人干扰素α2a注射液中对羟基苯甲酸甲酯和对羟基苯甲酸丙酯含量反相高效液相色谱检测方法的建立及验证  

Development and verification of reversed-phase high performance liquid chromatography for determination of methyl p-hydroxybenzoate and propyl p-hydroxybenzoate contents in human interferon α2a injection

作　　者：何珊 于雷[2] 贾丹 靳征 袁杰 史新昌[2] 裴德宁[2] HE Shan;YU Lei;JIA Dan;JIN Zheng;YUAN Jie;SHI Xin-chang;PEI De-ning(Shenyang Sunshine Pharmaceutical Co.,Ltd.,Shenyang 100271,Liaoning Province,China)

机构地区：[1]沈阳三生制药有限责任公司,辽宁沈阳100271 [2]中国食品药品检定研究院,北京100050

出　　处：《中国生物制品学杂志》2021年第8期984-988,共5页Chinese Journal of Biologicals

基　　金：“十三五”重大新药专项,创新生物技术药评价及标准化关键技术研究(2018ZX09101001)。

摘　　要：目的建立测定人干扰素α2a注射液中对羟基苯甲酸甲酯和对羟基苯甲酸丙酯含量的反相高效液相色谱(reversed-phase high performance liquid chromatography,RP-HPLC)法,并进行验证。方法色谱条件:色谱柱为C_(18)色谱柱(5μm,250 mm×4.6 mm),流动相为甲醇-1%冰乙酸(60∶40),流速为1 mL/min,检测波长为254 nm,柱温为25℃;使用标准曲线法进行计算,并验证方法的专属性、线性、准确度、精密度和耐用性。用建立的方法测定6批重组人干扰素α2a注射液中对羟基苯甲酸甲酯和对羟基苯甲酸丙酯的含量。结果供试品待测成分色谱峰之间、待测成分色谱峰与其他色谱峰之间分离度均大于1.5,方法专属性良好;对羟基苯甲酸甲酯和对羟基苯甲酸丙酯含量分别在0.002%~0.012%和0.000 2%~0.001 2%范围内线性关系良好;对羟基苯甲酸甲酯和对羟基苯甲酸丙酯含量的平均加标回收率分别为99.6%和99.2%,重复性检测的RSD分别为0.05%和0.07%;该方法对不同色谱柱品牌、不同流动相成分比例、不同柱温的耐用性良好;6批样品中对羟基苯甲酸甲酯和对羟基苯甲酸丙酯含量分别为0.058%~0.060%和0.005 7%~0.005 9%,与投料量非常接近。结论建立的测定人干扰素α2a注射液中对羟基苯甲酸甲酯和对羟基苯甲酸丙酯含量的RP-HPLC法操作简便,具有良好的专属性、线性、准确度、精密度和耐用性,可代替气相色谱法,用于人干扰素α2a注射液中对羟基苯甲酸甲酯和对羟基苯甲酸丙酯含量的测定。Objective To develop and verify a reverse-phase high performance liquid chromatography(RP-HPLC)for determination of methyl p-hydroxybenzoate and propyl p-hydroxybenzoate contents in human interferon(IFN)α2a injection.Methods C18 column(5μm,250 mm×4.6 mm)was adopted,serving methanol-1%glacial acetic acid(60∶40)as mobile phase at a flow rate of 1 mL/min,a detection wavelength of 254 nm,and a column temperature of 25℃.The result was calculated by standard curve method.The developed method was verified for specificity,linearity,accuracy,precision and durability,by which the methyl p-hydroxybenzoate and propyl p-hydroxybenzoate contents in six batches of IFNα2a injection were determined.Results All the resolutions between chromatographic peaks of the components to be determined and between the chromatographic peaks of the components to be determined and other chromatographic peaks were more than 1.5,indicating a good specificity of the method.The linear ranges of methyl phydroxybenzoate and propyl p-hydroxybenzoate contents were 0.002%~0.012%and 0.0002%~0.0012%respectively.The average spike recoveries of methyl p-hydroxybenzoate and propyl p-hydroxybenzoate were 99.6%and 99.2%,respectively.The RSDs in reproducibility test for methyl p-hydroxybenzoate and propyl p-hydroxybenzoate were 0.05%and0.07%respectively.The method showed good durability for different brands of chromatographic columns,different proportions of mobile phase components and different column temperatures.The contents of methyl hydroxybenzoate and propyl p-hydroxybenzoate in six batches of samples were 0.058%~0.060%and 0.0057%~0.0059%respectively,which were significantly closed to the feeding amounts.Conclusion The developed RP-HPLC method is simple,specific,linear,accurate,precise and durable,which may be used for the determination of methyl p-hydroxybenzoate and propyl phydroxybenzoate contents in human IFNα2a injection instead of gas chromatography.

关 键 词：人干扰素α2a注射液 对羟基苯甲酸甲酯 对羟基苯甲酸丙酯 反相高效液相色谱法 

分 类 号：R917[医药卫生—药物分析学]