Development and validation of a LC–MS/MS method for the detection of 38 benzodiazepines and 2 Z-drugs in blood

Abstract

Benzodiazepines and Z-drugs are commonly used as prescription medications to treat anxiety, epilepsy, insomnia, and alcohol withdrawal syndrome, but their use can lead to tolerance, dependence, and withdrawal reactions if taken against official guidelines. Furthermore, designer benzodiazepines, most of which lack clinical and toxicological data, have entered the illicit drug market as new psychoactive substances and are used for recreational purposes. Their abuse can cause confusion, memory loss, respiratory depression, and even death, especially when combined with other sedative-hypnotics or alcohol. Therefore, new qualitative and quantitative methods for benzodiazepines and Z-drugs are needed for use in forensic and clinical toxicology. This study explored the sample preparation and liquid-liquid extraction using 0.5 mL blood samples and 2 mL of extraction solvent for analysis of these drugs by liquid chromatography-tandem mass spectrometry and multiple reaction monitoring. The benzodiazepines were separated on a pentafluorophenylpropyl (PFPP) column using a mobile phase gradient consisting of A (water, 0.1 % formic acid, 5 % acetonitrile, and 20 mmol/L ammonium acetate) and B (acetonitrile) for 9 min. Validation steps confirmed that the method demonstrated good selectivity, sensitivity (limit of detection: 0.2 ng/mL, lower limit of quantification: 0.5 ng/mL), linearity (R² ≥ 0.99), accuracy, and precision (<20 %). Matrix effects ranged from 35 to 126 %, and recoveries ranged from 17 to 99 %, with 35 compounds having recoveries of more than 50 %. The method was successfully applied for the identification and quantification of benzodiazepines and nonbenzodiazepines in blood samples from 15 authentic poisoning cases. Ten analytes were detected: alprazolam (130.2–575.3 ng/mL), hydroxyalprazolam (2.3–37.3 ng/mL), clonazepam (11.7–773.2 ng/mL), lorazepam (63.4–166.9 ng/mL), 7-aminoclonazepam (17.4–385.3 ng/mL), oxazepam (2.6–964.1 ng/mL), diazepam (227.2 ng/mL), nordazepam(22.4 ng/mL), zolpidem (11.8–64.0 ng/mL), midazolam (70.2 ng/mL), and hydroxymidazolam (162.8 ng/mL).