Determining cyclooxygenase-2 activity in three different test systems utilizing online-solid phase extraction-liquid chromatography-mass spectrometry for parallel quantification of prostaglandin E(2), D(2) and thromboxane B(2).

Cyclooxygenase-2 (COX-2) catalyzes the formation of PGH2 from arachidonic acid. PGH2 is further converted to different prostaglandins (PG), such as PGE2, PGD2 and TxB2. In this study a rapid online-SPE-LC-MS method for the simultaneous quantification of PGE2, PGD2 and TxB2 streamlined for COX-2 enzyme assays is presented. Baseline separation of all analytes was achieved in only 7.1 min per sample, including sample preparation by online SPE. The method showed high sensitivity (LODs of 0.65-1.25 fmol on column) and accuracy (89-113%) in protein containing media. Because of online-SPE, no manual sample preparation was required, except for addition of IS solution, allowing to use the approach as rapid read-out in COX-2 activity assays. This was demonstrated by applying the method on three in vitro test systems: a cell-free enzyme assay, an assay using HCA-7 cells constitutively expressing COX-2 and primary human monocytes. In these assays, the potency of three popular drugs celecoxib, indomethacin and dexamethasone was successfully characterized with the new online-LC-MS method. The comparison of the results showed that the inhibitory effects of PG formation strongly depend on the test system. Thus we suggest that the modulation of COX-2 activity of a test compound should be at least characterized in two assay systems. With the online-SPE-LC-MS described in here we present a versatile tool as read-out for these types of assays.