Development of a flow injection chemiluminescent assay for the quantification of lipid hydroperoxides

An automated flow injection chemiluminescence (FICL) system for measuring lipid hydroperoxide (LOOH) concentrations in oils was developed. Initially, a crude oil-in-water emulsion (formed by mixing solvent-diluted oil with the aqueous-based CL compound, luminol, and the catalyst for the reaction, cytochrome c) was tested. The assay was rapid (60 samples per hour), reproducible (CV no greater than 10%, n = 3) and had a low sample requirement (1 mg of oil) because of its high sensitivity (0.5 nmol LOOH). CL intensity was influenced by the amount and type of oil under analysis. Owing to these factors, quantitative data were attainable only with a uniform oil concentration and with a calibrant derived from an oil equivalent to that under analysis. This method yielded quantitative data in good agreement with an iodometric titration assay for LOOH (r = 0.9204). A refinement of the first method consisted of replacing the luminol and cytochrome c: CL compounds with lucigenin, resulting in an assay insensitive to alpha-tocopherol. A monophasic reaction solution was devised to remove the effect of turbidity; however, the CL signal was still influenced by oil type. Therefore, quantitative data were still attainable only when the same type of oil was used for calibration.