Optimizing Absorbance for Ferric-Xylenol Assay

The FOX assay was carried out with some modifications of the previously described method (Gay and Gebicki 2002 (link); Pinto et al. 2007 (link)). The xylenol orange reagent containing 2.0 mM ferrous sulfate, 0.29 mM xylenol orange tetrasodium salt, and 440 mM perchloric acid in methanol/water (9:1) was freshly prepared. To find the optimum absorbance at which the concentration of the ferric-xylenol orange (Fe³⁺–XO) complex can be determined, 30 µL of cumene hydroperoxide (0–10.52 mM dissolved in water) and 150 µL xylenol orange reagent were mixed well. The mixtures were then incubated for 15 min at room temperature (~ 20 °C) and the absorption spectra of the mixtures were measured in the wavelength range of 400–650 nm using a Spectramax ID3 multi-detection microplate reader (Molecular Devices, LLC, San Jose, California, USA). When the absorbance of the samples exceeded 2.0, the samples were diluted in 75% methanol in water. The actual absorbance of the sample was then calculated, taking the dilution factor into account.