Development and validation of an HPLC-MS/MS method for the determination of arginine-vasopressin receptor blocker conivaptan in human plasma and rat liver microsomes: application to a metabolic stability study.

Purpose: To develop and validate a bio-analytical HPLC-MS/MS method for the determination of conivaptan (CVA) an arginine-vasopressin receptor blocker in human plasma and in rat liver microsomes (RLMs).

Methods: Analytes were separated on a reversed phase C18 column (50 mm × 2.1 mm, 1.8 μm). The mobile phase was a mixture of acetonitrile and 10 mM ammonium formate (40:60 v/v, pH 4.0) and was pumped isocratically for 4 min at a flow rate of 0.2 ml/min. Multiple reaction monitoring in positive ionization mode was used for the assay.

Results: The method yielded a linear calibration plot (r ² = 0.9977 and 0.9998) over 5-500 ng/ml with a limit of detection at 1.52 and 0.88 ng/ml for human plasma and RLMs, respectively. The reproducibility of detection of CVA in human plasma and RLMs was found to be in an acceptable range.

Conclusion: The method developed in this study is applicable for accurately quantifying CVA in human plasma and rat liver microsomal samples. The optimized procedure was applied to study of metabolic stability of CVA. Conivaptan concentration rapidly decreased in the first 2 min of RLMs incubation and the conversion reached a plateau for the remainder of the incubation period. The in vitro half-life (t_1/2) was estimated at 11.51 min and the intrinsic clearance (CL_in) was 13.8 ± 0.48 ml/min/kg.