UPLC-FLR测定长效人红细胞生长刺激素中唾液酸含量  

Determination of Sialic Acid in Long-acting Human Erythropoiesis Stimulating Protein by Ultra-performance Liquid Chromatography-Fluorescence Detection

作　　者：吕素娟 刘作龙 王玉营 王惠琰 王云飞 LYU Su-juan;LIU Zuo-long;WANG Yu-ying;WANG Hui-yan;WANG Yun-fei(Shandong Feng Jin Biopharmaceutical Co.,Ltd.,Yantai 264000,China)

机构地区：[1]山东丰金生物医药有限公司,山东烟台264000

出　　处：《食品与药品》2022年第5期416-420,共5页Food and Drug

摘　　要：目的建立超高效液相色谱-荧光检测法(UPLC-FLR)用于测定长效人红细胞生长刺激素中两种唾液酸组分N-乙酰神经氨酸和N-羟乙酰神经氨酸。方法将长效人红细胞生长刺激素原液经乙酸水解,4,5-亚甲二氧基-1,2-苯二胺盐酸盐试剂标记前处理,采用Acclaim RSLC 120 C_(18)色谱柱和流动相(甲醇:乙腈:超纯水=7:9:84)洗脱分离,采用荧光检测器测定各组分的荧光。使用唾液酸标准品外标法进行定量分析。结果此法专属性较强,N-乙酰神经氨酸准确度回收率为89.6%~95.1%,RSD为2.5%,N-羟乙酰神经氨酸回收率为90.9%~97.4%,RSD为2.9%;6份样品重复性RSD<1.0%。线性相关系数(r^(2))均>0.99,线性良好。12 h进样品回收率RSD<1.0%。结论此法可对长效人红细胞生长刺激素唾液酸N-乙酰神经氨酸和N-羟乙酰神经氨酸的含量进行准确分析。Objective To develop a method of ultra-performance liquid chromatography-fluorescence detection(UPLC-FLR)for the determination of N-acetylneuraminic acid and N-hydroxy acetylneuraminic acid in long-acting human erythropoiesis stimulating protein.Methods The long-acting human erythropoiesis stimulating protein bulk was firstly hydrolyzed with acetic acid solution,and secondly labeled with 4,5-methylenedioxy-1,2-phenylenediamine hydrochloride reagent.Then,the pretreated sample was eluted using mobile phase(methanol:acetonitrile:ultra-pure water=7:9:84)on an Acclaim RSLC 120 C_(18) column,and quantified by sialic acid external standard method with a fluorescence detector.Results The method displayed strong specificity.The accuracy of N-acetylneuraminic acid ranged from 89.6%to 95.1%with the RSD of 2.5%.The accuracy of N-hydroxy acetylneuraminic acid ranged from 90.9%to 97.4%with the RSD of 2.9%.The repeatability RSD of 6 samples was less than 1.0%.This method was also good at linearity with a linearity correlation coefficient(r^(2))above 0.99.The RSD of 12 h sample recoveries was less than 1.0%.Conclusion This method can accurately analyze the contents of N-acetylneuraminic acid and N-hydroxy acetylneuraminic acid in long-acting human erythropoiesis stimulating protein.

关 键 词：唾液酸 N-乙酰神经氨酸 N-羟乙酰神经氨酸 4 5-亚甲二氧基-1 2-苯二胺盐酸盐衍生 超高效液相色谱-荧光检测法 

分 类 号：R917[医药卫生—药物分析学]