Determination of bromazepam in human plasma by high‐performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study

Abstract

A simple method using a one-step liquid-liquid extraction (LLE) followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of bromazepam in human plasma, using lorazepam as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions: m/z 316 > 182 for bromazepam and m/z 321 > 275 for lorazepam. The method was linear over the studied range (1-100 ng ml(-1)), with r(2) > 0.98, and the run time was 2.5 min. The intra- and inter-assay precisions were 2.7-14.6 and 4.1-17.3%, respectively and the intra- and inter-assay accuracies were 87-111 and 75.8-109.5%, respectively. The mean recovery was 73.7%, ranging from 64.5 to 79.7%. The limit of quantification was 1 ng ml(-1). At this concentration the mean intra- and inter-assay precisions were 14.6 and 7.1%, respectively, and the mean intra- and inter-assay accuracies were 102.5 and 104%, respectively. Bromazepam stability was evaluated and the results showed that the drug is stable in standard solution and in plasma samples under typical storage and processing conditions. The method was applied to a bioequivalence study in which 27 healthy adult volunteers (14 men) received single oral doses (6 mg) of reference and test bromazepam formulations, in an open, two-period, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak plasma concentration), AUC(0-96) and AUC(0-inf) (area under the plasma concentration versus time curve from time zero to 96 h and to infinity, respectively) were within the range 80-125%, which supports the conclusion that the test formulation is bioequivalent to the reference formulation regarding the rate and extent of bromazepam absorption.

Benzodiazepine Metabolism: An Analytical Perspective

“…The cartridge sorbent is a C18 resin and 100 µL of sample are sufficient for one analysis. Another paper describes a method based on HPLC-MS/MS for bioequivalence studies [44]. It reports the use of LLE for the sample pre-treatment, a run time of 2.5 min and a LOQ of 1 ng/mL.…”

Development and validation of a stability indicating RP-HPLC-DAD method for the determination of bromazepam

“…These reported methods also reveal many analytical techniques for BMZ determination either alone or together with other benzodiazepines, in both pharmaceutical or biological matrices. These techniques include HPLC [13][14][15][16][17][18][19][20][21][22][23][24], LC-MS [25,26], GC-MS [27], TLC [28], capillary electrophoresis [29], spectrophotometry [30][31][32], ion-selective electrode [33][34][35], potentiometric [36] and voltammetric [37,38] methods.…”