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Abstract
Bromazepam was isolated from plasma samples by simple protein precipitation with acetonitrile. A high throughput isocratic reversed phase separation between bromazepam and nitrazepam (IS) was achieved within 2 minutes on a rapid resolution cartridge column Zorbax SB‐C18, using methanol/aqueous 0.1% formic acid 40/60 (v/v) as mobile phase. Detection was made by tandem mass spectrometry, using the multiple reaction monitoring mode (MRM). Both electrospray (ESI) and atmospheric pressure chemical (API) ionizations modes were considered, together with triple quadrupole (TQ) and ion trap (IT) mass analyzers. Ionization patterns are discussed; no major differences being observed between ESI and API. Higher sensitivity was obtained when using the TQ mass analyzer. Comparison between different ionization modes/mass analyzers types is made, based on bromazepam concentration/time profiles in real plasma samples. The best results were obtained in ESI/TQ conditions. Consequently, the method was fully validated, and then applied to a single dose (6 mg) open label, randomized, two period, two sequence, and crossover bioequivalence study of bromazepam in two commercially available tablet formulations. A low limit of quantification (LOQ) at 2.5 ng/mL level was obtained. Precision below 3% (expressed as relative standard deviation) and accuracy (expressed as % bias) within −7 and +11% were achieved. Pharmacokinetic parameters are presented, being in good accordance with literature data.