Simultaneous quantitation of 5- and 7-hydroxyflavone antioxidants and their binding constants with BSA using dual chiral capillary electrophoresis (dCCE) and HPLC with fluorescent detection

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 >0.9979) and 0.01–0.10 mg L−1 for 7-HF (r >0.9999). Limits of Detection were 0.500 µg L−1 and 0.025 µg L−1 for 5-HF and 7-HF respectively. Lower Limits of Quantification were 131.600 µg L−1 for 5-HF and 6.579 µg L−1 for 7-HF. Inter-assay imprecision was <10% 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 photo-excited molecules in biological systems, the binding constants of these HFs with serum albumin have been also estimated to be 1.910–2.019×105 L mol−1 and 2.390–2.500×105 L mol−1 for 5-HF and 7-HF respectively.