Determination of total flavonoid content by aluminum chloride assay: A critical evaluation

Flavonoids are important natural bioactive compounds. Quantitation of total flavonoid content (TFC) is widely performed using the aluminum chloride colorimetric assay against a flavonoid standard assuming equal responses from all flavonoids. The aim of this work was to critically evaluate the assay employing spike recovery in plant extracts and three authentic flavonoid standards (catechin, quercetin, and rutin). Due to the inherent variations in absorbance values at quantitation wavelengths between investigated flavonoids, the assay produced huge unacceptable differences in TFC. For trials involving AlCl3 alone in standard solutions, false-positive results were obtained (63–124%) when quercetin content was expressed as rutin equivalents. Conversely, false-negative results were found (26–42%) when rutin concentration was expressed as quercetin equivalents. Similarly, unacceptable spike recoveries were recorded (8–106%) when involving AlCl3 alone in standard solutions at all investigated wavelengths. For plant extracts, unacceptable differences (58–152%) in TFC were also obtained when either quercetin or rutin was used as a quantitation standard. When AlCl3 is used in conjunction with sodium nitrite, unacceptable high or low recoveries were noted depending on the quantitation standard used. The findings of this work provide conclusive evidence highlighting conceptual and methodological flaws in the AlCl3 colorimetric assay for the determination of TFC.