*2.5. Aminoglycosides*

These antibiotics (AGs) are widely used in veterinary medicine for bacterial and protozoan infections. In addition, AGs are frequent honey contaminants from non-EU countries, which is a big issue. As mentioned above, the use of antibiotics in beekeeping is not authorized in the EU [2] and, therefore, there are no European Community regulations about MRLs for these drugs in this product. To detect these compounds in other complex matrices, i.e., biological fluids and pharmaceutical samples, CE with LIF, C4D, or indirect detections, are requested [74]. Recently, Moreno-Gonzalez et al. detected nine AGs (three gentamicins, neomycin, apramycin, paromomycin, dihydrostreptomycin, spectinomycin, and streptomycin) in different types of honey using MISPE and FASS procedures as purification and pre-concentration steps, and a CZE-MS/MS analysis method [75]. MISPE also represented an interesting tool for the extraction and analysis of AGs in different animal-derived food (meat) [76].

In 2019, a microchip-CE-C4D method was developed using standard solution and was proposed as a good alternative to separate in a very short time (less than 1 min) AG sulfates (gentamicin sulfate, kanamycin sulfate, and streptomycin sulfate) in foodstuff with LOD values of 1–3 μg/mL [77].

#### *2.6. Macrolides*

Macrolides are bacteriostatic antibiotics with a broad spectrum of activity against Gram-positive and Gram-negative, particularly used in the treatment of respiratory diseases in bovines and pigs, but generally also added to animal feed. Like aminoglycosides, they are listed in Group B1-antibacterial substances under the Council Directive 96/23/EC [78] and possess the same mechanism of action consisting of the inhibition of bacterial protein synthesis, but they bind the 50S instead of 30S ribosomal unit. Their presence in food could cause problems to human health, in particular, to intestinal bacterial flora, and, in the last years, their use for long periods contributed to cause the development of antibiotic resistance [79,80]. An EU directive established MRL values in the range 40–200 μg/kg for erythromycin, tylosin (TYL), tilmicosin (TIL), and spiramycin [2,80].

For the evaluation of macrolides in food, HPLC-UV or HPLC-ESI/MS were the most frequently used methods [81,82]. However, off-line or on-line pre-concentration steps were the ideal approach to overcome the derivatization step required to obtain a high sensitivity by CE. Among the on-line pre-concentration techniques, FASS represented a promising solution, as demonstrated in the analysis of TIL, erythromycin, clarithromycin, roxithromycin, and tylosin residues in milk samples by a FASS-MEKC-UV method (LOD range of 0.002–0.004 mg/kg) [83]. CZE-DAD was recently proposed to analyze macrolides (TYL and TIL) in chicken fat samples, reaching LOQ values of a few μg/kg thanks to a preconcentration step that consisted of an evolution of the DLLME procedure: In fact, the extraction and the preconcentration occurred in an ionic liquid by using ultrasound instead of dispersive solvents (this procedure is named reverse ultrasound-assisted emulsification-microextraction, R-USAEME). Organic salts, which are more stable and less toxic than the organic solvents commonly used in DLLME, were used to prepare the ionic solution [84].
