Ingredient Analysis

Determination of the primary structure of insulin glulisine injection by UPLC-Q TOF MS/MS*

  • MAO Zi-juan ,
  • XU Yan-mei ,
  • QIAO Xiao-ning ,
  • GAO Yan-xia
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  • 1. Hebei University of Science and Technology, School of Chemical and Pharmaceutical Engineering, Shijiazhuang 050091, China;
    2. Hebei Institute for Drug and Medical device Control, NMPA Key Laboraory for Quality Control and Evaluation of Generic Drug, Shijiazhuang 050227, China

Received date: 2024-09-06

  Online published: 2025-11-13

Abstract

Objective: To determine the primary structure of insulin glulisine injection using ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q TOF MS/MS). was used to determine the primary structure of insulin glulisine injection. Methods: The primary structure of insulin glulisine injection was analyzed by UPLC-Q TOF MS/MS following reductive alkylation pretreatment using dithiothreitol and iodoacetamide. The amino acid sequence was determined on a Waters ACQUITY UPLC CSH C18 column (3 mm×150 mm, 1.7 μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Employing gradient elution at a flow rate of 0.3 mL · min-1. Data acquisition utilized positive electrospray ionization mode and MSE acquisition mode. The mass spectrometry parameters were optimized to determine the primary structure of insulin glulisine injection, with concurrent assessment of the method's applicability to isophane protamine recombinant human insulin injection. Results: The primary mass numbers of insulin glulisine chain A and chain B were accurate, and the amino acid sequence coverage was satisfactory. For b/y ions, the RSD of intensity for the five highest-intensity b/y ions per chain (A and B) was below 10.0%. The method demonstrated specificity and repeatability, with confirmed applicability to isophane protamine recombinant human insulin injection. Conclusion: The method is simple, rapid, sensitive and accurate, and can be used for the primary structure determination of insulin glulisine injection and other insulin analogues drug products.

Cite this article

MAO Zi-juan , XU Yan-mei , QIAO Xiao-ning , GAO Yan-xia . Determination of the primary structure of insulin glulisine injection by UPLC-Q TOF MS/MS*[J]. Chinese Journal of Pharmaceutical Analysis, 2025 , 45(6) : 921 -931 . DOI: 10.16155/j.0254-1793.2024-1116

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