High-performance liquid chromatographic determination of ribavirin in whole blood to assess disposition in erythrocytes

Ribavirin is an antiviral agent used in the treatment of chronic hepatitis C virus infection. One of the limitations associated with the use of ribavi rin is a reversible anemia caused by its accumulation in erythrocytes. Ther efore, it is of interest to determine ribavirin levels in erythrocytes, as well as in plasma, as these measurements may be predictive of hematotoxicit y. In the present study, we describe a high-performance liquid chromatograp hic (HPLC) assay for ribavirin in whole blood to estimate concentrations of free ribavirin and phosphorylated anabolites in erythrocytes. Since ribavi rin exists primarily as phosphorylated anabolites (mono-, di-, and triphosp hates) in erythrocytes, whole-blood extracts were initially dephosphorylate d with acid phosphatase. The enzyme-treated samples were subjected to pheny l boronic acid column extraction for cleanup. The purified fraction was ana lyzed by reversed-phase HPLC, which was optimized for determination of riba virin levels in whole blood. The recoveries of ribavirin from whole blood r anged from 63.1 to 90.7% at concentrations ranging from 1.67 to 40.0 mu M. Intra- and interassay variations estimated at these concentrations were 3.2 to 10.4 and 4.7 to 11.7%, respectively. This method was used to quantitate ribavirin in samples both treated and untreated with acid phosphatase to e stimate the extent of intracellular phosphorylation in erythrocytes. The me thod was also used to evaluate the effects of dipyridamole, a nucleoside tr ansporter inhibitor, on ribavirin disposition in erythrocytes in in vitro e xperiments.