Abstract
Despite growing acknowledgement of the role of oxidized fatty acids (oxFA) as cellular signaling molecules and in the pathogenesis of disease, developing methods to measure these species in biological samples has proven challenging. Here we describe a novel method utilizing HPLC-ESI-MS/MS to identify and quantify multiple full-length oxFA species in a regioisomer-independent manner without the need for time-consuming sample preparation or derivatization. Building on recent progress in the characterization of FA and their oxidation products by MS/MS, we employed positive-ion ionization by measuring sodium adducts in conjunction with Differential Energy Qualifier Ion Monitoring to unequivocally verify the presence of the hydroperoxide, hydroxide, and ketone oxidation products of linoleic and arachidonic acid. Our HPLC method achieved separation of these oxidized species from their unoxidized counterparts while maintaining regioisomer-independent elution, allowing quantification over a 5 log10 range with a lower limit of quantification of 0.1 picomoles. With a simple sample preparation and a runtime as low as 11 minutes, our method allows the rapid and facile detection and measurement of full-length oxFA in biological samples. We believe this approach will allow for new insight and further investigation into the role of oxFA in metabolic disease.