Hypertension plays a causative role in the onset of stroke, myocardial infarction, heart failure, peripheral arterial disease, and chronic kidney disease. Currently, antihypertensive drugs include thiazide diuretics, angiotensin converting enzyme (ACE) inhibitors, calcium channel blockers, β-blockers, and angiotensin II receptor antagonists. 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.

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.

The total chromatographic run time was 2.0 minutes and the elution of bisoprolol, enalapril, enalaprilat and IS (verapamil) occurred at ~1.01, 1.03, 0.96 and 1.09 minutes, 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.

The new bioanalytical method for simultaneous determination of bisoprolol and enalapril in the present of enalaprilat is considered a reliable, robust and patient‐friendly and is expected to be used widely. The application of incurred sample reanalysis in the validation protocol helped to ensure the validity of results and supports the use of the proposed method as a routine assay for bioequivalence studies, which will be very useful in therapeutic drug monitoring.