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

摘要

Human Milk Oligosaccharides (HMOs) are composed of 5 different monosaccharides: D-glucose, D galactose, L-fucose, -acetylneuraminic acid and -acetylglucosamine. Approximately 200 unique structures have been identified, ranging in degree of polymerization from 3 to 22. The diversity among individual mothers is considerable, ranging from as few as 23 and up to 130 different oligosaccharides. HMOs are known as beneficial for infant health and development, and have received increasing attention in recent years (Bode & Jantscher-Krenn 2012).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 groups in the molecule, pH and concentration of competing anions, while PAD has sensitivity for carbohydrates in the pmol-range (Lee 1990). In the present study, the aim is to develop analysis methods that can provide separation and quantification of the common structural constituents mentioned, as well as the disaccharide lactose and oligosaccharides such as lacto--triose II, lacto--tetraose and lacto--neotetraose, among others.As the basis for HPAE elution methods, the retention times and separation of various mono- and oligosaccharides were investigated as depending on eluent concentrations of NaOH and NaOAc, using a CarboPac PA1 column. The studied monosaccharides and several of the oligosaccharides 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: 383SLee YC. (1990) Anal. Biotechnol. 189: 151