High-performance liquid chromatographic separation of renin–angiotensin system peptides and most of their metabolic fragments

We describe here a gradient HPLC procedure for the separation, and quantification by UV absorption of renin tri- and tetradecapeptide substrates, angiotensins I, II, III, IV and V, angiotensin-derived peptides, and peptidase inhibitors including amastatin, bestatin, pepstatin, lisinopril, a renin peptide inhibitor, Z-Pro-prolinal, N-[1-(R,S)-carboxy-2-phenylethyl]-l-Ala-l-Ala-l-Phe-p-aminobenzoate, and phosphoramidon. Most peptides and peptidase inhibitors were baseline-resolved within 32 min. The overall intra- and inter-assay precisions ranged from 0.8 to 5.9 (n=6) and 2 to 13% (n=6), respectively. There was a linear relationship (correlation coefficients≥0.9660) between peak height and peptide amount injected. In conclusion, the present method when combined with a peptidase-inhibitor paradigm can lead to the identification of renin–angiotensin system metabolizing enzymes, and when combined with radioimmunoassay can enhance the specificity of angiotensin measurement.