超高效液相色谱-串联质谱法测定鸡血浆中利巴韦林及其主要代谢物

Simultaneous Determination of Ribavirin and Its Main Metabolites in Chicken Plasma by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry

建立了同时检测血浆中利巴韦林（Ribavirin）及其主要代谢物1H-1,2,4-三氮-3-甲酰胺（TCONH2）和1-β-D-呋喃基核糖-三氮唑-3-羧酸（RTCOOH）的超高效液相色谱-串联质谱（UPLC-MS/MS）分析方法。鸡血浆样品加入13C5-利巴韦林内标,以甲醇沉淀蛋白,经ZORBAX SB-Aq（100 mm×3 mm,1.8μm）色谱柱分离,以0.1%甲酸水-甲醇为流动相,梯度洗脱,采用正离子多反应监测模式（MRM）,内标法定量。结果表明：利巴韦林、TCONH2与RTCOOH的线性范围分别为5~5 000,5~5 000,50~5 000μg/L,相关系数（r2）分别为0.994 7,0.999 2,0.997 6。质控样品的加标回收率为84.1%~108.2%,批内相对标准偏差（RSD,n=6）为1.6%~13.4%,批间RSD（n=12）为3.8%~16.9%。样品的检出限（S/N=3）为2~10μg/L。本方法快速简便,有效排除了内源性干扰,方法的线性范围宽,重复性好,适用于血浆样品中利巴韦林及主要代谢产物的测定。

An ultra performance liquid chromatography tandem mass spectrometric（ UPLC -MS/MS） method was successfully developed for the simultaneous determination of ribavirin and its metabolites 1H-l, 2,4-triazole-3-carboxamide （ TCONH= ） and 1-fl-D-ribofuranosyl-1,2, 4-triazole-3-carboxylic acid（RTCOOH）. 13Cs-labelled-ribavirin was used as an internal standard in this approach. After pre- cipitation of proteins in chicken plasma by adding methanol, the pretreated sample was separated on a ZORBAX SB-Aq column（ 100 mm x 3 mm, 1.8 μm） by a gradient elution program using 0. 1% formic acid water solution and methanol as mobile phase. The target analytes were detected by elec- trospray ionization -mass spectrometry under positive MRM mode. The result indicated that the cali- bration curves were linear in the ranges of 5 - 5 000 μg/L for ribavirin and TCONH2, and 50 - 5 000 μg/L for RTCOOH, with correlation coefficients of 0. 994 7, 0. 999 2, 0. 997 6, respective- ly. The average recoveries of ribavirin, TCONH2 and RTCOOH were in the range of 84. 1% - 108.2% with intra-assay relative standard deviations（ RSDs, n =6） of 1.6% - 13.4% and inter-as- say RSDs（n = 12） of 3.8% - 16.9% . The limits of detection（LOD） for ribavirin, TCONH2 and RTCOOH in chicken plasma were 2 μg/L, 2 μg/L and 10 μg/L, respectively. With merits of rapid analysis, good repeatability and wide linearity range, the method could be a powerful tool for the study of ribavirin pharmacokinetics and metabolism in chicken plasma.