A sensitive and efficient method for determination of N-acetylhexosamines and N-acetylneuraminic acid in breast milk and milk-based products by high-performance liquid chromatography via UV detection and mass spectrometry identification
A sensitive and efficient method for determination of N-acetylhexosamines and N-acetylneuraminic acid in breast milk and milk-based products by high-performance liquid chromatography via UV detection and mass spectrometry identification
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高效液相色谱紫外检测和质谱鉴定测定母乳和乳制品中 N-乙酰己糖胺和 N-乙酰神经氨酸的灵敏、高效方法
DOI：
10.1016/j.jchromb.2015.12.032
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发表时间：
2016-02-01
影响因子：
3
通讯作者：
You, Jinmao
中科院分区：
文献类型：
--
作者：
Wu, Chuanxiang;Xia, Lian;You, Jinmao
建立了以1-（2-萘基）-3-甲基-5-吡唑啉酮（NMP）为柱前衍生试剂、紫外检测（HPLC-UV）和在线质谱鉴定的高效液相色谱法，用于测定最常见的N-乙酰己糖胺（N-乙酰-D-葡萄糖胺（GlcNAc）和N-乙酰-N-半乳糖胺（GalNAc））和N-乙酰神经氨酸（Neu 5Ac）。为了在不破坏Neu 5Ac或不使GlcNAc/GaINAc转化为GlcN/GalN的情况下获得最高的3种单糖的释放水平，采用响应面法（RSM）对影响N-乙酰己糖胺/Neu 5Ac从样品中释放的关键参数进行了研究。在优化的工艺条件下，产率最高。考察了关键参数对衍生、分离和检测的影响。在优化的条件下，三种单糖被快速、完整地标记，并且所有衍生物都表现出良好的基线分辨率和高的检测灵敏度。所开发的方法在0.25-12 μ M的校准范围内呈线性，R-2 > 0.9991。方法的检出限为0.48 - 2.01 pmol。三种单糖（GlcNAc，GalNAc和Neu 5Ac）的日内和日间精密度分别为3.07-4.02%和3.69- 4.67%。加标牛奶中的单个单糖回收率在81%-97%范围内。该方法的灵敏度，衍生化过程的设施和水解条件的可靠性表明，所提出的方法具有很高的潜力，用于常规痕量N-乙酰己糖胺和Neu 5Ac的生物样品分析。(C)2015 Elsevier B. V.版权所有。
A sensitive and efficient method of high performance liquid chromatography using 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) as pre-column derivatization reagent coupled with UV detection (HPLC-UV) and online mass spectrometry identification was established for determination of the most common N-Acetylhexosamines (N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-n-galactosamine (GalNAc)) and N-acetylneuraminic acid (Neu5Ac). In order to obtain the highest liberation level of the three monosaccharides without destruction of Neu5Ac or conversion of GlcNAc/GaINAc to GlcN/GalN in the hydrolysis procedure, the pivotal parameters affecting the liberation of N-acetylhexosamines/Neu5Ac from sample were investigated with response surface methodology (RSM). Under the optimized condition, maximum yield was obtained. The effects of key parameters on derivatization, separation and detection were also investigated. At optimized conditions, three monosaccharides were labeled fast and entirely, and all derivatives exhibited a good baseline resolution and high detection sensitivity. The developed method was linear over the calibration range 0.25-12 mu M, with R-2 > 0.9991. The detection limits of the method were between 0.48 and 2.01 pmol. Intra- and inter-day precisions for the three monosaccharides (GlcNAc, GalNAc and Neu5Ac) were found to be in the range of 3.07-4.02% and 3.69-4.67%, respectively. Individual monosaccharide recovery from spiked milk was in the range of 81%-97%. The sensitivity of the method, the facility of the derivatization procedure and the reliability of the hydrolysis conditions suggest the proposed method has a high potential for utilization in routine trace N-acetylhexosamines and Neu5Ac analysis in biological samples. (C) 2015 Elsevier B.V. All rights reserved.