Residue determination of two co-administered antibacterial agents : cephalexin and colistin : in calf tissues using high-performance liquid chromatography and microbiological methods

Abstract Residues of two antibacterial agents, cephalexin and colistin, co-administered by intramuscular injection to calves, were quantified in four different tissues (muscle, fat, liver and kidney) by column switching HPLC and by a microbiological method. For cephalexin assay, tissue samples with cephradin as internal standard were homogenized in a 5% trichloroacetic acid solution and filtrates were injected onto a concentration pre-column filled with LiChroprep RP-18 (25–40 μm). A clean-up step was incorporated by flowing a mobile phase (methanol—0.01 M phosphate buffer (pH 3.0); 15:85, v/v) through the enrichment column before elution on a LiChrospher RP-18e (5 μm) column with a methanol—phosphate buffer (30:70, v/v) at a flow rate of 1 ml min −1 . Spectrometric detection was at 260 nm. An additional “off-line” washing step of extracts with methylene chloride was operated to achieve higher selectivity in the case of liver and kidney samples. The limit for quantitative assay was 0.045 μg g −1 with relative standard deviations in the range 5–8% and recoveries within 70%. For microbiological assay of colistin, samples were homogenized in 0.1 M hydrochloric acid–acetonitrile mixtures (3:1, v/v, for kidney and liver; 3:2, v/v, for fat and muscle). The supernatants were assayed by the cylinder plate method after evaporation to dryness under vacuum. Bordetella bronchiseptica ATCC 4617 was chosen as test organism. After a 3-h diffusion step at room temperature, the medium was incubated at 37°C for 18 h and then the diameter of the growth inhibition zones was measured. Sensitivity reached 0.10–0.15 μg g −1 . Results from the analysed samples over a 7–28 day period after drug administration show that no cephalexin was found at concentrations higher than the quantitation limit in the four test tissues and that colistin was found in muscle (injection site only) for 15 days and in kidney for 21 days.