测定唾液酸含量的改良间苯二酚-盐酸法的建立及验证  

Development and validation of an improved resorcinol-hydrochloric acid assay for sialic acid content

作　　者：袁菲[1] 于旭博 孔素娟[1] 王晶[1] 王欣茹[1] 吴兵[1] 刘方蕾[1] 谢贵林[1] 

机构地区：[1]兰州生物制品研究所有限责任公司甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《中国生物制品学杂志》2016年第10期1087-1093,共7页Chinese Journal of Biologicals

基　　金：甘肃省科技重大专项(0801NKDA003);甘肃省"十二五陇药产业发展"专项

摘　　要：目的建立测定脑膜炎球菌多糖、多糖衍生物及结合物中唾液酸含量的改良间苯二酚-盐酸法,并进行验证。方法以《中国药典》三部（2015版）测定多糖中唾液酸含量的间苯二酚-盐酸法为基础进行改良,去除有机相萃取步骤,优化方法中的检测波长（300~900 nm）及煮沸时间（10、15、20、25、30、35和40 min）;并对建立方法的线性范围、中间精密度及准确度进行验证。比较改良法与《中国药典》法对样品唾液酸含量及其层析分部产物中唾液酸含量测定结果的差异,同时检测乳糖对改良法测定唾液酸含量的影响。结果方法中最佳检测波长为569 nm,最佳煮沸时间为30 min。唾液酸对照品浓度在5~40μg/ml范围内与569 nm吸收值呈良好的线性关系,R2均〉0.999;3位实验人员在3个不同时间对样品中唾液酸含量测定9次结果的CV均〈3%;外加5、10、20μg/ml浓度的N-乙酰神经氨酸的回收率均在95%~105%之间。改良法与《中国药典》法检测的样品唾液酸含量差异无统计学意义（P〉0.05）,且两种方法的层析分部图谱一致。乳糖溶液浓度超过100μg/ml时对唾液酸测定结果的干扰较大。结论本实验建立的改良唾液酸测定法具有良好的线性、精密度和准确度,可用于脑膜炎球菌多糖、多糖衍生物和结合物唾液酸含量的测定。Objective To develop and validate an improved resorcinol-hydrochloric acid assay for sialic acid content in meningococcal polysaccharides, polysaccharide derivatives and conjugates. Methods The resorcinol-hydrochloric acid assay for sialic acid content in polysaccharide in Chinese Pharmacopoeia（Volume Ⅲ, 2015 edition） was improved by removing the step of extraction in organic phase and optimizing the wavelength for detection（300 ~ 900 nm） and time for boiling（10, 15, 20, 25, 30, 35 and 40 min）, and verified for linear range, intermediate precision and accuracy. The determination results by the original and improved methods were compared, based on which the effect of lactose on determination of sialic acid content by the improved resorcinol-hydrochloric acid assay was evaluated. Results The optimal wavelength for detection in the improved method was 569 nm, while the optimal time for boiling was 30 min.The concentration of sialic acid reference within a range of 5 ~ 40 μg / ml showed good linear relationship to the A569value（each R^2〉0. 999）. All the CVs of nine determination results of sialic acid contents in samples by three personnel at three time points were less than 3%. All the recovery rates of N- acetylneuraminic acid at concentrations of 5, 10 and 20 μg / ml were 95% ~ 105%. The sialic acid contents determined by original and improved methods showed no significant difference（P〉0. 05）, while the chromatographic profiles of fractional samples were in agree-ment. Lactose at a concentration of more than 100 μg / ml showed significant interference to the determination result of sialic acid. Conclusion The improved resorcinol-hydrochloric acid assay showed good linearity, precision and accuracy,which might be used for determination of sialic acid content in meningococcal polysaccharide, polysaccharide derivatives and conjugates.

关 键 词：间苯二酚-盐酸法 唾液酸含量 

分 类 号：O656.33[理学—分析化学]