کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1212769 | 1494041 | 2016 | 10 صفحه PDF | دانلود رایگان |
• A HPLC-UV method for determination of GlcNAc, GalNAc and Neu5Ac.
• Parameters affecting liberation of N-acetylhexosamines from samples were optimized.
• The reagent of NMP was used to label ketone sugars directly for the first time.
• It was sensitive, efficient and reliable enough for GlcNAc, GalNAc and Neu5Ac analysis.
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 0.25–12 μM, with R2 > 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.
Journal: Journal of Chromatography B - Volume 1011, 1 February 2016, Pages 14–23