Determination of benzodiazepines and Z-hypnotic drugs in whole blood samples by GC–MS/MS: Method development, validation and application

[Display omitted] •GC–MS/MS-based method for the quantification of benzodiazepines and Z-hypnotic drugs.•Sensitive, specific, simple and fast GC–MS/MS-based method without derivatization.•Benzodiazepines and Z-hypnotic drugs in forensic and clinical toxicology.•Comparison of LC-MS/MS and GC–MS/MS in term of precision and accuracy for toxicology. Benzodiazepines (BZDs) and Z-drugs are widely used as anxiolytics, sedative hypnotics, anticonvulsants and muscle relaxants. “Designer benzodiazepines” (DBZDs) are a new psychoactive substance class consisting of benzodiazepine derivatives that are not allowed for medical use and are known for being used recreationally. From a toxicologist standpoint, the huge number of such substances implicate a necessity for developing fast and reliable methods to identify and quantify these substances in biological matrices, especially in blood. In this study, a method based on gas chromatography–tandem mass spectrometry (GC–MS/MS) was developed and validated for the determination of 28 benzodiazepine derivatives and 3 Z- drugs. Liquid–liquid extraction requiring the use of a 0.5 mL sample of whole blood and 1 mL of ethyl acetate was applied. No derivatization was necessary to obtain sensitivity and selectivity. All validation data met the established acceptance criteria in accordance with international forensic toxicology guidelines. The method was linear within the tested range of 1–100 ng/mL, 1–200 ng/mL, 1–250 ng/mL, 5–200 ng/mL and 10–250 ng/mL depending on the analyte. The LOD was in the range of 0.09 to 0.66 ng/mL, while the LOQ was between 1 and 10 ng/mL. The calculated mean accuracy ranged between 85.1% and 114.4% for intraday replicates and between 85.2% and 111.2% for interday replicates. Intraday precision (as CVs) ranged from 0.2% to 12.5%, while interday precision ranged from 2.0% to 14.5%. Recovery was between 83.8% and 111.2%. This study was the first attempt to apply GC–MS/MS for the multianalyte determination of benzodiazepines and Z-drugs without analyte derivatization. The developed method proved to be sensitive, specific, simple and fast. The analysis in MRM mode gives high sensitivity and selectivity. Thus, the presented method can be successfully utilized for clinical and forensic toxicology analysis, for interpretation ofresults and for the preparation of forensic toxicological opinions for courts of law.