## 3 Results and Discussion
### Parameter Optimization  
The parameter optimization was addressed to find out the best analytical conditions for achieving high recoveries and accurate and precise determinations of the NSAIDs in animal urine samples. In this way, extraction solvent and volume, sample pH, and NaCl effect were deeply investigated. First, the study dedicated its attention on the choice of organic extraction solvent. This issue plays a key role in the extraction efficiency. Chlorinated solvents are generally used because they show characteristics (higher density than water, low solubility in water) appropriate to obtaining high extraction efficiency and worthy gas chromatographic performance. Following these considerations, our attention was focused to five solvents: dichloromethane (CH\({}_{2}\)Cl\({}_{2}\); d = 1.3255 g mL\({}^{-1}\)), chloroform (CHCl\({}_{3}\); d = 1.4788 g mL\({}^{-1}\)), carbon tetrachloride (CCl\({}_{4}\); d = 1.5940 g mL\({}^{-1}\)), 1,2-dichloroethane (C\({}_{2}\)H\({}_{4}\)Cl\({}_{2}\); d = 1.2454 g mL\({}^{-1}\)), and 1,1,2,2-tetrachloroethane (C\({}_{2}\)H\({}_{2}\)Cl\({}_{4}\); 1.5953 g mL\({}^{-1}\)). Table 2 reports the results of the performance of a 300 \(\upmu\)L volume of each solvent on simulated urine samples spiked with 20 ng mL\({}^{-1}\) of each NSAID: Dichloromethane shows the best recoveries, ranging between 94.6% and 98.5% for IBP, NAP, and KPF, respectively, and 82.5% for ASA with a relative standard deviation (RSD, %) below 3.0. The recoveries are calculated as the accuracy (IS added before the extraction) [51]. The extraction recovery, defined as the percentage of the total analyte (n\({}_{0}\)), that was extracted to the sediment phase (n\({}_{\text{sed}}\)) has been determined according to the formula reported in a previous paper [36]. Over the extraction solvent choice, another quite important parameter is its volume, used to achieve the highest recoveries. The strength of the DLLME regards an extraction solvent volume as low as possible for obtaining good performance. [PERSON] and [PERSON] [39] highlighted that an extractionsolvent volume leads to a change in the sediment phase volume and therefore in the enrichment factors (EFs). For these reasons, the effect of different dichloromethane volumes (200, 250, 300 \(\mu\)L) were investigated (Table 3): a volume of 250 \(\mu\)L is sufficient to obtain good recoveries for all the NSAIDs, i.e., 94.2% for ASA, 100.1 for IBP, 99.8 for NAP, and 101.2 for KPF with RSDs \(\leq\)3.1. Another parameter influencing the extraction is the pH of the solution. In fact, it should be remembered that NSAIDs are weak acids. Particularly, ASA shows a pKa of 3.5 [52], IBP of 5.3 [53], NAP of 4.14, and KPF of 4.45 [54]. Solutions of simulated urine samples at different pH were tested for studying the best acidic conditions. Table 4 evidences that the best recoveries and RSDs are obtained at pH 3: in fact, they range between 93.5 and 100.1% and between 3.4 and 4%, respectively. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Compound** & \multicolumn{4}{c}{**Recovery (\%)**} \\ \hline  & **pH 2** & **pH 3** & **pH 4** & **pH 5** & **pH 6** \\ \hline ASA & 94.1 (5.6) & 93.5 (3.5) & 86.1 (4.0) & 82.0 (4.2) & 70.0 (4.3) \\ IBP & 100.2 (3.3) & 99.7 (3.4) & 97.2 (3.9) & 92.5 (4.1) & 82.2 (4.1) \\ NAP & 99.8 (3.8) & 100.1 (3.9) & 96.8 (4.1) & 91.0 (4.0) & 81.5 (4.0) \\ KPF & 101.0 (3.7) & 99.7(4.0) & 97.2 (4.2) & 90.2 (3.9) & 84.7 (4.3) \\ \hline \hline \end{tabular}
\end{table} 
Table 4: The effect of pH on the NSAID recoveries (%). The conditions were as follows: 9 mL of simulated urine samples spiked with NSAIDs (20 ng mL\({}^{-1}\) of each), 1 mL of acetone, 250 \(\mu\)L of CH\({}_{2}\)Cl\({}_{2}\), and 5 \(\mu\)L of IS (60 ng \(\mu\)L\({}^{-1}\)). In brackets are reported the RSDs (%); each analysis was in triplicate.