Analysis of Carbohydrates in a Tibetan Medicine Using New Labeling Reagent, 1-(2-Naphthyl)-3-Methyl-5-Pyrazolone, by HPLC with DAD Detection and ESI-MS Identification

Abstract

A new labeling reagent, 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP), coupling with liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS) for the detection of carbohydrates from a famous Tibetan medicine is reported. Carbohydrates were derivatized to their bis-NMP-labeled derivatives. The method, in conjunction with a gradient elution, offered a baseline resolution of carbohydrate derivatives on a reversed phase Hypersil ODS-2 column. The carbohydrates such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose, and fucose could be successfully detected by UV and ESI-MS. Derivatives showed intense protonated molecular ion at m/z [M + H]⁺ in positive ion mode. The mass to charge ratios of characteristic fragment ions at m/z 473.0 could be used for the accurately qualitative identification of carbohydrates; this characteristic fragment ion was from the cleavage of C2-C3 bond in the carbohydrate chain giving the specific fragment ions at m/z [MH–C _m H_2m+1O _m –H₂O]⁺ for pentose, hexose, and glyceraldehydes, and at m/z [MH–C _m H_2m−1O _m+1–H₂O]⁺ for alduronic acids, such as galacturonic acid and glucuronic acid (m = n − 2, n is carbon atom number of carbohydrate). Compared with the traditional 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent, currently synthesized NMP show the advantage of higher sensitivity to carbohydrate compounds with UV and ESI-MS detection.

Keywords:

• 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP)
• Carbohydrates
• Derivatization
• Herpetospermum pedunculosum (Ser.) Baill
• Liquid chromatography-mass spectrometry (ESI/MS)

ACKNOWLEDGMENTS

This work was supported by the Knowledge Innovation Program of Chinese Academy of Sciences and the National Science Foundation under Grant #20075016.

Notes

Manuscript 6288