A method for determining free azide ions by automatic analysis in the presence of a covalent cephalosporin azide

Abstract

An assay was devised in order to follow quantitatively the release of azide as free ions, N₃^–, from a cephalosporin azide when attacked by β-lactamase enzymes produced by specific strains of bacteria. Experiments were arranged to ascertain whether or not the azide cleavage occurred at the same rate as the rupture of the β-lactam ring. The assay was required to determine free azide at concentrations between 2 and 20 µg ml^–1; other experimental limitations were imposed by the requirements of enzymolysis.

The procedure adopted was based on the complete oxidation of azide ions to nitrogen by an excess of a standard aqueous solution of nitrite ions at pH 4·6. The residual nitrite was removed by diazotisation with 4-amino-salicylic acid, followed by coupling of the product with a second molecule of the 4-aminosalicylic acid. The reaction mixture was then rendered alkaline by the addition of tetramethylammonium hydroxide solution. The final yellow colour was stable and had an absorption maximum at 440 nm. It was not possible, however, to achieve sufficient operational reproducibility manually, and an Auto Analyzer system was therefore used.

The results show that cleavage of azide from the parent molecule occurs at the same rate as the rupture of the β-lactam ring and parallels the decline in microbiological potency.