Reversed-phase ion-pair liquid chromatography of the angiotensins.
The isocratic reversed-phase liquid chromatography of the angiotensins and a number of their synthetic analogues is described. Complete separation of 10 out of 12 peptides was achieved through a solvent optimization strategy with a total analysis time of about 20 min. The retention behavior of the angiotensins studied was described in terms of the hydrophobic contribution of their amino acid residues; there was good correlation between predicted and experimental retention for those peptides that were retained by a common mechanism. However, because ion-pair chromatography was required for good peak symmetry, retention was substantially modulated by the presence of acidic and basic residues. The limit of detection of these peptides was 3-5 pmol by UV absorbance at 214 nm. For those peptides containing a primary amino group the detection limit was improved by two orders of magnitude by fluorogenic derivatization with naphthalene-2,3-dicarboxaldehyde/cyanide to the corresponding N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives. The contribution of the CBI ring system to retention was also investigated.
Patel
H B
HB
Center for BioAnalytical Research, University of Kansas, Lawrence 66045-2504.
Stobaugh
J F
JF
Riley
C M
CM
D000595:Amino Acid Sequence; D000809:Angiotensins / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D007477:Ions; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000809:Angiotensins; D007477:Ions