## 2 Sample Preparation Methods
### Solid-Phase Extraction
Unlike traditional SPE sorbents, where the reverse phases are made up of silica that can only retain non-polar or moderately polar compounds, this type of polymeric adsorbent can retain a wide range of acidic to basic compounds, with low to high polarity, being very useful in the development of methods that consist of compounds with different physico-chemical properties. In addition, silica-based sorbents are unstable over a wide pH range and they contain free silanol groups, which can irreversibly bind to some analytes, such as tetracyclines [95]. The HLB sorbent is the SPE material that is most often (Table 1) used in the extraction of PhACs belonging to different therapeutic classes [10; 23; 45; 48; 60; 96]. In the analysis of complex matrices, such as wastewater, the extraction/concentration and cleanup process requires passing the sample through more than one type of SPE material. One example of this approach is the use of an anion exchange adsorbent, followed by an HLB adsorbent for the determination of antibiotics, such as fluoroquinolones, sulfonamides, and trimethoprim [97]. Mixed-mode sorbents were developed to simplify this type of analysis. This application combines two or more functional groups into a single cartridge. This combination allows multiple retention interactions between the sorbent and the analytes, improved cleanup, better reproducibility, and recovery, leading to, overall, more sensitive, precise, and accurate analytical methods [74]. An example of this application is a SPE cartridge with a cation exchange resin and an HLB copolymer is the mixed-mode/cationic-exchange (MCX) [98; 99], mixed-mode/anion-exchange (MAX) [26], and weak anion-exchange (WAX) [27]. These mixed-mode sorbents were used for reducing interference and matrix effects in the determination of PhACs in waters [92; 98; 100; 101; 102; 103]. Unfortunately, the sorption capabilities of these cartridges are often limited when compared to the HLB cartridge for large volumes of water or complex environmental matrices [88]. The matrix's complexity always affects the recovery of the PhACs, regardless of the sorbent used. The relative recoveries always show wide ranges of values due to the diversity of compounds under analysis (Table 1). This amplitude is higher for methods with a higher number of compounds, and when the number of therapeutic classes is also higher [37; 44; 51; 96; 99]. Sorbents with dual polarity (hydrophobic and hydrophilic) are the most versatile, so Oasis HLB is the most used sorbent [45; 104; 105]. For the analysis of PhACs belonging to the same therapeutic class, usually with similar polarity, more selective cartridges, both polar [26; 82] and non-polar [106] are chosen. When mixed-mode sorbents, such as Oasis MAX and WAX, are used, the sample pH is a crucial issue because this parameter can affect the sorption adsorbent capacity to a greater degree. Different ionic forms of the PhACs and the sorbent can be present, depending on the sample pH [107]. Unfortunately, the sorption capabilities of these cartridges are often limited when compared to the HLB cartridge. Regarding the use of an off-line or on-line SPE, one of the critical issues is the sample size, particularly the relative size of the water sample and injectable sample. This size depends on the expected concentration of PhACs in water samples. The enrichment factor should be high for drinking water with trace concentrations of PhACs. The water sample size is much larger than the injectable sample. Therefore, it is much more useful to opt for off-line SPE in order to obtain lower quantification limits. For wastewater, the enrichment factors can be lower, and the on-line SPE can be an advantageous option. However, coeluting of matrix interferences can be a significative drawback. In addition, on-line SPE coupled to ultra-performance liquid chromatography (UPLC) shows disadvantagesbecause of the elevated back pressure that is generated by the high flow rates used in small particle size columns (<2 \(\upmu\)m) (83).