免疫亲和富集-超高效液相色谱-质谱法检测人血浆中阿达木单抗浓度  

Determination of Adalimumab in Human Plasma by Immuno-Affinity Enrichment and UPLC-Q Exactive-Orbitrap MS

作　　者：丁肖梁[1,2] 朱圣雄 刘林生[1,2] 刘筱雪 缪丽燕[1,2] DING Xiaoliang;ZHU Shengxiong;LIU Linsheng;LIU Xiaoxue;MIAO Liyan(Department of Pharmacy,The First Affiliated Hospital of Soochow University,Suzhou 215006,China;Institute for Interdisciplinary Drug Research and Translational Sciences,Soochow University,Suzhou 215006,China)

机构地区：[1]苏州大学附属第一医院药学部,江苏苏州215006 [2]苏州大学药物研究与转化交叉研究所,江苏苏州215006

出　　处：《中国药学杂志》2025年第1期71-76,共6页Chinese Pharmaceutical Journal

基　　金：国家自然科学基金青年项目资助(82003857);苏州市姑苏卫生人才计划项目资助(GSWS2019001)。

摘　　要：目的基于超高效液相色谱-四极杆-静电轨道阱-高分辨质谱(UPLC-Q Exactive-Orbitrap MS)平台联合免疫亲和富集策略建立一种高效的阿达木单抗血药浓度定量分析方法。方法采用高分辨质谱的全扫描/二级离子扫描模式筛选候选特征肽,采用平行反应监测模式对特征肽进行定量分析;前处理采用蛋白A免疫磁珠捕获人血浆中免疫球蛋白和治疗性抗体,蛋白经高温变性和胰酶酶解成多肽后通过质谱分析;采用同位素标记的阿达木单抗作为内标。结果筛选得到位于阿达木单抗重链可变区GLEWVSAITWNSGHIDYADSVEGR肽段具有较好的特异性和选择性,可作为阿达木单抗定量分析的特征肽段。阿达木单抗在1~32μg·mL^(-1)内呈良好的线性关系,精密度、准确度和总误差均符合验证要求。31个临床真实样本同时采用该质谱方法和酶联免疫吸附试验(ELISA)方法检测,Passing-Bablok回归显示两种方法检测结果相关性较好,两种方法的浓度差均值为0.5μg·mL^(-1)。结论本研究建立了一种可用于抗体药物定量分析的通用型免疫亲和质谱方法,阿达木单抗血药浓度的质谱方法经验证可适用于临床样本检测。OBJECTIVE To establish an efficient method for quantification of adalimumab in human plasma based on UPLC-Q Exactive-Orbitrap MS platform combined with immuno-affinity enrichment strategy.METHODS Candidate surrogate peptides were screened by full MS/ddMS2 and the selective surrogate peptide was quantitatively analyzed by parallel reaction monitoring.Immunoglobulins and therapeutic antibodies in human plasma were extracted by magnetic beads coupled with protein A.The proteins were denatured at high temperature and digested by trypsin.Stable-isotope labeled adalimumab was used as internal standard.RESULTS The peptide GLEWVSAITWNSGHIDYADSVEGR in variable region of adalimumab heavy chain was selected as signature peptide,showing specificity and selectivity.Adalimumab demonstrated good correlation within the range of 1-32μg·mL^(-1).Precision,accuracy and total error all met the verification requirements.Thirty-one clinical samples had measurable adalimumab concentrations by the established method and ELISA method,yielding a good correlation.The mean of difference between the two methods was 0.5μg·mL^(-1).CONCLUSION The universal immuno-affinity mass spectrometry method established in the study is suitable for quantification of therapeutic antibodies,and can accurately and precisely determine adalimumab concentration,which provides a strategy for clinical monitoring.

关 键 词：阿达木单抗 质谱 治疗药物监测 

分 类 号：R969[医药卫生—药理学]