Limitations of the Tetramethylmurexide Assay for Investigating the Fe(II) Chelation Activity of Phenolic Compounds

Limitations of the colorimetric assay involving tetramethylmurexide (TMM) to determine the extent of complex formation between metal ions and phenolic compounds have been studied. Older literature reports using this method to determine bound Fe(II). Our study shows the TMM assay is inadequate when determining the Fe(II) chelation activity of phenolic preparations rich in tannin constituents on account of the high absorbance values derived by control samples (i.e., those that do not contain the TMM reagent). Phenolic test samples comprising the TMM reagent, iron ions, and tannins could not yield meaningful absorbance data on Fe(II) chelation activity. In our study, we investigated commercially available compounds, namely, sinapic acid, catechin, rutin, tannic acid, procyanidin B(2), as well as crude acetonic extracts of almonds, red lentil, buckwheat, and their low-molecular-weight and tannin fractions separated from the crude extracts by Sephadex LH-20 column chromatography. Even as little as 0.5 mg of tannins added per control sample resulted in high absorbance values to the extent of 0.4 for red lentil and almonds, and 1.3 for buckwheat. A strong correlation (r(2) = 0.98) between the content of condensed tannins, as determined by the vanillin reaction, and absorbance of control samples by the TMM assay was found for the plant extracts and their fractions. A more useful colorimetric assay to investigate the Fe(II) chelating ability of tannin-rich preparations may be the method that uses ferrozine.