Molecularly imprinted polymers and HPLC-MS/MS for determining penicillins in infant formulas

The dairy cattle may suffer from different infections relatively often, but the inflammation of the mammary gland is very important to the farmer. Mastitis is an animal welfare problem and a big economic problem because it deteriorates the milk quality. These infections are frequently treated with penicillins. However, their use may result in the presence of residues in milk and dairy products, such as milk powder and infant formulas, and it represents a potential risk for consumers. To avoid health risks for the consumer, the EU has defined safe maximum residue limits (MRL) through Commission Regulation (EU) No. 37/2010. Although LC–MS is a trustful option for confirmation and quantification of antibiotics, the analysis of real samples with complex matrices usually implies previous clean-up steps using common solid-phase extraction procedures. In this work, precipitation polymerization has been used and different MIP sorbents were tested and optimized for the solid-phase extraction (MISPE) of eight common penicillins (ampicillin, amoxicillin, oxacillin, penicillin G, penicillin V, cloxacillin, dicloxacillin and nafcillin). The detection was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and the applicability of these polymers as sorbents for the extraction of penicillins at MRL levels in infant formulas was proved.


Introduction
The term antibiotic refers to a very diverse range of chemical substances produced from bacteria or fungi in a natural, semisynthetic or synthetic way that possess antibacterial activity, by killing or inhibiting the growth of microorganisms. β-lactam antibiotics (BLAs) constitute one of the most widely used anti-microbial drug in veterinary medicine, especially to treat and prevent bacterial infections (respiratory, urinary or skin infections) of dairy cattle. This group of antibiotics can be classified into several groups (penicillins, cephalosporins, and more recently, carbapenems) according to their structural characteristics, but their unique structural feature is the presence of the fourmembered BLAs (2-azetidinone) ring [1]. In dairy cattle, in addition to digestive and respiratory diseases, the inflammation of the mammary gland is very problematic for the farmer. Mastitis, although an animal welfare problem, is a big economic problem because it increases the somatic cell count, a milk quality indicator [2]. These infections are frequently treated with penicillins, one of the most applied drugs in dairy cattle (see Figure 1). However, the incorrect use of these antibiotics may lead to residues in food products of animal origin, such as, milk, muscle, meat and kidney. It is well known that the improper use may have adverse effects on consumer health, including bacterial resistance to these drugs in humans, and also problems in dairy industry [3]. Bovine milk is a rich source of many nutrients that are necessary for the neonate and plays an important role during humans´ adult life, helping them to meet their nutritional requirements. The presence of penicillins in milk may represent a risk to consumer health, such as allergic reactions and anaphylactic shock in sensitive individuals [4]. To avoid health risks for the consumer due to residues, the EU has defined safe maximum residue limits (MRL) through Commission Regulation (EU) No. 37/2010. The limits implemented are shown in Table 1.  [6]. The applicability of MIP as selective sorbents for the extraction of some components of BLA group has already been reported [7]. However, it would be interesting to develop polymers that allow simultaneous For quantification, the most intense MRM transition was monitored along with a second transition for indentify confirmation (Table 2).

Preparation of polymers
The polymers were prepared by precipitation polymerization. OXA, AMOX and NAFC were used as template and MAA as functional monomer. As cross-linkers DVB, EGDMA and TRIM were tested, including different solvents in the polymerization mixture. The different combinations of template, monomer, cross-linker, initiator and porogen were as indicated in the table 3. The different polymerization mixtures were simultaneously introduced into a temperature controllable incubator equipped with a low-profile roller at 24 r.p.m. and 60ºC for 24 hours. The polymer particles were separated and cleaned by vaccum filtration through a nylon membrane filter of 0.45 µm of pore diameter, using 50 mL of acetonitrile and 50 mL of methanol. Then the imprint molecule was removed by Soxhlet extraction for 8 h using a methanol/acetic acid mixture (1:1). In each case, nonimprinted polymers (NIP) were prepared in the same way but without the addition of template.

Optimization of molecularly imprinted solid-phase extraction (MISPE)
Molecularly imprinted and non-imprinted polymers (0.05 g) were placed in empty SPE

Preparation of polymers and MISPE optimization
The selected polymerization technique was precipitation polymerization, which have an Also, some polymerization problems were found when using OXA and/or AMOX as templates in combination with DVB-80 in acetonitrile/toluene porogen, mainly due to the low solubility of these drugs in non-polar solvents (the mixture ACN/TOL presents the lower polarity of all the tested porogens). In this case, no polymerization was achieved, because it was not possible to dissolve the template in the polymerization mixture, which is a key factor in MIP synthesis. When using DVB as cross-linker, the combination of acetonitrile and toluene is required to obtain polymers with good performance and with the production of monodisperse, imprinted-polymer beads with well-developed, permanent pore structures [7]. Summing up, NAFC-MAA-EGDMA-ACN was selected as SPE polymer because it provided the higher retention capacity for eight penicillins studied (ampicillin, amoxicillin, oxacillin, penicillin G, penicillin V, cloxacillin, dicloxacillin and nafcillin), enabling the simultaneous extraction of these drugs at the level of interest in milk.

MISPE of infant formulas
Applicability of NAFC-based MIP in real samples was tested using infant formulas as analytical matrix. The matrix was selected based on the importance that food safety, and more precisely antimicrobials residues, has for vulnerable population, as babies and