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.
[1] 李晶, 梁成罡, 张慧, 等. 重组甘精胰岛素相关有关物质的结构确认及质量分析[J].中国药学杂志, 2008, 43(20):1588
LI J, LIANG CG, ZHANG H, et al. Structure identification and quality analysis of relative impurities of recombinant insulin glargine[J].Chin Pharm J, 2008, 43(20):1588
[2] 王绿音, 杨慧敏, 李晶, 等. 甘精胰岛素肽图分析方法研究[J].生物技术通讯, 2018, 29(5):667
WANG LY, YANG HM, LI J, et al. Research of peptide mapping method for insulin glargine [J].Lett Biotechnol, 2018, 29(5):667
[3] BRANGE J, LANGKJAER L, HAVELUND S, et al. Chemical stability of insulin. 1.Hydrolytic degradation during storage of pharmaceutical preparations[J].Pharm Res, 1992, 9(6):715
[4] BRANGE J, LANGKJAER L. Chemical stability of insulin. 3.Influence of excipients, formulation and pH[J].Acta Pharm, 1992, 4(3):149
[5] BRANGE J. Chemical stability of insulin. 4.Mechanisms and kinetics of chemical transformations in pharmaceutical formulation[J].Acta Pharm, 1992 4(4):209
[6] PETERS B, TROUT BL. Asparagine deamidation: pH-dependent mechanism from density functional theory[J].Biochemistry, 2006, 45(16):5384
[7] ROBINSON NE, ROBINSON AB. Molecular clocks[J].Proc Natl Acad Sci USA, 2001, 98(3):944
[8] EP 10.0. VolⅡ[S].2020: 2571
[9] USP 43-NF 38. VolⅠ[S].2020: 2342
[10] 中华人民共和国药典2020年版. 三部[S].2020:390
ChP 2020. Vol Ⅲ[S].2020: 390
[11] 董标, 董方霆, 王京兰, 等. 毛细管区带电泳监测牛胰岛素的去折叠过程[J].分析化学, 2001, 29(5):538
DONG B, DONG FT, WANG JL, et al. Monitoring the process of bovine insulin unfolding by capillary zone electrophoresis[J].Chin J Anal Chem, 2001, 29(5):538
[12] 王勇, 李水明, 何曼文.溶菌酶基质辅助激光解吸电离-串联飞行时间质谱分析中的不常见修饰及脱水反应[J].分析化学, 2013, 41(4):494
WANG Y,LI SM,HE MW. Uncommon modifications and dehydration reaction in the analysis of lysozyme by matrix-assisted laser desorption ionization tandem time of flight-mass spectrometry[J].Chin J Anal Chem, 2013, 41(4):494
[13] 孙瑞祥, 罗兰, 迟浩, 等. 自顶向下(top-down)的蛋白质组学—蛋白质变体的规模化鉴定[J].生物化学与生物物理进展, 2015, 42(2):101
SUN RX, LUO L, CHI H, et al. Top-down proteomics-the large-scale proteoform identification[J].Prog Biochem Biophys, 2015, 42(2):101
[14] CUI W, ROHRS HW, GROSS ML. Top-down mass spectrometry: recent developments, applications and perspectives[J].Analyst, 2011, 136(19):3854
[15] 陈晶, 付华, 陈益, 等.质谱在肽和蛋白质序列分析中的应用[J].有机化学, 2002, 22(2):81
CHEN J, FU H, CHEN Y, et al. Application of mass spectrometry on the sequencing of peptides and proteins[J].Chin J Organ Chem, 2002, 22(2):81
[16] 陈海平. 有关物质检查分析方法建立过程中破坏性试验的意义和存在的问题分析[J].中国新药杂志, 2008, 17(17):1548
CHENG HP. Effects of stressing test for determination of impurities during validation of the analytical procedure[J].Chin J New Drugs, 2008, 17(17):1548
[17] 中华人民共和国药典2020年版. 四部[S].2020: 457
ChP 2020. Vol Ⅳ[S].2020: 457
