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 100–10,000 ng/mL (0.412–41.2 μM) with a lower limit of quantification (LLOQ) of 100 ng/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 10,000 ng/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 10,000 ng/mL QC samples, respectively. Over-curve dilution QC at 400 μg/mL (1639 μM) for both viramidine and ribavirin are used to ensure the dilution accuracy (25 X dilutions) for monkey samples.
The method has been used to simultaneously determine the total concentrations of ribavirin and viramidine in monkey RBC following 5, 15, and 36 weeks dosing of viramidine or ribavirin (60 mg/kg). The concentrations of total ribavirin following ribavirin dosing are 1242 μM at week 5, 1257 μM at week 15, and 1146 μM at week 36. The concentrations of total ribavirin following viramidine dosing are 634 μM at week 5, 716 μM at week 15, and 683 μM at week 36. Only small amounts of viramidine are detected in RBC following viramidine dosing, 7.80 μM at week 5, 6.63 μM at week 15, and 10.4 μM at week 36. The results suggest that ribavirin levels in RBC were at steady state at week 5 of ribavirin or viramidine dosing. At steady state, ribavirin levels in RBC are approximately 2× after ribavirin dosing than viramidine dosing. The relatively small percentage of viramidine in RBC suggests that viramidine either poorly penetrated into RBC or was extensively converted to ribavirin following entry into RBC.