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, 3 μm)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1 000 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50 μL。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, 3 μm), the mobile phase A was buffer (7.05 g ammonium dihydrogen phosphate and 1.45 g diammonium hydrogen phosphate dissolved in 1 000 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 50 μL. 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.
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