A sensitive enzymic assay for benzylpenicillin

A method has been developed for the determination of sodium benzylpenicillin concentrations in the range 3·3–33 μg/ml. 6-Aminopenicillanic acid is released from the benzylpenicillin by the action of the enzyme penicillin acylase and is estimated from its reaction with fluorescamine at pH 4.7-Aminocephalosporanic acid shows a similar trend to 6-aminopenicillanic acid in its reaction at pH 4. The open β-lactam ring form of each compound shows little fluorescence with fluorescamine at pH but shows strong fluorescence in the pH range 7–9. 6-Aminopenicillanic acid and its open β-lactam ring form give different fluorescent responses to increasing volumes of a solution of the fluorigenic agent at pH 7·8. This effect can be used to estimate concentrations in a mixture of the two components providing other amino material is absent.