Procedure for Liquid Chromatographic Determination of Thiamphenicol in Bovine Serum and Milk

Abstract

We describe a method for measuring thiamphenicol (TP) by high-performance liquid chromatography (HPLC). The assay involves a simple extraction procedure using Extrelut-3 with ethyl acetate, evaporation of the extract, and redissolution in 40% acetonitrile. Wakosil-II 5C18 HG is used as a stationary phase with an eluting solvent of acetonitrile/water (40/60 by vol). TP is eluted from the column in about 2.3 min and is well separated from the other components present in serum or milk. Absorption of the eluent at 224 nm is monitored and measured. After extraction from a 1-ml sample, the antibiotic can be analyzed within 30 min. The within-day recoveries for bovine serum and milk spiked with 1 ppm TP were 92.9% and 93.5%, respectively, with coefficients of variation of 3.18% and 3.21%, respectively. The between-day recoveries for the 1 ppm samples were 91.9% and 92.3%, respectively, with coefficients of variation of 4.50% and 4.40%, respectively. The method is suitable for rapid and specific analysis of the drug in serum and milk, and will be applicable to other biological fluids, with a quantitative sensitivity of less than 0.1 ppm. We also analyzed TP by liquid chromatography-mass spectrometry with an atmospheric-pressure chemical-ionization interface system.