## 5 MIP Applications for the Extraction of Antibiotics in Milk
### Cephalosporins, Macrolides and Sulfonamides  
Cephalosporins are grouped into first, second, third, and fourth generations, with broader spectrum in each generation and closely related to cephalosporicns and carbapenems. They inhibit the bacterial cell synthesis. The maximum residue limit in milk according to the Commission Regulation (EU) No. 37/2010 is 100 \(\upmu\)g/kg for cephalexin (CFL), 60 \(\upmu\)g/kg for cephalpirin (CFP), 50 \(\upmu\)g/kg for cefazolin (CFZ) and 20 \(\upmu\)g/kg for QUI [1; 2; 3]. MIPs were only reported as sorbent materials in three MISPE protocols for the extraction of cephalosporicn antibiotics from milk samples. A MISPE-HPLC-UV method was developed for the determination of CFL. CFL (365.4 mg), TFMAA (560.2 mg) and EGDMA (3.9644 g) were dissolved in methanol (5 mL), the mixture was vortexed and sonicated for 5 min and purged under nitrogen stream. AIBN (1.5 mL) was added and the mixture was placed and sealed inside a glass ampule. The glass ampule was placed in a water bath at 60 \({}^{\circ}\)C for 14 h. The developed MIPs bulk was grinded and the template was removed with thermal annealing at 100 \({}^{\circ}\)C for 18 h, Soxhlet extraction with water-acetic acid (80:20, \(v/v\)) and methanol in an orbital shaker. The MIPs were washed with distilled water in order to remove methanol and dried under vacuum. CFX-MIPs (1 g) were packed into a SPE cartridge, washed with water to remove air, preconditioned with methanol (12 mL) and water (12 mL), loaded with defatted and deproteinized spiked milk extract (5 mL) and washed with methanol (12 mL). The analytes were eluted with water (12 mL), the eluate was lyophilized and the dry residue was reconstituted in methanol-acetate buffer (40:60, \(v/v\); 500 \(\upmu\)L). \begin{table}
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In comparison with the respective NIP, MIP adsorption capacity for CFL ranged between (2080-3620) \(\upmu\)g/g, while binding specificity study between CFL, ceftazidine, AMP and TC showed MIP selectivity over CFL. Furthermore, the developed MISPE cartridges were compared with commercially available C18 SPE cartridges and were found superior in terms of CFX selectivity and matrix interferences removal. Additional experiments showed that the synthesized MIPs were stable for 12 months without any CFX binding loss [80]. A MISPE-HPLC-UV method was developed for the determination of CFL and CFP, utilizing tributylammonium CFD salt as the template molecule. Tributylammonium CFD (173 mg), MAA (135 \(\upmu\)L), and EGDMA (1520 \(\upmu\)L) were dissolved in acetonitrile (2.20 mL) and methanol (200 \(\upmu\)L) and the mixture was sonicated. AIBN (28.5 mg) was added, the mixture was purged under nitrogen stream for 10 min, sealed and transferred in a water bath 60 \({}^{\circ}\)C for 20 h. The developed MIP monolithts were grinded and sieved. The MIP particles were washed with water-acetic acid (4:1, \(v/v\)) and methanol, finer particles were separated by sedimentations in acetone and the MIP particles were dried under vacuum. Tributylammonium CFD-MIPs (100 mg) were packed into a SPE cartridge, preconditioned with phosphoric acid (1.2 mM, 7.5 mL), loaded with deproteinized spiked milk extract (2 mL) and washed with acetonitrile (1 mL). The analytes were eluted with methanol-acetic acid (90:10, \(v/v\); 4 mL), the eluate was evaporated under nitrogen stream at 20 \({}^{\circ}\)C and the dry residue was reconstituted with mobile phase. In comparison with the respective NIP, MIP adsorption capacity for CFL was higher, while imprinting factors were 3 for CFD, 5.6 for CFP and 3.8 for CFL [81]. A MISPE-UHPLC-MS/MS method was developed for the determination of ceftiofur (THIO), CFZ, cephalonium (ALO), CFP, cefquinone (QUI) and CFL, utilizing sodium 7-(2-biphenylylcarboxamido)-3-methyl-3-cepheme-4-carboxylate as the surrogate template molecule.