Bromazepam determination in human plasma by high-performance liquid chromatography coupled to tandem mass spectrometry: a highly sensitive and specific tool for bioequivalence studies

A rapid, sensitive and specific method to quantify bromazepam in human plasma using diazepam as the internal std. (IS) is described. The analyte and the IS were extd. from plasma by liq.-liq. extn. using Et2O-hexane (80:20, vol./vol.). The exts. were analyzed by HPLC coupled to electrospray tandem mass spectrometry (MS/MS). Chromatog. was performed isocratically on a Genesis C18 anal. column (100 ´ 2.1 mm internal diam., film thickness 4 mm). The method had a chromatog. run time of 5.0 min and a linear calibration curve over the range 5.0-150 ng ml-1 (r2 > 0.9952). The limit of quantification was 5 ng ml-1. This HPLC/MS/MS procedure was used to assess the bioequivalence of two bromazepam 6 mg tablet formulations (bromazepam from Medley SA Industria Farmaceutica as the test formulation and Lexotan from Produtos Roche Quimico e Farmaceutico SA as the ref. formulation). A single 6 mg dose of each formulation was administered to 24 healthy volunteers (12 males and 12 females). The study was conducted using an open, randomized, two-period crossover design with a 3 wk washout interval. Since the 90% CI for Cmax, AUClast, AUC0-240 h (linear) and AUC(0-¥) ratios were all inside the 80-125% interval proposed by the US Food and Drug Administration, the bromazepam formulation from Medley is bioequivalent to the Lexotan formulation for both the rate and the extent of absorption.