# Pharmaceuticals in Coastal Waters: An UHPLC-TOF-MS Multi-Residue Approach
## 3 Results and Discussion
### Method Development
#### 3.1.1 Solid Phase Extraction  
The starting point for sample extraction, purification and concentration of water samples was an LC-MS/MS method published by [PERSON] and colleagues [14], which in turn was based on the work of [PERSON] and co-authors [13]. The procedures described for LC-MS-MS were tested and optimized for UHPLC-TOF-MS aiming to ultimately achieve the best recovery rates possible for all the pharmaceuticals of interest. Since estuarine, coastal and wastewater samples are complex matrices, clean-up is a key step in ensuring a clear final extract. Thus, spiked 500 mL samples (with the 63 compounds of interest and the internal standard) were submitted to a 3-step filtration sequence starting with 100 mm glass microfiber filter, followed by a 0.45 \(\upmu\)m cellulose nitrate filter and finally a 0.2 \(\upmu\)m polyamide filter. This ensured the removal of most of the co-extracted matter, including salt ions and dissolved organic matter that otherwise would be in the final extract and would lower greatly the recoveries due to ionization suppression effects and even compound losses [13]. This sequential filtration was compared with 1- and 2-step sequences and proved to produce the best recoveries when compared with spiked ultrapure water samples (to guarantee that no other interferent was present in the water) at the same levels. In addition, the use of internal standard can overcome the possible matrix effects that can compromise the accurate identification and quantification of the target compounds by correcting fluctuations due to sample extraction, chromatographic behavior and the ionization efficiency. The internal standard selection was based on the linearity and recovery studies. The physico-chemical differences among the 63 compounds, particularly regarding polarity, are presented in the Table 1 in terms of the coefficient of partition, logP. This parameter is given as an indicator of the polarity of the compounds targeted in the present method and is defined as the ratio of soluble concentration between two immiscible solvents, namely octanol and water. In practice, the more polar and hydrophilic the compound is, the lower is the logP. The presented logP in Table 1 comprises theoretical predicted values obtained in the database of [[https://go.drugbank.com](https://go.drugbank.com)]([https://go.drugbank.com](https://go.drugbank.com)); accessed on 1 February 2023 and calculated with the virtual computational Chemistry Laboratory, [[http://www.vcdab.org](http://www.vcdab.org)]([http://www.vcdab.org](http://www.vcdab.org)); accessed on 1 February 2023. Considering the range of polarities of the target compounds, ensuring the cartridges for clean-up and extraction is paramount. The previous methods were performed with Oasis MAX cartridges preconditioned with methanol and water. However, in the present work a comparison with Oasis HLB (200 mg) was performed since these reversed-phase cartridges are filled with polymeric sorbent which provides hydrophilic and lipophilic retention characteristics enhancing results for both polar and non-polar compounds. Another important feature of Oasis HLB is that preconditioning is not necessary in this type of SPE, reducing the volume of organic solvents needed and, thus, achieving a greener method. In addition, and most relevant in terms of saving time in sample processing, there is the sorbent being water wettable, which means that it can run dry without compromising retention and recovery. Given all these features, it became important to compare clean-up and extraction with both cartridges types, with and without acidifying the samples to pH 2. The minimum, maximum and mean recoveries are presented in Figure 1 and clearly show that Oasis HLB performed better with than Oasis Max, and that pH must be adjusted to 2 to ensure the best recoveries. Although WatersTM clearly states preconditioning is not required, Oasis HLB were tested with and without this step, and results clearly show that it is, in fact, unnecessary (Figure 2).