A single method to analyse residues from five different classes of prohibited pharmacologically active substances in milk.

In the European Union, the use of veterinary drugs belonging to the A6 group is prohibited in food-producing animals according to Commission Regulation (EU) No. 2010/37. The aim of this study was to improve the analytical control strategy by developing a single method to analyse residues of prohibited pharmacologically active substances in milk. For this, a single method was developed to analyse 16 prohibited pharmacologically active substances belonging to five different substance classes at required or recommended levels: nitroimidazoles at 3 μg kg, nitrofurans at 0.5 μg kg, chloramphenicol at 0.1 μg kg, dapsone at 5 μg kg and chlorpromazine at 1 μg kg. Milk sample preparation started with an acid hydrolysis combined with a derivatisation. These steps were followed by a clean-up consisting of a dispersive solid-phase extraction and a liquid-liquid extraction. Finally, the sample extracts were analysed by liquid chromatography combined with tandem mass spectrometry, operating alternately in the positive and negative mode. The method was fully validated according to Commission Decision 2002/657/EC for bovine milk and additionally validated for caprine milk. The validation proved that the method is highly effective to detect and confirm all 16 substances in bovine and caprine milk and, additionally to quantify 15 of these substances in bovine milk and 13 of these substances in caprine milk. This study resulted in a new multi-class method to detect, quantify and confirm the identity of 16 prohibited pharmacologically active substances belonging to five different substance classes in two types of milk.