Analysis of 2-Aminobenzoic Acid-Labeled Monosaccharides and Glycoprotein-Derived Oligosaccharides by Online Cleanup Liquid Chromatography in the Reversed-Phase and Hydrophilic Interaction Liquid Chromatography Modes

Abstract

Quantitative analysis of monosaccharides or glycoprotein glycans by high-performance liquid chromatography (HPLC) often involves labeling of the saccharide aldehyde groups with fluorescent tags to enhance sensitivity and selectivity. However, the methods required to remove the large excess of labeling reagents from the reaction mixture are time-consuming. Furthermore, these methods often hinder the quantitative analysis of the labeled samples. Here, we developed an online sample cleanup procedure for HPLC analysis of 2-aminobenzoic acid (2-AA)-labeled monosaccharides or oligosaccharides using a ten-port valve and mini columns. An online purification system using a combination of short HLB columns with the valve was proposed for the analysis of 2-AA-labeled monosaccharides utilizing reversed-phase modes. In the analysis of 2-AA-labeled glycans derived from glycoproteins, a short CN column with the valve was proposed utilizing hydrophilic interaction liquid chromatography (HILIC) modes. Optimized conditions enabled the direct injection of the diluted labeling reaction mixture into the chromatographic system without any prior removal of the excess labeling reagents. These methods were successfully applied to the analysis of various monosaccharides and N-linked glycans released from specific glycoproteins.

Analysis of Total Thiols in the Urine of a Cystathionine β-Synthase-Deficient Mouse Model of Homocystinuria Using Hydrophilic Interaction Chromatography

“…Pretreatment was followed by reduction with tris(2-carboxyethyl) phosphine (TCEP), deproteinization, and derivatization with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Since HILIC can separate highly polar compounds such as SBD-thiols [8,9], HILIC with fluorescence detection was developed to analyze the total homocysteine and its metabolite concentrations, in the plasma of CBS-wild type (CBS+/+, -WT), -heterozygous (CBS+/−, -Hetero), and -knockout (CBS−/−, -KO) mice [10].…”

Research of saccharides and related biocomplexes: A review with recent techniques and applications

“…LC detection and quantitative measurements of targeted saccharides are done with ultraviolet (UV), evaporative light scattering detectors (ELSDs), charged aerosol detectors (CADs), refractive index detectors (RIDs), contactless conductivity detectors (CCD4), or laser‐induced fluorescence (LIF). The RI and UV detectors are mostly utilized for screening saccharide profiles [84–144].…”

“…There are, for example, zwitterionic, carbohydrate, amide or amine-functionalized, and metal-organic complex-based materials among other phases [150]. Recently, zwitterionic charge modulated amine functionalized polymer [133], zwitterionic charge modulated amide functionalized silica (Table S1, [54,107,117]), copolymer-grafted silica, 2-(dimethylamino) ethylmethacrylate copolymerized with itaconic acid and acrylic acid (Table S1, [102]), and cyanopropyl functionalized (Table S1, [88]) sorbent materials were used in HILIC separations. In partitioning polar saccharides were transferred with organic solvent from the adsorption layer which consisted of water penetrating to the surface of the sorbent material.…”

“…N-glycans of immunoglobulin G Fc receptors influence efficiencies of mAbs and their variants [261,233,240,284,310]. These glycans are studied with IEXC and CE [88,162,181,193,226,229,240].…”

“…The phases with polymers of poly(succinimide) derivatives, polyethylene glycol/silica, "click" β-cyclodextrin, "click" saccharides ("click" maltose), "click" dipeptide, zwitterionic sulphobetaine bonded (ZIC-HILIC), CIEX bonded, or mix-mode RP-AEX bonded phases are informed. As examples of the recently used HILIC sorbents a combination of cyano and amide bonded phases (Table S1, [54,88]), ion exchange sorbent (Table S1, [19]), copolymer-grafted silica stationary phases (Table S1, [102]), cross-linked diol bonded (Table S1, [117]), zwitterionic sorbent (Table S1, [133]), and amide-RP sorbent for glycan mapping (Table S1, [137]) were used for determination of saccharide and saccharide-related compounds. Using HILIC the challenge is the material surface itself since it has a strong affinity to water and allows water-spreading on the surface.…”