Automated high-performance liquid chromatographic extraction and quantification procedure for lipoxygenase...

A number of methods have been used to measure various lipoxygenase metabolites in aqueous samples. These methods, however, suffer from three major limitations: first, they require extensive extraction and isolation from protein-containing media; second, mainly due to the first limitation, they have poor recoveries; and third, these methods usually require a two-step procedure, one for the actual extraction and the other for the quantification of the lipoxygenase metabolites. We have developed a fully automated high-performance liquid chromatographic method which circumvents these limitations. As a result, we are able to obtain high recoveries of various lipoxygenase metabolites from protein-containing samples (i.e. biological samples) while simultaneously quantifying each metabolite. The method employs a column venting technique, whereby the fatty acids are extracted by a pre-column and the proteins are vented to waste. The pre-column eluate is then directed through the analytical column which separates the lipoxygenase metabolites. The described method is reproducible and minimizes both the time and the cost involved in assaying a sample.