Development and Validation of an HPTLC Method for the Simultaneous Determination of Diosmin and Hesperidin in Different Citrus Fruit Extracts and Pharmaceutical Formulations

A high-performance thin layer chromatography (HPTLC) method for the simultaneous quantitative determination of diosmin and hesperidin in citrus fruit extracts and in pharmaceutical preparations was developed. Conditions were also optimized for best possible extraction of diosmin and hesperidin by using different solvents (water, methanol, and HMSO) for maximum recovery of these flavonoids. Methanol HMSO (1:1) was found to be best for highest possible recovery of target analytes. Moreover, extraction efficiency of targeted diosmin and hesperidin from plant matrix using methanol HMSO (1:1) were studied using cold percolation, Soxhlet, hot extraction, and ultra sonication techniques. For achieving good separation, a mobile phase of ethyl acetate methanol water acetic acid (25:2:2:1, v/v/v/v) was used. The densitometric determination was carried out at 330 nm in reflection/absorption mode. The calibration curves were linear in the range of 0.1-3 mu g per spot for diosmin and 0.25-7.5 mu g per spot for hesperidin. Recovery was also assessed by comparison with a previously reported high-performance liquid chromatography (HPLC) method. The quantitative results of both analytical methods did not show any statistical significance between each other.