Rapid reversed-phase high-performance liquid chromatographic determination of the regiospecificity of lipoxygenase products on linoleic acid

A new reversed-phase high-performance liquid chromatography method for the separation of regioisomeric products from lipoxygenase acting on linoleic acid was studied. The addition of salts to the mobile phase improved the retention and separation behaviour of 13-hydroperoxy-9,11-octadecadienoic acid and 9-hydroperoxy-10,12-octadecadienoic acid with respect to the results obtained with other mobile phases reported in the literature. The effect of the pH and ionic strength of the buffer on the retention times, capacity factor and separation factor of these lipoxygenase products were also studied. The pH optimum coincided with the pKa of linoleic acid (close to 7 depending on the fatty acid concentration). Phosphate concentrations close to 100 mM considerably reduced the retention times and led to better separation of the mixture of both products. Finally, this method was applied to the identification and separation of two linoleic acid hydroxides (13-hydroxy-9,11-octadecadienoic acid and 9-hydroxy-10,12-octadecadienoic acid) obtained by the reduction of their corresponding hydroperoxides.