HPTLC–MS method for the determination of benzodiazepines in urine samples

Misuse and abuse of prescription drugs has become a global health problem in the last few years as the illicit drug cartels have grown exponentially. The non-medical use of prescribed medications, such as benzodiazepines, synthetic opioids, stimulants and tranquilizers, has increased rapidly due to their easy availability. Nowadays benzodiazepines are not only misused/abused, but also play a significant part in facilitating various crimes such as robbery, date rapes, sexual harassment, which has made its forensic examination important and critical. Considering the gravity of these crimes, an attempt has been made to develop and validate simultaneous quantification method for seven benzodiazepines (clonazepam, lorazepam, alprazolam, diazepam, flurazepam, oxazepam and nitrazepam) using high-performance thin-layer chromatography–mass spectrometry (HPTLC–MS) from urine. Quantitative analysis of clonazepam, lorazepam, alprazolam, diazepam, flurazepam, oxazepam and nitrazepam was carried out using HPTLC–MS. MS operating in selective ion-monitoring mode was attempted for the accurate identification of the drugs under study. Small-volume liquid extraction technique using diethyl ether–chloroform (80:20, v/v) was used for the extraction of urine. Pre-coated HPTLC (silica gel G 60 F254) plates were developed using the mobile-phase chloroform–glacial acetic acid (9:1, v/v). The screening of HPTLC plates was carried out by ultraviolet light, and the m/z ratio of drugs was obtained by extracting spots from plates using a TLC–MS interface. Method validation was carried out according to the International Council for Harmonisation guidelines. The current HPTLC–MS method is simple, sensitive, precise and accurate, and it can be used for the detection and quantitative estimation of benzodiazepines in urine.