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

J Mass Spectrom. 2004 Nov;39(11):1348-55. doi: 10.1002/jms.667.


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.

MeSH terms

  • Anti-Anxiety Agents / analysis
  • Anti-Anxiety Agents / blood*
  • Anti-Anxiety Agents / pharmacokinetics
  • Bromazepam / analysis
  • Bromazepam / blood*
  • Bromazepam / pharmacokinetics
  • Chromatography, High Pressure Liquid / methods*
  • Chromatography, High Pressure Liquid / standards
  • Humans
  • Lorazepam / analysis
  • Lorazepam / blood
  • Plasma
  • Reproducibility of Results
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Spectrometry, Mass, Electrospray Ionization / standards
  • Therapeutic Equivalency


  • Anti-Anxiety Agents
  • Lorazepam
  • Bromazepam