LC–MS/MS for the simultaneous analysis of arachidonic acid and 32 related metabolites in human plasma: Basal plasma concentrations and aspirin-induced changes of eicosanoids

Eicosanoids play an important role in various biological responses and can be used as biomarkers for specific diseases. Therefore, we developed a highly selective, sensitive, and robust liquid chromatography–tandem mass spectrometric method to measure arachidonic acid and its 32 metabolites in human plasma. Sample preparation involved solid phase extraction, which efficiently removed sources of interference present in human plasma. Chromatographic separation was performed using a Luna C8-column with 0.5 mM ammonium formate buffer and acetonitrile as the mobile phase under gradient conditions. Detection was performed using tandem mass spectrometry equipped with an electrospray ionization interface in negative ion mode. The matrix did not affect the reproducibility and reliability of the assay. All analytes showed good linearity over the investigated concentration range (r > 0.997). The validated lower limit of quantitation for the analytes ranged from 10 to 400 pg/mL. Intra- and inter-day precision (RDS%) over the concentration ranges for all eicosanoids were within 16.8%, and accuracy ranged between 88.1 and 108.2%. This assay was suitable for the determination of basal plasma levels of eicosanoids and the evaluation of effect of aspirin on eicosanoid plasma levels in healthy subjects.