高效液相色谱法测定母乳中唾液酸含量

建立荧光高效液相色谱(fluorescence detector-high performance liquid chromatography，HPLC-FLD)测定母乳中唾液酸N-乙酰神经氨酸(N-acetylneuraminic acid，Neu5Ac)和N-羟乙酰神经氨酸(N-glycolyl neuraminic acid，Neu5Gc)含量的分析方法。利用酸水解法释放出母乳中的唾液酸，以4,5-亚甲二氧基-1,2-邻苯二胺盐(4,5-methylenedioxy-1,2-phenylenediamine dihydrochloride，DMB)为衍生化试剂，50℃避光衍生150min，采用荧光高效液相色谱仪检测。色谱条件：LiChrosorb RP-18柱(250mm×4mm，5μm)，流动相为甲醇-乙腈-超纯水(7:8:85)，流速0.9mL/min，进样体积10μL，柱温30℃，荧光检测器激发波长373nm，发射波长448nm。结果表明：唾液酸在50～400μmol/L范围内与唾液酸峰面积的线性关系良好，平均回收率为94.0%，精密度的相对标准偏差(relative standard deviation，RSD)为0.4%，稳定性RSD为1.0%，重复性RSD为0.8%，Neu5Ac的最低检出限为0.02μmol/L，Neu5Gc的最低检出限位0.03μmol/L。该方法简单、重复性好、灵敏度高，可广泛用于奶粉、牛奶及母乳中唾液酸含量测定。

Determination of Total Sialic Acid Content in Human Milk by HPLC-FLD

An analytical method was developed for determining total content of sialic acids including N-acetylneuraminic acid (Neu5Ac) and N-glycolyl neuraminic acid (Neu5Gc) in human milk. Sialic acid in human milk was released by acid hydrolysis, and derivatized with 4,5-methylenedioxy-1,2-phenylenediamine dihydrochloride at 50 ℃ for 150 min in a dark environment. The separation of sialic acid was achieved on LiChrosorb RP-18 column (250 mm × 4 mm, 5μm) using methanol-acetonitrile-water (7:8:85) as the mobile phase at a flow rate of 0.9 mL/min. Injection volume and column temperature were set at 10μL and 30 ℃, respectively. The fluorescence excitation and emission wavelengths were 373 nm and 448 nm, respectively. The results showed that the linear range of sialic acid was 50－400μmol/L. The average recovery rate was 94.0%. The precision, stability and repeatability RSDs were 0.4%, 1.0% and 0.8%, respectively. The limits of detection were 0.02μmol/L for Neu5Ac and 0.03μmol/L for Neu5Gc. Therefore, this developed method is characteristics of simple operation, and high reproducibility and sensitivity. It can be widely applied to determine total content of sialic acid in infant formula, cow milk and human milk.