*2.7.* β*-Lactam Antibiotics*

Penicillins and cephalosporines are classified as β-lactam antibiotics. They have the same action mechanism, but cephalosporines have a more extended spectrum. In particular, in veterinary medicine, ceftiofur and cefquinome are specifically used to treat respiratory diseases and exudative epidermitis, and meningitis, respectively. Cefquinome had MRL values more restrictive than those of ceftiofur (50–200 μg/kg vs. 100–6000 μg/kg) [2], but the use of ceftiofur must be carefully evaluated as its use could develop E. coli resistance [85,86].

CZE and MEKC modes were the best choice for the analysis of penicillins and cephalosporins in complex matrices [87,88]. For example, penicillin acid and penicillin G could be easily detected in milk by CZE-UV with a simple sample pre-treatment, which consisted of the deproteinization, extraction, and precipitation of milk protein with acetonitrile [89]. To achieve the best resolution, an on-line sample concentration step coupled with a CEC-MS method with a polymeric monolithic column was proposed by Liu et al. The injection in CE consisted of an anion selective injection (ASEI) performed by solubilizing the analytes in buffers with different pH values and promoting a stacking effect. This method was applied to analyze milk samples, obtaining a high sensitivity [90].

Regarding cephalosporins, Hancu et al. set-up a simple and rapid CZE-UV method, simply optimizing BGE composition and pH. The method was able to separate seven cephalosporins in 6 min and it was proposed for the analysis of pharmaceutical products and different complex matrices, obtaining very low LOD values; therefore, it could be useful for residue analysis [88].

### *2.8. Simultaneous Analysis of Di*ff*erent Antibiotics*

The separation and quantification of different drugs (macrolides and tetracycline antibiotics) in feedstuffs could be simultaneously carried out by CZE-UV, as demonstrated for TIL, TYL, TC, OTC, and DC with LOD values of 0.5–1 mg/kg [91]. The method sensitivity was good considering that OTC and TYL were added in feeds for growth promotion in a range from a few to 50 mg/kg, and that TIL was also often added as an antimicrobial and respiratory diseases agent at a concentration of a few hundreds of mg/kg [92]. The separation was performed in 15 min and, thus, this CE-method could be considered useful for rapid routine analysis.

In addition, β-lactams, tetracyclines, quinolones, amphenicols, and sulphonamides were simultaneously and rapidly (8 min) separated by CZE-UV in bovine raw milk. The combination of LLE and SPE extraction procedures before CE-analysis allowed LOQ values lower than MRLs to be obtained [93].

Another example of a simultaneous separation of different antibiotics (fluoroquinolones, tetracyclines, and β-lactams) in milk was proposed by Long et al. The use of ECL detector allowed trace to be detected, reaching LOD values of cents and thousands of μg/mL [23].

If carefully optimized, CE methods could be very promising in routine screening as good alternatives to LC-MS methods. For example, Kowalski et al. set-up a SPE procedure combined with a MEKC-UV method able to selectively and successfully separate sulfonamides (sulfamethazine, sulfamerazine, sulfathiazole, sulfachloropyridazine, sulfamethoxazole, sulfacarbamide, and sulfaguanidine) and amphenicol-type antibiotics (chloramphenicol, thiamphenicol, and florfenicol) in commercial poultry samples (muscle, liver, and skin with fat) [94].

More recently, the use of FASS combined with a micelle to the solvent stacking (MSS) approach allowed the set-up of a very sensitive CZE-UV method to detect sulfamethoxazole and trimethoprim (an antibacterial agent frequently used in combination with sulfamethoxazole for respiratory and urinary infections) in dairy products, chicken eggs, and honey. MSS consisted of an injection of a micellar solution plug prior to FASS, in order to obtain a focused sample zone with an increase in CE sensitivity [95].

In 2019, a microchip-CE with an LED-induced fluorescence detector was used as a promising platform to simultaneously analyze antibiotics in food. The chip was tested for chloramphenicol (CAP) and kanamycin (Kana) quantification in milk and fish samples, obtaining rapid analysis (3 min) with LODs of pg/mL. Unlike other microchip-CE methods, which had the disadvantages of complexity and low versatility, this platform used a simple strategy, named stir-bar assisted DNA multi-arm junctions recycling, which exploited the capacity of a gold bar with the DNA probe to capture antibiotics, allowing a multiplexed detection and increasing the method sensitivity without matrix interference [26].

To analyze fluoroquinolones and sulfonamides in environmental water, He et al. set-up an on-line preconcentration procedure with a pressure-assisted electrokinetic injection (PAEKI) [96]. PAEKI resolves FASI limits in the analysis of anionic molecules, which can be depleted when the voltage is applied in reverse mode. PAEKI parameters (pressure and voltage) were optimized, obtaining LOD values of a few μg/L and improving the results obtained with hydrodynamic or electrokinetic injections [96].

#### **3. Other Drugs**

#### *3.1. Estrogens*

Estrogens are widely used in intensive farming worldwide. In EU and United States livestock, the discharge of estrogens is about 83,000 kg/year, representing a very dangerous environmental pollutant. Their low water solubility and the fact that they can easily be degraded and transformed contribute to make them important water contaminants [97].

Wu et al. proposed the use of SPE and pressurized CEC (*p*-CEC)-AD to separate five estrogens (bisphenol-A, 4-tert-octylphenol, 4-*n*-nonylphenol, 2,4-dichlorophenol, and pentachlorophenol) in chicken eggs and milk powder samples. *p*-CEC is an advancement of CEC, in which the formation of typical air bubbles of CEC is prevented by setting a micro-HPLC pump at the inlet of the CE capillary. The set-up method was selective and exhibited a 100- to 500-fold higher sensitivity than CE-UV methods, with values comparable to GC-MS methods [98].

The first DLLME approach combined with MEKC-ESI/MS to extract and analyze estrogens (estriol, 17α-estradiol, 17β-estradiol, estrone, 17β-ethinylestradiol, and their main metabolites) in different milk samples and milk derivatives was set-up by D'Orazio et al. The method was simple, less expensive, and allowed a μg/L LOD level to be obtained [32]. DLLME and MEKC-UV were also used in the analysis of hexestrol, bisphenol A, diethylstilbestrol, and dienestrol, which are phenolic environmental estrogens (PEEs) frequently present in water as contaminants. In this case, after an optimization of DLLME procedure (type and volume of extraction solvent; volume of dispersive solvent; extraction

time; salt concentration), LOD values (0.3–0.6 μg/L) within the requirements of trace analysis in environmental water were obtained [33].

#### *3.2. Non-Steroidal Anti-Inflammatory Drugs (NSAIDs)*

NSAIDs are added to animal feed mainly to treat respiratory diseases and allergies, generally in association with antibiotics. In addition, they could also improve animal meat quality by reducing fats [99]. They were classified as group B substances and, for many of them, MRLs were established by the European Council in relation to different animals and food matrices [2].

Diclofenac was banned in dairy animals, and other commonly used molecules (ketoprofen, salicylic acid and salicylates, acetylsalicylic acid, and acetylsalicylates) were approved only for non-dairy animals or for animals not involved in egg production [99]. For ibuprofen and flurbiprofen, no MRL value was established, and this could represent a serious problem as NSAIDs cause important side effects (from gastrointestinal problems to cancer) in humans [100].

Regarding the analysis of NSAIDs in animal feed, LC-MS was the most widely used approach, reaching LOQ values around ng/mL [10,101]. Nevertheless, off-line and on-line stacking procedures could lead to promising sensitive CE methods. Alshana et al. set-up a rapid DLLME-FASS-CZE method to detect five main NSAIDs (etodolac, naproxen, ketoprofen, flurbiprofen, and diclofenac) and their derivatives in bovine milk with results similar to those obtained using conventional SPE-LC techniques. In fact, DLLME and FASS combined extraction and stacking procedures are able to develop a sensitive method with a 1000-fold decrease in LOQ values (μg/kg), in comparison to conventional CZE techniques [99]. Among NSAIDs, naproxen, ketoprofen, and clofibric acid are widely used in veterinary medicine and represent contaminants of emerging concern (CECs) for the aquatic environment. SPME is a rapid off-line pre-concentration step ideal for the analysis of highly polar compounds, as NSAIDs, in water samples, with high sensitivity. This was demonstrated by Espina-Benitez who set-up a rapid and economic synthesis of new coated fibers for SPME, developing a sensitive CZE-UV method [102].
