BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS DETERMINATION OF VERAPAMIL AND ENALAPRIL IN THE PRESENT OF ENALAPRILAT BY HPLC-MS/MS
Objective: The main purpose of this study was to develop a simple, precise, rapid and accurate method for the simultaneous quantification of verapamil 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 Ã— 2.1 mm, 5 Î¼m column. Samples were chromatographed in a gradient mode (eluent A (acetonitrileâ€“waterâ€“formic acid, 5: 95: 0.1 v/v), eluent B (acetonitrileâ€“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 Î¼l.
Results: The total chromatographic run time was 2.0 min and the elution of verapamil, enalapril, enalaprilat and IS (bisoprolol) occurred at ~1.09, 1.03, 0.96 and 1.01 min, respectively. A linear response function was established at 1-100 ng/ml for verapamil hydrochloride, 2-200 ng/ml for enalapril maleate, 1-100 ng/ml for enalaprilat dehydrate in human plasma. The % accuracy of LLOQ samples prepared with the different biological matrix lots were found 99.2 % for enalapril, 103.4 % for enalaprilat, 90.6 % for verapamil, which were found within the range of 80.00-120.00% for the seven different plasma lots. % CV for LLOQ samples was observed as 4.9%, 5.9 %, 9.0 % respectively, which are within 20.00% of the acceptance criteria. The % mean recovery for enalapril in LQC, MQC and HQC was 103.6 %, 100.1 % and 102.4 %, for enalaprilat in LQC, MQC and HQC was 103.2 %, 98.0 % and 101.1 %, for verapamil in LQC, MQC and HQC was 97.9 %, 96.1 % and 98.6 % respectively.
Conclusion: A new rapid method was developed for simultaneous determination of verapamil and enalapril in the present of enalaprilat in human plasma. The method was strictly validated according to the ICH guidelines. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for routine examination of verapamil and enalapril in the present of enalaprilat in human plasma.
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