# Assessment of Pesticide Contamination of Groundwater from Titu-Sarata Plain, Romania
## 2 Materials and Methods
### UHPLC-Orbitrap-MS/MS Analysis of OPs  
Sample preparation: 250 mL water sample was filtered using glass fiber filter to remove solid components before sample preparation, followed by the pH adjustment to 3 using concentrated acetic acid. Solid phase extraction (SPE) using StrataX (500 mg/6 mL) cartridges (Phenomenex, Aschaffenburg, Germany) was used to extract the pesticides from the water samples. The acidified water samples were passed through SPE cartridges preconditioned with 10 mL acetonitrile (ACN), followed by 10 mL water before the extraction, at a flow rate of 3-5 mL/min. Afterwards, 10 mL of ultrapure water was added to remove water-soluble interferences and the residual water in the column was dried under low-pressure vacuum for 10 min. The SPE cartridges were eluted with 10 mL methanol-methylene chloride mixture (1:1, \(v/v\)) at a flow rate of 1 mL/min, after which the solvent was removed under a stream of nitrogen at 40 \({}^{\circ}\)C, and the extract was reconstituted with acetonitrile-water (1:9, \(v/v\)), followed by UHPLC-MS/MS analysis.  
Quantitative UHPLC-Orbitrap-MS/MS analysis of pesticides: All samples were analyzed with Ultrahigh-Performance Liquid Chromatography coupled to a Q Exactive(tm) HF Hybrid Quadrupole-Orbitrap(tm) mass spectrometer (UHPLC-Orbitrap-MS/MS, Thermo Fisher Scientific, San Jose, CA, USA) using electrospray ionization (ESI). The samples were injected once each under ESI positive mode for organophosphorus and organophosphate pesticides, triazine pesticides, and carbamates and ESI negative mode for acid herbicides. For both ESI positive and negative modes, a reversed-phase Syncronis C18 column (50 mm \(\times\) 2.1 mm, 1.7 \(\upmu\)m) (Thermo Fisher Scientific, San Jose, CA, USA) was used with a binary mobile phase gradient consisting of (A) water and (B) methanol, both containing 0.1% formic acid and 5 mM ammonium fortiate. The injection volume was 10 \(\upmu\)L throughout the whole separation, the flow rate was 0.4 mL/min, the sampler compartment temperature was 15 \({}^{\circ}\)C, and the column temperature was 25 \({}^{\circ}\)C. The Orbitrap-MS/MS was operated in the data-dependent acquisition mode, with full scans acquired in the range of \(m/z\) 100-1000, at resolution of 70,000 FWHM and \(m/z\) 200, while the fragmentation was performed at a resolution of 35,000 FWHM. The MS detector was run under the following conditions: spray voltage 3.0 kV for negative mode and 3.5 kV for positive mode, capillary temperature 320 \({}^{\circ}\)C, auxiliary gas heater temperature 413 \({}^{\circ}\)C, sheath and auxiliary gas flow (N2) 48 and 11 (arbitrary units). The target pesticides were identified and quantified according to the spectral characteristics: mass spectra, accurate mass, and specific retention time, against external standard solutions covering a linear range between 0.1 and 100 \(\upmu\)g/L. Mass accuracy calibration of the high-resolution Orbitrap MS/MS was performed before the analysis, in both positive and negative ionization modes. Data processing was performed using Xcalibur 3.1 software package (Version 4.1) (Thermo Fisher Scientific, San Jose, CA, USA).  
The solvents (methanol, acetonitrile, and water) and reagents (formic acid and ammonium formate) used for sample preparation and mass spectrometric analysis were of LC-MS-grade purity, while methylene chloride was of HPLC gradient-grade purity, all reagents being purchased from Merck (Darmstadt, Germany). Reference-grade standard solutions were purchased from LGC Dr. Ehrenstorfer (Gibraltar, UK).