The assessment of withdrawal periods for milk is affected by the occurrence of data below the lower analytical quantification limit (BLQ data) and the resulting uncertainty. The current regulatory approach for dealing with BLQ residues is simple and easy: BLQ data (and missing data) are arbitrarily reassigned a value of one-half the LOQ before any calculation on the data with one of the three currently applicable methods. Here, we reconsider the determination of the withdrawal period of milk with data below the limit of quantification. Theoretical background on analytical limits and pharmacometric considerations will be established. Then, we analyze the uncertainty problems caused by the current approach and propose a calculation solution (maximum-likelihood estimation handling left-censored data) included in nonlinear mixed-effects modeling. Finally, we illustrate this issue using a case example.

Harmonization of the method for calculating the withdrawal period for milk dates from the 1990s. European harmonization has led to guidance with three accepted methods for determining the withdrawal period for milk that are currently applicable. These three methods can be used by marketing authorization holders, but, in some cases, their diversity can lead to very different withdrawal periods. This is particularly the case when concentrations in milk are nonmonotonic and heterogeneous, meaning that concentrations strictly increase and then strictly decrease with significant interindividual variability in the time to reach the maximal concentration. Here, we first describe the concepts associated with the different methods used in the harmonized approach currently applicable for the determination of milk withdrawal periods, and then, we propose the application of a modern pharmacometric tool. Finally, with a nonmonotonic heterogeneous dataset, we illustrate the usefulness of this tool in comparison with the three currently applicable methods and discuss the limitations and advantages of each method.

Chlortetracycline (CTC) is a broad-spectrum antibiotic used in veterinary medicine for pulmonary or digestive infections and having a regulatory maximum residue limit (MRL) necessitating an official analytical control method. The purpose of this study was to clarify the identification of different forms of CTC observed in standard solution, in spiked muscle samples and in naturally incurred muscle samples of pigs analysed by LC-MS/MS and to demonstrate the in vivo formation of 6-iso-chlortetracycline and 4-epi-6-iso-CTC as a metabolite of CTC and 4-epi-CTC in muscle. The six following forms were identified, all being isobaric with a protonated molecule at m/z 479 (precursor ion): the keto-enol forms of CTC and the keto-enol forms of 4-epi-chlortetracycline (4-epi-CTC), 6-iso-chlortetracycline (6-iso-CTC) and 4-epi-6-iso-chlortetracycline (4-epi-6-iso-CTC). The 6-iso-CTC and 4-epi-6-iso-CTC were observed only in incurred pig samples so were identified for the first time as metabolites of CTC and 4-epi-CTC. Identification of the different forms was obtained by comparing incurred muscle samples with standard solutions and with spiked samples. Then the differences between the features of the chromatograms obtained by LC-TQ-MS and the fragmentation study of the different forms of CTC obtained by LC-Q-TOF-MS helped us to support this identification. The extraction steps and the LC-MS/MS conditions developed to analyse muscle tissue samples are described. This clarification concerning the rigorous identification of chromatographic peaks allowed us to evaluate the relevance of our monitoring method with regard to the regulations in place in the European Union and could be of help to laboratories involved in official control of antibiotic residues in food of animal origin. Additional results are also presented highlighting the transformation of the CTC when prepared in a mixture with other antibiotics.