## 5 MIP Applications for the Extraction of Antibiotics in Milk
### Quinolones  
Quinolones constitute a broad-spectrum antibiotic class that includes OXO. Quinolones inhibit the bacterial DNA synthesis and the first quinolones were used for urinary tract infection treatment. More effective fluoroquinolones were synthesized by adding a fluorine atom in the basic heterocyclic aromatic structure of the quinolones. The maximum residue limit in milk according to the Commission Regulation (EU) No. 37/2010 is 100 \(\upmu\)g/kg for the sum of enrofloxacin (ENR) and ciprofloxacin (CIP), 30 \(\upmu\)g/kg for danofloxacin (DAN), while difloxacin (DIF) and FFC are not recommended for milk producing animals [1; 2; 3]. Multi-template MIPs used in MISPE protocols, a MIP column used in on-line MISPE, a PMME protocol, along with a DSPE protocol and a MSPE protocol were reported in the literature for the extraction of various quinolones from milk samples. A MISPE-HPLC-DAD method was developed for the determination of 10 quinolone antibiotics: flexoxacin (FLX), enoxacin (ENO), pefloxacin (PEF), norfloxacin (NOR), CIP, levofloxacin (LVFX), lomefloxacin (LOM), ENR, gatifloxacin (GAT) and sparflloxacin (SPA), utilizing multiple-template surface molecularly imprinted polymers (MTMIPs). CIP (165.5 mg), LVFX (180.6 mg) MAA (517.4 mg) were dissolved in toluene (6 mL) and the mixture was sonicated. TRIM (5.408 g) and AIBN (56 mg) were added and the mixture was sonicated, degassed under nitrogen stream for 10 min, sealed and placed in a water bath 60 \({}^{\circ}\)C for 24 h. The MIP bulk was grinded into particles of 54-74 \(\upmu\)m diameter and the template was removed by incubating the MIPs in methanol-acetic acid (8:2, \(v/v\)) under sonication followed by centrifuging at 6000 rpm. The MIP particles were washed with methanol until pH 7.0 and dried. MTMIPs (200 mg) were packed into a SPE cartridge, preconditioned with methanol (3 mL) and water (3 mL), loaded with pretreated spiked milk extract (1 mL) and washed with water (3 mL). The analytes were eluted with 4% ammonia in methanol (3 mL), the eluate was evaporated under nitrogen stream at 45 \({}^{\circ}\)C, the dry residue was reconstituted with mobile phase (1 mL) and filtered through a 0.22 \(\upmu\)m filter. In comparison with the respective NIP, MIP adsorption capacity for CIP acid LVFX was higher, with Q\({}_{\text{max}}\) values of 32.3 and 94.2 \(\upmu\)mol/g for CIP and 32.5 and 71.9 \(\upmu\)mol/g for LVFX. The developed MIPs could be successfully applied for the extraction and determination of 10 fluoroquinolone antibiotics by utilizing two template molecules rather than one and were reused for 20 extraction cycles with no extraction efficiency reduction [65]. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Analyte** & **Matrix** & **MIP Composite** & \begin{tabular}{c} **Polymerization** \\ **Technique** \\ \end{tabular}  & **Template-Monomer-Cross Linker** & **Extraction-Analysis** & **LOD, LOG-Co  
MTMIPs were also utilized for the development of a MISPE-HPLC-UV method for the determination of estriol, estrone, 17\(\beta\)-estradiol, ofloxacin (OFL), NOR and CIP. OFL (0.1 mmol) and 17\(\beta\)-estradiol (0.1 mmol) were dissolved in acetonitrile (15 mL), sufficient amount of MAA was added and the mixture was stirred at 50 rpm. Modified monodispersed P\({}_{\text{GMA/EDMA}}\) beads (0.8 g), EGDMA (3 mmol) and AIBN (0.1 g) were added and the mixture was transferred in a water bath at 65 \({}^{\circ}\)C under nitrogen atmosphere and continuous stirring at 120 rpm for 24 h.  
The P\({}_{\text{GMA/EDMA}}\) beads acted as support for the MIPs and were previously prepared by dispersion polymerization and chemically modified according to previous work. The developed MIPs were washed with water and methanol and the templates was removed by incubating the MIPs in acetic acid-methanol (80:20, \(v/v\); 50 mL) under continuous stirring for 24 h. The MIP particles were washed with distilled water and air-dried.