醋酸曲普瑞林缓释注射剂中杂质的检测分析

目的：对醋酸曲普瑞林缓释注射剂中的未知杂质进行检测和结构确证。方法：采用高效液相色谱法进行杂质分离检测，使用XBridge^? Peptide BEH C₁₈色谱柱（4.6 mm×250 nm，3.5 μm，300?），以磷酸-三乙胺缓冲液及乙腈-正丙醇为流动相体系，流速1 mL·min^-1，柱温35℃，检测波长210 nm；采用高效液相色谱-线性离子阱串联质谱法（HPLC-LTQ-MS/MS法）对未知杂质进行结构确证，采用Kromasil C₁₈预柱（2.1 mm），柱温30℃，检测波长210 nm，纯水（含0.05%甲酸）为流动相A，甲醇为流动相B，梯度洗脱，流速0.2 mL·min^-1；电喷雾电离源（ESI），正离子模式，扫描范围m/z 150~1 500，毛细管电压35 V，毛细管温度275℃，喷雾电压4 kV，鞘气速度35 arb，辅助气流速10 arb。结果：对3种来源的缓释制剂进行了检测，发现并确证了25种未知杂质的结构，并进行了来源归属，其中工艺杂质有缺失肽、插入肽、光学异构体，降解杂质有氧化杂质、还原杂质、酰化杂质等。结论：不同企业醋酸曲普瑞林缓释注射剂中的杂质与制剂工艺有关，曲普瑞林与辅料聚乳酸聚乙醇酸（PLGA）相互作用生成的酰化杂质，酰化位点为肽链4位丝氨酸残基上的游离羟基。

Objective: To study and confirm the structure of the impurity profiles in the sustained-release injection of triptorelin acetate. Methods: Impurities were detected and separated by high performance liquid chromatography (HPLC). The HPLC separation was carried out on XBridge^? Peptide BEH C₁₈ column (4.6 mm×250 nm,3.5 μm,300 ?),with a mobile phase consisting phosphate-triethylamine buffer and acetonitrile n-propanol system at a flow rate of 1 mL·min^-1,a column temperature of 35℃,a detection wavelength of 210 nm. The high performance liquid chromatography-multiple stage Orbitrap mass spectrometric method (HPLC-LTQ-MS/MS method) was developed to confirm the structure of its unknown impurify. The desalination of impurities was carried out on Kromasil C₁₈ pre-column (2.1 mm),with a mobile phase consisting of 0.05% formic acid aqueous solution (A) and methanol (B) at a flow rate of 0.2 mL·min^-1 by gradient elution. The MS settings were as follows:scan range,m/z 150-1,500;capillary voltage,35 V;capillary temperature,275℃;spray voltage,4 kV;sheath gas, 35 units;sweep gas,10 units. Results: The structures of 25 kinds of unknown impurities in the sustained-release injection of triptorelin acetate from three different sources were determined by the information of HPLC-MS-MS,and its origin was attributed to be the drug-excipient adducts related to the manufacturing processes. Process-related impurities were found in the production process including missing peptide and insert peptide ligands,optical isomers and degradation of impurities (oxide impurities,reduced impurities and acylated impurities). Conclusion: The impurities of the sustained-release injection of triptorelin acetate from different enterprises are all related to the manufacturing processes. The formation mechanism is identified to be the pharmaceutic adjuvant[poly (lactic-co-glycolic acid),PLGA] interaction with the 4'-OH Ser of triptorelin to generate acylated impurities.

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