Zorbax eclipse plus c18 column

In total 37 pesticides and 17 transformation products (TPs) were analyzed (Table S4) by direct injection of 80 µL into a liquid-chromatography (Agilent 1260 infinity series) system coupled to tandem mass spectrometry (Sciex Triple Quad 6500+) (LC-MS/MS). Additionally, the pharmaceutical ibuprofen was analyzed as a marker of raw wastewater in order to assess whether samples were impacted by combined sewer overflow. Chemical separation was performed on an Agilent Zorbax Eclipse Plus C18 column (Narrow Bore RR, 2.1 × 150 mm, 3.5 µm) with a Zorbax Eclipse XDB-C8 Guard Column (2.1 × 12.5 mm, 5 µm) according to Hermes et al. (2018) (link). Ionization was achieved using alternating electrospray positive ionization mode (ESI+) and negative ionization (ESI−). For compensation of matrix effects 26 stable isotopes labeled surrogate standards were used. Details about the LC gradient program (Table S5), ESI source parameters (Table S6) as well as the retention times and compound-specific MS/MS parameters (Table S7) of all measured analytes and surrogates are shown in the supporting information. The chosen analytical method did not allow to include the insecticide group of pyrethroids and the herbicide glyphosate into the pesticide monitoring.

The quality of B215 was determined by the amount of naringin in the extract. For analysis, 0.1 g of the B215 was mixed with 10 mL of methanol. The suspension was sonicated for 10 min and filtered through a 0.45 µm syringe filter prior to injection into the HPLC system. Agilent 1260 infinity II HPLC system (Agilent Technologies, Santa Clara, CA, USA) was equipped with a G7112B binary pump, a G7115A diode array detector (DAD) and G7129C autosampler. The chromatographic separations were performed on an Agilent Zorbax Eclipse Plus C18 column (4.6 mm × 250 mm, i.d., 5 µm) maintained at 25 °C. The mobile phases were 0.5% aqueous acetic acid (v/v, A), and acetonitrile (v/v, B). Gradient elution was performed as follows: 0–5 min, 5–18% B; 5–30 min, 18–18% B; 30–31 min, 18–100% B; 31–35 min, 100–100% B; 35–36 min, 100–5% B; 36–40 min, 5–5% B. The flow rate was 1.0 mL/min, and the injection volume was 10 µL. The eluents from the column were monitored at 280 nm for naringin. Each experiment was performed in triplicate.

The phenolic compounds confirmation analysis by HPLC (1260 Infinity ll, Agilent Technologies, Santa Clara, CA, USA) was accomplished by Agilent ZORBAX Eclipse Plus C18 column (250 × 4.6 mm, 5 μm). The phenolic compounds were separated under gradient conditions with a flow rate of 0.8 mL/min. Column temperature was 35 °C and volume injection was 10 µL. The wavelength was 280 nm. The solvents used in the elution process were an aqueous solution of 100% methanol (phase A) and 0.02% (v/v) formic acid (phase B). The samples were eluted according to the linear gradient, the phase-time program was as follows: 0–10% A (0–5 min), 10–20% A (5–10 min), 10–35% A (10–20 min), 35–40% (20–35 min), 40–75% A (35–40 min), 75–10% A (40–45 min). The peak areas of the samples were compared with those of the internal standards and quantified [26 (link)].

Analyses of the first ethanolic extract, combined fractions and reference substances were conducted on a Dionex UltiMate 3000 RS system equipped with a DAD detector and coupled to an LTQ XL linear ion-trap mass spectrometer with ESI ion source (allThermo Fisher Scientific, Waltham, MA, USA). A Zorbax Eclipse Plus C18 column, 2.1 × 100 mm and 1.8 µm particle size (Agilent, Santa Clara, CA, USA) served as stationary phase, while the mobile phase was made up of water +0.1% formic acid (A) and acetonitrile (B). Gradient elution at a flow rate of 0.25 mL/min started at 20% B, rising to 100% B at 20.0 min, followed by a plateau of 100% B to 23.0 min and a drop back to 20% B at 23.5 min, which was kept stable until 29 min for re-equilibration. Column temperature was 35 °C. Injection volumes were 2 µL for dry ethanolic extract in methanol (5 mg/mL) and 1 µL for reference substances dissolved in methanol (1 mg/mL) as well as subfractions of varying concentrations.
DAD-UV spectra were recorded in a wavelength range of 190 to 700 nm. Mass spectral detection was performed in the range of m/z 50 to 2000 amu, with conditions set as follows: source heater temperature 250 °C, sheath gas flow 50 arb (arbitrary units), auxiliary gas flow 8 arb, source voltage 4.0 kV for ESI negative mode and 4.2 kV for ESI positive mode.