Identification of isomeric disaccharides in mixture by the 1-phenyl-3-methyl-5-pyrazolone labeling technique in conjunction with electrospray ionization tandem mass spectrometry

• The method is based on PMP labeling in conjunction with ESI-CID-MS/MS.
• A novel empirical rule for the linkages and anomers was established completely.
• The method is useful to identify isomeric disaccharides in mixture directly.
• It was successfully exploited to analyze the unknown disaccharides in P. ginseng.
• This innovative method is simple, accurate and effective.

1-Phenyl-3-methyl-5-pyrazolone (PMP) labeling technique has hitherto proved to be a convenient and sensitive method for separating and detecting oligosaccharides. However, the detailed fragmentation of the derivatives by tandem mass spectrometry has been reported limitedly and no characteristic fragment ions for isomers have been detected. In this study, eight disaccharide isomers were labeled with PMP and analyzed by positive ion electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn). In comparison with the native disaccharides, PMP labeled disaccharides gave rise to more fragment ions in the tandem mass spectra. The distinctive diagnostic fragment ions formed from cleavage of CC bonds have been detected in the fragmentation of PMP-labeled disaccharide linkage isomers, allowing unambiguous assignment of the position of the glycosidic linkages. This feature is particularly useful for the structural determination of unknown isomeric disaccharides mixed together. In addition, the anomeric configurations can also be easily assigned based on the relative abundance ratios of the selected ion pairs. To verify the feasibility of the method used in the analysis of natural product, water soluble Panax Ginseng extract has been further investigated to identify its unknown disaccharides. The results confirmed that the PMP labeling technique in conjunction with ESI-MSn could offer a powerful and convenient tool for differentiation of structurally closely related isomers, even the unknown mixtures of isomeric disaccharides with different linkage types.

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