LC-MS/MS同时测定人血浆中阿莫西林和克拉维酸及其生物等效性研究
Simultaneous determination of amoxicillin and clavulanic acid by LC-MS/MS in human plasma and its application to a bioequivalence study
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摘要: 建立了同时测定人血浆中阿莫西林和克拉维酸浓度的LC-MS/MS方法。采用50%乙酸水溶液作为稳定剂，采用冰浴条件下蛋白沉淀后取上清液吹干复溶的方法进行血浆样品前处理。采用Hedera ODS-2(2.1 mm×150 mm)色谱柱进行梯度洗脱，水相为含0.2%乙酸的水溶液，有机相为甲醇。选择三重四极杆串联质谱仪(Triple Quad TM 6500+)，采用电喷雾离子化方式(ESI)，在多反应监测(MRM)模式下进行负离子检测，同时对阿莫西林(364.1→223.1)和克拉维酸(198.1→135.9)进行定量分析。结果表明，阿莫西林在20.0~5 000 ng/mL范围内线性关系良好，克拉维酸在10.0~2 500 ng/mL范围内线性关系良好，批内和批间准确度偏差均在±15.0%内，批内和批间精密度均小于15.0%，基质效应、回收率均满足接受标准，阿莫西林和克拉维酸在试验条件下稳定性良好。采用该方法对健康受试者服用受试制剂阿莫西林克拉维酸钾颗粒1袋(每袋颗粒中含有125 mg的阿莫西林和31.25 mg的克拉维酸钾)和参比制剂阿莫西林克拉维酸钾干混悬剂Augmentin®； 5 mL(每5毫升干混悬剂中含有125 mg的阿莫西林和31.25 mg的克拉维酸钾)进行阿莫西林和克拉维酸的血药浓度测定，进行非房室模型药代动力学参数的估算分析，计算阿莫西林和克拉维酸的药代动力学参数，评价受试制剂与参比制剂是否等效，评估饮食对阿莫西林和克拉维酸的药代动力学的影响。实验结果表明，该方法经考察符合生物样品的分析要求，可以应用于阿莫西林克拉维酸临床血药浓度的测定和生物等效性研究。Abstract: In this paper, we developed an accurate and sensitive LC-MS/MS method for the determination of amoxicillin and clavulanic acid in human plasma. A 50% aqueous acetic acid solution was used as a stabilizer, and the plasma samples were evaporated to dryness and resolved after protein precipitation on ice bathing and then were placed in an autosampler for injection. The gradient was eluted by Hedera ODS-2 column(2. 1 mm×150 mm). The aqueous phase was an aqueous solution containing 0. 2% acetic acid. The organic phase was methanol. The amoxicillin and clavulanic acid were detected under negative ion detection with electrospray ionization(ESI)in multiple reaction monitoring(MRM)mode of m/z 364. 1→223. 1 and 198. 1→135. 9 in the triple quagdrupole tandem mass spectrometer(Triple Quad TM 6500+). The concentration ranges of plasma from 20. 0 ng/mL to 5 000 ng/mL for amoxicillin and 10. 0 ng/mL to 2 500 ng/mL for clavulanic acid were good linear relationship. The accuracy deviation were ±15. 0% and precision were less than 15. 0% for the intra-assay and inter-assay. The matrix effect and recovery meeted the acceptance criteria, amoxicillin and clavulanic acid were stable under storage and processing conditions. Healthy subjects were given a test preparation of amoxicillin and clavulanate potassium granules 1 bag(125 mg/31. 25 mg/bag)and the reference preparation amoxicillin clavulanate potassium dry mix Suspension “Augmentin® ” 5 mL(125 mg/31. 25 mg/5 mL)was used to determine the plasma concentration of amoxicillin and clavulanic acid. The Phoenix WinNonlin 6. 4 software was used to estimate the pharmacokinetic parameters of non-compartmental models. The pharmacokinetic parameters of amoxicillin and clavulanic acid were statistically calculated and evaluated the bioequivalence. what′s more, we evaluated the diet on the pharmacokinetics of amoxicillin and clavulanic acid. The analytical method was rapid and sensitive, which was successfully employed in the bioequivalence study of amoxicillin(125 mg/bag)and clavulanate potassium granules(31. 25 mg/bag)for determining the concentration of amoxicillin and clavulanic acid.
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Keywords:
- amoxicillin /
- clavulanic acid /
- LC-MS/MS /
- bioequivalence /
- pharmacokinetics
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