## 5 MIP Applications for the Extraction of Antibiotics in Milk
### Tetracyclines
The loaded cartridge was washed with ultrapure water (3 mL) and methanol (3 mL) and dried under vacuum for 30 s. The analytes were eluted with methanol-acetic acid (9:1, \(v/v\); 1 mL) and the eluate was filtered through a 0.22 \(\upmu\)m nylon filter. Binding specificity study between CTC, TC, OTC, DC, sulfadimidine, CIP and AMX showed MIP selectivity over the studied tetracyclines, with recoveries higher than 87% for CTC, TC, OTC and DC, between (20-27)% for sulfadimidine, CIP and AMX, while NIP recoveries were between (20-23)% for all compounds. The developed MISPE protocol was compared with classic SPE protocols utilizing three commercially available SPE cartridges. The chromatograms demonstrated no matrix interference for the MISPE protocol in contrast with the SPE protocols where interfering peaks were observed. Furthermore, the developed MISPE cartridges could be reused for 30 extraction cycles with no extraction efficiency reduction [71]. The same laboratory developed three MISPE-HPLC-UV and an on-line MISPE-HPLC-UV method, utilizing molecularly imprinted polymer hybrid composite materials (MIP-HCMs) for the extraction of various tetracycline antibiotics. A MISPE-HPLC-UV method was developed for the determination of TC, CTC and DC, utilizing a hydrophilic MIP-HCM. DC (0.151 g), MAA (0.22 mL), DVB (0.2 mL) and styrene (0.6 mL) were dissolved in acetonitrile (2.25 mL) and the mixture was left to pre-polymerize for 30 min. KH-570 (1.176 mL) and AlBN (0.1 g) were added, the mixture was degassed by sonication for 2 min, purged under nitrogen stream and heated to 60 \({}^{\circ}\)C for 1 h and polymerization was carried out at 60 \({}^{\circ}\)C for 3 h. TEOS (2.95 mL) dissolved in HCl (0.23 mL) and ethanol (4.525 mL) was added and mixture was stirred 60 \({}^{\circ}\)C for 3 h. The developed MIP-HCM particles were filtered, dried 60 \({}^{\circ}\)C for 24 h and the template was removed by Soxhlet extraction with methanol-acetic acid (4:1, \(v/v\)) for 4 h. The MIP particles were treated with KH-570, washed with toluene (100 mL) and acetone (100 mL) and dried at 40 \({}^{\circ}\)C overnight. DC-MIP-HCMs (50 mg) were packed into a SPE cartridge, preconditioned with methanol (3 mL) and water (3 mL), loaded with pretreated spiked milk extract (5 mL), washed with methanol-water (20:80, \(v/v\); 2 mL). The analytes were eluted with methanol-acetic acid (60:40, \(v/v\); 2 mL), the eluate was evaporated under nitrogen stream and the dry residue was reconstituted with mobile phase. The developed MISPE protocol demonstrated good clean-up efficiency and high selectivity over TC, CTC and DC [72]. A MISPE-HPLC-UV method was developed for the determination of DC, TC and CTC in milk and honey samples. DC (0.151 g) and MAA (0.220 mL) were dissolved in acetone (4.588 mL) and the mixture was incubated at room temperature for 1 h. KH-570 (0.588 mL) and AlBN (75 mg) were added and the mixture was sonicated for 2 min, degassed under nitrogen stream for 5 min and stirred at 53.8 \({}^{\circ}\)C for 3 h. TEOS (2.731 mL) dissolved in ethanol (6 mL) and hydrolyzed with HCl (0.1 mL) was added and the mixture was left to react at 60 \({}^{\circ}\)C under continuous stirring for 3 h. The mixture was cooled, adjusted to pH 6.0 with ammonia solution and polymerization was carried out by heating at 80 \({}^{\circ}\)C for 3 h. The developed MIP-HCM was grinded into particles of (38.5-63) \(\upmu\)m size and the template was removed by Soxhlet extraction with methanol-acetic acid (4:1, \(v/v\)) for 24 h. The MIP-HCM particles were washed with methanol and dried at 120 \({}^{\circ}\)C for 24 h. DC-MIP-HCM particles (50 mg) were packed into an empty SPE cartridge, preconditioned with water-acetic acid (90:10, \(v/v\); 3 mL) and water (4 mL), dried for 2 min, further washed with cyclohexane (1 mL) and dried for 2 min. The MISPE cartridge was loaded with pretreated spiked milk extract (4 mL), dried for 3 min and washed with toluene-methanol (85:15, \(v/v\); 1 mL) and the analytes were eluted with methanol-acetic acid (60:40, \(v/v\); 3 mL).