*2.2. Fluoroquinolones*

Quinolones represent a class of drugs commonly used in veterinary fields, whose MRLs have been established in the range level of 100–500 μg/kg by the EU Council Regulation and FDA [2,3]. Fluoroquinolones (FQs) are synthetic quinolones, related to nalidixic acid, which act against Gram-negative and Gram-positive bacteria by inhibiting their DNA synthesis. FQs are commonly used for livestock growth and aquaculture and are toxic to human health because they can exhibit a direct toxicity, responsible for muscular and neuronal dysfunctions, or cause antibiotic resistance or allergies [46–48].

CE techniques also represented interesting platforms in the analysis of the third and fourth generation of FQs in different food matrices (water, milk, and animal muscle), but appropriate sample pre-treatments and detection systems (LIF and MS) became mandatory to obtain a high level of sensitivity [49]. To extract milk FQs, LLE and SPE were the most used procedures, but in the literature, examples of protein precipitation (PPT) followed by SPE are also present as a good alternative [50]. In particular, molecularly imprinted polymers (MIPs), which are selective and stable sorbent materials for SPE (MISPE technique) [51], allowed promising CZE-LIF or CZE-MS methods to be obtained in the animal foodstuff analyses. MIPs can be used both before CZE analysis and as an in-line-MISPE strategy, thus obtaining sensitive and selective methods for the analysis of different complex matrices (for example, pig kidney and bovine milk) [52,53]. In fact, by using advanced MIP technologies, the low CZE-UV sensitivity can also be overcome. Magnetic molecular imprinted polymers (MMMIPs) were prepared by combining ferroferric-oxide nanoparticles, and MIPs and had a rapid and efficient

binding capacity. These materials allowed a rapid and selective CZE-UV method to be obtained to separate fleroxacin, gatifloxacin, lomefloxacin (LOM), and norfloxacin in bovine milk samples [29]. The method sensitivity (LOD range: 12.9–18.8 μg/L) was slightly lower than, but comparable to, that of Springer et al. (7.5–11.6 μg/L), who separated ciprofloxacin (CIP), norfloxacin, and ofloxacin (OFL) in milk samples using a miniaturized SPE (made of carbon nanotubes as sorbents with high adsorption capacity and stability) to prepare samples [28].

Lara et al. set-up a CZE-MS/MS method for the simultaneous quantification (ng/kg level) of eight FQs, i.e., danofloxacin, sarafloxacin, CIP, marbofloxacin, enrofloxacin (ENRO), difloxacin, oxolinic acid, and flumequine, in chicken muscle samples. Two different sample preparation approaches were set-up; the first one consisted of pressurized liquid extraction (PLE), which was performed in an accelerated solvent extraction, followed by centrifugation, percolation, concentration, reconstitution, and filtration steps prior to the injection into the CE instrument; the second one was an in-line SPE, using a mixed-mode sorbent (RP and ion-exchange sorbents), in which different parameters, such as sample pH, volume, elution plug composition, and injection time, were optimized. The combination of in-line SPE-CE-MS/MS with PLE improved the selectivity and sensitivity, and this was particularly useful in multiresidue analysis [54].

Field-amplified sample stacking (FASS) with sweeping represented another on-line pre-concentration procedure used in combination with CE to rapidly quantify ENRO and CIP (LODs ng/mL) in milk and animal tissues. The FASS procedure consists of an electrokinetic injection of sample in a run buffer with high conductivity, creating an interface in which the difference in the electric field between the sample matrix and run buffer promotes sample stacking. The use of gamma-cyclodextrin in the sample matrix (sweeping procedure) could increase the FASS sensitivity, because it gave origin to micelles as in the MEKC technique, in which cyclodextrins are added to the background electrolyte (BGE) [55].

A different on-line preconcentration procedure, named field-enhanced sample injection (FESI), could increase CZE-UV sensitivity. FESI is useful for samples with low conductivity and consists of a careful optimization of the injection (pressure and time) of a water plug into the capillary in order to increase the capillary electric field by creating a sample stacking effect. The use of FESI in addition to a bubble cell capillary (with a longer window pathway than the traditional capillary) increased the method sensitivity for five different FQs (ENRO, CIP, LOM, fleroxacin, and OFL) in bovine milk samples [56].

Another CE mode, i.e., nonaqueous capillary electrophoresis (NACE)-UV, combined with a DLLME approach allowed the set-up of a selective method for the separation and quantification of eight FQs (omefloxacin, levofloxacin, marbofloxacin, CIP, sarafloxacin, ENRO, danofloxacin, and difloxacin) in water samples [57]. In NACE, BGE is added with organic solvents, mainly methanol or acetonitrile, and this promotes the separation of low-water-soluble molecules. In fact, even if the use of organic solvents could induce changes in the pKa values and mobility, the advantage consisting of an increased selectivity becomes fundamental [16]. The same CE-mode coupled with an in-line single-drop liquid-liquid-liquid microextraction (SD-LLLME) was used to separate CIP and ENRO in surface and groundwater samples. This setup allowed a reduction in the analysis time (as no sample pre-treatment was necessary) and in the volume of extraction solvent, as a buffer was used as a pH donor, a drop of NaOH as a high pH acceptor, and an organic solvent as a medium in which the analytes diffused [58].
