目的: 建立采用超高效液相色谱-串联质谱法(UPLC-MS/MS)测定人胰岛素及其类似物全序列的方法。方法:该方法以人胰岛素为主要研究对象,首先对其进行还原烷基化前处理,后进行UPLC-MS/MS分析。采用ACQUITY UPLC peptide BEH C18(100 mm×2.1 mm, 1.7 μm, 300Å)色谱柱,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(0～10 min,3%B→60%B;10～10.5 min,60%B→95%B;10.5～14 min,95%B;14～14.5 min,95%B→3%B;14.5～20 min,3%B),流速0.3 mL·min-1,柱温50 ℃;采用电喷雾离子源正离子(ESI+)扫描,通过优化质谱参数,对人胰岛素及其类似物进行“top-town”全序列的测定分析,并对人胰岛素的序列测定方法进行了深入的研究,同时考察该方法对门冬胰岛素和赖脯胰岛素的适用性。结果:该方法能够较好地覆盖人胰岛素A和B链的全序列,通过b/y离子分析,5个高强度b/y离子的离子强度RSD在20%之内,A链与B链一级质量数准确,其精密度、准确度良好;该方法可区分与其他胰岛素类似物的序列的不同,专属性良好,且对门冬胰岛素和赖脯胰岛素具有良好的适用性。结论:该方法操作简便,准确性好,专属性强,为胰岛素及其类似物的全序列测定提供了数据支持。
Objective: To establish a UPLC-MS/MS method to identify the complete sequence of insulin and its analogues. Methods: This method took human insulin as the main research object.It was first pre-treated by reductive alkylation and then analyzed by UPLC-MS/MS. ACQUITY UPLC peptide BEH C18(100 mm×2.1 mm, 1.7 μm, 300Å) column was adopted, 0.1% formic acid/water for solvent A and 0.1% formic acid/acetonitrile for solvent B as mobile phase. The gradient mode was 3%-60% solvent B over 10 min, 10-10.5 min, 60%B→95%B; 10.5-14 min, 95%B; 14-14.5 min, 95%B→3%B; 14.5-20 min, 3%B at a flow rate of 0.3 mL·min-1, with column temperature 50 ℃. The top-town complete sequencing analysis of human insulin and its analogues was carried out by ESI+ scanning and optimized mass spectrometry parameters. The sequencing method of human insulin was studied in depth,and the applicability of the method to insulin aspart and insulin lispro was also investigated. Results: The method could cover the whole sequence of A and B chains of human insulin well. The RSD of ion strength of five high strength b/y ions was within 20% by b/y ions statistical analysis, The first-order mass number of chain A and B was accurate, with good precision and accuracy; the method could also distinguish the sequence differences from other insulin analogues with good specificity and has good applicability to insulin aspartic and insulin lypro. Conclusion: The method is simple, accurate and specific,and provides data support for the complete sequence determination of insulin and its analogues.
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