IMPROVED HPLC RADIOIMMUNOASSAY FOR QUANTIFYING ANGIOTENSIN-II IN PLASMA

To measure the octapeptide angiotensin II (Ang II) in plasma, we devel oped a sensitive, specific assay that interfaces solid-phase extractio n, HPLC, and RIA. A reversed-phase HPLC system involving isocratic elu tion at 38 degrees C with a volatile mobile phase of acetonitrile and the ion-pairing reagent heptafluorobutyric acid produced baseline sepa ration of angiotensin peptides. Ang II was collected as a single fract ion, concentrated by evaporation to dryness, and measured by RIA after resuspension in RIA buffer. Even including column washing between sam ple injections to prevent carryover of plasma constituents, two plasma extracts could be processed per hour by HPLC. Assay validation experi ments demonstrated <2% cross-reactivity with Ang Ii-related peptides; a 75% recovery from plasma at physiological concentrations of Ang II; intra- and interassay precision (CVs) of 6.2% and 10.3%, respectively; and a lower limit of quantification of 1.3 ng/L. Two clinical protoco ls designed to measure plasma Ang II concentration under basal and sti mulated conditions confirmed the utility of the assay.