Determination of enzyme (angiotensin convertase) inhibitors based on enzymatic reaction followed by HPLC

For determination of levels of plasmatic inhibitor of ACE (angiotensin conv ertase) a simple method was used based on a combination of enzymatic reacti on followed by an HPLC determination of its product. The inhibitor (e.g. en alaprilat) was at first separated from the biological material by deprotein ation (methanol). Then, an aliquot of the sample was added to the reaction mixture containing a commercial ACE enzyme, its specific substrate FAPGG (N -(3-[2-furyl]acryloyl)-Phe-Gly-Gly and buffer (Tris-HCl, pH 7.5). Degree of inhibition of the conversion of this substrate to FAP (desGlyGlyFAPGG) by the inhibitor present in the sample is related to its amount by a simple do se-response relationship. The amount of the FAP was determined by an HPLC o n a RP-18 column with an acetonitril-nonylamine buffer (pH 2.4, adjusted wi th phosphoric acid) as a mobile phase with detection at 305 nm. Alternative ly, the activity of the endogenous ACE present in the plasma was measured. The substrate FAPGG was added to the plasmatic sample containing both the i nhibitor and endogenous ACE las the sample was not deproteinized in this ca se) and the reaction product was determined as above. Inhibitor concentrati on has been obtained from a dose-response curve expressing the interaction with inhibitor with an ACE enzyme. (C) 2001 Elsevier Science B.V. All right s reserved.