## 5 MIP Applications for the Extraction of Antibiotics in Milk
### Quinolones
MTMIPs (50 mg) were packed into a polypropylene cartridge, loaded with pretreated spiked milk extract (5 mL) and washed with water-methanol (9:1, \(v/v\); 1 mL). The analytes were eluted with 20% acetic acid in acetonitrile (3 \(\times\) 0.5 mL), the eluate was evaporated under nitrogen stream at 25 \({}^{\circ}\)C and the dry residue was reconstituted with of mobile phase (20 \(\upmu\)L). In comparison with the respective NIP, MIP adsorption capacity for OFL and 17\(\beta\)-estradiol was higher, with maximum adsorption capacity of 9000 and 6600 \(\upmu\)g/g respectively, while binding specificity study between OFL, 17\(\beta\)-estradiol, NOR, estriol and CAP showed SMIP selectivity over OFL and 17\(\beta\)-estradiol and imprinting factors were 6.7 for OFL, 12 for 17\(\beta\)-estradiol, 4.8 for NOR, 2.8 for estriol and 1.1 for CAP. The chromatograms demonstrated better peaks with no matrix interference for the MISPE protocol in contrast with the C18 SPE [66]. An on-line MISPE-HPLC-UV method was developed for the determination of OFL, LOM, CIP and ENR, utilizing a molecularly imprinted polymer hybrid composite monolithic column (MIP-HCMC). ENR (0.18 g) dissolved in toluene (0.5 mL) and ethanol (0.5 mL) was mixed with MAA (0.1 mL) and HEMA (0.15 mL) and the mixture was stirred at 52 \({}^{\circ}\)C under nitrogen stream for 1 h. TEOS (2.32 mL) dissolved in acetonitrile (3.55 mL) and HCl (0.10 mL) were added and the mixture was incubated for 1 h. 3-methacrylopropyltrimethoxysilane (KH-570) (0.58 mL), EGDMA (0.25 mL) and AIBN (0.06 g) were added, the mixture was sonicated for 2 min, transferred and sealed inside a stainless-steel column and polymerization was carried out at 53 \({}^{\circ}\)C for 12 h. The template was removed with methanol-acetic acid (4:1, \(v/v\)) and methanol and the column was connected to an on-line SPE-HPLC system. The MIP column preconditioned with water-acetonitrile (75:25, \(v/v\); 2 mL) and water-acetonitrile (90:10, \(v/v\); 2 mL), loaded with pretreated spiked milk extract, washed with methanol-water (7:93, \(v/v\); 1.5 mL) and the analytes were eluted with mobile phase. MIP dynamic adsorption capacity was 4.08 mg/g for ENR, while NIP adsorption capacity was and 2.44 mg/g [67]. A PMME-high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method was developed for the extraction and determination of CIP, DIF, DAN and ENR. PEF (24 mg), MAA (40 mg), di(ethylene glycol) dimethacrylate (DEGDMA) (240 mg) and AIBN (5 mg) were dissolved in methanol-water (10:3, \(v/v\); 0.8 mL) and the mixture was sonicated for 10 min. The mixture was transferred and sealed inside activated and modified fused-silica capillaries and polymerization was carried out at 65 \({}^{\circ}\)C for 16 h. The developed MIP monoliths were washed with methanol-trifluoroacetic acid (98:2, \(v/v\)) for 48 h by connecting the capillary columns with a HPLC pump. The needle of a syringe pinhead was replaced with a PEF-MIP monolith capillary and all the extraction steps (precondition, loading, washing and elution) were employed by means of a syringe infusion pump. The PEF-MIP monolith was preconditioned with 2% trifluoroacetic acid in methanol (2 mL) and phosphate buffer solution (25 mM, pH 6.0; 0.4 mL), loaded with pretreated spiked milk extract (1 mL) and washed with acetonitrile-phosphate buffer (15:85, \(v/v\); 0.2 mL). The analytes were eluted with 2% trifluoroacetic acid in methanol (0.4 mL), the eluent was evaporated under mild nitrogen stream at 50 \({}^{\circ}\)C and the dry residue was reconstituted with 100 \(\upmu\)L of mobile phase. In comparison with the respective NIP, MIP adsorption capacity for PEF was higher, with maximum adsorption capacity of 36 \(\upmu\)mol/g, while imprinting factors were 3.1 for PEF and 1 for FLU. Binding specificity study between fluoroquinolones (PEF, CIP, DAN, ENR, DIF) and quinolones (FLU, OXO)showed MIP selectivity over the fluoroquinolones, with recoveries up to 81.6% for the fluoroquinolones and less than 17.6% for the quinolones.