UPLC-MS/MS法测定犬血浆中阿莫西林和克拉维酸片剂的生物等效性研究  

Determination of amoxicillin and clavulanic acid tabletsin dog plasma by UPLC-MS/MS method and study onthe bioequivalence of the formulation

作　　者：徐一丹 郭凡溪[1] 刘利锋[1,2] 张冯奕驰 王俊丽 邹远鹏 余元倩 余祖功 XU Yidan;GUO Fanxi;LIU Lifeng;ZHANG-FENG Yichi;WANG Junli;ZOU Yuanpeng;YU Yuanqian;YU Zugong(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Nanjing Lanboto Animal Pharmaceutical Co.Ltd.,Nanjing 210032,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]南京朗博特动物药业有限公司,江苏南京210032

出　　处：《南京农业大学学报》2023年第6期1116-1124,共9页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31572569)。

摘　　要：[目的]本文旨在建立检测犬血浆中阿莫西林和克拉维酸片的UPLC-MS/MS方法,并进行犬口服国产阿莫西林克拉维酸钾片与参比制剂的生物等效性评价。[方法]以乙腈和甲酸水溶液为流动相,经HSS T3色谱柱梯度洗脱,阿莫西林和克拉维酸分别用电喷雾正、负离子模式进行多反应监测(MRM),血浆样品用乙腈沉淀蛋白、二氯甲烷萃取,对于克拉维酸血浆样品的预处理增加正己烷除脂的步骤,经方法学验证后分别检测血浆样品中阿莫西林与克拉维酸浓度。生物等效性试验采用单剂量、双处理、双周期随机交叉试验设计,选用26只健康成年比格犬,按顺序分别空腹口服参比制剂或受试制剂250 mg,再用建立的UPLC-MS/MS法检测血浆中阿莫西林和克拉维酸的浓度;采用Phoenix Winnonlin 8.1药动学软件的非房室模型计算药物的药动学参数,对C_(max)、AUC_(0-t)和AUC_(0-∞)进行对数转换后分析药物生物等效性。[结果]该方法专属性良好;阿莫西林在20~10000 ng·mL^(-1)、克拉维酸在50~5000 ng·mL^(-1)浓度范围内的信号响应值与浓度线性关系良好(R^(2)>0.99);批内、批间准确度偏差均在±15%以内,批内、批间精密度均在15%以内;基质效应符合要求,阿莫西林和克拉维酸在试验条件下稳定。参比制剂和受试制剂中:阿莫西林的C_(max)分别为(10934.87±3118.79)ng·mL^(-1)和(11101.66±3185.09)ng·mL^(-1),AUC_(0-t)分别为(30983.24±9395.40)ng·h·mL^(-1)和(31646.91±9319.93)ng·h·mL^(-1),AUC_(0-∞)分别为(31093.42±9425.85)ng·h·mL^(-1)和(31843.77±9290.25)ng·h·mL^(-1);克拉维酸的C_(max)分别为(3494.90±1574.11)ng·mL^(-1)和(3694.26±1701.65)ng·mL^(-1),AUC_(0-t)分别为(5652.61±2409.17)ng·h·mL^(-1)和(6041.81±2458.42)ng·h·mL^(-1),AUC_(0-∞)分别为(5730.52±2392.78)ng·h·mL^(-1)和(6122.80±2469.20)ng·h·mL^(-1),C_(max)、AUC_(0-t)、AUC_(0-∞)经对数转换后几何均值比的90%置信区间均落在80.00%~125.00%,T_(max)经非[Objectives]The paper aimed to establish a UPLC-MS/MS method for the determination of amoxicillin and clavulanic acid in dog plasma,and to evaluate the bioequivalence of domestic amoxicillin-clavulanate potassium tablets and reference preparations orally in dogs.[Methods]Multi reaction monitoring(MRM)of acetonitrile and aqueous formic acid were used as mobile phases and gradient elution was performed on a HSS T3 column.Amoxicillin and clavulanic acid were monitored by electrospray in positive and negative ion modes,respectively.Plasma samples were precipitated with acetonitrile and extracted with dichloromethane.For the plasma sample pretreatment method of clavulanic acid,the step of n-hexane degreasing was added.After methodological validation,the concentrations of amoxicillin and clavulanic acid in plasma samples were detected respectively.The bioequivalence study adopted a single-dose,two-treatment,and two-cycle randomized crossover trial design.Twenty-six healthy adult beagle dogs were selected.The reference preparation or the test preparation 250 mg were orally adminstrated on an empty stomach in order,respectively.The concentrations of amoxicillin and clavulanic acid in plasma were detected by the established UPLC-MS/MS method.Pharmacokinetic parameters of the drug were calculated using the non-compartmental model of the Phoenix Winnonlin 8.1 pharmacokinetic software.The bioequivalence of C max,AUC_(0-t)and AUC_(0-∞)was evaluated after logarithmic conversion.[Results]The specificity of the method was suitable for detecting amoxicillin and clavulanic acid.The signal response values of amoxicillin in the concentration range of 20-10000 ng·mL^(-1)and clavulanic acid in the concentration range of 50-5000 ng·mL^(-1)had a good linear relationship with concentration(R^(2)>0.99).Intra-batch and inter-batch accuracy deviations and precision were both within 15%.The matrix effect met the requirements.Amoxicillin and clavulanic acid were stable under the test conditions.In the reference preparation and test prepar

关 键 词：阿莫西林 克拉维酸 UPLC-MS/MS 犬 生物等效性 

分 类 号：S859.7[农业科学—临床兽医学]