Quantification of plant cell wall monosaccharides by reversed-phase liquid chromatography with 2-aminobenzamide pre-column derivatization and a non-toxic reducing reagent 2-picoline borane

In this report, we described a sensitive method for quantifying plant cell wall monosaccharides using chemical derivatization, reversed-phase high performance liquid chromatography separation with ultraviolet detection (HPLC-UV). Monosaccharides were derivatized with 2-aminobenzamide (2-AB) by reductive amination to increase the hydrophobicity and detected by ultraviolet absorption for HPLC-UV analysis. A non-toxic reductant, 2-picoline borane was utilized to replace the traditionally used sodium cyanoborohydride (NaCNBH3) to avoid the formation of toxic by-products. Experimental conditions were optimized using glucose as a model system to achieve a high reaction yield of 99%. Under the optimized conditions, we demonstrated that the derivatization yields of several saccharides with 2-AB using 2-picoline borane were all slightly higher than those observed using NaCNBH3. In plants, cell wall monosaccharides consist of arabinose, fucose, galactose, galacturonic acid, glucose, glucuronic acid, mannose, rhamnose, and xylose. Using our method, we successfully quantified these monosaccharides from Arabidopsis cell wall by HPLC-UV, and we obtained a good linearity at a wide dynamic range over five orders (1 pmol through 10 nmol of injection amount), a detection limit of ∼0.1 pmole, and a high precision and accuracy.