LC-MS/MS法测定大鼠血浆中奥贝胆酸的浓度  

Determination of obeticholic acid in rat plasma by liquid chromatography-tandem mass spectrometry(LC-MS/MS)

作　　者：张如月 谷元[2] 张爱杰[2] 董世奇[3] 李全胜[2] 魏广力[2] 司端运[2] 

机构地区：[1]天津医科大学研究生院,天津300070 [2]天津药物研究院新药安全评价有限公司,释药技术与药代动力学国家重点实验室,天津300193 [3]天津大学,天津300191

出　　处：《药学学报》2018年第2期271-277,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金青年科学基金资助项目(81503154).

摘　　要：本文建立了一种快速、灵敏、准确、可靠的LC-MS/MS分析方法测定大鼠血浆中奥贝胆酸浓度,并应用于奥贝胆酸大鼠灌胃给药后的药动学研究。血浆样品以甲基叔丁基醚提取后,经ACE Excel 2 Super C18色谱柱(50 mm×2.1 mm ID,1.7μm)分离,以乙腈-2 mmol·L^(-1)甲酸铵水溶液(含10%乙腈)作为流动相,梯度洗脱,流速0.2 m L·min^(-1)。电喷雾离子化源(ESI)负离子条件下,采用多反应监测模式(MRM)进行定量分析,监测离子对分别为奥贝胆酸m/z 418.9[M-H]^-→401.2,内标甘草次酸m/z 469.0[M-H]^-→425.2。大鼠血浆中奥贝胆酸在5~5 000 ng·m L^(-1)浓度内线性关系良好(r^2?0.99),定量下限为5 ng·m L^(-1);日内、日间精密度(RSD)小于9.82%,准确度(RE)在±6.90%以内;血浆中无内源性物质干扰,内标归一化后的基质效应为78.9%~82.5%,提取回收率为85.4%~88.5%;奥贝胆酸的血浆样品经室温放置24 h、-70℃3次冻融循环、-70℃冻存1个月,处理后的样品在进样器内放置24 h均稳定;药物浓度超出曲线范围的样品经空白血浆稀释10倍后,其准确度在±11.2%以内,精密度为7.25%。本方法经验证后,成功应用于大鼠灌胃给予奥贝胆酸后的药动学研究。A simple and sensitive method was developed for quantitation of obeticholic acid in rat plasma with liquid chromatography-tandem mass spectrometry （LC-MS/MS）. After liquid-liquid extraction by methyl tert-butyl ether, the chromatographic separation was carried out on an ACE Excel 2 Super C18 column （50 mm×2.1 mm ID, 1.7 μm） with a gradient mobile phase consisting of acetonitrile and 2 mmol·L-1 ammonium formate at a flow rate of 0.2 mL·min-1. The quantitation analysis was performed using multiple reaction monitoring （MRM） at the specific ion transitions of m/z 418.9[M-H]-→401.2 for obeticholic acid and m/z 469.0[M-H]^-→ 425.2 for glycyrrhetinic acid （internal standard） in the negative ion mode with electrospray ionization （ESI） source. This validated LC-MS/MS method yielded a good linearity over the range of 5 -5 000 ng·mL^-1 with the lower limit of quantitation （LLOQ） of 5 ng·mL^-1. The intra and inter-assay precisions （RSD） were all less than 9.82% and the accuracy （RE） was within ±6.90%. The extraction recovery of obeticholic acid was from 85.4% to 88.5%, and the matrix effect of obeticholic acid ranged from 78.9% to 82.5%. Stability test suggest that obeticholic acid in rat plasma was stable for 24 h on workbench, up to 1 month at -70℃, and after three cycles of freeze-thaw. Extracted samples were stable for more than 24 h in an auto-sampler at 6℃. The precision was less than 7.25%, and the accuracy was within ±11.2%, after being diluted 10 times by blank rat plasma. The method has been successfully applied to a pharmacokinetic study of obeticholic acid in rats following oral administration at the dose of 2.5 mg·kg^-1.

关 键 词：奥贝胆酸 LC-MS/MS 血药浓度 药代动力学 原发性胆汁性胆管炎 

分 类 号：R917[医药卫生—药物分析学]