水包油乳剂中聚山梨酯80含量高效液相色谱检测方法的建立、验证及与其他方法的比较  

Development and verification of a high-performance liquid chromatography method for determination of polysorbate 80 content in oil-in-water emulsions and its comparison with other methods

作　　者：贾兰馨 年悬悬 马宁 郑嘉昊 聂希霖 张哲罡 张家友 JIA Lanxin;NIAN Xuanxuan;MA Ning;ZHENG Jiahao;NIE Xilin;ZHANG Zhegang;ZHANG Jiayou(Second Laboratory of Viral Vaccine Research,Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,Hubei Province,China)

机构地区：[1]武汉生物制品研究所有限责任公司病毒性疫苗研究二室,湖北武汉430207 [2]国家联合疫苗工程技术研究中心,湖北武汉430207

出　　处：《中国生物制品学杂志》2025年第2期172-178,184,共8页Chinese Journal of Biologicals

基　　金：湖北省重点研发计划(2020DCC002)。

摘　　要：目的建立及验证水包油乳剂中聚山梨酯80含量的高效液相色谱(high-performance liquid chromatography,HPLC)检测方法,并与改良硫氰酸钴铵比色法进行比较,以期用于水包油乳剂中聚山梨酯80含量的检测。方法采用Eclipse Plus C8、TSKgel G2000SWXL、ChromCore SAA色谱柱检测聚山梨酯80标准品或供试品,确定最佳色谱柱,并优化色谱条件中的流动相B洗脱梯度(以50%、75%、90%作为流动相B第2阶段的洗脱梯度,检测聚山梨酯80标准品溶液;以90%、75%作为流动相B第2阶段的洗脱梯度,检测供试品溶液)及流速(0.8、1.0、1.2 mL/min),验证方法的专属性、线性范围、重复性、准确性、耐用性,并比较该方法与改良硫氰酸钴铵比色法的线性范围、重复性和准确性。结果确定采用ChromCore SAA色谱柱;流动相A为0.1%(v/v)乙酸水溶液,流动相B为0.1%(v/v)乙酸异丙醇溶液;柱温为25℃;上样量为10μL;梯度洗脱,最佳流动相B第2阶段洗脱梯度为75%;流速为1.2 mL/min。聚山梨酯80标准品溶液及供试品溶液均于3.1 min可见聚山梨酯80色谱峰,供试品溶液在6 min可见其他色谱峰;聚山梨酯80标准品溶液浓度在300~1000μg/mL范围内,与峰面积呈良好的线性关系,回归方程为:y=1.44 x+4.91,r=0.9998;6份供试品溶液聚山梨酯80含量RSD为1.13%;低、中、高含量样品的加标回收率为96.51%~104.82%;供试品溶液于室温放置0、2、4、6、8 h,聚山梨酯80含量RSD为2.38%。两种方法的线性关系均良好;HPLC法的重复性及准确性均优于改良硫氰酸钴铵比色法。结论建立的HPLC法优于改良硫氰酸钴铵比色法,具有良好的专属性、精密性、准确性、耐用性,为含有多种表面活性剂的生物制品中聚山梨酯80含量的检测提供了可靠方法。Objective To develop and verify a high-performance liquid chromatography(HPLC)method for the determination of polysorbate 80 in oil-in-water emulsions,and to compare it with the modified cobalt ammonium thiocyanate colorimetric method for the determination of polysorbate 80 in oil-in-water emulsions.Methods Eclipse Plus C8 column,TSKgel G2000SW_(XL)column and ChromCore SAA column were used to detect polysorbate 80 standard or test sample to determine the optimum chromatographic column,and the elution gradient of mobile phase B(50%,75% and 90% as the elution gradient of mobile phase B in the second stage to detect polysorbate 80 standard solution;90% and 75% as the elution gradient of mobile phase B in the second stage to detect test sample solution)and the flow rate(0.8,1.0,1.2 mL/min)were optimized.The specificity,linear range,repeatability,accuracy and durability of the method were verified,and the linear range,repeatability and accuracy were compared with those of the modified cobalt ammonium thiocyanate colorimetric method.Results The conditions of HPLC method were as follows:ChromCore SAA chromatographic column was adopted at a column temperature of 25℃,the mobile phase A was aqueous solution containing 0.1%(v/v)acetic acid,and the mobile phase B was isopropanol containing 0.1%(v/v)acetic acid,with an injection volume of 10μL and gradient elution with the optimum elution gradient of 75%for mobile phase B in the second stage at a flow rate of 1.2 mL/min.Polysorbate 80 chromatographic peak was observed at 3.1 min in both polysorbate 80 standard solution and test solution,and other chromatographic peaks were observed at 6 min in test solution.In the concentration range of 300-1000μg/mL,polysorbate 80 standard solution exhibited good linear relationship with the peak area,and the regression equation was:y=1.44 x+4.91,r=0.9998.The RSD of polysorbate 80 content in six test solutions was 1.13%.The spike recovery rates of low,medium and high content samples were 96.51%-104.82%.The RSD of polysorbate 80 content w

关 键 词：水包油乳剂 聚山梨酯80 高效液相色谱法 硫氰酸钴铵比色法 

分 类 号：R373.13[医药卫生—病原生物学]