Determination of Ribavirin in Rat Plasma by UPLC-MS/MS: Application to a Pharmacokinetic Study

An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine ribavirin in rat plasma using pirfenidone as the internal standard (IS). Sample preparation was accomplished through a protein precipitation procedure with acetonitrile to 0.1 mL plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C-18 column (2.1 x 50 mm, 1.7 mu m) with the mobile phase of acetonitrile and 0.1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. The injection volume was 2 mu L. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 245.1 -> 113.1 for ribavirin and m/z 186.2 -> 92.1 for IS. The linearity of this method was found to be within the concentration range of 5-1000 ng/mL with a lower limit of quantification of 5 ng/mL. Only 3.0 min was needed for an analytical run. The matrix effect was 102.2 to 107.3% for ribavirin. The intra-and inter-day precision (RSD%) were less than 8.0% and accuracy (RE%) was within +/- 9.3%. The recovery ranged from 78.5 to 84.0%. Ribavirin was sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the pharmacokinetic study of ribavirin in rats. The pharmacokinetic parameters were demonstrated as followed: t(1/2) was 3.60 +/- 1.28 h, Cmax was 588.52 +/- 47.64 ng/mL, and AUC(0 ->infinity) was 2475.24 +/- 250.51 ng/mL. h.