Application of one- and two-dimensional high-performance liquid chromatography methodologies for the analysis of C-glycosylflavones from fenugreek seeds

• Two-dimensional HPLC analysis of C-glycosylflavones in fenugreek seeds.
• Identification of new C-glycosylflavones in fenugreek seeds.

The composition of C-glycosylflavones in fenugreek seeds is complicated and comprises of flavone mono- and di-C-glycosides, derivatives of both apigenin and luteolin as well as their ester forms. In this paper the application of one- and two-dimensional HPLC techniques: off-line heart-cutting and on-line comprehensive for the analysis of flavone C-glycosides in fenugreek seeds is presented. For the identification of flavone C-glycosides two types of detectors: diode array detector and mass detector, coupled to HPLC were used. 18 flavones were separated and identified in the methanol extract from fenugreek seeds. The best separation was achieved in off-line heart-cutting mode. The presence of two new compounds in fenugreek seeds was shown, namely: vicenin-3 and isoschaftoside. Furthermore, five unknown C-glycosides, possibly derivatives of apigenin were revealed.