LC-MS/MS method for simultaneous determination of viramidine and ribavirin levels in monkey red blood cells.

A high performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) method has been developed for the simultaneous determinations of total viramidine (viramidine, viramidine monophosphate, viramidine diphosphate, and viramidine triphosphate) and total ribavirin (ribavirin, ribavirin monophosphate, ribavirin diphosphate, and ribavirin triphosphate) in monkey red blood cells (RBC). The method involves the addition of internal standards and perchloric acid, conversion of viramidine or ribavirin phosphorylated metabolites to viramidine or ribavirin, purification with an aminopropyl (NH2) solid phase extraction (SPE) cartridge, and LC–MS/MS analysis. The MS/MS is selected to monitor m/z 245→113, 250→113, 244→112, and 249→112 for ribavirin, [13C]ribavirin, viramidine, and [13C]viramidine, respectively, using positive electrospray ionization. The calibration curves are linear over a concentration range of 100–10,000ng/mL (0.412–41.2μM) with a lower limit of quantification (LLOQ) of 100ng/mL for both compounds. Mean inter-assay recoveries for ribavirin are 101%, 98.9%, and 96.0%, with coefficient of variance (%CV) values between 1.95 and 4.50% for 100, 1000, and 10,000ng/mL quality control (QC) samples, respectively. Mean inter-assay recoveries for viramidine are 96.3%, 101%, and 102%, with coefficient of variation (%CV) values between 3.61 and 7.22%, for 100, 1000, and 10,000ng/mL QC samples, respectively. Over-curve dilution QC at 400μg/mL (1639μM) for both viramidine and ribavirin are used to ensure the dilution accuracy (25 X dilutions) for monkey samples.