Application of a screening method for fentanyl and its analogues using UHPLC-QTOF-MS with data-independent acquisition (DIA) in MSE mode and retrospective analysis of authentic forensic blood samples.

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.

In vitro studies on flubromazolam metabolism and detection of its metabolites in authentic forensic samples.

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 (CLH ) 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 CLH 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.