A sensitive and specific method for the determination of total ribavirin in monkey liver by high-performance liquid chromatography with tandem mass spectrometry.
A sensitive and specific method using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the analysis of total ribavirin in monkey liver is developed and validated. In this method, ribavirin and its phosphorylated metabolites are extracted with perchloric acid. The metabolites are converted to ribavirin using acid phosphatase and further purified using a NH2 solid-phase extraction (SPE) cartridge prior to LC-MS/MS analysis. [13C]Ribavirin is added with the extraction solution as an internal standard to obtain better accuracy and precision of the analysis. The MS/MS was selected to monitor 245-->113 and 250-->113 transitions using positive electrospray ionization for ribavirin and [(13)C]ribavirin. The calibration curve is linear over a concentration of 1.0-100 microg/g with a limit of quantitation (LOQ) of 1.0 microg/g. Mean inter-assay accuracy for QC at 1.0, 10 and 100 microg/g are 108, 99.7 and 99.7%, respectively. Mean inter-assay precision (CV) for QC at 1.0, 10 and 100 microg/g are 5.34, 5.24 and 4.59%, respectively. Extractability of total ribavirin from liver has been confirmed with liver obtained from monkey dosed with [14C]ribavirin. The method has been proven to be useful in the determination of total ribavirin concentration in liver from monkeys in mass balance study (10 mg/kg) and in 28 days toxicology study (300 mg/kg/day). It is also used to determine the total ribavirin concentration in human livers from hepatitis C patients received dose of 600 mg ribavirin twice daily.
Yeh
Li-Tain
LT
Drug Development Department, Ribapharm Inc., 3300 Hyland Avenue, Costa Mesa, CA 92626, USA. lyeh@valeant.com
Nguyen
Mai
M
Lourenco
David
D
Lin
Chin-Chung
CC
D000818:Animals; D000998:Antiviral Agents / Q000032:analysis*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008099:Liver / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D012254:Ribavirin / Q000032:analysis*; D012680:Sensitivity and Specificity
D000998:Antiviral Agents; D012254:Ribavirin