Dionex ultimate 3000 lc system

Water samples were collected from different tanks at nine separate occasions during the exposure and stored in darkness at −20°C until analysis. EE₂ concentrations were analyzed in single or duplicate samples as previously described in Volkova et al. (2015b (link)). Briefly, water samples (100 mL) were extracted on 100 mg Strata X-33μ Polymeric Reversed Phase cartridges, reconditioned with MeOH. Dionex ultimate 3000 LC system (Thermo Scientific, San Jose, CA, USA), coupled to a triple quadruple mass spectrometer (TSQ Vantage, Thermo Scientific, San Jose, CA, USA) was used for quantitation of EE₂ content. The quantification range was 0.5–100 ng/L, using EE₂-d4 as internal standard.

10 µL of each sample was loaded into a Dionex UltiMate 3000 LC System (Thermo Scientific Bremen, Germany) equipped with a C-18 column (Acquity UPLC -HSS T3 1. 8 µm; 2.1 x 150 mm, Waters) coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Scientific) operating in negative ion mode. A step gradient was carried out using solvent A (10 mM TBA and 15 mM acetic acid) and solvent B (100% methanol). The gradient started with 5% of solvent B and 95% solvent A and remained at 5% B until 2 min post injection. A linear gradient to 37% B was carried out until 7 min and increased to 41% until 14 min. Between 14 and 26 min the gradient increased to 95% of B and remained at 95% B for 4 min. At 30 min the gradient returned to 5% B. The chromatography was stopped at 40 min. The flow was kept constant at 0.25 mL/min and the column was placed at 40°C throughout the analysis. The MS operated in full scan mode (m/z range: [70.0000-1050.0000]) using a spray voltage of 4.80 kV, capillary temperature of 300°C, sheath gas at 40.0, auxiliary gas at 10.0. The AGC target was set at 3.0E+006 using a resolution of 140000, with a maximum IT fill time of 512 ms. Data collection was performed using the Xcalibur software (Thermo Scientific). The data analyses were performed by integrating the peak areas (El-Maven - Polly - Elucidata) (37 ).

Tocopherols were analyzed according to Pignitter et al. [31 (link)]. Briefly, 0.05 g of oil sample was dissolved in 1 mL 2-propanol, vortexed and filtered through a nylon filter (0.22 µm). Analysis was carried out by means of a UHPLC (Dionex Ultimate 3000 LC system, Thermo Fisher Scientific, Vienna, Austria) with a flow rate of 1 mL/min using a C18 column (Kinetex EVO, 150 × 4.6 mm, 5 µm, Phenomenex). For separation of the tocopherols, a methanol gradient was used, starting with 75% methanol and 25% double-distilled water, reaching 100% methanol after 10 min and holding the plateau for 3 min. The initial conditions were reached again at 15 min. The tocopherol homologs were detected at a wavelength of 295 nm and quantitated using the standard addition method. Total tocopherol content was calculated by summing up the concentrations of α-, γ- and δ-tocopherol homolog. Limit of detection of α-, γ- and δ-tocopherol was determined by a signal-to-noise ratio of 3 with 0.30 µg/mL, 0.53 µg/mL and 0.23 µg/mL, respectively.

A total of 10 µL of each sample was loaded into a Dionex UltiMate 3000 LC System (Thermo Scientific, Bremen, Germany) equipped with a C-18 column (Acquity UPLC -HSS T3 1. 8 µm; 2.1 × 150 mm, Waters, Milford, MA, USA) coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Scientific), operating in negative ion mode. A step gradient was carried out using solvent A (10 mM TBA and 15 mM acetic acid) and solvent B (100% methanol). The gradient started with 5% of solvent B and 95% solvent A and remained at 5% B until 2 min post injection. A linear gradient to 37% B was carried out until 7 min and increased to 41% until 14 min. Between 14 and 26 min the gradient increased to 95% of B and remained at 95% B for 4 min. At 30 min the gradient returned to 5% B. The chromatography was stopped at 40 min. The flow was kept constant at 0.25 mL/min and the column was placed at 40 °C throughout the analysis. The MS operated in full scan mode (m/z range: (70.0000–1050.0000)) using a spray voltage of 4.80 kV, capillary temperature of 300 °C, sheath gas at 40.0, auxiliary gas at 10.0. The AGC target was set at 3.0 × 10⁶ using a resolution of 140,000, with a maximum IT fill time of 512 ms.

The qualitative identification of saikosaponins in XG was carried out using a Q Exactive™ hybrid quadrupole-orbitrap mass spectrometer equipped with a Dionex Ultimate 3000 LC system (Thermo Fisher Scientific, USA). Analytes were detected at 30°C on a Waters CORTECTS C18 column (4.6 mm × 150 mm, 2.7 μm). The mobile phase was comprised of 0.01% acetic acid (A) and acetonitrile (B) with gradient elution program: 0∼9 min, 5–25% B, 9∼16 min, 25–35% B, 16∼23 min, 35–60% B, and 23∼30 min, 60–80% B. The flow rate was 0.3 mL·min⁻¹. The heated ESI (H-ESI) source was operated, and MS parameters were optimized as follows: spray voltage, 3.5 kV; sheath gas flow, 40 L/min; aux gas flow, 10 L/min; capillary temperature, 320°C; aux gas heater temperature, 350°C. A full MS/dd-MS² (data dependent MS²) method was used for acquisition. The full scan range was from 100 to 1500 in negative ion mode at a resolution of 70000 and 17,500 for MS² scan. MS/MS spectra were fragmented by high-energy collision-induced dissociation (HCD) of normalized collision energy (NCE) values at the levels of 10%, 20%, and 30%. Date acquisition and processing were accomplished with Xcalibur software (version 4.2, Thermo Fisher Scientific, USA).