采用荧光高效液相色谱法测定人和大鼠血清中的单-O-乙酰化N-乙酰神经氨酸。

Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography.

作者信息

Hara S, Yamaguchi M, Takemori Y, Furuhata K, Ogura H, Nakamura M

机构信息

Faculty of Pharmaceutical Sciences, Fukuoka University, Japan.

出版信息

Anal Biochem. 1989 May 15;179(1):162-6. doi: 10.1016/0003-2697(89)90218-2.

DOI:10.1016/0003-2697(89)90218-2

PMID:2757191

Abstract

A simple, rapid, and highly sensitive fluorometric high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and its mono-O-acetyl derivatives in human and rat sera is described. The neuraminic acids, released by hydrolysis of serum in 2 M acetic acid, are converted with 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives without the occurrence of O-acetyl migration and de-O-acetylation. The derivatives are separated isocratically within 25 min by reversed-phase chromatography using a TSK gel ODS-120T column. The limits of detection are 57-192 fmol in a 10-microliters injection volume at a signal-to-noise ratio of 3. This sensitivity permits precise determination of the neuraminic acids in 5 microliters of human and rat sera.

摘要

描述了一种简单、快速且高灵敏度的荧光高效液相色谱法，用于测定人和大鼠血清中的N-乙酰神经氨酸及其单-O-乙酰基衍生物。血清在2M乙酸中水解释放出的神经氨酸，与α-酮酸的荧光试剂1,2-二氨基-4,5-亚甲二氧基苯反应，转化为高荧光衍生物，且不会发生O-乙酰基迁移和脱O-乙酰化。使用TSK凝胶ODS-120T柱通过反相色谱在25分钟内等度分离衍生物。在信噪比为3时，10微升进样体积的检测限为57 - 192飞摩尔。这种灵敏度允许精确测定5微升人和大鼠血清中的神经氨酸。