An UPLC-MS/MS Method for the Quantitation of Ledipasvir in Rat Plasma: Application to a Pharmacokinetic Study

An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine ledipasvir in rat plasma using diazepam 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 C18 column (2.1 mm 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 889.8 -> 96.1 for ledipasvir and m/z 285.2 -> 193.1 for IS. The linearity of this method was found to be within the concentration range of 5-500 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 103.5 to 107.8% for ledipasvir. The intra-and inter-day precision (RSD%) were less than 9.5% and accuracy (RE%) was within +/- 11.5%. The recovery ranged from 78.9 to 85.6%. Ledipasvir was sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the pharmacokinetic study of ledipasvir in rats. The pharmacokinetic parameters were demonstrated as followed: t(1/2) was 4.65 +/- 1.23 h, C-max was 187.24 +/- 19.45 ng/mL, and AUC(0 -> 8) was 986.64 +/- 292.57 ng/mL.h.