UPLC法Hilic模式下测定促胰岛素分泌肽类似物(Ex4c)有关物质

Determination of related substances of insulinotropic peptide analogue (Ex4c) in Hilic mode by UPLC

目的:建立UPLC法Hilic模式下测定促胰岛素分泌肽类似物(Ex4c)有关物质的方法。方法:色谱柱为Waters ACQUITY UPLC BEH Amide C18(150 mm×3.0 mm,1.7μm),流动相A为0.1%三氟乙酸水溶液,流动相B为0.085%三氟乙酸乙腈溶液,进行梯度洗脱,流速为0.6 m L·min^(-1),柱温为55℃,检测波长为214 nm,进样量为2μL。结果:Ex4c主峰与相邻杂质峰均能够分离,分离度均符合规定;Ex4c对酸、碱、氧化、高温均不稳定,主峰与各降解杂质峰均能够分离,且主峰的峰纯度均符合要求。Ex4c及各杂质在各自的线性范围内线性良好(相关系数r≥0.997 7,n≥5);杂质B,C,D的检测限分别为10.7,9.9和9.8 ng;低、中、高3种浓度(n=9)的平均回收率分别为109.73%,110.28%和92.97%。结论:经方法学验证,所建立的方法专属性、线性、准确度、重复性好,本法可用于Ex4c有关物质检查。

Objective： To establish a method for determination of related substances of insulinotropic peptide analogue （Ex4c） in Hilic mode by UPLC.Methods： The Waters ACQUITY UPLC BEH Amide C_（18） （150 mm × 3.0 mm,1.7 μm） column was used.The mobile phase A was 0.1% trifluoroacetic acid aqueous solution,and mobile phase B was 0.085% trifluoroacetic acid in acetonotrile.A gradient elution was performed at a flow rate of 0.6 m L·min^（-1）,while the column temperature was maintained at 55 ℃,and the detection wavelength was set at UV 214 nm with a 2 μL injection.Results： The main substance and its adjacent impurities could be separated adequately with a well-required resolution.Ex4c was not stable to acid,alkali,oxidation and high temperature,and the main peak was able to be effectively separated with the degradation impurities with qualified purity.The calibration curves of Ex4c and three impurities were linear in the concentration range （correlation coefficient r≥0.997 7,n≥5）.The detection limits of impurity B,C,D were 10.7,9.9 and 9.8 ng,respectively.The average recovery rates were 109.73%,110.28% and 92.97% respectively at low,medium and high concentrations （n = 9）.Conclusion： The method is validated that it has good specificity,linearity,accuracy,and repeatability.This method can be used for the related substance control of Ex4c.