## 5 MIP Applications for the Extraction of Antibiotics in Milk
### Cephalosporins, Macrolides and Sulfonamides
Template molecule (0.24 mmol), 15-crown-5 ether (0.24 mmol) and \(N\)-3,5-bis(trifluoromethylomethyl)phenyl-\(N^{\prime}\)-4-vinylphenyl urea (VPU) (0.48 mmol) were dissolved in acetonitrile-dimethylsulfoxide (0.8:1, \(v/v\); 550 \(\upmu\)L) and sufficient amount of DVB and 2,2\({}^{\prime}\)-azobis(2,4-dimethylvaleronitrile) was added. The mixture was mixed with silica beads (5 g) by stirring inside a glass vial. The vial was sealed and purged under nitrogen stream for 5 min and polymerization was carried out at 60 \({}^{\circ}\)C for 24 h. The developed particles were shaken with ammonium hydrogen dilluoride aqueous solution (3 M, 3 \(\times\) 140 mL) for 24 h, washed with water, methanol-trifluoracetic acid (99:1, \(v/v\); 1 L) and methanol (0.5 L) and dried under vacuum at 60 \({}^{\circ}\)C for 24 h. The MIP particles were suspended in methanol-water (80:20, \(v/v\)) in order to remove finer particles. MIPs (20 mg) were packed into a SPE cartridge preconditioned with methanol (10 mL) and phosphate buffer (0.05 M, pH 7.5; 10 mL), loaded with pretreated milk extract (10 mL) and washed with HEPES buffer (2:98, \(v/v\); 5 mL). The analytes were eluted with 0.1% trifluoroacetic acid in methanol (1 mL) and the eluate was diluted with water (800 \(\upmu\)L). In comparison with the respective NIP, MIP extraction recoveries for the studied cephalospororins ranged between (74-95)%, while NIP recoveries ranged between (0-8)% for CFZ, ALO, CFP, QUI and CFL [82]. Macrolides constitute a medium-spectrum semi-synthetic antibiotic class, with a macrocyclic lactone chemical structure, discovered in 1950. Macrolides inhibit the bacterial protein synthesis. ERY is the first macrolide antibiotic, with similar spectrum and uses to penicillin, administered as an alternative to people allergic to penicillin. Newer macrolide antibiotics include azithromycin and clarithromycin, which is used stomach ulcer treatment. The maximum residue limit in milk according to the Commission Regulation (EU) No. 37/2010 is 20 \(\upmu\)g/kg for ERY and 200 \(\upmu\)g/kg for spiramycin (SPI) [1; 2; 3]. Only two MISPE applications for the extraction of macrolides from sheep milk samples were reported in the literature. The same laboratory developed two MISPE-HPLC-DAD methods were reported for the determination of SPI and ERY in sheep milk samples. For the first method, SPI (0.02 mmol) and MAA (2 mmol) were dissolved in acetonitrile (7 mL) and the mixture was sonicated for 5 min. EGDMA (10 mmol) and AIBN (5.1 mmol) were added and the mixture was sonicated for 10 min, degassed under nitrogen stream for 7 min and placed in a water bath at 50 \({}^{\circ}\)C for 4 h. The developed MIP bulk was grinded into particles of (200-355) \(\upmu\)m size and the template was removed by Soxhlet extraction with methanol (80 mL) for 20 h. SPI-MIPs (200 mg) were packed into a syringe barrel, preconditioned with methanol (3 \(\times\) 2 mL) and acetonitrile (3 \(\times\) 2 mL), loaded with deproteinized spiked milk extract (1 mL) and washed with acetonitrile (3 \(\times\) 2 mL). The analytes were eluted with acetic acid in methanol (3 \(\times\) 2 mL), the eluate was evaporated under nitrogen stream and the dry residue was reconstituted with 1 mL of NaH\({}_{2}\)PO\({}_{4}\)-acetonitrile (7:3, \(v/v\); 1 mL). In comparison with the respective NIP, MIP adsorption capacity for SPI was higher, with Q\({}_{\text{max}}\) values of 1270 and 3560 \(\upmu\)g/g, while binding specificity study between SPI, ERY, josamycin, ivermectin and tylosin hemitartrate showed MIP selectivity over SPI and the imprinting factor was 3.4 for SPI [83]. For the second method, the same MIP preparation protocol was followed for the preparation of ERY-MIPs. ERY-MIPs (200 mg) were packed into an empty SPE cartridge, preconditioned with methanol (3 \(\times\) 2 mL) and acetonitrile (3 \(\times\) 2 mL) and loaded with pretreated spiked milk extract (1 mL). The loaded cartridge was treated with hexane (6 \(\times\) 1 mL), in order to remove the milk fat.