Determination of tilmicosin in bovine and porcine sera by liquid chromatography

A liquid chromatographic (LC) assay is described for determining tilmicosin in bovine and porcine blood sera. Tilmicosin is isolated from the serum matrix and purified by solid-phase extraction with C18 sorbent. Sample is analyzed by LC using a gradient system with a phenyl reversed-phase column that separates tilmicosin from the matrix in 30 min. Tilmicosin is measured by UV absorbance at 280 nm. Validation of assay included evaluation of accuracy, precision, linearity, specificity, sensitivity, range, and sample stability. The method has a limit of quantitation of 0.1 ppm and a validated range of 0.1 to 10.0 ppm. Recoveries were 91-95% for bovine serum and 85-93% porcine serum. The limit of detection was 0.05 microgram/mL. Limits of detection and quantitation were based on 3 and 6 times the baseline noise of control serum samples, respectively. Relative standard deviations of precision samples (n = 6) were 2% or less for both sera. The method has better specificity and analysis time than previous microbiological methods for tilmicosin in sera.