## 3 Results and Discussion
### Extraction and Clean-Up Optimisation
#### 3.2.1 Extraction  
Various modifications of the QuEChERS technique were used to isolate pharmaceuticals from wastewater. Depending on the variant, 10-106% recoveries were obtained for all compounds tested (Figure 1). Recovery values above 100% may result from matrix effects that cause signal enhancement, as well as inherent analytical variability. Such occurrences are common in complex matrices like wastewater and are accounted for in the method validation. The original procedure involves using acetonitrile as the extraction solvent (Figure 1). The original procedure involves the use of acetonitrile as the extraction solvent [42; 68; 69]. This variant (trial 1) yielded 25-88% recoveries, with satisfactory recoveries (above 70%) obtained for only seven compounds. The average recovery for this variant was 60%. Figure 1: Cont. Acidic conditions can accelerate the desalting process and increase the stability of compounds in acetonitrile, as demonstrated for pesticides [70]. Furthermore, at acidic pH, the anionic sites of the matrix and analytes become protonated, leading to electrostatic repulsion between them, increasing the extraction efficiency [71]. Acidification of acetonitrile with formic acid (trial 4) significantly improved the recoveries of fluoxetine (from 62% to 72%), indapamide (from 67% to 85%), and ramipril (from 46% to 75%). However, it negatively affected the recovery of valsartan (from 74% to 59%). The use of ethyl acetate instead of acetonitrile (trial 7) improved the recoveries of ciprofloxacin (from 61% to 79%), fluoxetine (from 62% to 100%), indapamide (from 67% to 107%), torasemide (from 68% to 80%), and venlafaxine-O-desmethyl (from 58% to 75%). The choice of buffers directly impacts the recovery rates of pharmaceuticals during extraction--it allows pH adjustment to ensure that as many compounds as possible are  
Figure 1: The recoveries of the pharmaceuticals obtained in different extraction trials. obtained in the neutral state. This procedure minimizes the presence of charged forms that could increase their affinity for water during drug extraction [72]. In the QuEChERS technique, there are two different standards regarding the type of buffer [68; 73]: the US standard AOAC Official Method 2007.01 [74], which includes the use of an acetate buffer (pH about 4.8), and the European Committee for Standardisation (CEN) Standard Method EN 15662 [75], which includes the use of a citrate buffer (pH about 5-5.5), weaker in terms of ionic strength. For their comparison, an extraction with acetonitrile was performed. The acetate buffer-based method (trial 2) yielded satisfactory recoveries for only two beta-blockers (atenolol and metoprolol) and an antiarrhythmic drug (propafenone). The use of citrate (trial 3) yielded about twice the recovery of levonorgestrel (but still less than 70%) and fluoxetine, and nearly three times better for indapamide compared to the acetate buffer version (trial 2). No significant difference in recoveries (greater than 5%) was observed for bisoprolol, doxycycline, escitalopram, duloxetine, metoprolol, ramipril, serntraline, torasemide, and valsartan. However, the mean recovery rate for acetonitrile extraction (trial 3) with citrate buffer was higher compared to the version without buffer (trial 1) (67 and 60%, respectively). The use of EDTA in the extraction of pharmaceuticals allows higher recoveries to be achieved by increasing the ionic strength, potentially increasing their solubility in the organic solvent [1]. Due to their strong chelating properties, EDTA and its salts can interact more strongly with metals in the matrix and thus exchange with analytes at active sites or mask adsorption sites [71]. Therefore, adding EDTA or its salts in the extraction process prevents the complexation of analytes with cations such as Mg\({}^{2+}\) or Ca\({}^{2+}\). Improvements in extraction efficiency by preventing chelate formation have been observed for fluoroquinoline and tetracycline compounds--for example, [76; 77].