Enzymic Determination of Peroxides in Non-aqueous Media

Abstract

A fast enzymic flow injection procedure was developed for the determination of peroxides in non-aqueous samples. The biochemical reaction is effected in a flow injection system using a non-covalently immobilized horseradish peroxidase reactor, followed by spectrophotometric monitoring of p-anisidine. The method provides a limit detection of 0.9 µM for hydrogen peroxide, 2.6 µM for tert-butyl hydroperoxide and 2.0 µM for benzoyl peroxide with a maximum sampling frequency of 60 h^–1. The enzyme reactor exhibits enhanced stability in water-saturated toluene, being stable for more than 4 months, and during this period an average number of 250 reactions can be performed with 1 mg of enzyme (220 purpurogallin units). The method permits the determination of peroxide in olive oil and margarine samples without any chemical pre-treatment or extraction of the sample. An average recovery of 98% was found for the determination of hydrogen peroxide in 12 different types of olive oil samples spiked with known amounts of H₂O₂, indicating the applicability of the procedure to real sample analysis. The procedure was also applied to the determination of H₂O₂ in four olive oils and a margarine sample and the results were comparable to those obtained by the reference method.