# Pharmaceuticals in Coastal Waters: An UHPLC-TOF-MS Multi-Residue Approach
## 3 Results and Discussion
### Validation  
Once separation and optimization of the UHPLC-TOF conditions were achieved, the quality parameters were studied to ensure suitable identification, confirmation and quantification of the target analytes. Therefore, validation was based on specificity, selectivity, precision, linearity, limit of detection (LOD) and limit of quantification (LOQ). The summarized results of the validation are detailed in Table 1. Prior to validation protocol, several samples were analyzed to select blank samples to be used during the process. For that, coastal waters were collected far from urbanization or industrial regions. Those samples were then used for spiking purposes and for blank interferences analysis.  
Specificity and selectivity--that is, the capacity of the method to discriminate between the target analytes and interferences that might have similar behavior--were confirmed by analyzing 20 blank samples of coastal water to verify the absence of interference above a signal-to-noise ratio of 3 at the retention time of the target analytes that could compromise their detection and identification. Additionally, in the spiked blank samples, all identification criteria were fulfilled without false-negative results, proving once more the specificity of the method.  
Linearity was determined in a concentration range of 0.8 to 6 ng/L through direct injection of standard solutions. However, for some compounds where the LOD and the LOQ were found to be out of the validation concentration range (ciprofloxacin, danofloxacin, enoxacin, enrofloxacin, marbofloxacin, oxolinic acid and ibuprofen), extra calibration  
Figure 4: Number of compounds detected for each chromatographic column tested.  
concentration points were added from 6 ng/L until 12 ng/L. Repeatability (within-day precision) and reproducibility (inter-day precision) were established for each analyte as relative standard deviation (RSD). Repeatability ranged from 1.4% for ofloxacin and 17.2% for fenofibrate whereas reproducibility was between 2.5% for sulfadimin and 20.9% for amoxicillin. Regarding the LOD of the method, values were between 0.01 ng/L for flumequine, spiramycin, tilmicosin, azithromycin, fluoxetine and atenol and 8.92 ng/L corresponding to atorvastatin. Taking a closer look at the LOD of the 5 substances from the EU Watch List, the values were much lower than requested by the legislation. Specifically, the LOD were 0.11 ng/L for amoxicillin, 3.47 ng/L for ciprofloxacin, 0.03 ng/L for sulfamethoxazole, 0.80 ng/L for trimethoprim and 0.02 ng/L for venlafaxine, while the maximum acceptable LOD regarded in the EU 2020/1161 are, respectively, 70 ng/L, 89 ng/L, 100 ng/L (both sulfamethoxazole and trimethoprim) and 6 ng/L.  
As for the LOQ, fluoxetine and atenol presented 0.03 ng/L while atorvastatin had the highest value of 29.73 ng/L. However, all the other analytes presented values below 12 ng/L for both LOD and LOQ.  
Looking at recoveries, values were between 80.6% and 112.6%, well within the acceptable range for a multi-residue method and in accordance with specific guidelines for validation of analytical methods as stated in the Commission Implementing Regulation (EU) 2021/808.