## 5 MIP Applications for the Extraction of Antibiotics in Milk
### Penicillins  
Penicillins are the oldest antibiotics class. They belong to the group of \(\beta\)-lactams, along with cephalosporors, and are bactericidal. Penicillins include aminopenicillins such as ampicillin (AMP) and amoxicillin (AMX) that have a broader spectrum of action in comparison with natural penicillins, penicillinase-resistant penicillins such as oxacillin (OXA) and penicillin G (PEN G) structure-based penicillins. The maximum residue limit in milk according to the Commission Regulation (EU) No. 37/2010 is 4 ug/kg for AMP, AMX and benzylpenicillin and 30 ug/kg for OXA, coloxacillin (CLOX), dicloxacillin (DICLOX) and nafcillin (NAFC) [2; 3]. MIPs were reported as sorbent materials for the extraction of penicillins from milk samples. Synthesized MIPs were mainly employed in MISPE protocols, along with a report of a MSPE protocol and a report of a MSPD protocol. A MISPE-LC-MS/MS method was developed for the determination of AMP in cow milk samples. AMP (0.01 g) was mixed in acetonitrile (10 mL) and the mixture was briefly vortexed at 50 \({}^{\circ}\)C. MAA was added and the mixture was vortexed for 1 min and sonicated for 5 min. EGDMA and 2% hydrogen peroxide were added and the mixture was vortexed for 1 min and sonicated for 2 min. The mixture was purged under nitrogen stream for 3 min and polymerization was carried out under UV light lamp at 28 \({}^{\circ}\)C for 24 h. The developed MIP bulk was grinded into particles of 300 \(\upmu\)m size, filtered and washed with ethanol and methanol. The template was removed by incubating the MIPs in methanol-acetic acid (9:1, \(v/v\)) in a water bath at 50 \({}^{\circ}\)C for 6 h. The MIP particles were dried and kept inside a desiccator. AMP-MIPs (350 mg) were packed into a syringe barrel, preconditioned with acetonitrile (5 \(\times\) 2 mL), loaded with deproteinized and defatted spiked milk extract (20 g) and washed with acetonitrile (2 \(\times\) 2 mL). The analytes were eluted with methanol-acetic acid (99.4:0.6, \(v/v\); 3 \(\times\) 2 mL), the eluate was evaporated under nitrogen stream at 30 \({}^{\circ}\)C and the dry residue was reconstituted with water-KH\({}_{2}\)PO\({}_{4}\)-acetic acid (89:10:1, \(v/v/v\); 1 mL). In comparison with the respective NIP, binding specificity study between AMP, AMX, OXA and PEN G showed MIP selectivity over AMP and recoveries were 110% for AMP, 21.8% for AMX, 33.3% for OXA and 15.4% for PEN G [59]. A second MISPE-LC-MS/MS method was reported for the determination of benzylpenicillin. Benzylpenicillin (0.7 g), MAA (1.4 g), TRIM (2.4 g) and AIBN (0.07 g) were dissolved in acetonitrile (70 mL) and the mixture was placed in a water bath, under nitrogen atmosphere at 75 \({}^{\circ}\)C for 20 h. The developed MIPs were dried at 60 \({}^{\circ}\)C for 48 h and the template was removed by Soxhlet extraction with methanol-water (95:5, \(v/v\)). Benzylpenicillin-MIPs (50 mg) were packed into an empty SPE cartridge, washed with methanol (50 mL) and methanol-water mixture (95:5, \(v/v\); 15 mL) and preconditioned with methanol-acetonitrile mixture (50:50, \(v/v\); 5 mL). The cartridge was loaded with pretreated spiked milk extract and washed with acetonitrile (2 mL). The analytes were eluted with methanol-water (50:50, \(v/v\); 6 mL), the eluate was evaporated under nitrogen stream at 60 \({}^{\circ}\)C and the dry residue was reconstituted with water-acetonitrile mixture (1:1, \(v/v\); 1 mL) and internal standard (1 mL). The reconstituted eluate was filtered through a 0.22 \(\upmu\)m filter. In comparison with the respective NIP, MIP adsorption capacity for benzylpenicillin was higher, with Q\({}_{\text{max}}\) values of 0.13 and 0.82 \(\upmu\)mol/g, while MIP clean-up recoveries were up to 88% and NIP recoveries (0-11)%. The developed MISPE protocol was compared with a classic sample preparation protocol applied in benzylpenicillin-positive milk samples, showing that MIPSE is a good alternative [60].