HPLC法测定普瑞巴林纯度及其10个工艺杂质

Determination of purity and ten process impurities in pregabalin API by HPLC

目的:建立HPLC法检测苯乙胺工艺普瑞巴林原料药中的有关物质及纯度。方法:采用Inertsil ODS-3(150 mm×4.0 mm,3μm)色谱柱,以缓冲液(7.05 g磷酸二氢铵和1.45 g磷酸氢二铵溶于1000 mL水中)-甲醇-乙腈(900∶80∶20)为流动相A,乙腈为流动相B,进行梯度洗脱,流速0.8 mL·min^(-1),检测波长210 nm,柱温30℃,进样量50μL。结果:普瑞巴林峰与各杂质及降解产物峰能够完全分离(分离度>2.0),供试品溶液在48 h内稳定性良好;其他单杂(用普瑞巴林进行验证)、内酰胺、4-烯普瑞巴林、5-烯普瑞巴林、三聚物、3-异丁基戊二酸(PGB-3)、3-异丁基戊二酸单酰胺(PGB-5)、R-苯乙胺、4-异丁基-2,6-哌啶二酮(PGB-5B)、单酰胺苯乙胺(PGB-5C)、二酸苯乙胺(PGB-5D)的定量限分别为0.05%、0.01%、0.01%、0.03%、0.01%、0.05%、0.03%、0.01%、0.01%、0.01%和0.01%;其他单杂(用普瑞巴林进行验证)、内酰胺、4-烯普瑞巴林、5-烯普瑞巴林、三聚物、PGB-3、PGB-5线性相关系数均>0.99,范围为LOQ~各杂质指标150%;内酰胺、4-烯普瑞巴林、5-烯普瑞巴林、三聚物、PGB-3、PGB-5平均回收率(n=9)分别为100.6%(RSD=0.56%)、100.2%(RSD=0.38%)、100.5%(RSD=0.46%)、101.1%(RSD=1.1%)、100.0%(RSD=0.63%)、100.0%(RSD=0.54%);重复性和中间精密度符合规定。经检测,3批普瑞巴林原料药6个月加速和60个月长期稳定性各个杂质检测结果均符合质量标准。结论:该方法简便快速,灵敏度高,专属性强,可用于苯乙胺工艺普瑞巴林原料药中有关物质及纯度的检测。

Objective:To establish an HPLC method for determination of related substances and purity in pregabalin API.Methods:The analytical column was an Inertsil ODS-3(150 mm×4.0 mm,3μm),the mobile phase A was buffer(7.05 g ammonium dihydrogen phosphate and 1.45 g diammonium hydrogen phosphate dissolved in 1000 mL water)-methanol-acetonitrile(900∶80∶20)and the mobile phase B was acetonitrile,the whole carried out by gradient elution at a flow rate of 0.8 mL·min^(-1),the detection wavelength was set at 210 nm,the column temperature was 30℃and the injection volume was 50μL.Results:Pregabalin was separated completely from the impurities and degradation products(the resolution>2.0).The test solution was stable for at least 48 h.The LOQs of other impurities(verification was performed by using pregabalin),lactam,4-alkene pregabalin,5-alkene pregabalin,trimer,3-isobutylglutaric acid(PGB-3),3-isobutyl glutaric acid monoamide(PGB-5),R-phenethylamine,4-isobutyl-2,6-piperidinedione(PGB-5B),monoamide phenylethylamine(PGB-5C)and phenylethylamine adipate(PGB-5D)were 0.05%,0.01%,0.01%,0.03%,0.01%,0.05%,0.03%,0.01%,0.01%,0.01%and 0.01%.The linear correlation coefficients of other impurities(verification was performed by using pregabalin),lactam,4-alkene pregabalin,5-alkene pregabalin,trimer,PGB-3 and PGB-5 were all more than 0.99.The linear ranges were LOQ-150%of impurities'specification,respectively.The average recoveries(n=9)of lactam,4-alkene pregabalin,5-alkene pregabalin,trimer,PGB-3 and PGB-5 were 100.6%(RSD=0.56%),100.2%(RSD=0.38%),100.5%(RSD=0.46%),101.1%(RSD=1.1%),100.0%(RSD=0.63%),100.0%(RSD=0.54%).The repeatability and intermediate precision completely met the requirements.The impurities contents in three batches of pregabalin API 6 months accelerate and 60 months long-term stability test completely met the requirements,respectively.Conclusion:This method is simple,rapid,sensitive and specific to be used for the determination of related substances and purity in phenethylamine process pregabalin API.