Comprehensive validation of a liquid chromatography–tandem mass spectrometry method for the confirmation of chloramphenicol in urine including stability of the glucuronide conjugate and efficiency of deconjugation

• Efficiency of deconjugation process was demonstrated.
• Stability of the conjugate in urine matrix was demonstrated for three months.
• Urine matrix effects were significantly reduced by MIP cleanup.
• Validation results demonstrated the reliability of the analytical method.

This paper describes a method to reveal the illegal use of chloramphenicol (CAP) in animals intended for human consumption based on the detection of free CAP and chloramphenicol-glucuronide (CAP-glu) in urine. It details the different steps of the method, including hydrolysis of CAP-glu, extraction and cleanup with molecularly imprinted polymers and detection by LC–MS/MS, as well as the validation design. The efficiency of chloramphenicol release during the hydrolysis step and the stability of CAP-glu in urine samples stored at −20 °C were also investigated. These verifications were important to ensure the method’s suitability for checking CAP misuse in veterinary medicine. Validation results were fully compliant with the qualitative and quantitative criteria required by European regulations. Intraday relative standard deviations were all below 7.5%, while interday relative standard deviations were below 6.9%. Recoveries lay between 93.3 and 104.6%. Purification appears very effective since no matrix effect was demonstrated. CAP-glu was found to be stable for at least 3 months, and the mean recovery following deconjugation was assessed to be 79.4%. The decision limits (CCa) were all found to be lower than 0.1 μg/kg.