Analysis of Plasma Angiotensins by Reversed Phase HPLC and Radioimmunoassay

Abstract

The analysis of biologically active angiotensin peptides in blood plasma by high performance liquid chromatography in a weakly non-polar reversed phase (C₂) chromatographic system combined with quantification of chromatographically isolated peptides by radioimmunoassay has been developed. This system is able to resolve each of seven closely-related peptides of the angiotensin group. The chromatographic system was applied to plasma samples which have been prepared for chromatographic analysis by C₁₈ cartridge extraction. Samples were reconstituted in HPLC solvent prior to injection into the HPLC system. Separated angiotensin were collected by fraction collector and the volatile components of the solvent system were blown off under an air stream. The content of several of the various angiotensin peptides in the fractions was then determined by radioimmunoassay using an appropriate antiserum. Antiserum to angiotensin II (octapeptide) was used to quantify the biologically active components angiotensin II, angiotensin III (hepta-peptide) and C-terminal hexapeptide. Recovery of angiotensin II in the C₁₈ cartridge extraction has been assessed at 85.0 ± 0.9% (mean ± SEM) using I¹²⁵-labelled angiotensin II, and 82.2 ± 4.45% using synthetic unlabelled angiotensin II. Recovery of standard preparations of angiotensin II in the HPLC system have been estimated at 67.5 ± 6.08%. The application of this technique to evaluating some components of the angiotensin in normal plasma is presented.