Fig. 4. Ionophore-challenged B. amphitrite larval sample showing generation of prostaglandin (PG)-like material prepared in the absence of the COX inhibitor, indomethacin. (A) HPLC-MS trace showing characteristic peak generation in the m/z 351 and 353 ion channels for the eicosanoid standards, PGE₂, PGD₂, PGF₂ and trioxilin A₃. (B) Larval sample showing peaks with prostaglandin-like masses (I-IV) on HPLC-MS prepared in parallel and run under identical HPLC-MS conditions to the standards in A. (C) HPLC fractions collected from material shown in B that was subsequently lyophilised, resuspended in buffer and assayed on a total PG Screening Assay Kit (Cayman Chemicals, Ann Arbor, MI, USA) designed to react with all classical PGs. This immunoreactivity was significantly reduced when barnacle samples were pre-incubated with indomethacin (20 µmol l^-1) prior to ionophore challenge (not shown).