Ribavirin-induced hemolytic anemia is one cause for cessation of combination therapy with alpha interferon 2b and ribavirin for hepatitis C infection. Determining cellular ribavirin levels in blood, including the levels of its phosphorylated metabolites, might be useful for predicting ribavirin-induced anemia, because the metabolites accumulate in erythrocytes. We simplified an assay method developed previously to make it suitable for routine monitoring of cellular ribavirin. Whole blood diluted with a sixfold volume of ice-cold distilled water was subjected to acid phosphatase digestion to convert phosphorylated ribavirin metabolites to free ribavirin. The resulting mixture, spiked with an internal standard, was treated by phenyl boronic acid column extraction, followed by reverse-phase high-performance liquid chromatography analysis. The calibration curve for ribavirin levels in whole blood was linear at concentrations of 5.3 to 1,024 M (r 2 ؍ 0.9999). Validation coefficients of variation for intra-and interday assays were 2.9 to 5.8% and 4.3 to 8.3%, respectively. We tested this method by monitoring blood ribavirin concentrations in two hepatitis C patients receiving alpha interferon 2b-plusribavirin combination therapy.Ribavirin, a guanosine analog broad-spectrum antiviral agent, is used for hepatitis C virus (HCV) elimination in combination with alpha interferon 2b (2, 3, 13). Polyethyleneglycolalpha interferon 2a combined with oral ribavirin also brings substantial benefit to HCV patients (12,14). Despite the strong beneficial effects of these combination therapies in severe HCV infection, loss of hemoglobin occurs in a substantial population of HCV patients. Progressive loss of hemoglobin leads to anemia, which is counteracted by reducing the ribavirin dose or prematurely discontinuing the combination therapy (1,5,12). Current studies suggest that the excessive accumulation of ribavirin in erythrocytes is responsible for the anemia (8, 11). Once incorporated into erythrocytes, ribavirin is converted into phosphorylated metabolites by intracellular phosphorylation (9). The phosphorylated metabolites decrease intracellular ATP levels, resulting in the reduction of erythrocyte integrity, which is followed by extravascular hemolysis via the reticuloendothelial system (4). It should therefore be possible to predict the occurrence of ribavirin-induced anemia by determining the ribavirin concentrations in blood cells and plasma.A high-performance liquid chromatography (HPLC) method was previously developed to determine ribavirin levels in order to assess the disposition of ribavirin in erythrocytes (7). Since phosphorylated metabolites are the main form of intracellular ribavirin (7, 8), whole-blood samples were treated with acid phosphatase prior to column extraction and analysis. However, the dephosphorylation procedure used in a previous study (7) was too tedious to use for routine monitoring of cellular ribavirin. In the present study, we simplified the dephosphorylation procedure. The modified method was ...