Improved spectrophotometric assay for β-lactam residues in kidney tissue

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-NIµ-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.