Improved determination of milk oligosaccharides using a single derivatization with anthranilic acid and separation by reversed-phase high-performance liquid chromatography.
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 360nm 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.
Leo
Fiame
F
Graduate School of Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
Asakuma
Sadaki
S
Nakamura
Tadashi
T
Fukuda
Kenji
K
Senda
Akitsugu
A
Urashima
Tadasu
T
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008895:Milk, Human / Q000737:chemistry*; D009844:Oligosaccharides / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D062367:ortho-Aminobenzoates / Q000737:chemistry
D009844:Oligosaccharides; D062367:ortho-Aminobenzoates; C031385:anthranilic acid