Automated enzyme-inhibition assay for clavulanic acid in biological samples

Abstract

Clavulanic acid is a B-lactamase inhibitor [l, 21 with low intrinsic antibiotic activity. The conventional cup-plate assay is unsuitable for its determination in biological fluids because of the low sensitivity to clavulanic acid of test organisms such as Staphylococcus aureus and Bacillus subtilis. HPLC methods for the determination of clavulanic acid in plasma and urine have been reported [3-51, but these methods are unsuitable for the measurement of low nanogram concentrations of clavulanic acid which are encountered in single-dose pharmacokinetic studies. A synergistic plate assay has been reported [6] in which bacterial growth is inhibited by penicillin G in regions where B-lactamase is inhibited by clavulanic acid. The measurement of residual penicillin G resulting from p-lactamase inhibition offers potential for a selective and sensitive chemical assay for clavulanic acid in biological fluids. Penicillin G is hydrolysed by B-lactamase and residual intact penicillin is measured chemically using the Bundgaard reaction [7]. If a B-lactamase inhibitor is introduced into the system, less penicillin G is hydrolysed resulting in a higher measured penicillin concentration. The measured increase in penicillin concentration is a function of the inhibitor concentration. An autoanalyser method based on this principle has been developed for the determination of clavulanic acid in plasma and urine. The continuous-flow incubation manifold is combined with a penicillin autoanalyser (Wadds and Legg, unpublished observations) for measurement of the substrate concentration.