title: Development and validation of a useful HPLC-UV method for quantification of total and phosphorylated-ribavirin in blood and erythrocytes of HCV+ patients.
abstract: 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 320,000 ng/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.
authors.affiliation: Laboratory of Clinical Pharmacology and Pharmacogenetic, Department of Infectious Diseases, University of Turin, Amedeo di Savoia Hospital, Turin, Italy. antonio.davolio@unito.it
authors.forename: Antonio
authors.initials: A
authors.lastname: D'Avolio
authors.forename: Amedeo
authors.initials: A
authors.lastname: De Nicolò
authors.forename: Marco
authors.initials: M
authors.lastname: Simiele
authors.forename: Stefano
authors.initials: S
authors.lastname: Turini
authors.forename: Danilo
authors.initials: D
authors.lastname: Agnesod
authors.forename: Lucio
authors.initials: L
authors.lastname: Boglione
authors.forename: Jessica
authors.initials: J
authors.lastname: Cusato
authors.forename: Lorena
authors.initials: L
authors.lastname: Baietto
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Cariti
authors.forename: Andrea
authors.initials: A
authors.lastname: Calcagno
authors.forename: Mauro
authors.initials: M
authors.lastname: Sciandra
authors.forename: Giovanni
authors.initials: G
authors.lastname: Di Perri
authors.forename: Stefano
authors.initials: S
authors.lastname: Bonora
mesh_terms: D000743:Anemia, Hemolytic / Q000139:chemically induced; D000998:Antiviral Agents / Q000008:administration & dosage / Q000009:adverse effects / Q000493:pharmacokinetics*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016903:Drug Monitoring / Q000379:methods; D004912:Erythrocytes / Q000378:metabolism; D005240:Feasibility Studies; D006526:Hepatitis C / Q000188:drug therapy*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000008:administration & dosage; D010766:Phosphorylation; D011786:Quality Control; D011994:Recombinant Proteins / Q000008:administration & dosage; D012254:Ribavirin / Q000008:administration & dosage / Q000009:adverse effects / Q000493:pharmacokinetics*
chemical_list: D000998:Antiviral Agents; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D012254:Ribavirin