Determination of the monosaccharide types and absolute configurations in polysaccharides with (S)-(-)-1-phenylethylamine based on UPLC-MS/MS

The structural characterization of polysaccharides requires precise determination of their monosaccharide composition, including the differentiation of D- and L-configurations. Current analytical methods can identify monosaccharide types but often fail to distinguish between enantiomeric configurations, posing a significant challenge for accurate polysaccharide structural elucidation. This study introduces a robust, accurate, and reproducible ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method to address this limitation. By utilizing ( S )-(−)-1-phenylethylamine (S-PEA) as a chiral derivatizing reagent, monosaccharide enantiomers are converted into diastereomers through the introduction of a new chiral center. This approach enables the simultaneous determination of D/L configurations for aldoses, amino sugars, alditols, and uronic acids, achieving excellent chromatographic baseline separation. Method validation demonstrated excellent linearity (R 2  > 0.9900) within the range of 2.5–160 μg/mL. The limits of detection (LOD) and quantification (LOQ) ranged from 0.04 to 0.11 μg/mL and 0.13–0.35 μg/mL, respectively, indicating high sensitivity. Accuracy was confirmed with recovery rates between 97.23% and 100.67%, while intra- and inter-day precision RSDs were below 3.54%. The method was successfully applied to characterize the monosaccharide composition of six plant-derived polysaccharides, demonstrating its efficacy in identifying both structural types and absolute configurations. These findings establish the proposed method as a valuable tool for advancing the comprehensive structural analysis of polysaccharides.