药学学报, 2018, 53(2): 271-277
张如月, 谷元, 张爱杰, 董世奇, 李全胜, 魏广力, 司端运. LC-MS/MS法测定大鼠血浆中奥贝胆酸的浓度[J]. 药学学报, 2018, 53(2): 271-277.
ZHANG Ru-yue, GU Yuan, ZHANG Ai-jie, DONG Shi-qi, LI Quan-sheng, WEI Guang-li, SI Duan-yun. Determination of obeticholic acid in rat plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS)[J]. Acta Pharmaceutica Sinica, 2018, 53(2): 271-277.

张如月1,2, 谷元2, 张爱杰2, 董世奇3, 李全胜2, 魏广力2, 司端运2
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
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,1.7 μm)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 mL·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·mL-1浓度内线性关系良好(r2 > 0.99),定量下限为5 ng·mL-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。
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     
Determination of obeticholic acid in rat plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
ZHANG Ru-yue1,2, GU Yuan2, ZHANG Ai-jie2, DONG Shi-qi3, LI Quan-sheng2, WEI Guang-li2, SI Duan-yun2
1. Tianjin Medical University Graduate School, Tianjin 300070, China;
2. State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin 300193, China;
3. Tianjin University, Tianjin 300191, China
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.
Key words:    obeticholic acid    LC-MS/MS    plasma concentration    pharmacokinetics    primary biliary cholangitis   
收稿日期: 2017-09-15
DOI: 10.16438/j.0513-4870.2017-0841
基金项目: 国家自然科学基金青年科学基金资助项目(81503154).
通讯作者: 司端运,Tel/Fax:86-22-84845261,E-mail:sidy@tjipr.com
Email: sidy@tjipr.com
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谷元  在本刊中的所有文章
张爱杰  在本刊中的所有文章
董世奇  在本刊中的所有文章
李全胜  在本刊中的所有文章
魏广力  在本刊中的所有文章
司端运  在本刊中的所有文章

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