The validation of a new high throughput method for determination of chloramphenicol in milk using liquid–liquid extraction with low temperature partitioning (LLE-LTP) and isotope-dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS)

Abstract

Chloramphenicol (CAP) is an antibiotic banned for treatment of food-producing animals. The minimum required performance limit (MRPL) of 0.3 μg kg⁻¹ for analytical methods was set by the European Community for the detection of its residues in different matrices, including milk. A highly sensitive analytical method using isotope-dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) and liquid–liquid extraction with low temperature partitioning (LLE-LTP) was developed and validated for CAP residues in milk. Selected validation parameters such as selectivity, working range and linearity, trueness (recoveries), precision (repeatability and intermediate precision), limit of detection (LOD) and limit of quantification (LOQ) were evaluated. The validation procedures were based on the new Eurachem Guide and the European Union Commission Decision 2002/657/EC. The matrix effect was evaluated by the ion suppression test and by comparison of the matrix-matched analytical curve and the solvent standard analytical curve. A linear working range between 0.1 and 5.0 μg kg⁻¹ was observed. The homoscedasticity was demonstrated by the Cochran test, the coefficient of determination (R²) was higher than 0.99 and the residual plot was free of trends. The limits of detection and quantification were 0.015 μg kg⁻¹ and 0.05 μg kg⁻¹, respectively. Mean recoveries evaluated at three levels (0.3, 1.0 and 3.0 μg kg⁻¹) ranged from 94% to 114% with RSDs lower than 6.7% (repeatability). For intermediate precision, different analytes were compared and the RSDs were lower than 7.3%. The method was accurate and reproducible, and was successfully applied to the evaluation of CAP residues in milk samples.