Chemical Perspective and Drawbacks in Flavonoid Estimation Assays
Authors: Mammen Denni 
Published: May 2022

Colorimetric or spectrophotometric methods have been used over the past few decades for rapid and convenient estimation of certain classes of flavonoids in fruits, vegetables, grains, raw herbal material, herbal formulations, and nutraceuticals. This has resulted in a surge in the numbers of research articles discussing the use of these methods for comparison between numbers of samples of the same kind, such as analysis to find differences between various tea samples, food articles, raw drug powders, etc. However, these methods are not selective since several factors influence color development. Also, the reagents used to form the colored complex are not specific to a certain class of compounds. There are studies performed where all compounds belonging to a particular class do not react uniformly to the reagents used in the method. Chelation using AlCl3 was used to develop deep yellow-colored complexes of the flavonoids and absorbance was subsequently measured at 420 nm, using quercetin as the standard. In a modification, potassium acetate was added after the addition of AlCl3, and the absorbance was measured at 415 nm, again against standard quercetin solutions, wherein only flavones and flavonols were estimated. A study conducted by our team proves that all flavonoids do not form complexes that absorb at 420 nm, and each flavonoid shows variation in absorption maxima. Only flavonoids with o-dihydroxy systems show good results, while others absorb at either higher or lower wavelengths. This research work has been one of the top 20 most downloaded articles in flavonoid chemistry since its date of publication. Catechins, flavanones, and anthocyanins cannot be estimated using this method, due to either inability to bind with AlCl3 in an appropriate manner or due to differences in absorption maxima of the complex formed. Flavanones like naringenin, naringin, and hesperidin have been estimated using the 2,4-dinitrophenyl hydrazine method. The method does not work for flavonols and flavones. Estimation of catechins in tea samples has been described where caffeine is removed from solution using extraction by chloroform, and the absorbance of the aqueous phase is taken at 274 nm. The technique however is flawed since the aqueous extract will also contain phenolic acids like gallic, protocatechuic, and syringic acids, and a good amount of flavonols such as quercetin and kaempferol, which also absorb around 274 nm. These phenolic acids and flavonols need to be removed before the estimation of catechins. The reaction of flavanols like catechin and epicatechin with vanillin in presence of H2SO4 yields redcolored complexes that show absorptions around 500 nm, but certain matrices interferences of proanthocyanins. Many flavonoid compounds occur in the form of glycosides, where the presence of sugar molecules like glucose, rhamnose, galactose, etc. can hamper complex formation responsible for color development. The effect of hydrolysis can yield better results to remove the sugar moieties, and the aglycones can be estimated. Another widely used method is the Folin-Ciocalteu method for estimation of phenolics, developed by Folin and Denis in 1915, and modified by Singleton and Rossi in 1965, where a blue-colored complex due to reduction of molybdenum by phenolate ions formed in a basic medium. One major drawback of this method is that the absorption maxima of the complex formed varies between 620 and 765 nm. Studies also confirm that this assay is not specific to only phenolics, but can also react to interferences of ascorbic acid, reducing sugars, certain metals, amino acids, and reducing agents like NaHSO3. Most results published in thousands of research papers worldwide are erroneous due to a lack of knowledge of the actual chemical reactions taking place in the estimation methods, and how the flavonoids react with the reagents.