Determination of 14 benzodiazepines and hydroxy metabolites, zaleplon and zolpidem as tert-butyldimethylsilyl derivatives compared with other common silylating reagents in whole blood by gas chromatography–mass spectrometry

The most common commercially available silylating reagents, N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), N,O-bis-(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane (BSTFA + 1% TMCS) and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) were evaluated to achieve optimal derivatization conditions for analyzing various benzodiazepines based on gas chromatography–electron impact ionization–mass spectrometry (GC–EI–MS). Sensitivity, repeatability, retention times and stability of the derivatives, as well as specificity of mass fragmentation, were studied in detail. Also other parameters affecting the derivatization chemistry of benzodiazepines are discussed. tert-Butyldimethylsilyl (TBDMS) derivatives proved to be more stable, reproducible and sensitive than corresponding trimethylsilyl (TMS) derivatives for the GC–EI–MS analysis of benzodiazepines. Based on the TBDMS derivatives, a rapid, reliable, sensitive and quantitative GC–MS method was developed for the determination of 14 benzodiazepines and two hydroxy metabolites, as well as two non-benzodiazepine hypnotic agents, zolpidem and zaleplon, using 500 μl of whole blood. The method was completely validated in terms of accuracy, intra- and interday precision, limit of detection (LOD), limit of quantitation (LOQ), linearity, selectivity and extraction efficiency; these were all within the required limits, except for the accuracy of nitrazepam at a medium concentration level.