Analysis of 2- and 3-series prostanoids by post-HPLC ELISA

Measurement of cyclooxygenase products of both arachidonic acid and eicosapentaenoic acid is of broad interest due to the wide and diverse biological activities of these compounds. We developed a technique combining (i) solid-phase extraction, (ii) reversed-phase HPLC separation using a high-capacity column, and (iii) post-HPLC enzyme-linked immunosorbant assay (ELISAs) employing monoclonal antibodies for quantification of all main 2- and 3-series prostanoids within one run. Thermal and chemical stress on these labile agents was reduced by use of ionic pair reagent and high buffer strength for processing at neutral pH, alpha-tocopherole as antioxidant, and freeze-drying evaporation during critical steps of the analytical procedure. With these techniques, mixtures of 2- and 3-series prostanoids (thromboxane (Tx)B2/3, 6-keto-prostaglandin (PG)F1 alpha/2 alpha, PGF2 alpha/3 alpha, PGF2/3) were recovered from Krebs-Henseleit buffer with linear characteristics for each compound in the range between 50 and 500 pg/ml. Percentages of overall recovery ranged between 45 and 90% for the different prostanoids with satisfying reproducibility. Application of the technique to a perfused rabbit lung and human platelets challenged with free eicosapentaenoic acid demonstrated marked generation of TxB3; quantities of this trienoic prostanoid as assessed by HPLC/ELISA were well correlated to those obtained by GC/MS. Thus, convenient quantification of 2- and 3-series prostanoids in media of biological interest is achieved by combined chromatographic and immunological techniques.