Critical Examination of the Suitability of the Folin-Ciocalteu Reagent Assay for Quantitative Analysis of Polyphenols—The Case of Olive-Mill Wastewater

Abstract

The suitability of the Folin-Ciocalteu reagent (FCR) and of HPLC for analyzing polyphenols is evaluated. FCR assay is commonly used although its flows, such as overestimating polyphenol content due to interference by oxidizable species, were previously reported. The wide range of oxidizable species present in biological systems seriously compromises this assay’s reliability. Adding small amounts of protein to olive-mill wastewater (OMWW) significantly increased the apparent polyphenol content indicated by the FCR assay. The commonly used “reference” polyphenol as a standard for “total polyphenols” quantification is problematic since each polyphenol responds differently to the FCR. Conversely, HPLC may underestimate polyphenol content. No single HPLC protocol is likely to detect the whole myriad of polyphenols which may be present in a polyphenol-containing system. In analyzing 5 OMWW samples both by FCR assay and HPLC, the polyphenol content indicated by the FCR assay was up to six-fold higher than that determined by HPLC.