# Pharmaceuticals Removal from Wastewater with Microalgae: A Pilot Study
## 2 Materials and Methods
### Analysis of Pharmaceuticals
#### 2.3.2 Experimental Procedure  
The analytical procedure for the identification and quantification of 63 pharmaceuticals of 8 different therapeutic classes already validated (analgesic, antibiotics, anticonvulsants, antidepressants, antihyperstines, beta-blockers, lipid regulators, and anti-inflamatories) was based on previous studies [7; 18].  
Samples were frozen at \(-20\)\({}^{\circ}\)C after being collected and stored this way until the day of analysis. Then, water samples were defrosted and acidified with formic acid until pH 2 and two consecutive filtration steps were performed with 0.45 and 0.22 \(\upmu\)m pore size filters. For the extraction procedure, 500 mL of water was measured and added to 500 \(\upmu\)L of internal standard solution comprising 10 mg/mL of sulfameter. The next step was a purification with solid phase extraction (SPE) cartridges, achieved with Oasis HLB (200 mg, 6 mL). After the water sample was loaded in the cartridges, a washing step followed with 5 mL of methanol:water (10:90), and then drying at low vacuum pressure. The elution was performed with methanol, 6 mL, and this extract was evaporated under N\({}_{2}\) flow at 40 \({}^{\circ}\)C, and then redissolved with 500 \(\upmu\)L of 0.1% of formic acid (mobile phase A) and filtered with a PVDF Mini-uniprep TM filter (0.45 \(\upmu\)m). The detection and quantification were performed by injecting 10 \(\upmu\)L of this final extract into the UHPLC-ToF-MS system. Thisequipment comprises UHPLC Nexera X2 Shimadzu coupled with a high-Resolution mass spectrometer, a Time-of-Flight Triple TOFTM 5600+ from Sciex. The UHPLC equipment is composed of a vacuum degasser, an autosampler with a programmed temperature (10 \({}^{\circ}\)C), a binary pump, and a compartment for the chromatographic column at a controlled temperature (40 \({}^{\circ}\)C). The column used in this method was a reverse-phase one, Acquity UPLC HSS T3 1.8 \(\mu\)m, 2.1 \(\times\) 100 mm (Waters). The mobile phases used were A--formic acid 0.1% (\(v/v\)) in water and B--acetonitrile, at a flow rate of 500 \(\mu\)L/min and following the gradient as described: the first 5 min from 97% to 40% [A], from 5 to 9 min from 40% to 0% [A], until 10 min from 0% back to 97% [A], and maintained this way till the end of the chromatographic run of 12 min total.  
For the detection, the mass spectrometer ToF-MS operated with ionization in positive mode and an electrospray ion source (ESI+) working in full-scan data acquisition mode from 100 to 920 Da. For acquisition, the software used was the Analyst(r) TF software and for data analysis, three software were used: PeakViewTM, LibraryViewTM, and Multi-QuantTM all from SCIEX (Framingham, MA, USA). In terms of criteria of identification, the parameters evaluated were the exact mass, with a maximum acceptable error of 5 ppm, relative retention time deviation below 1% (Commission Regulation (EU) 2021/808), and isotope pattern similarity with a maximum of 10% difference (between the obtained and the theoretical).  
The developed method was previously validated, and the obtained limits of detection ranged between 0.01 and 3.47 ng/L (Table S1, Supplementary Materials).