## 5 MIP Applications for the Extraction of Antibiotics in Milk
### Amphenicols  
Amphenicols are broad-spectrum antibiotics class that includes chloramphenicol (CAP), thiamphenicol (TAP) and florfenicol (FFC). CAP is the oldest amphenicol antibiotic and was originallyisolated in 1947 from _Streptomyces venezuelae_ cultures. It inhibits bacterial proteins synthesis; thus, it is effective against many bacteria strains and used for clinical purposes. CAP, TAP and FFC are used for veterinary purposes, for bacterial infection prevention and treatment. However, high toxicity and unwanted effects can be observed and their use in food-producing animals is illegal in most countries. The maximum residue limit in milk according to the Commission Regulation (EU) No. 37/2010 is 50 \(\mathrm{\SIUnitSymbolMicro g}\)/kg for TAP, while FFC is not recommended for milk-producing animals and CAP is prohibited [1; 2; 3]. MIPs were reported as sorbent materials for the extraction of amphenicol antibiotics from milk samples. Synthesized MIPs were mainly employed in MISPE protocols, with reports of polymer monolith microextraction (PMME) protocols. Furthermore, commercially available MISPE cartridges were employed for the extraction of CAP. A MISPE-liquid chromatography-mass spectrometry (LC-MS/MS) method was developed for the determination of CAP. TAP was used as a dummy template for the preparation of the MIPs. A mixture of TAP (0.25 mmol), MAA (1 mmol) and methanol (40 mL) was left for 30 min and EGDMA (5 mmol) and AIBN (30 mg) were added. The mixture was sealed and purged with nitrogen for 10 min and placed in a water bath at 60 \({}^{\circ}\)C for 24 h. The developed MIPs were collected and the template was removed by Soxhel extraction with methanol-acetic acid (90:10, \(v/v\)). The MIP particles were washed with methanol and dried under vacuum at 50 \({}^{\circ}\)C. TAP-MIPs (30 mg) were dispersed in acetone (3 mL) and packed into a SPE cartridge, washed with methanol-acetic acid (90:10, \(v/v\)) and preconditioned with methanol (3 mL) and water (1 mL). The cartridge was loaded with milk extract (10 mL) and washed with water (1 mL). The analytes were eluted with methanol-acetic acid (90:10, \(v/v\); 3 mL), the eluate was concentrated under nitrogen stream and re-dissolved in water. In comparison with the respective NIP, MIP adsorption capacity for CAP was higher, with maximum adsorption capacities (Q\({}_{\mathrm{max}}\)) of 1188.49 and 2775.94 \(\mathrm{\SIUnitSymbolMicro g}\)/g, while imprinting factors were 1.45 for CAP and 1.35 for TAP [48]. A MISPE-high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method was developed for the determination of CAP in milk and shrimp samples. CAP (1 mmol), EGDMA (5 mL), AIBN (120 mg) and required amount of 2-(diethylamino)ethyl methacrylate (DEAEM) were dissolved in octanol-chloroform (2:1, \(v/v\); 15 mL) and added in a flask that contained polyvinyl alcohol 1788 (4 g) dissolved in double distilled water (100 mL). Polymerization was carried out under nitrogen atmosphere and continuous stirring at 70 \({}^{\circ}\)C and for 24 h. The developed MIP microspheres were filtered and washed with double distilled water, methanol and acetone and the template was removed by Soxhel extraction with methanol-acetic acid (90:10, \(v/v\)). The MIP microspheres were washed with methanol and dried under vacuum at 70 \({}^{\circ}\)C for 12 h. CAP-MIP microspheres (100 mg) were suspended in isopropanol-methanol (2:1, \(v/v\); 2 mL) and packed into a SPE cartridge, washed with methanol (5 mL) and preconditioned with phosphate buffer (0.05 M, pH 7.0). The cartridge was loaded with milk sample extract (10 mL), washed with methanol-phosphate buffer (40:60, \(v/v\); 3 \(\times\) 1 mL) and the analytes were eluted with methanol (2 \(\times\) 1 mL). In comparison with the respective NIP, MIP microsphere adsorption capacity for CAP was, with maximum adsorption capacity of 222 \(\mathrm{\SIUnitSymbolMicro g}\)/g, and higher recoveries for CAP, FFC and TAP [49]. A MISPE-liquid chromatography-mass spectrometry (LC-MS) method was developed for the determination of CAP.