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

A high-performance liquid chromatographic method with fluorescence detectio n was developed to simultaneously analyze endothelins, a class of vasoactiv e peptides, in plasma samples. Sample preparation for HPLC analysis was car ried out by initial stabilization of blood and plasma samples against trans formation of big endothelins to mature endothelins and breakdown of mature endothelins by serine proteases, as well as oxidative modifications of endo thelins. Deproteinization of plasma samples was achieved with acidified ace tone, and the samples were further purified on molecular weight cutoff filt ers. Endothelins were separated on a reversed phase LC-318 column by gradie nt elution using a mobile phase containing acetonitrile and water (0.1% tri fluoroacetic 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 a nalyte amounts in the range of 1 to 100 pmols. Recoveries of endothelins fr om spiked plasma samples analyzed ranged from 60-95%. Under optimized condi tions the HPLC-fluoreseence method was determined to be sensitive and speci fic for the analysis of big endothelin-1, endothelin-1, endothelin-2, and e ndothelin-3 in plasma. Simultaneous measurement of these endothelins by the HPLC method should permit a better understanding of their specific roles a nd relationships under various pathological conditions. (C) 2001 Elsevier S cience.