Non-destructive distinction of single seed for Medicago sativa and Melilotus officinalis by capillary electrophoresis with laser-induced fluorescence detection.

Flavonoids are a class of natural polyphenolic compounds with great health benefits, and the development of methods for their analysis is of continuing interest. In this work, apigenin, kaempferol and formononetin were selected as the typical representatives of flavone, flavonol and isoflavone, three subclasses of flavonoids. Fluorescence studies revealed that tetraborate complexation could significantly sensitize the weak intrinsic fluorescence of flavonoids in solution, with a maximum of 137-fold for kaempferol. Subsequently, an integrated strategy of derivatization and separation was proposed for the universal analysis of flavonoids by capillary electrophoresis (CE) with 405 nm laser-induced fluorescence (LIF) detection. Using a running buffer consisting of 20 mM sodium tetraborate, 10 mM SDS and 10% methanol (pH 8.5), the dynamic derivatization was realized in the capillary, and the baseline separation was achieved within 10 min, with the detection limits of 0.92-35.46 nM (S/N=3) for the total of 9 flavonoids. The developed CE-LIF method was employed to the quantitative analysis of some flavonoids in Medicago sativa (alfalfa) plants and granulated alfalfa with the recoveries of 80.55-94.25%. Combined with the principal component analysis, the developed method was successfully applied to the non-destructive distinction of single seed for alfalfa and Melilotus officinalis (sweet clover), two forage grass seeds with very similar apparent morphology. Furthermore, this method was used to continuously monitor the substance metabolism during the soaking process at the level of single seed.