Extraction, Isolation, and Quantitative Determination of Flavonoids by HPLC

Abstract

Flavonoids are natural products, more precisely secondary metabolites with low molecular weight produced by plants. Unlike primary metabolites, they are non-essential for plant survival. These secondary metabolites are biologically active and more than 10,000 structural variants of flavonoids have been reported such as flavonols, flavones, flavanones, anthocyanidins, catechins, biflavins and many more. Majority of these compounds act as antioxidants and exist as sugar conjugates in food. Flavonoids are omnipresent in vegetables and fruits with broad range of properties. Plant-derived flavonoids are essential dietary parts of animal system and are known to have an expansive scope of properties including antimicrobial, antiviral, anticancer, antiatherosclerosis and antithrombotic activity. Hence, it is imperative to estimate flavonoid sources in food, which will indubitably lead to a new era of flavonoids in foods or pharmaceutical supplements. Different traditional methods like UV/Visible absorption spectroscopy and separation techniques like thin-layer chromatography (TLC), paper electrophoresis and polyamide chromatography were commonly used in earlier times for flavonoid separation. Evidently, TLC is still a preferable method for flavonoid analysis, because of its simplicity, rapidity and versatility. On the other hand, majority of recently published work refers to qualitative and quantitative determination of flavonoids by high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-nuclear magnetic resonance (LC-NMR) and capillary electrophoresis (CE). The separation, identification and quantification of flavonoids can be performed in one operation by pairing HPLC to UV/Vis, MS or NMR detectors, especially if the study aims at determining individual flavonoids. Furthermore, HPLC enables the separation and quantification of the flavonoids simultaneously without requiring a preliminary derivatization. These factors have contributed to make HPLC analysis of flavonoids a commonly used technique.