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

ABSTRACT 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.