Assigning glucose or galactose as the primary glycosidic sugar in 3‐O‐mono‐, di‐ and triglycosides of kaempferol using negative ion electrospray and serial mass spectrometry

Kaempferol 3‐O‐β‐glucopyranoside, kaempferol 3‐O‐β‐galactopyranoside and higher glycosides of these two flavonoids with α‐rhamnose at C‐2 and/or C‐6 of the primary sugar were studied by negative ion electrospray ionisation and serial mass spectrometry in a three‐dimensional (3D) ion trap mass spectrometer. Kaempferol 3‐O‐β‐glucopyranoside and kaempferol 3‐O‐α‐rhamnopyranosyl(1→6)‐β‐glucopyranoside could be distinguished from their respective galactose analogues by differences in the ratio of the radical aglycone ion Y₀ – H]^?? to the rearrangement aglycone ion Y following MS/MS of the deprotonated molecules. Kaempferol 3‐O‐rhamnopyranosyl(1→2)‐β‐glucopyranoside and kaempferol 3‐O‐α‐rhamnopyranosyl(1→2)α‐rhamnopyranosyl(1→6)]‐β‐glucopyranoside could be distinguished from their respective galactose analogues by differences in the product ion spectra of the (M – H) – rhamnose]^? ion following serial mass spectrometry. In the triglycoside, it was deduced that this ion resulted from the loss of the rhamnose substituted at 2‐OH of the primary sugar by observing that MS/MS of deprotonated kaempferol 3‐O‐β‐glucopyranosyl(1→2)α‐rhamnopyranosyl(1→6)]‐β‐glucopyranoside showed the loss of glucose and not rhamnose. Thus the class of sugar (hexose, deoxyhexose, pentose) at C‐2 and C‐6 of the primary sugar can be determined. These observations aid the assignment of kaempferol 3‐O‐glycosides, having glucose or galactose as the primary glycosidic sugar, in LC/MS analyses of plant extracts, and this can be done with reference to only a few standards. Copyright © 2009 John Wiley & Sons, Ltd.