Yeh Li-Tain, Nguyen Mai, Lourenco David, Lin Chin-Chung
Drug Development Department, Ribapharm Inc., 3300 Hyland Avenue, Costa Mesa, CA 92626, USA.
J Pharm Biomed Anal. 2005 Jun 1;38(1):34-40. doi: 10.1016/j.jpba.2004.12.006. Epub 2005 Jan 19.
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.
开发并验证了一种使用高效液相色谱-串联质谱法（LC-MS/MS）分析猴肝中利巴韦林总量的灵敏且特异的方法。在该方法中，用高氯酸提取利巴韦林及其磷酸化代谢物。使用酸性磷酸酶将代谢物转化为利巴韦林，并在LC-MS/MS分析之前，使用NH2固相萃取（SPE）柱进一步纯化。将[13C]利巴韦林作为内标添加到提取溶液中，以获得更高的分析准确度和精密度。采用正电喷雾电离，选择MS/MS监测利巴韦林和[(13)C]利巴韦林的245→113和250→113跃迁。校准曲线在1.0-100μg/g浓度范围内呈线性，定量限（LOQ）为1.0μg/g。1.0、10和100μg/g质量控制样品的批间平均准确度分别为108%、99.7%和99.7%。1.0、10和100μg/g质量控制样品的批间平均精密度（CV）分别为5.34%、5.24%和4.59%。用给予[14C]利巴韦林的猴的肝脏证实了从肝脏中提取总利巴韦林的能力。该方法已被证明可用于在质量平衡研究（10mg/kg）和28天毒理学研究（300mg/kg/天）中测定猴肝脏中的总利巴韦林浓度。它还用于测定接受每日两次600mg利巴韦林剂量的丙型肝炎患者人肝脏中的总利巴韦林浓度。
