Rapid determination of cephalosporins with an immobilized enzyme reactor and sequential subtractive spectrophotometric detection in an automated flow-injection system

Abstract

A flow-injection system is described for the rapid automated determination of different cephalosporins in aqueous solution. Measurement is based on a selective enzymatic cleavage of the cephalosporin-β-lactam ring in a small immobilized enzyme reactor, which contains highly purified cephalosporinase covalently bound to activated glass beads. Samples are injected into a phosphate buffer stream. The opening of the β-lactam ring is accompanied by a significant decrease in the ultraviolet (u.v.) absorbance. The difference between the absorbances of untreated and degraded cephalosporin is measured at 254 nm and is related linearly to cephalosporin concentration over the range 10–800 μg ml −1. Several commercially available cephalosporins including some β-lactamase-resistant types were examined. The accuracy of the enzyme reactor/flow injection system was evaluated by comparison with h.p.l.c. results; the correlation was good. The relative standard deviation, evaluated from 15 consecutive injections of the same sample, was 0.7%; 2 μg ml −1 cephalosporin C was the minimal detectable concentration. A single determination took about 2 min; sample throughput is 30 h −1. Different β-lactamases were examined for enhancement of substrate selectivity.