HPLC–MS/MS method for the intracellular determination of ribavirin monophosphate and ribavirin triphosphate in CEMss cells

A sensitive and specific method using high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) for the determination of ribavirin monophosphate (RBV-MP) and ribavirin triphosphate (RBV-TP) in cells has been developed and validated. In this method, ribavirin phosphorylated metabolites were extracted and separated by anion exchange solid phase extraction (SPE). The RBV-MP and RBV-TP fractions were dephosphorylated using acid phosphatase and further purified by phenyl boronate SPE prior to HPLC–MS/MS analysis. ¹³C₅-uridine was added as internal standard to obtain better accuracy and precision of the analysis. The MS/MS detector was optimized at multiple reaction monitoring (MRM) using positive electrospray ionization to detect 245 → 113 and 250 → 133 transitions for ribavirin and internal standard, respectively. The calibration curve was linear over a concentration range of 0.01–10 μg/mL with a limit of quantitation of 0.01 μg/mL. Mean inter-assay accuracy and precision for RBV-MP and RBV-TP quality control samples at 0.03, 0.3 and 8 μg/mL were 5% and 10%, respectively. This method was successfully used for the in vitro determination of RBV-MP and RBV-TP in CEM_ss cells cultured with RBV.