Validation of an LC-MS/MS Method for the Quantification of 13 Designer Benzodiazepines in Blood

Abstract

The misuse of designer benzodiazepines, as an alternative to prescription benzodiazepines and for drug-facilitated sexual assaults, has emerged as a growing threat, due in part to the ease of purchasing these drugs on the internet at low prices. Causing concern for safety is the lack of dosage information resulting in users self-medicating, often leading to unintended overdoses, coma or death at higher doses. With limited published data regarding the quantification of designer benzodiazepines in forensic cases, a method was validated for the determination of 13 designer benzodiazepines in postmortem blood, to add to the in-house method that already included a limited number of common designer benzodiazepines. The developed method included 3-hydroxyphenazepam, clobazam, clonazolam, delorazepam, deschloroetizolam, diclazepam, flualprazolam, flubromazepam, flubromazolam, flunitrazolam, meclonazepam, nifoxipam and pyrazolam in 0.5 mL postmortem blood using liquid chromatography-tandem mass spectrometry. The analytes were treated with solid phase extraction before undergoing separation on a C18 column and analyzed on the mass spectrometer in electrospray positive mode using multiple reaction monitoring. The linear range of the calibration curve was 1–200 ng/mL and up to 500 ng/mL for 3-hydroxyphenazepam, clobazam, flubromazepam and pyrazolam. The limits of detection and quantitation were 0.5 ng/mL (signal-to-noise ratio >3) and 1 ng/mL, respectively. The calculated bias, intra-day imprecision, relative standard deviation (RSD) and inter-day imprecision RSD were ±12%, 3–20% and 4–21%. Matrix effects ranged from −52% to 33% with RSD values ranging from 3–20%, indicating consistent effects throughout multiple sources. Recovery ranged from 35 to 90%, where only two compounds were <50%. Other parameters tested included carryover, stability, interference and dilution integrity, which all yielded acceptable results. With the application of this method to blood specimens from the New York City Office of Chief Medical Examiner, this validated method proved to be simple, reproducible, sensitive and robust.

A fluorine turns a medicinal benzodiazepine into NPS: the case of flualprazolam

“…Both medicinal and illegal drugs were also detected in blood samples, with buprenorphine being the most common finding, followed by alcohol, pregabalin and gabapentin, or psychoactive substances such as amphetamine and morphine [28]. A single femoral blood concentration of 1.94 ng/mL has also been reported by Mei et al [40], when a newly developed method including flualprazolam was applied for the investigation of forensic cases. In nine flualprazolam-related cases that were investigated in UK between April and December 2019, flualprazolam found was considered to be a contributing factor and not the compound responsible for these deaths, as numerous other drugs, such as heroin metabolites, cocaine and other NPS, were detected [3].…”

“…Flualprazolam has been identified in numerous postmortem blood samples collected from drug addicts [3,21,28,40]. The analysis of 171 samples found positive for flualprazolam in USA was conducted by Papsun et al [21], between August 2019 and February 2020.…”

“…The isolation of flualprazolam from blood or urine samples has been performed by liquid-liquid extraction with butyl acetate [28], a mixture of methyl tert-butyl ether/1-chlorobutane (60:40, v/v) [21], ethyl acetate/heptane (4:1, v/v) or with methyl tert-butyl ether [29] after alkalinization. For the solid-phase extraction (SPE) of flualprazolam, mixed-mode (strong cation exchange and hydrophobic) [16] and Preconditioned Clean Screen XCEL I columns [40] have been used. Protein precipitation with acetonitrile or different acetonitrile/methanol mixtures has been used in some methods, prior to SPE [16] or before the injection of sample into the liquid chromatography (LC) system [11,41].…”

“…Most of the published methods use LC coupled to tandem mass spectrometry (MS/MS) [3,16,21,29,40,41] or quadrupole time-of-flight (QTOF)-MS [11]. Both techniques have also been used for the screening of blood or urine samples [3,21,29].…”

“…Both techniques have also been used for the screening of blood or urine samples [3,21,29]. For the chromatographic separation, C18 [3,11,21,40], biphenyl [41] and phenyl-hexyl (Ph6) [16,29] columns have been used.…”