Improved spectrophotometric assay for β-lactam residues in kidney tissue

Abstract

This paper describes a detection system for β-lactams using a commercially prepared carboxypeptidase enzyme (CPase) and a substrate system in which lactic acid is cleaved from a synthetic peptide, Nα-Nε-diacetyl-L-lysyl-d-alanyl-d-lactic acid. The lactate is itself oxidized by lactate dehydrogenase to form NADH. Oxidized NAD⁺ is regenerated by diaphorase with the simultaneous reduction of the colourless 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride hydrate (INT) indicator substrate to produce a red–mauve colour that is proportional to CPase activity. The presence of β-lactams decreases the intensity of colour produced. The lower limit of detection for benzyl penicillin (Pen G) by this system is 20 ng g^–1 compared with 50 ng g^–1 by the same assay but using an R-d-ala-d-ala substrate from a commercial kit.