High-Performance Liquid Chromatographic Determination of Ribavirin in Whole Blood To Assess Disposition in Erythrocytes

Abstract

Ribavirin is an antiviral agent used in the treatment of chronic hepatitis C virus infection. One of the limitations associated with the use of ribavirin is a reversible anemia caused by its accumulation in erythrocytes. Therefore, it is of interest to determine ribavirin levels in erythrocytes, as well as in plasma, as these measurements may be predictive of hematotoxicity. In the present study, we describe a high-performance liquid chromatographic (HPLC) assay for ribavirin in whole blood to estimate concentrations of free ribavirin and phosphorylated anabolites in erythrocytes. Since ribavirin exists primarily as phosphorylated anabolites (mono-, di-, and triphosphates) in erythrocytes, whole-blood extracts were initially dephosphorylated with acid phosphatase. The enzyme-treated samples were subjected to phenyl boronic acid column extraction for cleanup. The purified fraction was analyzed by reversed-phase HPLC, which was optimized for determination of ribavirin levels in whole blood. The recoveries of ribavirin from whole blood ranged from 63.1 to 90.7% at concentrations ranging from 1.67 to 40.0 μM. 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 estimate the extent of intracellular phosphorylation in erythrocytes. The method was also used to evaluate the effects of dipyridamole, a nucleoside transporter inhibitor, on ribavirin disposition in erythrocytes in in vitro experiments.

Simultaneous determination of ribavirin and ribavirin base in monkey plasma by high performance liquid chromatography with tandem mass spectrometry

“…Unfortunately, very low recovery was obtained for both analytes due to their extremely polar nature. Although a phenyl boronic acid (PBA) cartridge could be used to isolate ribavirin from biological matrix through an exclusive extraction of poly-ol species via interaction with surface-bound borate groups [27,28,32,33], ribavirin base was expected to be present in the wash waste during the loading step in SPE due to its lack of poly-ol groups in the molecule. Thus, protein precipitation with 10% methanol in acetonitrile was employed.…”

“…have been reported for the determination of ribavirin in biological matrices [24][25][26][27][28][29][30][31][32][33][34]. However, separate methods using HPLC-UV or HPLC with radioactivity detection were used for the measurement of ribavirin base in these samples [22,31,34].…”

Erythrocyte ribavirin concentration for assessing hemoglobin reduction in interferon and ribavirin combination therapy

“…Once incorporated into erythrocytes via the equilibrate nucleoside transporter 1 (ENT-1), ribavirin is converted into phosphorylated metabolites by intracellular phosphorylation [9,11,12]. Since the phosphorylated metabolites are not substrates for ENT-1, they cannot be effluxed out of the erythrocytes and so accumulate internally.…”

“…Although the mechanism of ribavirin-induced anemia has remained unclear, it has been speculated that highly accumulated ribavirin in erythrocytes reduces erythrocyte life span [8][9][10]. Once incorporated into erythrocytes via the equilibrate nucleoside transporter 1 (ENT-1), ribavirin is converted into phosphorylated metabolites by intracellular phosphorylation [9,11,12].…”

Human uridine-cytidine kinase phosphorylation of ribavirin: a convenient method for activation of ribavirin for conjugation to proteins

“…The anemia is likely due to accumulation of ribavirin in erythrocytes. Once transported into erythrocytes and phosphorylated to its active form, ribavirin is sequestered within the cells at concentrations estimated to be up to 9-fold higher than those in plasma [4]. Although the mechanism has not been fully clarified, it is believed that ribavirin phosphates impair ATP-dependent transport systems by competing with ATP located on the erythrocyte cell membrane, resulting in membrane destabilization.…”