Development and validation of a useful HPLC-UV method for quantification of total and phosphorylated-ribavirin in blood and erythrocytes of HCV+ patients
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- 作者：Antonio D&rsquo ; Avolio ; antonio.davolio@unito.it ; Amedeo De Nicolò ; Marco Simiele ; Stefano Turini ; Danilo Agnesod ; Lucio Boglione ; Jessica Cusato ; Lorena Baietto ; Giuseppe Cariti ; Andrea Calcagno ; Mauro Sciandra ; Giovanni Di Perri ; Stefano Bonora
- 关键词：HPLC&ndash ; UV ; Anemia ; HCV ; Intracellular ; Ribavirin
- 刊名：Journal of Pharmaceutical and Biomedical Analysis
- 出版年：2012
- 出版时间：July, 2012
- 年：2012
- 卷：66
- 期：Complete
- 页码：376-380
- 全文大小：296 K
文摘
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 (r2 = 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.