Fluorimetric determination of ampicillin by use of non-toxic catalysts. Estimation of beta-lactamase activity and parameters

A fluorescence assay based on the use of biological reducing agents as cata lysts rather than heavy metal ions has been developed for estimation of amp icillin concentrations. The assay is based on the conversion of ampicillin to its penicilloate, by treatment with sodium hydroxide, then neutralization, dilution with 0.5 M a cetate buffer at pH4 and heating for 30 min at 100 degrees C in the presenc e of ascorbic acid (0.5 mg) and EDTA (50 mu M). Reduced glutathione, cystei ne and N-acetylcysteine also catalysed the development of fluorescence. A p ractical sensitivity range of 0.5-50 mu M ampicillin was used. The assay wa s used to estimate ampicillin in some biological solutions to which the ant ibiotic has been added. Milli, blood serum, trypticase soy broth and nutrie nt broth containing 25 mu M antibiotic assayed at 18.5, 21.7, 16.5 and 14.7 mu M, respectively, with standard deviations between 1.2 and 0.7%. The low results were attributed to binding of some ampicillin by proteins or pepti des which were removed by pretreatment. Urine containing 5 mM ampicillin as sayed at 4.97 mM with a standard deviation of 3%. A modification of the pro cedure was used to measure beta-lactamase activity against ampicillin in se veral organisms in a fixed time assay. Kinetic parameters of a commercial b eta-lactamase preparation from Bacillus cereus could also be determined by an additional modification. In both instances a correction was required for the intrinsic fluorescence of ampicillin remaining in the solution. The pr eparation examined had a Michaelis constant (K-m) of 0.32 mM, a maximum vel ocity (V-max) of 5398 mu mol ampicillin hydrolysed mg(-1) min(-1) an appare nt catalytic constant (K-cat, turnover number) of 20 220 s(-1) and a K-cat/ K-m ratio of 0.63 x 10(7) M-1 s(-1) The major advantage of using this assay technique is that toxic metals are not used in the development of fluorescence so it is more environmentally a cceptable. The technique is useful for examining beta-lactamase activity ag ainst ampicillin and might be useful for pharmaceutical products-for determ ining available therapeutic levels and for monitoring the activity of penic illin acylase against the penicilloate of ampicillin.