Development and Validation of an HPLC-MS/MS Method for the Determination of Benzodiazepines in Hair. Comparison of...

Benzodiazepines are widely prescribed psychoactive drugs frequently encountered in forensic and clinical toxicology. Hair analysis is a useful tool for retrospective drug monitoring, but its effectiveness depends on highly sensitive and reliable analytical methods due to the complexity of the matrix and the typically low concentrations of analytes, especially after single-dose exposure. This study aimed to develop, validate, and compare two LC-MS/MS methods for the simultaneous identification and quantification of 38 benzodiazepines and their metabolites in 25 mg of hair. Both methods used identical procedures for sampling, washing, and instrumental analysis, differing only in the extraction step. In the first method, 500 μL of M3 solution was added to cut hair and incubated at 100°C for 60 min. In the second, methanol was added to pulverized hair and incubated overnight at 50°C. Extracts were evaporated, reconstituted in 500 μL of M3, and 3 μL injected into the LC-MS/MS system. Validation with spiked hair and quality control samples showed limits of quantification ranging from 2 to 46 pg/mg, with linear calibration curves up to 200 pg/mg. Imprecision was ≤ 20% (n = 9), and most analytes showed process efficiencies above 70%. Application to 72 real samples demonstrated that the M3 extraction provided lower detection limits than the methanol method, making it more suitable for identifying single-dose exposure. Both methods are suitable for benzodiazepine screening in forensic toxicology, particularly when sample amounts are limited.