Simultaneous determination of cross-reactive leukotrienes in biological matrices using on-line liquid chromatography...

Sulfidopeptide leukotrienes, which are important biomarkers for several diseases, are commonly measured by microtiter plate immunoassays. These immunoassays, however, cannot distinguish between several structurally similar leukotrienes and their cross-reactive metabolites and, therefore, need extensive sample handling and fractionation by means of liquid chromatography (LC). This paper describes the development and automation of a continuous-flow immunochemical detection (ICD) system and its subsequent on-line coupling to LC. The on-line LC-ICD system based on fluorescence-labeled leukotriene E4 (LTE4) was used to determine sulfidopeptide leukotrienes and their cross-reactive metabolites in a single run. Furthermore, biological matrices, e.g., urine and human cell extracts, were analyzed, the only sample pretreatment being on-line solid-phase extraction (SPE) on a novel RP-C4 restricted-access support. The determination limit of LTE4 in urine was 0.2 ng/mL (800 fmol; injection volume, 2000 microL; signal-to-noise ratio, 10). The system was linear from 0.2 to 1.0 ng/mL LTE4. Using nonlinear curve-fitting, the range could be expanded to 2.5 ng/mL. It is shown that, besides quantitation of known analytes, on-line LC-ICD is useful in the discovery of cross-reactive LTE4 metabolites.