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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


Subrahmanyam Talari, Anuradha V* and Ratna Kumari S  


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 of verapamil and trandolapril.

Objective: The 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.

Method: Separation was carried on column Symmetry C18 column (150x4.6mm, 3.5µm) 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 2.4ng/mL to 48ng/mL(r2 = 0.9993 ± 0.018) for verapamil and 10pg/mL to 200pg/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% at different QC concentration levels. Precision and recovery study results were done within the acceptable limit. An electro spray 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 been successfully applied to explore verapamil (1.2mg/kg) with its internal standard (D6-Verapamil), trandolapril (0.005mg/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 bio analytical 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 bio analysis.


UPLC-MS/MS, Verapamil, Trandolapril, development, validation, rat plasma


Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, AP, Department of Chemistry, Vignan Degree College, Guntur, AP, Department of Chemistry Vignans Nirula Engineering college, Guntur, AP

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