The Folin–Ciocalteu assay revisited: improvement of its specificity for total phenolic content determination

TLDR

Phenolics, known for their potent free‑radical neutralizing ability, are routinely quantified in food research, yet their total phenolic content (TPC) measurements are often skewed by reducing interferants such as ascorbic acid, prompting the development of methods to enhance the Folin–Ciocalteu assay’s specificity. This review aims to evaluate existing strategies to improve Folin–Ciocalteu assay specificity and proposes a simple modification that simultaneously quantifies TPC and ascorbic acid reducing activity. The proposed approach combines solid‑phase extraction, correction for ascorbic acid activity, and oxidative pre‑treatment, and details how these steps are implemented to separate interferants and refine TPC estimation. Experimental data show that the modified assay yields corrected TPC values, and the discussion highlights the advantages and drawbacks of each method.

Abstract

This study presents a review of the Folin–Ciocalteu (F–C) assay for total phenolic content (TPC) determinations and describes different approaches to improve its specificity. Phenolics are regarded as the molecules with the highest potential to neutralize free radicals. Therefore, their quantification is a common practice in different areas of food research. However, when determining TPC in plant food extracts, the presence of reducing interferants [ascorbic acid (AA)] produces inaccurate estimations of TPC values. Different methodologies have been proposed to improve the specificity of the F–C assay. These methodologies include: (i) the use of solid phase extraction (SPE) cartridges to separate interferants from phenolics; (ii) the calculation of a corrected TPC value based on the AA reducing activity present in the extract; and (iii) the pre-treatment of extracts with oxidative agents prior to TPC quantification. These methods are described in detail in the present study. Likewise, their advantages and disadvantages are discussed based on new experimental data. A simple modification of the F–C assay procedure is proposed to quantify both the TPC value and the AA reducing activity in plant food extracts. Values obtained by the modified F–C assay can be used to estimate a corrected TPC value.