DETERMINATION OF PYRIDONECARBOXYLIC ACIDS IN PLASMA BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A simple qualitative and quantitative determination for pyridonecarbox ylic acids including nalidixic acid (NA), oxolinic acid (OA) and pipem idic acid (PPA) in chicken plasma was carried out by microbiological, spectrophotometric, thin-layer chromatographic (TLC) and reverse-phase high-performance liquid chromatographic (HPLC) methods. As a test org anism for bacteriological bioassay, Bacillus subtilis ATCC-6633 was th e most sensitive of seven organisms investigated. Using the cup and th e disc methods, a standard curve was obtained by determining the relat ionship between various drug concentrations and the diameter of the in hibition zone. The three drugs had two strong UV absorbance wavelength s (257 and 330 nm) on spectrophotometry. TLC analysis using a silica g el 60 F254 plate was investigated, and a solution of methanol:chlorofo rm:acetic acid (3:1:1, v/v/v) was found to be the most suitable solven t for separation. The minimum concentration of drug detectable by this method was 0.5 mug/ml for NA, 0.075 mug/ml for OA and 0.39 mug/ml for PPA. For HPLC analysis, a solution of acetonitrile:0.2 M phosphoric a cid (1:1, v/v) was superior, and simultaneous determination of all thr ee drugs was possible under the HPLC conditions used. The lowest measu rable amount of drug in chicken plasma was 0.01 mug/g. Recovery from e xtracts spiked with each drug at a known concentration was close to 10 0% for NA and OA, but only about 50% for PPA.