Novel and Nonroutine Benzodiazepines and Suvorexant by LC–MS-MS

Abstract

Benzodiazepines are a commonly prescribed class of drugs that have the potential for abuse. The Palm Beach County Sheriff’s Office received drug seizure submissions that included novel and/or non-routine benzodiazepines of increasing prevalence from 2017 to 2019. This prompted the development of a method of analysis for these compounds in biological specimens. The method tests for 16 novel and non-routine benzodiazepines and suvorexant in whole blood by liquid chromatography–tandem mass spectrometry (LC–MS-MS). The target analytes included bromazepam, clobazam, clonazolam, clotiazepam, diclazepam, estazolam, etizolam, flualprazolam, flubromazepam, flubromazolam, loprazolam, lormetazepam, phenazepam, prazepam, suvorexant, tetrazepam, and triazolam. The method uses 200 µL of sample, protein precipitation, and an instrument run-time of 8 min. The limit of detection was either 1 or 5 ng/mL and the limit of quantitation was either 5 or 25 ng/mL depending on the analyte. The method was validated for quantitative analysis for 15 out of the 17 analytes. Flubromazepam and prazepam were validated for qualitative identification only. A quadratic calibration model (r2 > 0.990) with 1/x weighting was used for all analytes for quantitative analysis. The calibration range was either 5-100 or 25–500 ng/mL depending on the analyte. The coefficient of variation of replicate analyses was within 14% and bias was within ± 14%. The method provides a sensitive, efficient, and robust procedure for the quantitation and/or qualitative identification of select novel and non-routine benzodiazepines and suvorexant using LC–MS-MS and a sample volume of 200 µL.

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

“…The results indicated that flualprazolam contributes to impairment, as in a mono-substance case, the concentration found was 15 ng/mL and the person's driving ability was described as 'considerably impaired'. In two other DUID cases reported by Garcia et al [41], flualprazolam was detected at concentrations of 5.4 and 45 ng/mL, respectively. Cannabis and/or ethanol were also consumed by the drivers.…”

“…Many of NPS benzodiazepines give positive results to commercial urine immunoassays [42]. Flualprazolam has been shown to cross-react with commercially available urine benzodiazepine immunoassay kits, such as immunochromatographic tests (cutoff 150 ng/mL of nordiazepam) [11] or immunochemical assays (enzyme-linked immunosorbent assay, ELISA) (cutoff 10 ng/mL of clonazepam) [41]. The cross-reactivity of flualprazolam to benzodiazepine-specific lateral flow immunoassay test strips that were designed to detect alprazolam in drug samples has also been evaluated.…”

“…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]. When urine samples were analyzed, their incubation with β-glucuronidase enzyme preceded extraction [11,16].…”

“…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.…”