ENANTIOSELECTIVE IMMUNOAFFINITY EXTRACTION FOR SIMULTANEOUS DETERMINATION OF OPTICALLY-ACTIVE BUFURALOL AND ITS METABOLITES IN HUMAN PLASMABY HPLC

A combined method of immunoaffinity extraction with high-performance l iquid chromatography has been developed for the enantioselective deter mination of bufuralol and its metabolites in human plasma. The antibod ies having high affinity toward the asymmetric center at the C-1 posit ion of bufuralol and its 1'-oxidized metabolites and low affinity to t heir antipodes were elicited by immunization of rabbits with immunogen s, (1R)- and (1S)-1'-oxobufuralol O-carboxymethyloxime-bovine serum al bumin conjugates, respectively. 0.5 ml Of the immunoaffinity adsorbent (7.6 mg.ml(-1) for anti-(1S)-antibody and 28.8 mg.ml(-1) for anti-(1R )-antibody) prepared by immobilization of an antibody was capable of r etaining up to 1 mu g of (R)- and (S)-bufuralol and up to 500 ng of ot her metabolites. The adsorbates were recovered quantitatively by eluti on with methanol-10 mM ammonium acetate buffer (pH 5) (95:5, v/v) with out any interfering peaks on the high-performance liquid chromatogram. The proposed method was evaluated to be useful for the simultaneous d etermination of optically active bufuralol and its metabolite in plasm a with acceptable recovery and precision. (C) 1998 Elsevier Science B. V. All rights reserved.