醋酸亮丙瑞林杂质谱研究和有关物质HPLC分析方法建立

目的：通过对醋酸亮丙瑞林的杂质谱研究，建立基于杂质谱的有关物质HPLC分析方法。方法：采用全覆盖键合硅胶色谱柱[Sepax GP-C₁₈（150 mm×4.6 mm，3 μm）]，以三乙胺缓冲液-正丙醇乙腈混合溶液为流动相，等度洗脱90 min，流速0.9 mL·min^-1，柱温35℃，检测波长220 nm，进样量20 μL。结果：醋酸亮丙瑞林与各已知杂质及强制破坏产生的降解产物分离良好；杂质B（2-D-His-leuprorelin）和杂质M（缺失肽4-Ser-leuprorelin）质量浓度在0.001~0.02 mg·mL^-1范围内呈良好的线性关系（r² ≥ 0.999，n=6），且校正因子（相对于醋酸亮丙瑞林）均为1.09；杂质B和M的平均回收率（n=9）分别为96.1%和108.3%，RSD分别为1.6%和3.0%；重复性试验杂质B、杂质M和总杂质RSD（n=6）分别为0.85%、1.9%和1.7%；各考察项下进样精密度试验中峰面积的RSD（n=5）最大值为1.4%，保留时间RSD（n=5）最大值为0.34%。经检测表明，稳定工艺的3批产品中，单个杂质均不大于鉴定限0.5%，总杂质均不大于0.6%。结论：本法可用于醋酸亮丙瑞林有关物质的检测。

Objective: To study the impurity profile of leuprorelin acetate and develop an HPLC method for the determination of related substances.Methods: The analysis was conducted on a full coverage chemically bonded silica column[Sepax GP-C₁₈ column(150 mm×4.6 mm, 3 μm)]with a mobile phase of triethylamine buffer, n-propyl alcohol and acetonitrile mixed solution by isocratic at the flow rate of 0.9 mL·min^-1 for 90 min. The column temperature was 35℃, the detection wavelength was 220 nm and the injection volume was 20 μL.Results: The known impurities and degraded products by forced degradation were completely separated from leuprorelin acetate. The calibration curves of impurity B(2-D-His-leuprorelin)and impurity M(deletion 4-Ser-leuprorelin)were linear in the self-concentration range of 0.001-0.02 mg·mL^-1(r² ≥ 0.999, n=6)and the correction factors were 1.09. The average recoveries of the impurities B and M(n=9)were 96.1% and 108.3%, respectively, and the RSDs (n=9)were 1.6% and 3.0%, respectively. The RSDs of repeatability(n=6)for impurity B, impurity M and total impurities were 0.85%, 1.9% and 1.7%, respectively. The RSD of peak area(n=5)for all validation items was no more than 1.4%, and the RSD of retention time was no more than 0.34%. The results showed that all the impurities were less than identification limit of 0.5% and the total impurities level was no more than 0.6% in the three batches leuprorelin acetate.Conclusion: The established method is suitable for the determination of the related substances in leuprorelin acetate.

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