Development and validation of a useful UPLC-MS/MS method for quantification of total and phosphorylated-ribavirin in peripheral blood mononuclear cells of HCV+ patients

The current standard-of-care therapy in HCV consists in ribavirin (RBV) plus pegylated-interferon-α 2a or 2b and, for HCV-1 infected patients, also directly acting antivirals (DAAs). Despite the increase in the number of patients who reach sustained virological response (SVR) for HCV-1, a great inter-individual variability in the response to therapy remains. Whether new drugs are available in combination with RBV and Peg-IFN for HCV-1, the treatment of the other viral genotypes remains the same: this issue highlights the lasting importance of RBV and Peg-IFN in anti-HCV treatment. Moreover, a strong limiting factor to the usefulness of anti-HCV treatment remains the occurrence of adverse events, first of all hemolytic anemia, which have increased with the addition of DAAs, but is mainly an RBV-dependent effect. For these reasons, the monitoring of RBV exposure in the various compartments should be important. Since the routinely determination of RBV in the target cells as the hepatocytes is impracticable for of its invasiveness, the quantification in easier to obtain cells could be a good choice. In this work, we developed and validated an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay method to quantify RBV concentrations in peripheral blood mononucleated cells (PBMCs). QCs were prepared with RBV and RBV-monophosphate (RMP). Each sample was divided into two aliquots, which undergone the same extraction procedure: one was treated with acid phosphatase to convert RBV phosphorylated metabolites into free RBV, the other one was not-treated. The extracts were analyzed with reverse-phase column with UPLC-MS/MS. Calibration curves fitted a least squares model (weighed 1/X) for ribavirin levels in a range from 0.1ng to 200ng (mean r2=0.9993). Accuracy, intra-day and inter-day precision of the methods were in accordance with FDA guidelines. Moreover, phosphorylated QCs were used to assess the correct determination of total RBV concentration. We tested this method by monitoring RBV concentrations in PBMCs from 20 HCV+ patients, receiving alpha interferon-plus RBV combination therapy. This method showed to be reliable, precise, accurate and suitable for evaluation of intracellular RBV concentrations

Development and validation of a useful HPLC-UV method for quantification of total and phosphorylated-ribavirin in blood and erythrocytes of HCV+ patients

Ribavirin-induced hemolytic anemia is the main cause of discontinuation of the combination therapy with alpha-interferon-2b and ribavirin for the treatment of hepatitis C virus (HCV) infection. The determination of intracellular ribavirin levels in blood, including the levels of its phosphorylated metabolites, might be useful for predicting ribavirin-induced anemia, which could be caused by the accumulation of these molecules within the erythrocytes. In this work, we simplified and validated a previously developed assay method, to make it suitable for routine monitoring of cellular ribavirin. Whole blood diluted with a five-fold volume of ice-cold distilled underwent a process of acid phosphatase digestion to convert phosphorylated ribavirin metabolites to free ribavirin. The resulting mixture, spiked with an internal standard, was treated with a protein precipitation protocol in acetonitrile, followed by reverse-phase high-performance liquid chromatography analysis. The calibration curve for ribavirin levels in whole blood was linear at concentrations from 625 to 320,000ng/mL (r(2)=0.998). Accuracy, intra-day and inter-day precision for ribavirin and phosphorylated-ribavirin quality controls were all below 9.0%. We tested this method by monitoring blood ribavirin concentrations in 13 HCV+ patients, receiving alpha interferon-plus ribavirin combination therapy