2.5.1. Sample Preparation

The extraction and purification of amphenicols and metabolites from livestock and poultry meat were performed using our self-built method [13]. Briefly, pork, beef, lamb and chicken samples were chopped and homogenized at 10,000 r/min using an HM6300 intelligent homogenizer (Lab Precision Beijing Technology Co., Ltd., Beijing, China). After homogenization, 5 g (accuracy, 0.01 g) of each livestock and poultry meat sample were placed in a 50 mL centrifuge tube. Then, 10 μL of 1.0 μg/mL mixed internal working standard solution and 15 mL of ethyl acetate with 2% ammonia were added and vortexed for 1 min. The mixture was centrifuged at 4 ◦C for 5 min at 10,621 g in a refrigerated centrifuge (D-16C, Sartorius Lab Instruments GmbH & Co. KG, Goettingen, Germany), and the supernatant was collected. The extraction operation was repeated with another 15 mL of ethyl acetate with 2% ammonia, and the supernatants were combined. The supernatants were evaporated to dryness at 40 ◦C with a nitrogen evaporator (N-EVAPTM-112, Organomation Associates Inc., Berlin, MA, USA). Subsequently, the residue was reconstituted with 5 mL of acetone: n-hexane (1:9, *v/v*), purified by a CNW Si solid-phase extraction (SPE) column and defatted with ACN-saturated hexane. Finally, the solution was filtered through a 0.22 μm filter membrane and injected into the ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) system analysis.
