Determination of ceftiofur and its metabolite desfuroylceftiofur in bovine serum and milk by ion-paired liquid chromatography

Abstract

A simple and sensitive liquid chromatographic method has been developed for the simultaneous determination of ceftiofur and its metabolite desfuroylceftiofur in bovine serum and milk. The method involved an ultrafiltration of diluted serum/milk with an equal volume of 50% acetonitrile through a 10 000 dalton molecular mass cut-off filter. Separation of ceftiofur and desfuroylceftiofur from the other serum/milk components was performed by ion-paired (octane and dodecanesulfonate) liquid chromatography using a reversed-phase column eluted with acetonitrile—water solution. The ultraviolet—visible absorbance of the column effluent was monitored in 200–350 nm range of a photodiode-array detector or at λ max 289.6 nm for ceftiofur, λ max 265.8 nm for desfuroylceftiofur and λ max 271.4 nm for dimer of desfuroylceftiofur. Recoveries of ceftiofur from bovine milk spiked with 1 and 10 μg/ml were 95.9 and 97.0% with coefficients of variation of 3.69 and 2.51%, respectively. Recovery of ceftiofur from bovine serum spiked with 10 μg/ml was 90.4% with a coefficient of variation of 5.29%. A correlation coefficient of 0.9992 occurred with ceftiofur in aqueous solutions ( n = 5, in duplicates). The limit of detection was estimated to be approximately 50 ppb (ng/ml). Additionally, this paper documents the presence of a ceftiofur metabolite in bovine serum under in vitro and in vivo conditions. The metabolite was identified as desfuroylceftiofur together with its dimer 3,3'-desfuroylceftiofur disulfide by thermospray liquid chromatography—mass spectrometry.