Improved determination of milk oligosaccharides using a single derivatization with anthranilic acid and separation by reversed-phase high-performance liquid chromatography |
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Authors: | Fiame Leo Sadaki Asakuma Tadashi Nakamura Kenji Fukuda Akitsugu Senda Tadasu Urashima |
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Institution: | 1. Graduate School of Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan;2. Intensive Grazing Research Team, National Agricultural Research Center for Hokkaido Region, 1 Hitsujigaoka, Toyohira-ku, Sapporo 062-8555, Japan;3. Department of Bio-Resource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan |
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Abstract: | An improved analytical scheme for human milk neutral oligosaccharides determination was developed, in which, the oligosaccharides were pooled in two fractions (pools 1 and 2) after gel filtration, and then were quantitatively derivatized with a single fluorescent reagent, 2-anthranilic acid. Separation was by reversed-phase HPLC on an ODS-100Z column with a mobile phase of 50 mM ammonium acetate pH 4.0 and 150 mM citrate buffer pH 4.5 and monitored by a fluorescence detector at 360 nm excitation and 425 nm emission wavelengths. The method improved on the separation of neutral tetra- and hexa-saccharide isomers, namely, lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) as well as of lacto-N-difucohexaose I (LNDFH I) and lacto-N-difucohexaose II (LNDFH II). The separation of trisacccharide isomers, 3-fucosyllactose (3-FL) and 2′-fucosyllactose (2′-FL) was also successful. Limits of detection and quantification were in the range of 1–10 ng/l and 2–30 ng/l, respectively. The methods’ accuracy was good with its precision at <20% RSD and <1% RSD, respectively, for oligosaccharide concentration and retention time. The recoveries were in the range of 80–100%. This method was successfully applied to the separation and determination of representative neutral oligosaccharide contents in Samoa women milk. |
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Keywords: | Neutral oligosaccharides Anthranilic acid Derivatization HPLC Lacto-N-fucopentaose III 2&prime -Fucosyllactose |
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