An automated high-performance liquid chromatography fluorescence method for the analyses of endothelins in plasma samples.

A high-performance liquid chromatographic method with fluorescence detection was developed to simultaneously analyze endothelins, a class of vasoactive peptides, in plasma samples. Sample preparation for HPLC analysis was carried out by initial stabilization of blood and plasma samples against transformation of big endothelins to mature endothelins and breakdown of mature endothelins by serine proteases, as well as oxidative modifications of endothelins. Deproteinization of plasma samples was achieved with acidified acetone, and the samples were further purified on molecular weight cutoff filters. Endothelins were separated on a reversed phase LC-318 column by gradient elution using a mobile phase containing acetonitrile and water (0.1% trifluoroacetic acid) and were analyzed by fluorescence detection (lambda(Ex), 280 nm; lambda(Em), 340). Limit of detection values were in the range of 0.2-0.5 pmol. Linear (R(2), 0.99) calibration curves were established for analyte amounts in the range of 1 to 100 pmols. Recoveries of endothelins from spiked plasma samples analyzed ranged from 60-95%. Under optimized conditions the HPLC-fluorescence method was determined to be sensitive and specific for the analysis of big endothelin-1, endothelin-1, endothelin-2, and endothelin-3 in plasma. Simultaneous measurement of these endothelins by the HPLC method should permit a better understanding of their specific roles and relationships under various pathological conditions.