Mass Spectrometry and NMR Spectroscopy-Based Structural Characterization of Polysaccharide From Gastrodia elata Blume

Structural characterization of complex carbohydrates remains challenging. This study employed methylation, acid hydrolysis, 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization, and ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) to determine monosaccharides, glycosidic linkages, and sequence of polysaccharides. Combined with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) and nuclear magnetic resonance (NMR), the structure of two fractions (GEP-60 and GEP-60S) obtained from Gastrodia elata Blume was characterized. Both fractions contained mainly glucose, and GEP-60 was homogeneous polysaccharide (25.5 kDa), while GEP-60S was mixture of lower molecular weight (DP ≤ 24) fractions. Their primary glycosidic linkage was →4)-α-Glc p -(1→, with respective percentages of 78.41 ± 2.18% and 79.72 ± 1.98%. Sequence analysis identified both contained [→4)Glc(1→] 4 , [→4)Glc(1→] 5 , and [→4)Glc(1→] 6 . NMR confirmed that both polysaccharides possessed main chain composed of →4)-α-Glcp-(1→ and substituted with α-Glcp-(1→ branches. Taken mass spectrometry and NMR spectroscopy results together, polysaccharides in G. elata Blume were α-1,4-glucan with different molecular weight containing branches substituted at C-6, with occasional substitution also observed at the C-2 and C-3. These findings provided comprehensive structural insights into G. elata Blume polysaccharides and validated feasibility of UPLC–MS/MS as an alternative strategy for analyzing plant polysaccharides.