Analysis of human milk oligosaccharides using high-performance anion-exchange chromatography with pulsed amperometric detection

Human Milk Oligosaccharides (HMOs) are composed of 5 different monosaccharides: D-glucose, D-galactose, L-fucose, N-acetylneuraminic acid and N-acetylglucosamine. Approximately 200 unique structures have been identified, ranging in the degree of polymerization from 3 to 22. The diversity among different mothers is considerable, ranging from as few as 23 and up to 130 different oligosaccharides. HMOs are known to be beneficial for infant health and development, and have received a lot of attention in recent years (Bode & Jantscher-Krenn 2012).
Syntheses of this unique family of lactose-based molecules necessitates analysis methods that can provide separation and quantification of the common structural constituents mentioned, as well as the disaccharide lactose (Galβ1-4Glc) and oligosaccharides such as lacto-N-triose II (GlcNAcβ1-3Galβ1-4Glc), lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc) and lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), among others.
High-performance anion-exchange chromatography (HPAE) with pulsed amperometric detection (PAD) is an analysis method highly suited for carbohydrates. HPAE with alkaline eluents results in retention of neutral carbohydrates depending on the number of charged group in the molecule, pH and concentration of competing anions, while the PAD has sensitivity for carbohydrates in the pmol-range (Lee 1990).
As a basis for the development and optimisation of HPAE elution methods, the parameter space was investigated in terms of eluent concentrations and temperature, using a CarboPac PA1 column. Specifically, the retention times and separations of various mono- and oligosaccharides, as depending on the eluent concentrations of NaOH and NaOAc, was studied. The investigated monosaccharides and several of the disaccharides exhibited maximum retention at distinct concentrations of NaOH. In addition, the different species showed varying relations between eluent composition and retention time, resulting in varying separation and even changes in elution order with different elution conditions.

References:
Bode L, Jantscher-Krenn E. (2012) Adv. Nutr. 3: 383S
Lee YC. (1990) Anal. Biotechnol. 189: 151