A sensitive enzymic assay for benzylpenicillin

A method has been developed for the determination of sodium benzylpenicillin concentrations in the range 3.3-33 micrograms/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 beta-lactam ring form of each compound shows little fluorescence with fluorescamine at pH 4 but shows strong fluorescence in the pH range 7-9. 6-Aminopenicillanic acid and its open beta-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.