利用LC-MS/MS法解析化学修饰型胰岛素类似物的氨基酸序列  

Identification of amino acid sequence of chemically modified insulin analogues by LC-MS/MS

作　　者：胡馨月 丁晓丽[1] 孙悦[1] 秦希月 张慧[1] 李晶[1] 梁成罡[1] HU Xin-yue;DING Xiao-li;SUN Yue;QIN Xi-yue;ZHANG Hui;LI Jing;LIANG Cheng-gang(Division of Hormone,National Institutes for Food and Drug Control,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs,Bejing 102629,China;Jilin University,Changchun 130015,China)

机构地区：[1]中国食品药品检定研究院激素室,国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室,国家药品监督管理局化学药品质量研究与评价重点实验室,北京102629 [2]吉林大学,长春130015

出　　处：《中国新药杂志》2023年第3期301-310,共10页Chinese Journal of New Drugs

基　　金：中国食品药品检定研究院中青年发展研究基金课题项目(2022A3)。

摘　　要：目的:德谷胰岛素和地特胰岛素是以长链脂肪酸化学修饰为代表的第3代胰岛素类似物,有必要建立专属性强、灵敏度高的LC-MS/MS法测定化学修饰型胰岛素类似物的氨基酸序列。方法:氨基酸序列测定方法的色谱参数为ACQUITY UPLC peptide BEH C18色谱柱(100 mm×2.1 mm, 1.7μm, 300A),流动相A为0.1%甲酸/水溶液,流动相B为0.1%甲酸/乙腈溶液,梯度洗脱,流速0.3 mL·min^(-1);质谱参数为MSE采集模式,碰撞能量为30~40 eV。结果:德谷胰岛素和地特胰岛素序列覆盖率良好,化学修饰位点的碎片离子y1&可检出,中高强度离子的离子强度RSD在15%之内,可有效区分与其他未修饰胰岛素的序列。结论:建立了重复性好、专属性强和耐用性好的脂肪侧链修饰型胰岛素类似物氨基酸序列LC-MS/MS方法,为该类产品序列结构的研究方法提供了借鉴意义。Objective: Insulin degludec and insulin detemir are the third generation of insulin analogues that are chemically modified on long chain aliphatic acid. It is necessary to establish a specific and sensitive liquid chromatography-tandem mass spectrometry method for the determination of amino acid sequences of chemically modified insulin analogues. Methods: The separation was achieved by an ACQUITY UPLC peptide BEH C18column(100 mm×2.1 mm, 1.7 μm, 300 ?) with a mobile phase of 0.1% FA/H2O as Solvent A and 0.1% FA/acetonitrile as Solvent B in a gradient mode. The flow rate was set at 0.3 mL·min^(-1). MSEwas used as the detection mode of mass spectrometry, and the collision energy was 30~40 eV. Results: The coverage rate of insulin degludec and insulin detemir sequences was good as expected, and the fragment ion y1& was detected by chemical modification. RSD% of ionic strength of medium and high strength ions was within 15%. Both insulin sequences can be effectively distinguished it from other unmodified ones. Conclusion: A liquid mass spectrometry method for amino acid sequences of adipose-side chain modified insulin analogs with good repeatability, specificity and durability was established, providing reference for the research method of sequence structure of this kind of products.

关 键 词：脂肪侧链修饰型胰岛素类似物 德谷胰岛素 地特胰岛素 LC-MS/MS法 氨基酸序列定位 酶切 

分 类 号：R917[医药卫生—药物分析学]