*2.3. Tetracyclines*

Tetracyclines are widely used as economic broad-spectrum antibiotics against both Gram-positive and Gram-negative bacteria. MRLs were established by the Commission Regulation (EU) and ranged from 100 to 600 μg/kg, depending on animal tissues or the food sample [2]. In the literature, many works aimed to analyze and quantify these antibiotics. A simple SPE procedure with a NACE-LIF method allowed a very sensitive separation of chlortetracycline (CTC), tetracycline (TC), oxytetracycline (OTC), and doxycycline (DC) in feeds and milk with pg/mL LOD values [59]. In the same year, Deng et al. proposed a simple CZE-enhanced chemiluminescence (ECL) method to monitor TC residues over time in crucian carp muscle of fish samples, with a sensitive detection under MRL values [60].

Recently, a field-amplified sample injection (FASI) procedure in CZE-UV was set-up for the detection of four tetracyclines in pig farms' wastewaters with results comparable to those obtained by HPLC-UV methods [61]. FASI pre-treatment is particularly suitable for large amounts of water samples and is based on a difference in electrical conductivity between the sample and the background electrolyte, which causes a stacking effect responsible for the increase in peak efficiency and method sensitivity.

The combination of two pre-concentration procedures could be useful to determine drug residues, even when present at a concentration below MRL limits. Islas et al. recently set-up a SPE step followed by a large-volume sample stacking (LVSS) approach before a CZE-UV analysis of TC. LVSS consisted of a stacking procedure with an on-line series of polarity switches (PS) and enabled an improvement in sensitivity and reproducibility, particularly useful for low-concentration analytes in complex matrices, such as milk samples (Figure 1) [62]. The same pre-concentration approach (SPE-LVSS-PS) was previously used to determine five TC residues (metacycline-MTC, OTC, TC, CTC, and DC) in water samples, reaching a high sensitivity (ng/L) in a short time (10 min) [63]. The LVSS approach alone, after a careful optimization of stacking conditions (sample zone length and stacking time), allowed a CZE-UV method to be obtained with a sensitivity around 10 ppb, a sensitivity level very similar to that obtained by using ED or LIF detectors. This method was used to detect TC, CTC, OTC, and DC in tap water samples [64].

**Figure 1.** Solid phase extraction (SPE)-large-volume sample stacking (LVSS)-capillary electrophoresis (CE) method applied to milk sample for the detection of tetracyclines (TCs). Electropherograms of (**a**) standard TC sample (10 mg/L) analyzed by CE, (**b**) standard TC sample (1 mg/L) analyzed by LVSS-CE, (**c**) blank milk sample, and (**d**) real milk sample analyzed by SPE-LVSS-CE method [62].

Another promising extraction approach consisted of matrix solid-phase dispersion (MSPD), which required the addition of sorbents to the sample with a consequent elution step before the analysis. Mu et al. set-up an economic MSPD-CZE-UV method to rapidly (about 6 min) separate TC, OTC, and DC in milk samples [65]. In order to improve CE sensitivity, a functionalized β-cyclodextrin-ionic liquid was added in-line; unlike the conventional β-cyclodextrin, it acted as an additive able to form a complex with TCs and as a capillary coating agent. The method proposed by Zhou et al. was developed to separate four TCs in about 30 min by using CE with amperometric detection (AD) [66].

Tetracyclines can also be present in honey, representing a serious problem, as the EU Commission did not admit to the use of antibiotics in honey and did not establish MRLs for bee products [2]. Casado-Terrones et al. set-up an SPE procedure followed by a CZE-UV method to simultaneously and rapidly (16 min) determine eight tetracyclines in honey with LOD values of 23.9–49.3 μg/kg [67].
