Simultaneous determination of 7 benzodiazepines in human plasma by UHPLC-MS/MS

The study was designed to develop the method for determination of 7 benzodiazepines concentration in human plasma. UHPLC-MS/MS was adopted to analyze plasma with protein precipitated by acetonitrile. Citalopram was used as an internal standard. Plasma samples were separated on CORTECS UHPLC C₁₈ column with the mobile phase of aqueous solution (0.01% formic acid)-methanol (0.01% formic acid) at a flow rate of 0.3 mL·min^-1. Multiple reaction monitoring (MRM) mode was performed in combiation with electrospray ionization source operating in the positive ionization mode. The liner calibration curve of midazolam, nitrazepam, estazolam, clonazepam, lorazepam, triazolam and diazepam were obtained in the concentration range of 1.05-840 (r=0.999 4), 2.06-824 (r=0.998 1), 2.02-1 616 (r=0.994 7), 6.18-2 472 (r=0.997 9), 6.12-2 448 (r=0.997 4), 3.02-2 416 (r=0.990 2), 1.02-816 (r=0.998 8) ng·mL^-1, respectively. The lowest detection limit were 0.02, 0.52, 0.51, 1.55, 0.77, 0.76, 0.02 ng·mL^-1, respectively. The RSD of inter-day and intra-day were less than 10.81%. The relative recovery was 81.46%-106.53%. The method was successfully applied to clinical analysis of blood samples from patients.