A Critical Evaluation of the Effect of Sorbitol on the Ferric–Xylenol Orange Hydroperoxide Assay

Measurement of hydroperoxide concentration by the ferric–xylenol orange assay has the advantages of simplicity, convenience, and inertness to oxygen (Gay, C., et al. (1999) Anal. Biochem. 273, 149–155). However, its sensitivity is limited by the molar absorption coefficients, which are less than 50,000 M−1 cm−1 for most hydroperoxides. An earlier report showed that this could be significantly enhanced by the inclusion of 100 mM sorbitol in the assay solution, resulting in an increase of the apparent absorption coefficient for H2O2 from 4.46 × 104 to 2.24 × 105 M−1 cm−1 (Wolff, S. P. (1994) Methods Enzymol. 233, 182–189). It was claimed that the technique was also valid for other hydroperoxides, such as butyl and cumyl. In an attempt to extend this modification to a wider range of hydroperoxides, we have confirmed the enhancement of the assay of H2O2 by sorbitol. However, the sensitivity of the measurements of butyl, cumyl, amino acid, protein, and human blood serum hydroperoxides was only approximately doubled by the inclusion of sorbitol. A mechanism explaining the difference in the assay of H2O2 and the organic hydroperoxides is proposed.