UPLC-MS/MS测定人血浆中阿莫西林浓度及其在生物等效性研究中的应用  

Determination of amoxicillin concentration in human plasma by UPLC-MS/MS and its application in bioequivalence study

作　　者：唐维英 朱恒怡 李莎 杨姗姗 吴强 TANG Wei-ying;ZHU Heng-yi;LI Sha;YANG Shan-shan;WU Qiang(Sichuan Institute for Drug Control,NMPA Key Laboratory for Technical Research on Drug Products in Vitro and in Vivo Correlation,Chengdu 611731;Sichuan Shangrui Analysis and Testing Co.Ltd,Chengdu 610093)

机构地区：[1]四川省药品检验研究院国家药品监督管理局药物制剂体内外相关性技术研究重点实验室,成都611731 [2]四川尚锐分析检测有限公司,成都610093

出　　处：《中南药学》2024年第12期3253-3258,共6页Central South Pharmacy

摘　　要：目的 建立一套样品和沉淀剂用量更少,操作更简便的超高效液相色谱-串联质谱法(UPLC-MS/MS)测定人血浆中阿莫西林浓度,并应用于国产制剂阿莫西林胶囊与其参比制剂的生物等效性研究。方法 采用Waters Acquity UPLC BEH C18色谱柱(2.1 mm×50 mm,1.7 μm),以0.05%甲酸水溶液(A)-含0.1%甲酸的75%甲醇水溶(B)为流动相,流速0.35 mL·min^(-1)进行梯度洗脱,进样量1 μL,柱温 35℃,以ESI离子源、正离子MRM模式测定阿莫西林(m/z 366.1→114.0),阿莫西林-d4(m/z 370.2→114.0)作为内标。血浆样品依次加入内标、甲醇沉淀蛋白后,取上清液稀释后进样检测。结果 阿莫西林在20~8000 ng·mL^(-1)与测定值线性关系良好,定量下限为20 ng·mL^(-1),质控样品批内、批间精密度CV≤3.9%,准确度相对偏差在标示值-1.9%~3.9%,提取回收率、专属性、基质效应、稳定性等各项指标均符合《中国药典》四部通则中生物样品定量分析方法验证指导原则。本方法被成功应用于健康受试者口服250 mg阿莫西林胶囊的生物等效性研究,在餐后给药状态下,受试制剂和参比制剂C_(max)分别为(4922.16±967.16)和(5172.19±905.07)ng·mL^(-1),AUC_(0~12 h)分别为(14 165.22±1686.79)和(13 869.53±1939.31)h·ng·mL^(-1);在空腹给药状态下,受试制剂和受试制剂C_(max)分别为(6087.36±1766.53)和(5697.95±1768.73)ng·mL^(-1),AUC_(0~12 h)分别为(15 081.27±2342.12)和(14 564.04±2364.83)h·ng·mL^(-1),受试制剂与参比制剂C_(max)、AUC_(0~12 h)几何均值比的90%置信区间均落在80.00%~125.00%。结论 该方法灵敏度高、前处理过程简便,适用于血浆样品中阿莫西林的高通量分析;受试制剂阿莫西林胶囊与参比制剂具有生物等效性。Objective To establish a UPLC-MS/MS method with less sample and precipitant and easy operation to determine amoxicillin concentration in human plasma,and to use it in the bioequivalence study of amoxicillin capsules and their reference preparations.Methods A Waters Acquity UPLC BEH C18 (2.1 mm×50 mm,1.7 μm) column was used with the mobile phase consisting of 0.05% formic acid water (A) and 75% methanol solution (containing 0.1% formic acid) (B) in gradient elution.The flow rate was 0.35 mL·min^(-1) with the injection volume of 1 μL and the column temperature was 35℃.Amoxicillin (m/z 366.1→114.0) was detected by ESI ion source and positive ion MRM scanning mode,with amoxicillin-d4 (m/z 370.2→114.0) as the internal standard.After adding the internal standard,the plasma protein was precipitated by methanol,the supernatants were diluted and measured.Results The linearity ranged 20~8000 ng·mL^(-1) and the lower limit of quantification was 20 ng·mL^(-1).The intra-day and inter-day precision CV of quality-control samples was ≤3.9%,and the accuracy ranged -1.9%~3.9% in terms of relative error.The recovery rate,specificity,matrix effect and stability all met the guiding principles for verification of quantitative analysis of biological samples in the General Rules of Chinese Pharmacopoeia (four edition).The method was successfully applied to a bioequivalence study of amoxicillin orally disintegrating capsules containing 250 mg in healthy volunteers.In the fed test,the C_(max) of the test or reference formulation of amoxicillin capsules was (4922.16±967.16) and (5172.19±905.07) ng·mL^(-1);AUC_(0~12 h) was (14 165.22±1686.79) and (13 869.53±1939.31) h·ng·mL^(-1).In the fasting test,the C_(max) of the test or reference formulation of amoxicillin capsules was (6087.36±1766.53) and (5697.95±1768.73) ng·mL^(-1);AUC_(0~12 h) was (15 081.27±2342.12) and (14 564.04±2364.83) h·ng·mL^(-1).The 90% confidence intervals of the geometric mean ratios of C_(max),and AUC_(0~12 h) between the test and the referen

关 键 词：阿莫西林 抗菌药 血浆浓度 生物等效性 超高效液相色谱-串联质谱法 

分 类 号：R96[医药卫生—药理学]