Objective: To optimize the analysis method of related substances of insulin glargine injection, demonstrate the scientific limits for the current standards and identify the main related substances. Methods: The separation was performed on Thermo scientific Bio-Basic-C18 column (250 mm×4.6 mm, 5 μm). Sodium chloride, phosphate buffer and acetonitrile were mixed in a 18.4 g:250 mL:250 mL ratio, and added with water to 1 000 mL to be used as mobile phase A; sodium chloride, phosphate buffer, acetonitrile were mixed in a 3.2 g:250 mL:650 mL ratio,and added with water to 1 000 mL to be used as mobile phase B; gradient elution program was used at the flow rate of 1.0 mL·min-1. The detection wavelength was 214 nm and the column temperature was 35 ℃. The maximum content of related substances was collected and identified by LC-MS. The chromatographic separation was carried out on a column ACQUITY UPLC BEH C18 column(100 mm×2.1 mm, 1.7 μm, 300Å) with the mobile phase consisting of 0.1% formic acid aqueous solution (A)-0.1% formic acid acetonitrile solution (B) in a gradient mode. The column temperature was set to 50 ℃. The mass spectra was acquired with MSE mode. The primary mass spectrum energy was 40 V. Results: The RP-HPLC method for the analysis of related substances has good methodological verification results, and could effectively separate and accurately determine the related substances in the samples.It was identified that the main related substance of insulin glargine injection was the 3B-succinimide-insulin glargine,which was deaminated degradation product of asparagine at the third position of chain B. Conclusion: The optimized RP-HPLC method could be effectively used for the analysis of related substances in insulin glargine injection. The current national standards for related substances limits are scientific and reasonable, which could meet the quality control of insulin glargine injection during the shelf life.
DING Xiao-li, CHEN Ying, HU Xin-yue, LI Jing, ZHANG Hui, LIANG Cheng-gang
. Study on analytical methods of related substances in insulin glargine injection*[J]. Chinese Journal of Pharmaceutical Analysis, 2022
, 42(1)
: 23
-32
.
DOI: 10.16155/j.0254-1793.2022.01.03
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