A sensitive and specific method for the determination of total ribavirin in human red blood cells by liquid chromatography-tandem mass spectrometry

A sensitive and specific method using high-performance liquid chromatography (LC)-tandem mass spectrometry (MS) for the analysis of total ribavirin in human red blood cells (RBC) is developed and validated. The method involves the addition of an internal standard and perchloric acid, the conversion of ribavirin phosphorylated metabolites to ribavirin, purification with a solid-phase exchange cartridge, and LC-MS-MS analysis. The MS-MS is selected to monitor m/z 245-113 for ribavirin and m/z 250-113 for [13C]ribavirin using positive electrospray ionization. The calibration curve is linear over a concentration of 100-10,000 ng/mL with a limit of quantitation of 100 ng/mL. Mean interassay accuracy for quality control (QC) at 100, 1000, and 10,000 ng/mL are 101.8%, 99.4%, and 98.8%, respectively. Mean interassay precision (%CV) for QC at 100, 1000, and 10,000 ng/mL are 5.0%, 5.0%, and 2.5%, respectively. Extractibility of total ribavirin from RBC is confirmed with RBC obtained from a [14C] ribavirin-dosed monkey. The method is used to determine the free and total ribavirin concentration in human RBC obtained from hepatitis C patients treated with ribavirin.