Improved determination of milk oligosaccharides using a single derivatization with anthranilic acid and separation by reversed-phase high-performance liquid chromatography

Determination of Sialyl and Neutral Oligosaccharide Levels in Transition and Mature Milks of Samoan Women, Using Anthranilic Derivatization Followed by Reverse Phase High Performance Liquid Chromatography

“…In our previous paper, we described a simple method that involves pre-column labeling with anthranilic acid prior to reverse phase high performance liquid chromatography (RP-HPLC) for determination of neutral HMOs. 1) The results suggested that there is a difference in the patterns of neutral oligosaccharides between Samoan and other (Italian, Mexican, and Japanese) women, though our sample size for Samoan women's milk was rather small. The main difference was the relatively low concentrations of 2 0 -fucosyllactose (2 0 -FL) and lacto-Nfucopentaose I (LNFP I) in the milk of the Samoan women, even though these are the most abundant oligosaccharides in other women's milks.…”

“…We used RP-HPLC analysis following AA derivatization of oligosaccharide fractions separated by gel filtration and solid phase anion exchange. A variety of methods have been described for the determination of neutral oligosaccharides alone, whereas the one used both in our previous study 1) and here is suitable for the determination of both sialyl and neutral oligosaccharides. Its major advantages are high sensitivity, reproducibility, high resolution, and protection against loss of sialic acid and fucose.…”

“…To date, there are no studies other than our previous one 1) concerning differences between ethnic groups with respect to milk oligosaccharides. In this study we attempted to establish whether there are differences between the milk oligosaccharides of Samoan women and those of other groups.…”

Analysis and role of oligosaccharides in milk

“…Chromophoric active tags such as 1-phenyl-3-methyl-5-pyrazolone (PMP), 2-aminopyridine (PA) and 2-aminobenzoic acid (2-AA) are commonly used for the analysis of HMO (40)(41)(42)(43). These labels serve an additional purpose of providing a chromophore for spectrophotometric detection with HPLC.…”

Oligosaccharides in Milk: Their Benefits and Future Utilization

“…Milk oligosaccharides can be quantifi ed using reverse -phase or normal -phase HPLC subsequent to pre-or post -column labeling techniques. Derivatizations are often performed by condensation with 2-aminopyridine (PA) (Asakuma et al, 2008), 1-methyl-3-phenyl-5-pyrazolone (PMP) (Asakuma et al, 2007(Asakuma et al, , 2008, anthranilic acid (Leo et al, 2009(Leo et al, , 2010, or benzoic anhydride (Chaturvedi et al, 2001) to the reducing end of the sugar aldehyde. They can also be quantifi ed by high pH anion exchange chromatography (HPEAC-PAD) without derivatization (Thurl et al, 1996;Coppa et al, 1999;Kunz et al, 1996).…”