Development and Validation of a UPLC-MS/MS Method for the Simultaneous Determination of Verapamil and Trandolapril in Rat Plasma: Application to a Pharmacokinetic Study

Background: Verapamil is an excellent drug used for the medication of hypertension and trandolapril. It is an angiotensin-converting-enzyme inhibitor. Hence, it is an interesting method to develop a novel and reliable MS/UPLC strategy for the simultaneous development of verapamil and trandolapril. Objectives: This research study aims to develop a new, rapid, and sensitive UPLC-MS/MS method for the simultaneous estimation of verapamil and trandolapril in rat plasma using D6- verapamil and D6-trandolapril. Methods: Separation was carried on column Symmetry C18 column (150x4.6 mm, 3.5 μm) using isocratic elution with a buffer containing 1mL of formic acid in 1L of water and the mixture of two components like Buffer and Acetonitrile in the ratio of 80:20 as mobile phase with 1mL/min flow rate at ambient temperature. Results: Analysis was performed within 5 minutes over a good linear concentration range from 2.4 ng/mL to 48 ng/mL (r² = 0.9993 ± 0.018) for verapamil and 10 pg/mL to 200 pg/mL (r² = 0.9993 ± 0.006) for trandolapril. The extraction recoveries and matrix effect of verapamil and trandolapril were 98.45, 99.95, 98.12, 99.66% and 98.27, 99.89, 97.78, 99.23% respectively, at different QC concentration levels. Precision and recovery study results were determined within the acceptable limit. An electrospray ionization source was used to study verapamil and Trandolapril at m/z 454.72→182.16, 430.25→201.48, and IS for m/z 460.18→ 324.39, 436.28 → 340.52, which were ion pairs of mass analysis. This method has successfully been applied to explore verapamil (1.2 mg/kg) with its internal standard (D6-Verapamil), trandolapril (0.005 mg/kg) with its internal standard (D6-Trandolapril) extracted from rat plasma using liquid-liquid extraction. Conclusion: This manuscript focuses on the consistent evaluation of the key bioanalytical validation parameters, and the following are discussed: accuracy, precision, sensitivity, selectivity, standard curve, limits of quantification, range, recovery, and stability. These validation parameters are described, together with illustrations of validation methodology applied in the case of chromatographic methods used in bioanalysis.