hepatocytes; the top three metabolites in urine specimen were completely different from those of hepatocytes. M3 was reported as the predominant metabolite in several previous works and M4 was listed as a minor metabolite in only one work, but, in this work, M4 has been found to be the major metabolite in all of the authentic urine, lung, liver and kidney specimens. The M3 plus M4 metabolites in lung or kidney were found most recommendable to prove AB-FUBINACA consumption, when urine specimen is lacking.

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.

Sensitive quantification of 5F-NNEI and characterization of its several metabolites in authentic urine and/or serum specimens obtained from three individuals by LC–QTRAP-MS/MS and high-resolution LC–Orbitrap-MS/MS

A synthetic cannabinoid 5F-NNEI and its metabolites in authentic human specimens have not been reported yet. The aim of this study is, firstly, to establish a sensitive quantification method of 5F-NNEI in human serum and urine specimens, and, secondly, the characterization of its metabolites in authentic urine specimens obtained from three individuals. These compounds were extracted from β-glucuronide-hydrolyzed and unhydrolyzed urine and/or serum specimens via liquid-liquid extraction. The identification and quantification were performed using liquid chromatography (LC)–QTRAP tandem mass spectrometry (MS/MS), and the characterization of the metabolites was done using LC–high-resolution-MS/MS. The limits of detection for unchanged 5F-NNEI were 6 pg/mL with the quantification range of 10–2000 pg/mL in serum, and 3 pg/mL with the quantification range of 5–1000 pg/mL in urine. The devised method was applied to quantify 5F-NNEI in authentic serum and/or urine specimens from three individuals. The levels of 5F-NNEI were 11.6 and 3680 pg/mL in serum specimens of cases 1 and 2, respectively, and were 5.07 and 7.90 pg/mL in urine specimens of cases 2 and 3, respectively. Several metabolites of 5F-NNEI produced by amide hydrolysis, defluorination, hydroxylation, carboxylation and/or glucuronization were tentatively identified in the urine specimens by the LC–high-resolution-MS/MS. A synthetic cannabinoid, 5F-NNEI, was identified and quantified in its unchanged form, and its several metabolites were tentatively identified in authentic specimens obtained from three individuals, for the first time to our knowledge.

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.