Determination of arachidonic acid by on-line solid-phase extraction HPLC with UV detection for screening of cytosolic phospholipase A2α inhibitors

Abstract

An on-line solid-phase extraction (SPE)-liquid chromatographic method with ultraviolet detection at 200nm for screening of inhibitors of cytosolic phospholipase A(2)α (cPLA(2)α) was developed and validated. cPLA(2)α was isolated from porcine platelets. Enzyme activity was determined by measuring the release of arachidonic acid from a phospholipid substrate using automated on-line sample clean up on a trap column followed by isocratic back-flush elution on a RP18 analytical column. While the use of a conventional RP18 column for trapping the analyte led to peak broadening only after a few runs due to pollution of the column by binding of components present in the enzyme preparation, the application of a turbulent flow column (TurboFlow Cyclone™) resulted in sharp peaks even after a plurality of injections. Interestingly, for sample introduction a turbulent flow of the mobile phase produced by high flow rates was not necessary to maintain good peak shapes. The same result could also be achieved applying low flow rates (0.5 mL/min). Several known cPLA(2)α inhibitors were used to validate the test system.