## 5 MIP Applications for the Extraction of Antibiotics in Milk
### Penicillins
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A MISPE-HPLC-DAD method was developed for the determination of OXA, CLOX and DICLOX, utilizing 2-biphenylylpenicillin as the surrogate template molecule. 2-Biphenylylpenicillin (0.25 mmol), \(N\)-(2-aminoethyl)methacrylamide (EAMA) (1 mmol) and 15-crown-5 ether (0.5 mmol) were dissolved in acetonitrile-dimethylsulfoxide (60:40, \(v/v\); 0.7 mL) and mixed with TRIM (5 mmol) and \(N\),\(N^{\prime}\)-azo-bis(2,4-dimethyl)valeronitrile. Silica (6 g) was added to the above mixture (2.5 mL), the mixture was stirred, sealed and purged under nitrogen stream for 5 min and polymerization was carried out at 50 \({}^{\circ}\)C for 24 h. The developed MIP particles were treated three consecutive times with ammonium hydrogen difluoride aqueous solution (3 M, 150 mL) for 8 h, washed with water, methanol-trifluoroacetic acid (99:1, \(v/v\); 1 L), and methanol (0.5 L) and dried under vacuum at 50 \({}^{\circ}\)C for 24 h. The MIPs were suspended in methanol-water (80:20, \(v/v\)) in order to remove finer particles. MIPs (20 mg) were packed into a SPE cartridge, equilibrated with methanol (10 mL) and 2-[4-(2-hydroxyethyl)]-1-piperazineallylethanesulfonic acid buffer (0.1 M, pH 7.5; 10 mL), loaded and rinsed with water-acetonitrile (80:20, \(v/v\); 3 mL). The analytes were eluted with 0.05 M trifluoroacetic acid in methanol (1 mL) and the eluate (0.4 mL) was diluted with water (0.6 mL). In comparison with the respective NIP, MIP adsorption capacity for CLOX was 124 umol/g and NIP adsorption capacity was 31 umol/g, while binding specificity study between OXA, CLOX, DICLOX, AMX, AMP, doxycycline, PEN G and NAFC showed MIP selectivity over OXA, CLOX and DICLOX and imprinting factors were 90.5 for OXA, 80.8 for CLOX and 74.9 for DICLOX [61]. A MISPE-HPLC-MS/MS was developed for the determination of eight penicillin antibiotics, AMP, AMO, OXA, PEN G, penicillin V (PEN V), CLOX, DICLOX and NAFC in baby formula samples. NAF, MAA, EGDMA and AIBN were dissolved in acetonitrile and the mixture was placed inside an incubator at 60 \({}^{\circ}\)C and stirring at 24 rpm for 24 h. The developed MIPs were collected and filtered through a 0.45 um filter using acetonitrile (50 mL) and methanol (50 mL). The template was removed by Soxhlet extraction with methanol-acetic acid (1:1, \(v/v\)) for 8 h. NAFC-MIPs (0.05 g) were packed into an empty SPE cartridge, loaded with pretreated spiked baby formula extract (0.3 g) and washed with acetonitrile (6 \(\times\) 1 mL). The analytes were eluted with methanol with 1% acetic acid (3 \(\times\) 1 mL), the eluate was evaporated under nitrogen stream and the dry residue was reconstituted with mobile phase (100 \(\upmu\)L) [62]. A magnetic solid-phase extraction (MSPE)-LC-MS/MS method was developed for the determination of PEN V, AMX and OXA. PENV (1 mmol) was dissolved in water-ethanol (9:1, \(v/v\); 10 mL) and MAA (8 mmol) by stirring for 30 min and EGDMA (20 mmol) were added. The mixture was mixed with Fe\({}_{3}\)O\({}_{4}\) particles (1 g) and oleic acid (1 mL) and sonicated for 30 min in order to form a pre-polymerization mixture. Polyvinylpyrrolidone (0.4 g) was dissolved in methanol (100 mL) inside a three-necked round-bottomed flask, the mixture was purged under nitrogen stream, stirred at 300 rpm and heated at 60 \({}^{\circ}\)C. Then, the pre-polymerization solution and AIBN (1 g) were added into the flask and polymerization was carried out at 60 \({}^{\circ}\)C for 24 h. The developed MMIPs were collected with a magnet, washed with methanol-acetic acid (8:2, \(v/v\)) and the template was removed by washing the MMIPs with methanol. The MMIPs were washed three times with water and dried at 60 \({}^{\circ}\)C. PENV-MMIPs (100 mg) were preconditioned with methanol (3 mL) and water (3 mL), dispersed in a flask that contained milk sample (2 mL) and hydrochloric acid aqueous solution (pH 5.0, 18 mL) and sonicated for 5 min.