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A high performance liquid chromatography procedure for the determination of ceftiofur and all its desfuroylceftiofur metabolites in plasma has been developed and validated using reverse phase liquid chromatography. Following a derivatization method that converts ceftiofur and all desfuroylceftiofur metabolites to desfuroylceftiofur acetamide, separation was attained on a Symmetry C18 column and quantification occurred using UV detection at 265 nm. The mobile phase was a mixture of (A) 0.1% trifluroracetic acid (TFA) in water and (B) 0.1% TFA in acetonitrile. The mixture was pumped at a starting gradient of 90% A and 10% B and was adjusted to 75% A and 25% B over 25 min, and back to initial conditions over 3 min. with a flow-rate of 1.0 ml/min. The procedure produced a linear curve over the concentration range of 0.1 – 100 μg/mL with a lower limit of quantification of 0.1 μg/mL. Intra-assay variability ranged from 0.7 to 4.5% and inter-assay variability ranged from 3.6 to 8.8% for desfuroylceftiofur acetamide, respectively, and the average recovery was 99%. This method could be useful to those investigators dealing with small sample volumes, particularly when conducting pharmacokinetics studies which require multiple sampling from the same animal.

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