title: 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.
abstract: 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-d-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/GalNAc 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 0.25-12μM, with R(2)>0.9991. The detection limits of the method were between 0.48 and 2.01pmol. 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.
authors.affiliation: Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu, PR China.
authors.forename: Wu
authors.initials: W
authors.lastname: Chuanxiang
authors.affiliation: Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu, PR China. Electronic address: xialian01@163.com.
authors.forename: Xia
authors.initials: X
authors.lastname: Lian
authors.affiliation: Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu, PR China.
authors.forename: Liu
authors.initials: L
authors.lastname: Lijie
authors.affiliation: Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu, PR China.
authors.forename: Qu
authors.initials: Q
authors.lastname: Fengli
authors.affiliation: Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu, PR China.
authors.forename: Sun
authors.initials: S
authors.lastname: Zhiwei
authors.affiliation: Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu, PR China.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Xianen
authors.affiliation: Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal University, Qufu, PR China; Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining, 810001, PR China. Electronic address: jmyou6304@163.com.
authors.forename: You
authors.initials: Y
authors.lastname: Jinmao
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006595:Hexosamines / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D008895:Milk, Human / Q000737:chemistry*; D019158:N-Acetylneuraminic Acid / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006595:Hexosamines; D019158:N-Acetylneuraminic Acid
keywords: 1-(2-naphthyl)-3-methyl-5-pyrazolone; HPLC-UV-MS/MS; Milk-based products; N-acetylhexosamines; N-acetylneuraminic acid; Pre-column derivatization