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.

Enantioselective immunoaffinity extraction for simultaneous determination of optically active bufuralol and its metabolites in human plasma by HPLC.

A combined method of immunoaffinity extraction with high-performance liquid chromatography has been developed for the enantioselective determination of bufuralol and its metabolites in human plasma. The antibodies having high affinity toward the asymmetric center at the C-1 position of bufuralol and its 1'-oxidized metabolites and low affinity to their antipodes were elicited by immunization of rabbits with immunogens, (1R)- and (1S)-1'-oxobufuralol O-carboxymethyloxime-bovine serum albumin 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 retaining up to 1 microgram of (R)- and (S)-bufuralol and up to 500 ng of other metabolites. The adsorbates were recovered quantitatively by elution with methanol-10 mM ammonium acetate buffer (pH 5) (95:5, v/v) without any interfering peaks on the high-performance liquid chromatogram. The proposed method was evaluated to be useful for the simultaneous determination of optically active bufuralol and its metabolite in plasma with acceptable recovery and precision.

Residues of doxycycline and oxytetracycline in eggs after medication via drinking water to laying hens.

Doxycycline (DOTC) and oxytetracycline (OTC) were dissolved in drinking water (0.5 g/l) and supplied to laying hens for 7 consecutive days. Eggs laid were collected daily during and after medication, and the antibiotic concentrations in the yolk and albumin were determined by the cup-plate method with Bacillus cereus var. mycoides ATCC 11778. The concentrations of both antibiotics were increased in yolk day by day with the advance in medication, reached peaks 2 days after withdrawal and then declined gradually. Mean peak concentrations in the yolk were 6.70 micrograms/g for DOTC and 1.42 micrograms/g for OTC. Peak concentrations in the albumin occurred in the middle stage of medication, where the mean values were 12.24 micrograms/g for DOTC and 1.03 micrograms/g for OTC. DOTC was detected in albumin until 24 days after withdrawal and for 2 days more in yolk than in albumin. OTC was detected in yolk until 9 days after withdrawal. The depletion period of OTC was shorter for the albumin, where the residue disappeared in all eggs 6 days after withdrawal. In spite of similarities between DOTC and OTC in structure, DOTC was deposited in higher concentrations and lasted for a longer period in eggs. This characteristic was considered due to its greater lipophilicity, closely correlated with oral absorption and tissue penetration.