Simultaneous Determination of Buspirone, Alprazolam, Clonazepam, Diazepam, and Lorazepam by LC‐MS/MS: Evaluation of Greenness Using AGREE Tool

Anxiolytic drugs such as buspirone, alprazolam, clonazepam, diazepam, and lorazepam are used to treat anxiety disorders. Measurement of drug plasma concentrations is important in the treatment of the patient. This research developed a rapid, reliable, and cost-effective LC-MS/MS method to quantify buspirone, alprazolam, clonazepam, diazepam, and lorazepam in human plasma for therapeutic drug monitoring. Furthermore, compliance with the International Council for Harmonisation (ICH) M10 Bioanalytical Method Validation and Study Sample Analysis ensured the method's reliability. Before injection into the LC-MS/MS system, the analytes and an internal standard were extracted from plasma through salt-assisted liquid-liquid microextraction (SALLME). The lower limit of quantification (LLOQ) was determined as 0.5, 2.5, 2, 30, and 10 ng/mL for buspirone, alprazolam, clonazepam, diazepam, and lorazepam, respectively. The calibration curve ranges are 0.5-50 ng/mL for buspirone, 2.5-250 ng/mL for alprazolam, 2-200 ng/mL for clonazepam, 30-3000 ng/mL for diazepam, and 10-1000 ng/mL for lorazepam, respectively, with correlation coefficients > 0.99. Its suitability for therapeutic drug monitoring was demonstrated using the method to determine drug concentration levels in real patients. The developed method has been observed to achieve a high analytical greenness metric approach and software (AGREE) score for the greenness profile.