In this article we present two novel uses of the sensitive techniques HPLC fluorescence and dCCE for both
the quantitation and binding studies of the 5- and 7-HFs extracted from the plant Alfalfa with Albumin.
Ultrasonic extraction method as an extra energy source is used to enhance the extraction efficiency and
speed up. The two antioxidants could be easily separated and quantified after a 10.0-min run time.
Multiple calibration curves for their analysis exhibited consistent linearity and reproducibility in
the range of 0.20–2.00 mg L
1 for 5-HF (r 40.9979) and 0.01–0.10 mg L
1 for 7-HF (r 40.9999). Limits
of Detection were 0.500 mg L
1 and 0.025 mg L
1 for 5-HF and 7-HF respectively. Lower Limits of
Quantification were 131.600 mg L
1 for 5-HF and 6.579 mg L
1 for 7-HF. Inter-assay imprecision
was o10% for both flavones. Mean recovery was 104.76% (range 90%–110%) for 5-HF and 93.18%
(range 90%–110%) for 7-HF. Since the intermolecular hydrogen atom transfer in the excited triplet state as
well as in the excited singlet state might play an important role in the quenching process of photoexcited
molecules in biological systems, the binding constants of these HFs with serum albumin have
been also estimated to be 1.910–2.019105 L mol
1 and 2.390–2.500105 L mol
1 for 5-HF and 7-HF
respectively.