Formation of isoindole derivatives of sulfidopeptide leukotrienes by reaction with o-phthalaldehyde and separation by reverse phase high performance liquid chromatography

A method is described for the formation of fluorescent conjugates of the sulfidopeptide leukotrienes (LTC₄, LTD₄, and LTE₄) by reaction of the primary amine moiety of these metabolites with o-phthalaldehyde. Separation of the fluorescent derivatives was achieved by reverse-phase high performance liquid chromatography in less than 30 minutes using a convex gradient of methanol-50 mM Na Acetate-5% Tetrahydrofuran pH 5.5. Detection limits realized under the conditions described were 0.35 ng, 3.8 ng and 3.7 ng for LTC4, LTD4, and LTE4, respectively. This represents an increased sensitivity over detection of these metabolites by ultra-violet spectroscopy. The leukotriene-OPA derivatives are fully stable for 50 minutes at 23°C and for at least 4 hours at 0°C. The method is applied to the detection of LTC4 generated by zymosan stimulated murine peritoneal macrophages.