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

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 nonroutine 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 nonroutine 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 nonroutine benzodiazepines and suvorexant using LC-MS-MS and a sample volume of 200 µL.