## 3 Results and Discussions  
### Method Development  
As previously mentioned, azithromycin, erythromycinycin, fluoxetine and sotalol possess dissimilar physical-chemical properties (solubility, polarity, partition coefficient), and the necessity of their separation, identification and quantification in the same and time-effective analytical run and under the same experimental conditions, choice of the column and nature of the eluent are determining factors. During the method development, four HPLC columns were tested for the separation of target pharmaceuticals. In the preliminary phase, a mobile phase made of 10% water and 90% ACN in MeOH was tested as eluent. The first column was a pentafluorophenyl (PFP) column from waters. Columns made of PFP as stationary phase are originally designed for separation of cyclic compounds [32] and have already been used successfully for the separation of macrolides (among them, azithromycin, and erythromycincin) [33]. The elution peaks of the four compounds were differentiated with a moderate resolution, but sotalol could only be detected using ESI in negative mode. We then tested two other columns from waters: Cortes UPLC(tm) C18 and UPLC(tm) BEH C18. The first one is made of 1.6 um solid-core particles, while the second one is made of 1.7 um porous particles having a bridged ethyl-siloxane silica hybrid (BEH) structure. Both are expected to improve resolution and efficiency of separation and have been successfully used for determination of pharmaceuticals (except sotalol) in water matrices [17; 27; 34; 35]. These columns were assessed with a gradient elution 0.1% ammonium formate (AF) used as aqueous eluent, while 0.1% to 0.3% AF in ACN were tested as organic elements, based on the assumption that shifting pH to acid will serve a better separation of targeted molecules. The 0.3% AF was quickly abandoned because this amount trusted the capillary used for electro-spraying. Moreover, these two columns were found to be unsuitable, due to the pronounced peak broadening of azithromycin and erythromycincin. Finally, the highest resolution combined to the lowest retention times for analyte separations (see Appendix A) were achieved using a PHENOMENEX Luna Omega 1.6 um Polar C18 50 \(\times\)2.1 mm at 40 \({}^{\circ}\)C, thus selected for the further method development. This stationary phase column is able to provide a selectivity within a wide elution window and increased retention for both polar and non-polar analytes. The C18 ligand provides hydrophobic interactions while a polar modified particle surface provides enhanced polar retention and aqueous stability. Using a mobile phase consisting of A: 0.1% FA in ultra-pure water and B: 0.1% FA in UHPLC/MS grade allowed us to avoid tailing and fronting of chromatographic peaks. Elution mode was performed as non-isocratic gradient for standard separation and quantification of analytes at a flow rate of 0.4 mL min\({}^{-1}\). A multi-step gradient was thus used as follows, initial conditions: 2% B; 0.0-3.0 min, 70% B; 3.0-3.5 min, 95% B; 3.5-4.0 min, return to initial conditions; 2% B; 4.0-7.0 min, re-equilibration of the column. Before injection, samples in vials were acidified with 0.1% FA. The SEI source contained two individual orthogonal sprays. One spray was for the column eluent while the other was for the internal standard (lock mass). During each chromatographic run, leucine enkephalin (2 mg L\({}^{-1}\), C\({}_{28}\)H\({}_{37}\)N\({}_{5}\)O\({}_{7}\), MW 555.27, waters Q-ToF product) was used for lock-mass correction to obtain accurate masses for each organic component eluting from the column. A solution of sodium formate (HCOONa, waters Q-ToF product) was infused daily in the ESI source to calibrate the instrument. The optimum mass spectrometric parameters were found using a 2 kV capillary voltage, 500 \({}^{\circ}\)C desolvation temperature, 150 \({}^{\circ}\)C source temperature, 10 L h\({}^{-1}\) cone gas flow rate and 1000 L h\({}^{-1}\) desolvation gas flow rate.