ANALYSIS OF 2-SERIES 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, α-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)F1α/2α, PGF2α/3α, PGE2 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. © 1993 Academic Press, Inc.