Determination of angiotensin metabolites in human plasma by fluorimetric high-performance liquid chromatography using a heart-cut column-switching technique.

Fluorimetric column-switching HPLC method with naphthalene-2,3-dialdehyde (NDA) was developed for the determination of endogenous angiotensin (ANG) metabolites in human plasma. After one-step extraction to clean up the ultrafiltered plasma sample on the reversed HPLC system, the zone of the retention time of each ANG analyte was subjected to the NDA-derivatization. After putting into a first Phe-ODS (for ANG (3-4) and (5-8) determinations) or ODS column (for ANG I and II determinations), the heart-cut of the retention time of the NDA-ANG was separated on a second ODS column with a mobile phase containing 5 mM ion-pair reagent. Complete separation and good detection were accomplished within 2 h. Good linearity of the regression equation for all ANG analytes with the correlation coefficient of >0.993 as well as good reproducibility (C.V.<4.0%). Good agreement of the range of ANG II plasma level between the present (25-47 fmol/ml in plasma) and the radioimmunoassay methods (28-52 fmol/ml in plasma) indicated that the column-switching method could be applicable for the determination of endogenous smaller ANGs as well as for ANG I or II in plasma.