Total flavonoid content revised: An overview of past, present, and future determinations in phytochemical analysis

Flavonoids represent an important research topic in the analytical chemistry of secondary plant metabolites. During habitual laboratory determinations, preliminary quantitative analysis is often associated with in vitro colorimetric assessment. Total flavonoid content (TFC) is used as screening method with high relevance in the chemical analysis of plants and derived products, being typically applied before HPLC-MS phytochemical profiling. Its importance stems from affordability, simplicity, rapidity, and low cost. The AlCl₃ assay, with or without NaNO₂ addition, is the most used method in the present, although less frequently used methods (using 2,4-dinitrophenylhydrazine, dimethylamino-cinnamaldehyde, or diethylene glycol) show potential for complementary and specific determinations. Given the prevalence of research papers focusing on a single method for "total flavonoid" determination, we identified the need for an objective and critical comparison of existing methodologies. Moreover, a special notice is dedicated to the past and the future of in vitro TFC determinations, in the context of recent advances in flavonoid research. The focal point of this review is to serve as a basis for laboratory protocol reorganization regarding TFC determination, as a powerful tool before mass spectrometry, as well as to present a potential complementary analysis protocol applicable to biological samples. Among the methods found in the literature, SBC was the only assay providing accurate determinations of TFC.