Synthetic cannabinoids (SCs) are the most chemically diverse group of new psychoactive substances. This group has been associated with several intoxications, many with fatal outcomes. Although advancements have been achieved in pharmacology, metabolism, and detection of these compounds in recent years, these aspects are still unresolved for many SCs. The aim of this study was to investigate the in vitro potency of 14 indole-and indazole-based SCs by applying a stable CB1 or CB2 receptor activation assay and correlating the activity with their structure. The half-maximal effective concentration (EC 50 ) of 5-chloropentyl, 5-bromopentyl, and 5-iodopentyl JWH-122 analogs varied from 74.1 to 283.7 nM for CB1 and 7.05 to 23.4 nM for CB2, where the addition of a chlorine atom enhanced the potency at CB1 compared with the bromo and iodo analogs. AM-2201 was the most active at CB1 within this naphthoylindole family, with an EC 50 of 23.5 nM but with the lowest efficacy (E max 98.8%). Within the indole-3-carboxamide derivatives, 5F-MDMB-PICA was the most active compound, with a CB1/CB2 EC 50 of 3.26/0.87 nM and an E max around three times higher than JWH-018. ADB-FUBINACA was the most potent tested SC overall, with a CB1/CB2 EC 50 of 0.69/0.59 nM, and an E max around 3-fold higher than that for JWH-018 at CB1. The data obtained in this study confirm how small differences in the structure of SCs might lead to large differences in their activity, especially at CB1, which may be correlated with differences in their toxic effects in humans.

Synthetic cannabinoids (SCs) represented 45% of new psychoactive substances seizures in Europe (data from 2016). The consumption of SCs is an issue of concern due to their still unknown toxicity and effects on human health, the great variety of compounds synthetized, and the continuous modifications being made to their chemical structure to avoid regulatory issues. These compounds are extensively metabolized in the organism and often cannot be detected as the intact molecule in human urine. The monitoring of SCs in forensic samples must be performed by the analysis of their metabolites. In this work, a workflow for the comprehensive study of SC consumption is proposed and applied to 5F‐APP‐PICA (also known as PX 1 or SRF‐30) and AMB‐FUBINACA (also known as FUB‐AMB or MMB‐FUBINACA), based not only on the elucidation of their metabolites but also including functional data using the NanoLuc approach, previously published. Both cannabinoids were completely metabolized by human hepatocytes (12 and 8 metabolites were elucidated by high resolution mass spectrometry for 5F‐APP‐PICA and AMB‐FUBINACA, respectively) and therefore suitable consumption markers are proposed. The bioassays revealed that 5F‐APP‐PICA presented lower activity than AMB‐FUBINACA at CB1 and CB2 receptors, based on the half maximal effective concentration (EC50) and the maximum response (Emax). These results are in agreement with the different intoxication cases found in the literature for AMB‐FUBINACA.

The steady appearance of new fentanyl analogues and the associated overdose deaths require the development of sensitive screening approaches to detect these compounds in biological samples and seizures. We developed a targeted screening method to detect 50 4‐anilidopiperidine‐related fentanyl analogues in whole blood using ultra‐high performance liquid chromatography quadrupole time‐of‐flight mass spectrometry in data‐independent acquisition mode. Sample preparation was performed using protein precipitation on a fully automated robotic setup. Thirteen analogues were selected to validate the method. A small matrix ion enhancement effect (110–123%) was observed for all of the compounds; the recovery ranged from 67% to 81% and the process efficiency from 81% to 98%. Limit of detection was within 0.0005–0.001 mg/kg and limit of identification ranged from 0.001 to 0.005 mg/kg. In the retrospective analysis of 2339 forensic blood samples, the major finding was fentanyl (n = 56), followed by alfentanil (n = 5) and remifentanil (n = 1). Identification of 34 fentanyl analogues was based on the predicted product ions resulting from common fentanyl‐specific collision‐induced cleavages, particularly on the product ion result of the fragmentation on the C‐N bond between the phenylamide moiety and the piperidine ring. The proposed hypothesis was supported by the targeted analysis of 16 fentanyl analogues using this method and available published mass spectral data sources for fentanyl analogues. A targeted screening method for 50 fentanyl analogues was successfully validated and implemented to analyse authentic blood samples, where identifying targeted fentanyl analogues was tentatively achieved without using reference standards.

Flubromazolam is a triazole benzodiazepine with high potency and long-lasting central nervous system depressant effects; however, limited data about its pharmacokinetics are available. Here, we report in vitro studies of the human flubromazolam metabolism analyzed by liquid chromatography high-resolution mass spectrometry (LC-HRMS). In vitro investigations were carried out in pooled human liver microsomes (pHLM) and recombinant cytochrome P450 (CYP)-enzymes. To confirm those metabolites detected in vitro, authentic samples obtained from two forensic cases were also analyzed by LC-HRMS. Additionally, determination of the unbound fraction of flubromazolam in pHLM and in plasma was performed by equilibrium dialysis with subsequent prediction of its hepatic clearance (CL ) using well-stirred and parallel-tube models. Additional findings obtained by routine screening methods of these forensic cases are also reported. Studies using incubations with nicotinamide adenine dinucleotide phosphate-fortified pHLM with or without uridine 5'-diphosphoglucuronic acid and incubations with CYP-enzymes identified the main metabolic pathway of flubromazolam as hydroxylation on the α- and/or 4-position mediated by CYP3A4 and CYP3A5, with subsequent glucuronidation of the hydroxylated metabolites as well as of the parent drug. Further, α-hydroxy-flubromazolam and its corresponding glucuronide were detected in vivo together with the N-glucuronide of flubromazolam. The predicted CL of flubromazolam using the well-stirred and parallel-tube models were 0.42 and 0.43 mL/min/kg, respectively. Based on the data presented here, flubromazolam is primarily metabolized by CYP3A4/5 with a high protein-binding and a predicted low clearance. Analysis of authentic samples suggested that analytical targets for flubromazolam should be the compound itself and α-hydroxy-flubromazolam. Copyright © 2016 John Wiley & Sons, Ltd.

New synthetic opioids continue to appear as novel psychoactive substances (NPS) on illicit drug markets. Isotonitazene emerged in mid‐2019, becoming the most prevalent NPS opioid in the United States within a few months. Notification by the Drug Enforcement Administration of its intent to schedule isotonitazene in mid‐2020 led to its decline in popularity and replacement with a new NPS opioid: brorphine. Brorphine is a potent synthetic opioid, but little information was previously available regarding its toxicity or involvement in impairment and death. Our laboratory developed an assay for the identification and quantitative confirmation of brorphine using standard addition. Quantitative analysis was performed using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). In vitro and in vivo metabolism studies were performed using pooled human liver microsomes and authentic biological specimens, respectively, with analysis by liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC‐QTOF‐MS). Brorphine was confirmed in 20 authentic forensic cases, commonly found in combination with fentanyl (100%) and flualprazolam (80%). The average concentration of brorphine in blood was 2.5 ± 3.1 ng/mL (median: 1.1 ng/mL, range: 0.1–10 ng/mL). The average concentration of brorphine in urine was 4.6 ± 7.6 ng/mL (median: 1.6 ng/mL, range: 0.2–23 ng/mL). The majority of cases originated from Midwestern states. Metabolism was verified to included N‐dealkylation and hydroxylation. Detailed case histories and autopsy findings are presented herein. The prevalence of brorphine continues to increase in the United States. Forensic scientists should remain aware of the ongoing emergence of new opioids, especially those outside a standard scope of toxicology testing.