DMBA衍生/超高效液相色谱法测定鱼肉中唾液酸含量

Determination of Sialic Acid in Fish by Ultra High Performance Liquid Chromatography with DMBA Derivatization

建立了同时检测鱼肉中N-乙酰神经氨酸(Neu5Ac)、N-羟乙酰神经氨酸(Neu5Gc)和酮基-脱氧壬酮糖酸(KDN)3种核心唾液酸的超高效液相色谱-荧光检测(UPLC-FLD)方法。冷冻干燥后的样品在8 mL 0.6 mol/L盐酸溶液中80℃酸解20 min,采用4,5-二甲基-1,2-苯二胺(DMBA)衍生试剂进行衍生。前处理后的样品经Waters Acquity UPLC BEH C_(18)柱(100 mm×2.1 mm,1.7μm)分离,以纯水-乙腈为流动相梯度洗脱,荧光检测器检测。结果表明,Neu5Ac、Neu5Gc和KDN在0.02~5.0μg/mL范围内呈良好的线性关系(r^(2)>0.999),定量下限(S/N=10)分别为0.20、0.20、0.10μg/g;加标回收率为90.8%~103%,相对标准偏差(RSD)为2.8%~5.0%。该方法分析时间短,4 min内可完成3种唾液酸的分离,分离效果好,且方法灵敏度、准确度高,适用于鱼肉中唾液酸的测定。

An ultra high performance liquid chromatography-fluorescence detection(UPLC-FLD)method was developed for the simultaneous determination of 3 core kinds of sialic acids(SAs)including N-acetylneuraminic acid(Neu5Ac),N-glycolylneuraminic acid(Neu5Gc)and 2-keto-3-deoxynononic acid(KDN)in fish.The freeze-dried samples were acid hydrolyzed with 8 mL 0.6 mol/L hydrochloric acid solution for 20 min at 80℃,followed by derivatization with 4,5-dimethylbenzene-1,2-diamine(DMBA).The derivative conditions for DMBA were optimized by single factor test.All the target compounds were separated on an Waters Acquity UPLC BEH C_(18) column(100 mm×2.1 mm,1.7μm)by gradient elution,using pure water and acetonitrile as eluents,then detected by UPLCFLD.Under the optimized conditions,there were good linear relationships for Neu5Ac,Neu5Gc and KDN in the range of 0.02-5.0μg/mL(r^(2)>0.999),with their limits of quantitation(S/N=10)of 0.20μg/g,0.20μg/g and 0.10μg/g,respectively.The spiked recoveries for three SAs at three lev⁃els ranged from 90.8%to 103%,with relative standard deviations(RSDs)of 2.8%-5.0%.The analy⁃sis was rapid,and it could be achieved within 4 min with good separation and sharp peak shapes.With the characteristics of high accuracy and good precision,the method was suitable for the simultane⁃ous determination of Neu5Ac,Neu5Gc and KDN in fish.