## 5 MIP Applications for the Extraction of Antibiotics in Milk
### Cephalosporins, Macrolides and Sulfonamides
The analytes were eluted with 0.5% acetic acid in methanol (3 \(\times\) 2 mL), the eluate was evaporated under gentle nitrogen stream at 40 \({}^{\circ}\)C and the dry residue was reconstituted with NaH\({}_{2}\)PO\({}_{4}\)-acetonitrile (7:3, \(v/v\); 1 mL). In comparison with the respective NIP, binding specificity study between ERY, SPI, roxithromycin, josamycin, ivermectin and tylosin hemitartrate showed MIP selectivity over ERY with recoveries higher than 98% for ERY and between (15-35)% SPI, josamycin, ivermectin and tylosin hemitartrate. The developed MIPs were applied for more than 200 extraction rounds without any performance and stability drop [84]. Sulfonamides constitute synthetic antibiotic class that inhibit the bacterial DNA synthesis and include sulfamethoxazole (SMO), SDZ and SMZ. They contain the sulphonamide functional group (-S(=O)\({}_{2}\)-NH\({}_{2}\)) and are used for bacterial and fungal infection treatment. The maximum residue limit in milk according to the Commission Regulation (EU) No. 37/2010 is 100 \(\upmu\)g/kg for all sulfonamide analogues [2; 3]. MIPs were reported as sorbent materials in a MISPE, a MSPE, a PMME and a SBSE protocol for the extraction of sulfonamides from milk and other matrices. A MISPE-HPLC-UV method was developed for the determination of SMO and SDZ in egg and milk samples. Acrylamide-functionalized silica nanoparticles (50 mg), SMO (50 mg), AM (40 mg), EGDMA (3 mL) and AIBN (30 mg) were dissolved in acetonitrile (50 mL) and the mixture was sonicated for 10 min and left at room temperature for 3 h. Polymerization was carried out under nitrogen stream and continuous stirring at 60 \({}^{\circ}\)C for 24 h and the mixture was left to age at 85 \({}^{\circ}\)C for 3 h. The developed MIP nanoparticles were washed with acetonitrile and the template was removed by Soxhlet extraction with methanol-acetic acid (9:1, \(v/v\)). The MIP nanoparticles were washed methanol and dried under vacuum at 60 \({}^{\circ}\)C. SMO-MIP nanoparticles (100 mg) were added to pretreated spiked milk extract (50 mL) and the mixture was incubated for 45 min at room temperature and centrifuged at 3000 rpm for 10 min. The supernatant was discarded and the nanoparticles were collected. The analytes were eluted with methanol-acetic acid (9:1, \(v/v\)), the eluate was evaporated under nitrogen stream and the dry residue was reconstituted with methanol (1 mL). In comparison with the respective NIP, MIP adsorption capacity for SMO was higher, with Q\({}_{\text{max}}\) values of 5634 and 20,210 and \(\upmu\)g/g, while binding specificity study between SMO, SDZ, SMZ, sulfadimethoxine, sulfararazine and sulfaracter showed MIP selectivity over SMO and the imprinting factor was 21.52 for SMO. The SiO\({}_{2}\)-SMO-MIP nanoparticles were compared with SMO-MIPs prepared by bulk polymerization in terms of SMO binding capacity and demonstrated three times higher capacity. Furthermore,SMO adsorption equilibrium was achieved in 45 min with the SiO\({}_{2}\)-SMO-MIP nanoparticles and 10 h with the SMO-MIPs, proving that the developed MIP nanoparticles had higher number of imprinted sites and improved accessibility by the analytes [85]. A MSPE-HPLC-UV method was developed for the determination of SMO in milk and honey samples. SMO (150 mg) and MAA (225 \(\mu\)L) were dissolved in acetonitrile (30 mL) and the mixture was stored for 12 h in a refrigerator. A second mixture was prepared by dissolving vinyl-modified magnetic carbon nanotubes (CNTs) (100 mg) in acetonitrile (30 mL), EGDMA (1 mL) and AIBN (40 mg) were added, and the mixture was sonicated 30 min and degassed under nitrogen stream for 10 min. The two mixtures were combined and stirred and left to pre-polymerize at 50 \({}^{\circ}\)C for 6 h, polymerize at 60 \({}^{\circ}\)C for 24 h and left to age at 75 \({}^{\circ}\)C for 6 h under nitrogen stream and continuous stirring. The template was removed by incubating the developed MIPs in methanol-acetic acid (9:1, \(v/v\)) and the MIP particles were washed with methanol and dried under vacuum.