A HPLC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS DETERMINATION OF BISOPROLOL AND ENALAPRIL IN THE PRESENT OF ENALAPRILAT IN HUMAN PLASMA

Abstract

Objective: A highly specific, sensitive and rapid HPLC-MS/MS method has been developed and validated for the simultaneous quantification of bisoprolol and enalapril in the present of enalaprilat in human plasma.

Methods: Analytes were extracted from plasma using a protein precipitation extraction method. Chromatography was achieved on Discovery C18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile-water–formic acid, 5: 95: 0.1 v/v), eluent B (acetonitrile–formic acid, 100: 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.400 ml/min into the mass spectrometer ESI chamber. The sample volume was 5 μl.

Results: The total chromatographic run time was 2.0 min and the elution of bisoprolol, enalapril, enalaprilat and IS (verapamil) occurred at ~1.01, 1.03, 0.96 and 1.09 min, respectively. A linear response function was established at 0.5-50 ng/ml for bisoprolol fumarate, 2-200 ng/ml for enalapril maleate, 1-100 ng/ml for enalaprilat dehydrate in human plasma. The intraday and interday accuracy and precisions were in the range of 0.311 %-0.647 % and 0.364 %-0.572 % for bisoprolol, 0.321 %-0.747 % and 0.390 %-0.673 % for enalapril, 0.221 %-0.547 % and 0.264 %-0.773 % for enalaprilat, respectively.

Conclusion: A new rapid method was developed for simultaneous determination of bisoprolol and enalapril in the present of enalaprilat in human plasma. The method was strictly validated according to the ICH guidelines. The information thus obtained from the study can be used for the full pharmacokinetic profiling in individuals.