Determination of renin activity in human plasma by column-switching high-performance liquid chromatography with...

A column-switching high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the determination of the renin activity in human plasma. The method is based on the quantification of the enzymatically produced angiotensin I. Angiotensin I liberated from a synthetic substrate (tridecapeptide of human angiotensinogen) and [Val5]-angiotensin I as an internal standard are converted into fluorescent derivatives by reaction with benzoin. The derivatives are separated from various interfering substances by column-switching HPLC using three reversed-phase columns. The limit of detection (signal-to-noise ratio = 3) of the renin activity is 2.7 pmol of angiotensin I formed per h per ml of plasma, which corresponds to approximately 820 fmol of angiotensin I injected. The column-switching method in combination with pre-column derivatization for the fluorimetric detection permits the sensitive and selective determination of the enzymatically formed angiotensin I. Hence low activities of renin in normal human plasma are readily measured.