目的: 优化甘精胰岛素注射液有关物质分析方法,论证现行标准规定限度科学性,并对主要有关物质进行鉴别。方法:采用Thermo scientific Bio-Basic-C18色谱柱(250 mm×4.6 mm,5 μm),以氯化钠-磷酸盐缓冲液-乙腈(18.4 g∶250 mL∶250 mL,加水至1 000 mL)为流动相A,氯化钠-磷酸盐缓冲液-乙腈(3.2 g∶250 mL∶650 mL,加水至1 000 mL)为流动相B,梯度洗脱,流速1.0 mL·min-1,检测波长214 nm,柱温35 ℃。利用该方法收集含量最大有关物质组分并利用LC-MS方法进行鉴别。分析色谱柱为ACQUITY UPLC BEH C18 Column(100 mm×2.1 mm,1.7 μm,300Å),以0.1%甲酸水溶液为流动相A,0.1%甲酸乙腈溶液为流动相B,进行梯度洗脱,柱温为50 ℃。质谱数据采集条件为MSE模式,一级质谱能量40 V。结果:该RP-HPLC有关物质分析方法,方法学验证结果良好,能够有效分离并准确测定样品中的有关物质;经鉴定,甘精胰岛素注射液的主要有关物质为B链第3位天冬酰胺的脱-NH3降解产物,即3B-琥珀酰亚胺-甘精胰岛素。结论:优化后的RP-HPLC方法能够有效用于甘精胰岛素注射液有关物质分析,现行国家标准有关物质限度规定科学合理,能够满足甘精胰岛素注射液货架期内的质控需求。
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.
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