Elsevier

Analytical Biochemistry

Volume 347, Issue 2, 15 December 2005, Pages 182-192
Analytical Biochemistry

Mass spectrometric identification and quantification of glycosyl flavonoids, including dihydrochalcones with neutral loss scan mode

https://doi.org/10.1016/j.ab.2005.09.020Get rights and content

Abstract

We developed a strategy for determination and quantification of glycosyl flavonoids using liquid chromatography–triple quadrupole mass spectrometry with neutral loss scan at 15 and 30 eV collision energy in the positive ion mode. The fragmentation patterns of glycosyl flavonoids at 15 and 30 eV showed that fragmentation of sugar moiety depended on the type of glycosidic bond to aglycone, the site of C-glycosylation, and the type of aglycone. C-Glycosyl dihydrochalcones especially stood out because they produced [M+H−162]+ even at 15 eV such as O-glycoside in spite of C-glycoside. C-Glycosides were classified according to (i) the intensity ratio A of [M+H−150]+ to [M+H−120]+ at 30 eV and (ii) the intensity ratio B of [M+H−120]+ at 15 eV to one at 30 eV. The 8-C-glycosides were A < 1 and B < 1, the 6-C-glycosides were A > 1 and B < 1, and the C-glycosyl dihydrochalcones were A > 1 and B  1. Therefore, the intensity ratios of the neutral loss scan of 120 and 150 Da at 30 eV and those of 120, 162, and 308 Da at 15 eV allowed sequential distinction among these three types of C-glycosides as well as between O- and C-glycosides. Our method was applied for analysis of Rooibos tea, and the identified glycosides could be quantified specifically by the selected reaction monitoring method.

Section snippets

Materials

Aspalathin and nothofagin were provided by the Medical Research Council of South Africa (Tygerberg, South Africa). Vitexin, orientin, isoorientin, isoquercitrin, rutin, hyperoside, luteolin-7-O-glucoside, and phloretin were purchased from Extrasynthese (Genay, France). Isovitexin was obtained from Indofine Chemical (Hillsborough, NJ, USA). Daidzin, acetonitrile (for LC–MS), and methanol (for HPLC) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Formic acid was obtained from

Fragmentation patterns of C- and O-glycosyl flavonoids in the positive mode

The theoretical fragmentation pattern of glycosyl flavonoid is shown in Fig. 1. The cleavage and nomenclature were described according to the system adopted by Waridel et al. [11]. O-Glycosyl flavonoid for rutin, which is a disaccharide composed of rhamnosyl glucose (rutinose), produced the Y0+ and Y1+ ions by fragmentation of the O-glycosidic bond. The Y0+ ion is neutral loss of 308 Da (rutinose), and the Y1+ ion is 146 Da (rhamnose) as loss of terminal sugar moiety. Although both ions appeared

Conclusion

We confirmed that collision-induced dissociation analysis at two levels of collision energy in the positive ion mode could distinguish sequentially not only between O- and C-glycoside but also the C-glycosidic site. In addition, it was remarkable that C-glycosyl dihydrochalcone showed characteristic fragmentation, which occurred even at low collision energy and produced Y0+ ion corresponding to neutral loss of 162 Da in spite of C-glycoside.

These results allowed effective identification of

Acknowledgment

We thank Yoshikazu Ohtsuka, Department of Pediatrics and Adolescent Medicine, Juntendo University School of Medicine, Tokyo, for kindly providing the glycosyl flavonoid standards and Rooibos tea samples.

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