The enantioselective immunoaffinity extraction of an optically active ibuprofen-modified peptide fragment.
Acyl glucuronides are known to produce the covalently bound protein adducts which may be the cause of hypersensitivity and toxic responses to acidic drugs. The structural analysis of the drug-protein adducts is therefore needed. From this point of view, we developed an enantioselective immunoaffinity extraction method, which employs an immobilized antibody to specifically isolate peptide fragments that have been modified with optically active ibuprofen. Rabbits were immunized with (S)-ibuprofen coupled to bovine serum albumin through a beta-alanine group. The elicited antibody strongly recognizes the asymmetric center and the isobutylphenyl moiety of (S)-ibuprofen and its conjugates but has a low affinity for their anti podes. A 0.5-mL aliquot of the immunosorbent (11.5 mg of IgG/mL gel) prepared by immobilization of the antibody was capable of retaining up to 1 microg of (S)-ibuprofen. When a mixture of substance P with (R)- and (S)-ibuprofen-modified substance P was loaded on the immunosorbent, the (S)-ibuprofen-modified substance P was selectively retained. The modified peptide was quantitatively recovered by elution with 10 mM ammonium acetate buffer (pH 5.0)/methanol (5:95, v/v). The proposed method would be useful for the structural characterization of optically active ibuprofen-modified human serum albumin.