Oligosaccharide analysis by graphitized carbon liquid chromatography–mass spectrometry

Abstract

Structural analysis of complex mixtures of oligosaccharides using tandem mass spectrometry is regularly complicated by the presence of a multitude of structural isomers. Detailed structural analysis is, therefore, often achieved by combining oligosaccharide separation by HPLC with online electrospray ionization and mass spectrometric detection. A very popular and promising method for analysis of oligosaccharides, which is covered by this review, is graphitized carbon HPLC-ESI-MS. The oligosaccharides may be applied in native or reduced form, after labeling with a fluorescent tag, or in the permethylated form. Elution can be accomplished by aqueous organic solvent mixtures containing low concentrations of acids or volatile buffers; this enables online ESI-MS analysis in positive-ion or negative-ion mode. Importantly, graphitized carbon HPLC is often able to resolve many glycan isomers, which may then be analyzed individually by tandem mass spectrometry for structure elucidation. While graphitized carbon HPLC-MS for glycan analysis is still only applied by a limited number of groups, more users are expected to apply this method when databases which support structural assignment become available.

Online enzymatic sequencing of glycans from Trastuzumab by phospholipid‐assisted capillary electrophoresis

“…Electrospray ionization can be coupled online with a chemical separation, but the electrospray buffer must be compatible with the separation buffer. Separation methods that have been typically used for glycans are chromatographic and utilize separation columns based on reversed phase, hydrophilic interaction, graphitized carbon, and anion exchange stationary phases [20,28,29]. Capillary separations of glycans coupled to MS, particularly CE, have several advantages over traditional chromatography [30][31][32].…”

Analysis and role of oligosaccharides in milk

“…Despite the advancements in liquid chromatography in normal phase (34,35) and reverse phase (36)(37)(38) as well as in capillary electrophoresis (37,38), there remains no single separation method sufficiently effective for separating HMO mixtures. The problem lies in the heterogeneity of the structures.…”

Immobilization of Styrene-acrylamide Co-polymer on Either Silica Particles or Inner Surface of Silica Capillary for the Separation of D-Glucose Anomers

“…Various glycosylation analysis strategies including pretreatments of samples, chromatography, and mass spectrometry have been well introduced in some review articles. [4][5][6][7][8][9][10][11] HPLC and HPCE methods have been developed for the high-resolution and reproducible quantitation of carbohydrates. 12,13 The high performance anion exchange chromatographypulsed amperometry (HPAEC-PAD) system was developed for common use in separation and detection of saccharides.…”

“…C18 columns are somewhat useful for separation of derivatized saccharides, but still show poor performance in separation of isomers. Two highly prevailing types of stationary phase for separation of saccharides are porous graphite carbon (PGC) columns 8,11 and hydrophilic interaction chromatography (HILIC) columns. 5 PGC columns show good performance in separation of saccharide isomers, but their separation efficiency is known to be inferior to that of C18 columns.…”