A Sensitive Derivatization Method for the Determination of the Sugar Composition after Pre‐column Reductive Amination with 3‐Amino‐9‐ethylcarbazole (AEC) by High‐Performance Liquid Chromatography

Abstract

3-Amino-9-ethylcarbazole (AEC) was employed for monosaccharide derivatization. The derivatives can be analyzed by high-performance liquid chromatography (HPLC) with an ultraviolet detection (wavelength: 254 nm). Monosaccharides were quantitatively derivatized with 3-amino-9-ethylcarbazole (AEC) at 70 for 60 min. The ℃ method was linear for all samples over the concentration range tested (r＞0.999), the precision was found to be satisfactory (R.S.D.＜3%), and the recovery ratios were ＞98.62%. The stability analysis showed R.S.D. was between 1.81%-3.16%. Detection limits for the samples (D-glucose, L-xylose, D-mannose, L-arabinose, and L-rhamnose) ranged from 0.06 to 1.97 ng/mL (S/N＝3). Under the optimized derivatization and HPLC conditions, five monosaccharides were well separated using a narrow bore C18 column (250 mm×4.6 mm) with 0.1 mol/L ammonium acetate containing 25% acetonitrile at a flow rate of 0.5 mL/min. As an application, the method has been successfully applied to the determination of monosaccharide compositions of three polysaccharides SPPA-1, SPPB-1 and SPPC-1 of Spirulina platensis. This method also has potential application to oligosaccharide or glycan analyses.

Detection of carbohydrates using a pre-column derivatization reagent 1-(4-isopropyl) phenyl-3-methyl-5-pyrazolone by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry

“…The ESI-MS/MS 2 condition was carried out as described in Fig. 4b. Man, Fuc and trace amount of GalN, Gal, Xyl, Ara, in which the molar ratio of Man, Glc and Rha is 0.51:1.79:3.79 that is consistent with Zhang's results [20].…”

“…They can be divided into two groups. The first group comprises some reductive amination reagents with aromatic amino-groups, some of them are typical, such as 2-aminopyridine (2-AP) [4], 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) [18], 2-aminoacridone (AMAC) [19] and 3-amino-9-ethylcarbazole (AEC) developed in our lab and so on [20], which introduce an aromatic amine-groups to the aldehyde group at the reducing end of saccharide before HPLC or CE separation. Such reagents are used widely on account of their derivatives appearing the stronger UV or fluorescence absorbance than native carbohydrates and high stability.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…The compounds formed (Zhang, Huang, & Wang, 2007e). A method for gas-phase preparation of fluorescent 2-AP derivatives that avoids the need for removal of the excess of reagent has given glycan profiles (from chicken ovalbumin) comparable to that obtained by traditional liquid-phase preparation (Nakakita et al, 2007b).…”

Analysis of Selected Sugars and Sugar Phosphates in Mouse Heart Tissue by Reductive Amination and Liquid Chromatography-Electrospray Ionization Mass Spectrometry

“…Although the optimized reaction conditions for glucose had been determined previously, 26 the optimal derivatization conditions had not been determined for other sugars or for any of the sugar phosphates targeted in this study. In order to develop an LC/MS-based method for precise and accurate quantitation of all of the C3 to C6 reducing sugars and sugar phosphates in biological samples, we optimized the AEC derivatization conditions for these compounds that were detected in the mouse heart tissue.…”