A quantitative semi-automated turbidimetric bioassay for cefazaflur, using Streptococcus faecium as the indicator, is described. Assays were run at pH 6.5 -7 for 3.75 hours at 37°C using 2-12,ug cefazaflur per ml assay broth for standards.The dose response line was plotted point to point using the natural log of the absorbance vs natural log of the concentration. This assay is both accurate and precise and is more rapid than traditional plate assays for antibiotics.Cefazaflur, SK&F 59962, is a cephalosporin with a broad spectrum of in vitro and in vivo antibacterial activity. The pharmacokinetics have been studied in several species of laboratory animals and in man1,2). Plate assays for antibiotics, while acceptable, may not provide adequate sample throughout during development of a new compound. The AutoturbR, a semiautomated system for doing photometric assays, has been shown capable of producing large numbers of precise assays of vitamins or antibiotics including several l-lactams3,4). This system is best suited for the assay of bulk or formulated samples rather than samples of body fluids from clinical specimens. This paper details our experience with the Autoturb® for assay of cefazaflur. Included in the study are (1) dose-response relationship, (2) effect of pH, (3) effect of incubation time, (4) assay precision as a function of pH, (5) standard response line plotting parameters and (6) a comparison with two chemical assays.
Materials and MethodsSodium cefazaflur samples were prepared fresh daily in 1 % phosphate buffer, pH 6.0. The standard substance was kept in a dessicator at -20°C until used. The indicator culture used was Streptococcus faecium ATCC 10541. Culture conditions, media, and operation of the Autoturb® were described previously.') Separate standard response lines were prepared from data obtained from each of the four sampling loops of the Autoturb(D. Results from test samples from each loop were calculated using the standard response line prepared from that loop. Data were analyzed point to point using a plot of the natural log of the absorbance versus the natural log of the concentration.Chemical Assays: The cefazaflur content of samples was determined chemically using the automated hydroxylamine assay described for cefazolin') and by high pressure liquid chromotography (HPLC) described as follows: The HPLC assay was run at ambient temperature on a Chromatronix 3100 chromatograph using a strong anion-exchange resin column (DuPont 820960005) one meter long and 2.1 mm ID. The column was operated at 1,500 PSI and a flow rate of 1.3 ml/min. An ultraviolet detector operating at 254 nm was used. The mobile phase was 1.42% anhydrous sodium sulfate in distilled water adjusted to pH 3.5 with acetic acid. Samples were introduced to the column using a 20 Id loop. Under these conditions, the retention time for cefazaflur was about 11 minutes.
