Abstract

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.

Keywords: UPLC-MS/MS, verapamil, trandolapril, development, validation, rat plasma.

Graphical Abstract