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.
HU Xin-yue, DING Xiao-li, CHEN Ying, ZHANG Hui, LI Jing, LIANG Cheng-gang
. Analysis and study on the complete sequence of insulin and its analogues by UPLC-MS/MS*[J]. Chinese Journal of Pharmaceutical Analysis, 2022
, 42(1)
: 13
-22
.
DOI: 10.16155/j.0254-1793.2022.01.02
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