Characterization of goat colostrum oligosaccharides by nano-liquid chromatography on chip quadrupole time-of-flight mass spectrometry and hydrophilic interaction liquid chromatography-quadrupole mass spectrometry

Abstract

A detailed qualitative and quantitative characterization of goat colostrum oligosaccharides (GCO) has been carried out for the first time. Defatted and deproteinized colostrum samples, previously treated by size exclusion chromatography (SEC) to remove lactose, were analyzed by nanoflow liquid chromatography-quadrupole-time of flight mass spectrometry (Nano-LC-Chip-Q-TOF MS). Up to 78 oligosaccharides containing hexose, hexosamine, fucose, N-acetylneuraminic acid or N-glycolylneuraminic acid monomeric units were identified in the samples, some of them detected for the first time in goat colostra. As a second step, a hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS) methodology was developed for the separation and quantitation of the main GCO, both acidic and neutral carbohydrates. Among other experimental chromatographic conditions, mobile phase additives and column temperature were evaluated in terms of retention time, resolution, peak width and symmetry of target carbohydrates. Narrow peaks (wh: 0.2-0.6min) and good symmetry (As: 0.8-1.4) were obtained for GCO using an acetonitrile:water gradient with 0.1% ammonium hydroxide at 40°C. These conditions were selected to quantify the main oligosaccharides in goat colostrum samples. Values ranging from 140 to 315mgL(-1) for neutral oligosaccharides and from 83 to 251mgL(-1) for acidic oligosaccharides were found. The combination of both techniques resulted to be useful to achieve a comprehensive characterization of GCO.

Keywords: Goat colostrum; Hydrophilic interaction liquid chromatography; Mass spectrometry; Nanoflow liquid chromatography–quadrupole-time of flight mass spectrometry; Oligosaccharides.

Fig. 1

Nano-LC-Chip-Q-TOF MS profiles of goat colostrum oligosaccharides: CS1, CS2, CS3, CS4, and CS5.

Fig. 2

MS/MS spectra of: (a) hexosyl-lactose 3_0_0_0_0 (m/z 505.176), (b) 2′-FL (m/z 489.181), and (c) 3′-SL (634.218). Light blue circle: hexose; dark blue circle: glucose; yellow circle: galactose; red triangle: fucose; violet diamond: Neu5Ac. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Extracted ion chromatographic profile of CS5 oligosaccharides obtained using BEH amide column and 0.1% ammonium hydroxide as mobile phase additive. (a) ESI interface working under positive polarity and (b) under negative polarity. (1) 3′-Sialyl-lactose; (2) sialyl-lactosamine; (3) glycolyl-neuraminyl-lactosamine; (4) 6í-sialyl-lactose; (5) 2′-fucosyl-lactose; (6) galactosyl-lactoses; (7) di-hexosyl-lactoses.