荧光高效液相色谱法测定血清中总唾液酸含量的分析

【目的】 探讨荧光高效液相色谱法测定血清中总唾液酸含量的最佳条件。 【方法】 按优化实验的原则,将人体血清分别用三氟乙酸、硫酸、盐酸80℃水解,酸化浓度分别为0.05、0.10、0.15、0.20 mol/L,酸解时间分别为30、60、90、120 min,经50℃避光衍生150 min后进行色谱分析。 【结果】 用0.10 mol/L TFA溶液80℃水解60 min测定的唾液酸浓度最佳,唾液酸的最适宜浓度在0.625~80 μmol/L范围内,其平均回收率为94.0%,精密度、稳定性、重复性试验的相对标准差分别为1.3%、1.1%和1.0%。Neu5Ac的最低检出限为0.02 μmol/L,16~19岁健康青少年血清中唾液酸(Neu5Ac)含量的平均值为(2.12±0.15) mmol/L。 【结论】 荧光高效液相色谱法测定唾液酸简单、重复性好、灵敏度高,可广泛用于人体血清唾液酸含量测定。

Analysis of the total sialic acid content in serum determinated with fluorometric high-performance liquid chromatography

【Objective】 To determine the optimum conditions for total sialic acid analysis in serum using the fluorometric high-performance liquid chromatography (HPLC). 【Methods】 The sialic acid standard operating fluid contained N-acetylneuraminic acid (Neu5Ac),N-glycolylneuraminic acids (Neu5Gc) and ketodeoxynonulosonic acid (KDN).The human plasma would be hydrolyzed with the trifluoroacetic acid (TFA),sulphuric acid and hydrochloric acid at different concentration of 0.05,0.10,0.15 mol/L and 0.20 mol/L at 80 ℃ and at varying time of 30,60,90 min and 120 min respectively.The hydrolyzed sample was treated with 4,5-Methylenedioxy-1,2- phenylenediamine dihydrochloride (DMB,Sigma) derivative reagent at 50 ℃ for 150 min away from light. 【Results】 The maximum release of sialic acid was obtained with an equal volume of 0.1 mol/L TFA for 60 min at 80 ℃.The optimum detection limit of sialic acid was in 0.625~80 μmol/L,the average recovery rate was 94.0% and the relative standard deviation (RSD) for the precision,stability and repeatability test were 1.3%,1.1% and 1.0%,respectively.The minimum detection limit of Neu5Ac was 0.02 μmol/L and the average content of sialic acid in human serum was (2.12±0.15) mmol/L in 16~19 years old healthy adolescents. 【Conclusion】 The total sialic acid content determinated with fluorometric HPLC method is a simple,high sensitivity and good repeatability,which can be widely used in determination of sialic acid concentration in serum.