**6. Detection and quantification of antibiotics into the environment**

#### **6.1. Sample preparation techniques**

cause the sharing of resistant plasmids to nonresistant organisms [18, 99]. Another possibility of occurrence of bacterial resistance is that low concentrations of ATB residues transferred to the soil by the application of contaminated animal manure favor the selection of resistant populations [109]. However, the direct introduction of resistant microorganisms derived from feces of animals treated with ATBs seems to be more important to resistance [99] than induction

It is important to remember that there is a large reservoir of ATBs-resistant bacterial genes in the soil. However, according to Schmidt e al. [110], it is not known whether this occurs naturally or due to the use of veterinary ATBs. As an example, in a study by Esiobu et al. [102], isolated bacteria of a garden soil fertilized with dairy cattle manure showed 70% resistance to ampi‐

The exposure intensity of bacteria to ATBs agents influences the amplitude of its resistance, and the exposure intensity usually depends on the origin of the treatments by which bacteria were submitted. Costanzo et al. [111] indicated that bacteria from a sewage treatment plant reactor were resistant to ciprofloxacin, tetracycline, ampicillin, trimethoprim, erythromycin, and sulfamethoxazole antibiotics, while bacteria isolated from the effluent receiver water‐ course showed resistance to erythromycin and ampicillin. This same study showed that erythromycin, clarithromycin, and amoxicillin ATBs, at a concentration of 1.000 µg L-1,

In aquaculture, the intensive use of ATBs provides a selective pressure for the creation of bacteria resistant to drugs and genes resistant to transmitted pathogens of fish and other bacteria in the aquatic environment. From these resistant bacteria, resistance genes can be spread by horizontal gene transfer and transfer to human pathogens. Drug-resistant pathogens present in the aquatic environment can directly reach humans. The horizontal gene transfer can occur in the aquaculture environment, in the food chain, or in the human intestinal tract. Among ATBs commonly used in aquaculture, several are classified by the World Health Organization (WHO) as extremely important for use in humans. The occurrence of ATBs resistance in human pathogens severely limits the therapeutic options in human infections. Taking into account the rapid growth and the importance of the aquaculture sector in many regions of the world, due to the widespread, intense and often irregular use of ATBs in this animal production area, efforts are necessary to prevent the development and spread of

Another issue in aquaculture regarding bacterial resistance needs to be highlighted, that is, if bacterial populations are resistant to a certain ATB used in this sector, or the producer changes the ATB or increases the dose in anticipation of a more efficient control. However, these two practices make such microorganisms to become resistant to this new applied molecule over time. Increasing the dose may also have negative effects on native aquatic biota of where the

In the study of Akinbowale et al. [113], isolated bacteria from water samples and organisms used in aquaculture showed widespread resistance to ampicillin, amoxicillin, cephalexin, and

due to the presence of ATB residues on the environment.

126 Emerging Pollutants in the Environment - Current and Further Implications

cillin, penicillin, tetracycline, vancomycin, and streptomycin ATBs.

decreased more significantly the rate of bacterial denitrifying.

bacterial resistance in order to reduce the risk to human health [112].

creation is installed.

The determination of antibiotics in environmental samples is a difficult task due to the high complexity of the analyzed matrices and the low concentrations of these compounds in the samples [120].

The sample preparation step affects all the other steps of the test and therefore is critical for unambiguous identification, confirmation, and quantification of antibiotics. It includes the isolation and/or preconcentration of interest compounds from the matrix and also properly provides the compounds for the separation and detection. Sample preparation takes typically more than 70% of total analysis time.

Chromatographic methods are usually preferred in the analysis of organic molecules, which causes the need to have an initial sample preparation, a process of extraction, which is normally a liquid–liquid extraction, followed by a clean-up process, which is usually a solid-phase extraction (SPE). Comparing the analysis by ultrafast chromatography with the conventional, sample pretreatment processes are more laborious and time consuming, as it requires an even purer extract. For this reason, many new sample preparation techniques have been developed, and there is a continuing interest in this area.

A quick search in the scientific literature showed that more than 1300 articles on analysis of antibiotic residues were published during the period of 2004–2015, and liquid extraction (LE) and liquid–solid extraction (LSE) were the most popular sample treatment techniques, which were used in 30% and 60% of the reported studies, respectively. The LE includes all techniques based on liquids, such as liquid–liquid extraction (LLE), liquid–liquid microextraction, and pressurized liquid extraction (PLE). LSE includes solid-phase extraction (SPE) and all other procedures based on extraction absorbers, such as solid-phase microextraction (SPME), stirbar sorptive extraction (SBSE), restricted access material (RAM), turbulent flow chromatogra‐ phy (TFC), dispersive solid-phase extraction (DSPE), and matrix solid-phase dispersion (MSPD). Other techniques for some specific applications are the microwave-assisted extraction (MAE), the ultrasound-assisted extraction (UAE), extraction based on immune affinity, and the technique that use molecular imprinted polymers (MIP).

There have been many changes in sample preparation with the advent of mass spectrometry. Previously, methods of analysis were able to analyze residues of only a limited number of compounds (usually a single class of drugs); but with mass spectrometry, now there is the possibility of residue analysis of many compounds in a single analysis. Although mass spectrometry allows the use of simple and generic cleaning methods, the effective removal of matrix constituents is necessary since these may affect the performance of the mass spectrom‐ eter (MS), in particular, by ion suppression.

There was also the migration of manual sample preparation to faster techniques with auto‐ mated processes. The automated preparation of samples can be made online (with sample preparation directly connected to the chromatographic system) or offline (sample preparation is automated, but the sample has to be manually transferred to the chromatographic system).

Most analytical methods developed for the antibiotic determination in water use offline solidphase extraction (SPE) and LC-MS/MS [121–127]. Some studies, however, point toward developing methods with the SPE-LC-MS/MS system, which allows reduction in the sample amount, lower preparation time, and consequently, increase in productivity, in addition to less sample manipulation, decreasing contamination chances [128–132]. The SPE-online system has also been used for the determination of pesticides, hormones, explosives, phar‐ maceuticals, and personal care products [133–137].

Among the advantages of SPE-online method, it is possible to highlight the small sample volume requirements, making it easier to transport and store. As in most cases, the sampling sites are too distant from laboratories. Several sample preparation steps, such as evaporation and reconstitution, are eliminated, and there is less need for sample handling and processing, which lead to a reduction in analysis time and analyst's interference, minimizing errors, losses, and sample contamination, which is reflected in better method accuracy and precision values, in addition to a significant reduction in the consumption of organic solvent, contributing to the "green chemistry" [138, 139].

Chromatographic methods are usually preferred in the analysis of organic molecules, which causes the need to have an initial sample preparation, a process of extraction, which is normally a liquid–liquid extraction, followed by a clean-up process, which is usually a solid-phase extraction (SPE). Comparing the analysis by ultrafast chromatography with the conventional, sample pretreatment processes are more laborious and time consuming, as it requires an even purer extract. For this reason, many new sample preparation techniques have been developed,

A quick search in the scientific literature showed that more than 1300 articles on analysis of antibiotic residues were published during the period of 2004–2015, and liquid extraction (LE) and liquid–solid extraction (LSE) were the most popular sample treatment techniques, which were used in 30% and 60% of the reported studies, respectively. The LE includes all techniques based on liquids, such as liquid–liquid extraction (LLE), liquid–liquid microextraction, and pressurized liquid extraction (PLE). LSE includes solid-phase extraction (SPE) and all other procedures based on extraction absorbers, such as solid-phase microextraction (SPME), stirbar sorptive extraction (SBSE), restricted access material (RAM), turbulent flow chromatogra‐ phy (TFC), dispersive solid-phase extraction (DSPE), and matrix solid-phase dispersion (MSPD). Other techniques for some specific applications are the microwave-assisted extraction (MAE), the ultrasound-assisted extraction (UAE), extraction based on immune affinity, and

There have been many changes in sample preparation with the advent of mass spectrometry. Previously, methods of analysis were able to analyze residues of only a limited number of compounds (usually a single class of drugs); but with mass spectrometry, now there is the possibility of residue analysis of many compounds in a single analysis. Although mass spectrometry allows the use of simple and generic cleaning methods, the effective removal of matrix constituents is necessary since these may affect the performance of the mass spectrom‐

There was also the migration of manual sample preparation to faster techniques with auto‐ mated processes. The automated preparation of samples can be made online (with sample preparation directly connected to the chromatographic system) or offline (sample preparation is automated, but the sample has to be manually transferred to the chromatographic system). Most analytical methods developed for the antibiotic determination in water use offline solidphase extraction (SPE) and LC-MS/MS [121–127]. Some studies, however, point toward developing methods with the SPE-LC-MS/MS system, which allows reduction in the sample amount, lower preparation time, and consequently, increase in productivity, in addition to less sample manipulation, decreasing contamination chances [128–132]. The SPE-online system has also been used for the determination of pesticides, hormones, explosives, phar‐

Among the advantages of SPE-online method, it is possible to highlight the small sample volume requirements, making it easier to transport and store. As in most cases, the sampling sites are too distant from laboratories. Several sample preparation steps, such as evaporation

and there is a continuing interest in this area.

128 Emerging Pollutants in the Environment - Current and Further Implications

the technique that use molecular imprinted polymers (MIP).

eter (MS), in particular, by ion suppression.

maceuticals, and personal care products [133–137].

Besides these, the automated preparation of samples has the advantage of performing the clean-up, concentration, and separation of the compound in a closed system. This reduces the sample preparation time, and the whole sample becomes available for analysis, leading to a reduction in detection limits. It also decreases the analyst procedural errors, thereby improving accuracy and reproducibility. Moreover, in automated sample preparation, the cost is also reduced, using less solvent and less personnel. Other advantages include reduced risk of sample contamination and elimination of analyte disposal losses by evaporation or degrada‐ tion during sample preconcentration.

Automatic methods also have some disadvantages, such as increase in initial capital expen‐ diture and risk of increased service downtime due to equipment breakdowns, which require parallel processes to be made in order to reduce the laboratory inactivity.

For the determination of antibiotics in environmental, soil, sediment, and manure solid matrices, among others, different procedures are performed, which involve several techniques, such as accelerated solvent extraction (ASE), pressurized liquid extraction (PLE), ultrasoundassisted extraction (UAE), and microwave-assisted extraction (MAE). The solvent choosing is critical to ensure selectivity and minimize the extraction of other matrix constituents. Better diffusion of the solvent in the matrix interstices by mixing the sample and quartz sand is essential for best performance. The correct use of pH also increases extraction efficiency, and pH acids are generally more indicated because they favor electrostatic repulsion between antibiotics and sediment surface, which are both protonated [140].

Prior to antibiotic chromatographic determination, postextraction sample purification is often necessary to remove interfering (e.g., coextracted organic matter or organic solvent) and to achieve lower quantification limits. The adoption of this strategy leads to substantial improve‐ ment in method selectivity, where the cleaning is carried out in most cases by solid-phase extraction (SPE). Used adsorbents differ in composition, chemical properties, and affinity with the analyte [140].

Currently, EAU followed by filtration or centrifugation is the most common procedure. Yang et al. [57] developed a method with EAU using a mixture of acetonitrile and citrate buffer (50:50 v/v) placed in an ultrasound bath for 15 min, repeating this process three times. The extract was then purified by using SPE cartridges in series, SAX (6 mL, 500 mg), and HLB (6 mL, 200 mg); the procedure obtained good recoveries for 14 antibiotics studied in the sediment of Pearl rivers in Guangdong province, China, and was also used by Zhou et al. [141], which increased the number of analyzed antibiotics.

Antibiotic extraction from sediment and the transfer of these to an aqueous solution can provide the use of the SPE-online system, increasing the sensitivity of the method, facilitating the preparation of samples, and reducing purification steps, as shown by Monteiro [46] in his PhD study. In this study, the procedure proposed by Yang et al. [57] was used, but the purification step using SPE with HLB cartridges was replaced by SPE-online system, in which a semipreparative precolumn with C8 adsorbent was used, obtaining optimal results for antibiotics of the class of tetracyclines, fluoroquinolones, sulfonamides, and phenicols in sediments collected from tank-nets fish farms.

As for biological samples, such as fish and aquatic plants, for example, one of the main problems for quantitative analysis of pharmaceuticals is that the analyte is typically bound to proteins and peptides, with the consequent need for cleavage of these structures before analysis. Enzymatic digestion is widely accepted as a sample preparation method for analyz‐ ing compounds in biological matrices. However, these methods are labor intensive and significantly prolong the examination time [142]. Most methodologies use extraction proce‐ dures based on liquid–liquid extraction, with relatively polar solvents and subsequent extraction purification using solid-phase cartridges [143, 144].

Another technique that has been highlighted for the extraction and purification of antibiotics in biological matrices is QuEChERS (quick, easy, cheap, effective, rugged, and safe), which was initially developed by Anastassiades et al. [145] for the determination of pesticide residues in food. It has been adapted and has been used for the determination of other compounds, including antibiotics in fish [54, 146].

The extraction procedure should be appropriate to the intended analysis and the reality of laboratory, so factors such as reagents consumption, availability of skilled labor force, and equipment acquisition are crucial. Simple and rapid methods stand out in this context because they are less dependent on high investments [147, 148].

### **6.2. Confirmatory and quantitative methods**

For the determination of medicaments, different analytical methods are reported in literature, which are primarily valid for biological matrices, such as blood and tissue [148–150], and some modifications in these methods may be sufficient to environmental samples. However, residual drug analysis in WWTP effluents, rivers, subsoils, sediments, soil, and sludge waters still require the development of more sensitive methods for the detection of concentrations in µg L-1 and ng L-1 range.

Separations in environmental chemistry generally involve the two most recognized chroma‐ tographic techniques: high-performance liquid chromatography (HPLC) and gas chromatog‐ raphy (GC).

Knowledge of physical and chemical properties of the analytes is of utmost importance to avoid problems in quantification, which can be related to side reactions, impurities, or degradation in their structure during the analytical method application. For example, tetra‐ cycline antibiotics may irreversibly interact with residual metal ions present in sorbents of solid-phase extraction cartridges based on modified silica with alkyl groups (C18, C8, etc.), and certain metals can catalyze the ring opening of β-lactams. This problem can be solved by adding a chelating agent to the matrix to be extracted (Na2EDTA, for example) or by replacing the cartridges sorbent by polymeric material [57].

The solubility of the analytes in the environmental sample and in the eluting solvent as well as in the mobile phase to be used also deserves attention. For example, some antibiotics form water-insoluble lipophilic complexes in the presence of alkali metal cations. Furthermore, penicillin patterns undergo methanolysis when solubilized in methanol, and should be prepared in acetonitrile or another compatible solvent [57].

During the detection step, when using a mass spectrometer (MS), there may be some sort of fragmentation that is characteristic of the analyte, depending on its pH range. For example, the erythromycin in acidic solution has a mass loss of 18 Da, which corresponds to the loss of a water molecule [57].

One of the critical parameters to be observed during antibiotic determination is referred to the sample pH, because in many cases, the medium pH determines the chemical form of the analyte in solution and thus interferes in the extraction efficiency. For example, the low recovery percentage of quinolones extraction process was improved after acidifying the solution in 2.5 pH. However, it is important to choose a pH range in which degradation of the analytes will not occur [57].

Furthermore, the pH of the mobile phase needs adjustment, in which its value depends on the p*K*<sup>a</sup> of the compounds to be analyzed. The recommended buffer concentration is in the range between 2 and 20 mmol L-1 to avoid solubility problems in the mobile phase and to facilitate the ionization mode when using the MS detector [128].

The most commonly used stationary phases in HPLC for separation of organic compounds are of reverse phase (RP) type, which are silica based with C18 groups. Stationary phases with C8 groups may be used for β-blockers and antibiotics (tetracyclines, penicillins, sulfonamides, and macrolides) [128], [57].

The mobile phases used in the RP-HPLC are mixtures of methanol–water (MeOH:H2O) or acetonitrile–water (ACN:H2O) with adjustments of the chromatographic strength and mobile phase selectivity to the obtainment of enough resolution to occur the separation of all chro‐ matographic peaks in minimum analysis time. The addition of modifiers, such as formic acid, ammonium acetate, ammonia, etc., is performed in order to favor the process of analytes ionization by medium pH adjustment, improving their interactions with the mobile phase and the stationary phase. Medium pH control may also be performed by using buffered mobile phases.

When the mass spectrometer detector (MS) and the electrospray ionization process (ESI) are used, modifiers may also be used in order to favor the process of analytes ionization.

#### **6.3. Detectors for HPLC**

PhD study. In this study, the procedure proposed by Yang et al. [57] was used, but the purification step using SPE with HLB cartridges was replaced by SPE-online system, in which a semipreparative precolumn with C8 adsorbent was used, obtaining optimal results for antibiotics of the class of tetracyclines, fluoroquinolones, sulfonamides, and phenicols in

As for biological samples, such as fish and aquatic plants, for example, one of the main problems for quantitative analysis of pharmaceuticals is that the analyte is typically bound to proteins and peptides, with the consequent need for cleavage of these structures before analysis. Enzymatic digestion is widely accepted as a sample preparation method for analyz‐ ing compounds in biological matrices. However, these methods are labor intensive and significantly prolong the examination time [142]. Most methodologies use extraction proce‐ dures based on liquid–liquid extraction, with relatively polar solvents and subsequent

Another technique that has been highlighted for the extraction and purification of antibiotics in biological matrices is QuEChERS (quick, easy, cheap, effective, rugged, and safe), which was initially developed by Anastassiades et al. [145] for the determination of pesticide residues in food. It has been adapted and has been used for the determination of other compounds,

The extraction procedure should be appropriate to the intended analysis and the reality of laboratory, so factors such as reagents consumption, availability of skilled labor force, and equipment acquisition are crucial. Simple and rapid methods stand out in this context because

For the determination of medicaments, different analytical methods are reported in literature, which are primarily valid for biological matrices, such as blood and tissue [148–150], and some modifications in these methods may be sufficient to environmental samples. However, residual drug analysis in WWTP effluents, rivers, subsoils, sediments, soil, and sludge waters still require the development of more sensitive methods for the detection of concentrations in

Separations in environmental chemistry generally involve the two most recognized chroma‐ tographic techniques: high-performance liquid chromatography (HPLC) and gas chromatog‐

Knowledge of physical and chemical properties of the analytes is of utmost importance to avoid problems in quantification, which can be related to side reactions, impurities, or degradation in their structure during the analytical method application. For example, tetra‐ cycline antibiotics may irreversibly interact with residual metal ions present in sorbents of solid-phase extraction cartridges based on modified silica with alkyl groups (C18, C8, etc.), and certain metals can catalyze the ring opening of β-lactams. This problem can be solved by adding a chelating agent to the matrix to be extracted (Na2EDTA, for example) or by replacing

sediments collected from tank-nets fish farms.

130 Emerging Pollutants in the Environment - Current and Further Implications

including antibiotics in fish [54, 146].

µg L-1 and ng L-1 range.

raphy (GC).

extraction purification using solid-phase cartridges [143, 144].

they are less dependent on high investments [147, 148].

**6.2. Confirmatory and quantitative methods**

the cartridges sorbent by polymeric material [57].

Spectrophotometric absorption detectors in the ultraviolet range (UV) and by fluorescence were initially used in HPLC equipment for the analysis of compounds, which are absorbed in the ultraviolet region or are fluorescent. The UV detection was used for the determination of antibiotics from fluoroquinolones class in environmental matrices (hospital effluent) [149], and the fluorescence detection was used for the determination of antibiotics in water, sediment, and fish farm plants samples since they are lower cost equipment comparing to HPLC-MS. However, the limits of detection values obtained for UV detectors are much higher, in the range of µg L-1 to ng L-1, in comparison to the MS detector, that can achieve detection limits in the order of ng L-1 to pg L-1 when used in series (MS/MS), offering also the possibility of confirmation of the analyzed compounds. For environmental analyzes for the purpose of screening, with detection limits in the concentration range of µg L-1, the UV detector can be optimally utilized, besides being used when the concentration of the analyte in the matrix is high, as is the case of the publications cited above [149, 151].

The wide use of HPLC-MS/MS in environmental chemistry is due to the fact that most USEPA official methods use this separation and detection mode [140] due to good limits of detection and the possibility of structural confirmation of the analyzed compounds, besides the robust‐ ness of the method.

The electrospray ionization process is the most used in the detection by mass spectrometry (MS) because it is a more versatile ionization form that works for analytes with median polarity to very polar and poorly volatile, as is the case for most drugs, or thermally labile analytes, such as certain antibiotics, when compared with the atmospheric pressure chemical ionization (APCI), which uses heating in the range of 300°C–400°C for analytes thermal desorption [57].

Several recent studies [152–155] used analytical methods based on HPLC separation with mass spectrometry detection in series with ESI ionization (HPLC-ESI-MS/MS) for the determination of antibiotics in aqueous matrices. This can be explained by the versatility of this ionization mode, which can be used for analytes with polarities ranging between medium and high, with better detectability by ESI.

The most used mass analyzers for analytes detection are the triple quadrupole (QqQ) for sequential mode (mass in series), the time of flight (TOF), and the ion trap. The TOF type analyzer was used in the determination of drugs (analgesics, antibiotics, β-blockers, and antiepileptics) in surface water, groundwater, and wastewater samples due to having higher detectability, linear dynamic range, and mass accuracy than triple quadrupole type analyzers (QqQ), although the best detection limits were found for the QqQ type analyzer [128].

Recently, the quadrupole time-of-flight hybrid analyzer type (Q-TOF) has been used for providing better resolution and detectability than the conventional quadrupole, thus being applied for identification and quantification of drugs unknown metabolites [57, 128, 139].

In recent years, capillary electrophoresis (CE) has become a popular technique because of its simplicity, high separation power, short analysis time, and low consumption of sample and solvents [138, 139]. Among different CE modes, micellar electrokinetic chromatography (MEKC) [140], which makes use of micellar solutions of ionic surfactants, has proved to be a very attractive technique for separating different medicament classes, including antibiotics, nonsteroidal and steroidal anti-inflammatory agents, and analgesics [141]. However, capillary electrophoresis with UV-Visible detection has not been applied for drug residue analysis in small parts levels per billion (µg L-1) due to its lower intrinsic detectability [46].

#### **6.4. Trends in liquid chromatography applied to the study of antibiotics**

and fish farm plants samples since they are lower cost equipment comparing to HPLC-MS. However, the limits of detection values obtained for UV detectors are much higher, in the range of µg L-1 to ng L-1, in comparison to the MS detector, that can achieve detection limits in the order of ng L-1 to pg L-1 when used in series (MS/MS), offering also the possibility of confirmation of the analyzed compounds. For environmental analyzes for the purpose of screening, with detection limits in the concentration range of µg L-1, the UV detector can be optimally utilized, besides being used when the concentration of the analyte in the matrix is

The wide use of HPLC-MS/MS in environmental chemistry is due to the fact that most USEPA official methods use this separation and detection mode [140] due to good limits of detection and the possibility of structural confirmation of the analyzed compounds, besides the robust‐

The electrospray ionization process is the most used in the detection by mass spectrometry (MS) because it is a more versatile ionization form that works for analytes with median polarity to very polar and poorly volatile, as is the case for most drugs, or thermally labile analytes, such as certain antibiotics, when compared with the atmospheric pressure chemical ionization (APCI), which uses heating in the range of 300°C–400°C for analytes thermal desorption [57].

Several recent studies [152–155] used analytical methods based on HPLC separation with mass spectrometry detection in series with ESI ionization (HPLC-ESI-MS/MS) for the determination of antibiotics in aqueous matrices. This can be explained by the versatility of this ionization mode, which can be used for analytes with polarities ranging between medium and high, with

The most used mass analyzers for analytes detection are the triple quadrupole (QqQ) for sequential mode (mass in series), the time of flight (TOF), and the ion trap. The TOF type analyzer was used in the determination of drugs (analgesics, antibiotics, β-blockers, and antiepileptics) in surface water, groundwater, and wastewater samples due to having higher detectability, linear dynamic range, and mass accuracy than triple quadrupole type analyzers (QqQ), although the best detection limits were found for the QqQ type analyzer [128].

Recently, the quadrupole time-of-flight hybrid analyzer type (Q-TOF) has been used for providing better resolution and detectability than the conventional quadrupole, thus being applied for identification and quantification of drugs unknown metabolites [57, 128, 139].

In recent years, capillary electrophoresis (CE) has become a popular technique because of its simplicity, high separation power, short analysis time, and low consumption of sample and solvents [138, 139]. Among different CE modes, micellar electrokinetic chromatography (MEKC) [140], which makes use of micellar solutions of ionic surfactants, has proved to be a very attractive technique for separating different medicament classes, including antibiotics, nonsteroidal and steroidal anti-inflammatory agents, and analgesics [141]. However, capillary electrophoresis with UV-Visible detection has not been applied for drug residue analysis in

small parts levels per billion (µg L-1) due to its lower intrinsic detectability [46].

high, as is the case of the publications cited above [149, 151].

132 Emerging Pollutants in the Environment - Current and Further Implications

ness of the method.

better detectability by ESI.

The trends in high-performance liquid chromatography applied to the study of antibiotics include the use of ultra-performance liquid chromatography (UHPLC), two-dimensional liquid chromatography (LC-LC), and hydrophilic interaction chromatography (HILIC).

The UHPLC is one of the latest advances in liquid chromatography, using stationary phases with smaller particle diameters (approximately 2µm) compared to those employed in HPLC. The use of these particles, in addition to high linear velocities of the mobile phase (MP), allows to reduce the analysis time while maintaining resolution and separation efficiency, providing less broad peaks (5–10 s), besides drastically reducing the analysis time to approximately 10 min or less [136].

An example of UHPLC applied to the study of antibiotics is the work done by Zhou et al. [156]. A robust and sensitive method with UHPLC-MS/MS was developed for the simultaneous determination of multiclass of antibiotic residues in several environmental matrices (surface water, pond wastewater, effluent, sediment, sludge, and manure). The analytical method applied SPE with HLB cartridges for water samples and ultrasound extraction for solid samples followed by cleanup using SAX-HLB cartridges. The method was successfully applied to the analysis of environmental samples collected from a WWTP of a swine farm. The detection of several antibiotics with high concentrations in the analyzed samples indicates that WWTP and animal farms are two major sources of antibiotic residues in the environment.

Two-dimensional liquid chromatography is a good alternative when performing analysis in complex samples, such as biological samples, for example, because with the increase of a dimension, there is also the increase of peak capacity, and subsequently the separation process is enhanced [157, 158]. A very interesting example of this technique was the method developed by Wang et al. [159], which analyzed 14 antibiotics in urine with two-dimensional liquid chromatography coupled with Q-TOF mass spectrometer detector, quadrupole time-of-flight hybrid analyzer, where detection limits of 0.04 to 1.99 ng mL-1 were obtained. The method was used to identify the antibiotics in urine of children, but it can be a great tool for use in envi‐ ronmental matrices, especially biological matrices such as fish, crustaceans, aquatic plants, and more.

The term HILIC was proposed by Alpert in 1990 as an acronym for "hydrophilic-interaction chromatography" for the separation of polar solutes. This technique has also been also called "hydrophilic-interaction liquid chromatography," and "aqueous normal phase." In a simple way, it can be said that HILIC is an HPLC form very similar to liquid chromatography "in normal phase," using a column in hydrophilic stationary phase ("normal"), but with an eluent comprising water, buffer, and a high concentration of organic solvent that is miscible with water (typical of a "reverse phase"). The elution order obtained in an HILIC system will be practically opposite to that obtained when employing the reversed phase (RP) mode. Retention is directly proportional to the polarity of the solute and inversely proportional to the polarity of the mobile phase [160].

The HILIC was applied to extract and quantify spectinomycin and lincomycin in manure supernatant liquid from swine and farmland erosion water treated with manure [161]. It was also successfully applied in the determination of oxytetracycline in environmental waters [162], and the results showed that there was no irreversible adsorption of oxytetracycline in the stationary phase, which shows that the silica column may be used in the determination of tetracycline antibiotics in HILIC mode with no peaks distortion, providing a different under‐ standing of what was previously found in the literature.

As prospects for the area of liquid chromatography applied to the study of antibiotics, the identification of metabolites and processing products as well as even lower detection ranges and quantification limits is highlighted.

HPLC is also closely linked to applied environmental legislation because today, for the vast majority of antibiotics, there is no maximum permissible concentration established by the legislation.

Studies on bioremediation and advanced processes of wastewater treatment, such as chemical and photochemical oxidation through advanced oxidation processes (AOP), ozonolysis, nanofiltration, reverse osmosis, membrane bioreactors, etc., are likely to use high-performance liquid chromatography as key tool in monitoring antibiotic degradation processes in artificial or natural environments.
