超高效液相色谱法测定Ex4c的有关物质

Determination of Related Substances in Ex4c by UPLC

摘要: 目的：建立超高效液相色谱法(UPLC)测定聚乙二醇化促胰岛素分泌肽类似物原料Ex4c有关物质的方法。方法：采用Waters ACQUITY UPLC® Peptide CSH™C18 column (130 Å, 2.1 × 150 mm, 1.7 mm)为色谱柱；以0.05%三氟乙酸水溶液-四氢呋喃(9:1)为流动相A，0.05%三氟乙酸乙腈溶液-四氢呋喃(9:1)为流动相B；进行梯度洗脱。柱温45℃，检测波长为214 nm，进样量为2 ml，流速为0.3 mL∙min−1。结果：杂质A、B、C、D线性良好，相关系数r均 ≥ 0.995；4种杂质的检测限范围为4.0~5.6 ng；平均回收率为96.3%~101.1%。结论：经方法学验证，所建立的方法简便、准确、重复性好，可用于测定聚乙二醇化促胰岛素分泌肽类似物原料Ex4c的有关物质，有效地控制药品质量。

Abstract: Objective: To establish a method for the determination of related substances in Ex4c (the raw ma-terial of the pegylation of glucagon-like peptide-1) by UPLC. Methods: The UPLC method was de-veloped by using a Waters ACQUITY UPLC® Peptide CSH™C18 column (130 Å, 2.1 × 150 mm, 1.7 mm) with a gradient elution system. The mobile phase A was 0.05% trifluoroacetic acid aqueous solution/tetrahydrofuran (9:1), and the mobile phase B was 0.05% trifluoroacetic acid in acetoni-trile/tetrahydrofuran (9:1). The column temperature was maintained at 45˚C while the detection wavelength setting at 214 nm. Furthermore, the sample injection was 2 ml via keeping a flow rate of 0.3 mL∙min−1. The developed method was validated. Results: The resolution between Ex4c and adjacent impurities, between Ex4C and the known impurities were greater than 1.5. All four known impurities exhibited good linear relationships with their related coefficients ≥ 0.995. And their detection limits ranged from 4.0 ng to 5.6 ng, comprising 96.3%~101.1% of recovered material. Conclusion: The validation results showed that the established method was simple, accurate and reproducible. It is capable of determinating impurities in Ex4c (the raw material of the pegylation of glucagon-like peptide-1) and can be effectively used for quality control of this drug.