该文建立了高效阴离子交换色谱-脉冲安培法检测母乳和婴儿配方粉中常见唾液酸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)的方法。利用酸水解法释放母乳和婴儿配方粉中的唾液酸,用离子色谱前处理柱纯化婴儿配方粉水解液。采用Dionex CarboPac PA20高效阴离子色谱柱分离,以NaOH和乙酸钠为淋洗液进行梯度洗脱,流速0.5 mL/min,进样体积10 μL,柱温30 ℃,脉冲安培检测器检测,优化了酸水解时间和婴儿配方粉纯化条件。所测2种组分在该实验条件下能够很好的分离,且在较宽的浓度范围内离子峰面积呈良好的线性关系(R2>0.999)。Neu5Ac和Neu5Gc的检出限分别为3和2 μg/L,定量限为10和6 μg/L。母乳和婴儿配方粉的加标回收率在87.65%~102.33%,稳定性的相对标准偏差在1.86%~5.21%。该方法样品处理简单,具有较低的检出限与定量限,较宽的线性范围,较好的稳定性,良好的重复性与回收率,能广泛用于乳基唾液酸的检测。
A method for the determination of N-acetylneuraminic acid (Neu5Ac) and N-glycolyneuraminic acid (Neu5Gc) in human milk and infant formulas was established by high-performance anion-exchange chromatography with pulsed amperometric detection. The sialic acids in human milk and infant formulas were released by the acid hydrolysis method, and the hydrolysate of infant formulas was purified by an ion chromatography pretreatment column. And Dionex CarboPac PA20-high performance anion chromatographic column was used for separation, gradient elution was conducted with sodium hydroxide and sodium acetate as eluents with the flow rate of 0.5 mL/min, injection volume of 10 μL, the column temperature of 30 ℃ and pulsed amperometric detector for detection. Furthermore, the acid hydrolysis time and the purification conditions of infant formulas were optimized, and the results showed that Neu5Ac and Neu5Gc could be separated well under the experimental conditions. And the ion peak area had a good linear relationship in a wide range of concentration (R2>0.999). The detection limits of Neu5Ac and Neu5Gc were 3 and 2 μg/L, and their limits of quantification were 10 and 6 μg/L, respectively. The spiked recoveries of sialic acids in human milk and infant formula were 87.65%-102.33%, the RSDs of stability were 1.86%-5.21%. This method developed here has the advantages of simple sample treatment procedure, low detection limit and quantitation limit, wide linear range, good stability, good repeatability and recovery rate. It can be widely used for the determination of sialic acids in milk substrate.
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