Acquity ultra high performance liquid chromatography system

All reagents were of analytical grade and were used directly as received. The reagents were purchased from Sigma-Aldrich, unless stated otherwise. LC-MS analyses of synthesized compounds were performed on a Waters ACQUITY ultra high performance liquid chromatography system. Eluents A (0.1% HCOOH in water (v/v)) and B (0.1% HCOOH in acetonitrile (v/v)) were used in a linear gradient (100% A to 95% B) in a run time of 2.5 at a flow rate of 0.6 ml/min.

In this study, an ACQUITY ultra-high-performance liquid chromatography system (Waters, Milford, MA, USA) was coupled with a QE high-resolution mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm) (Waters, USA). The mobile phases consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient elution was as follows: 0–2 min, 5–20% B; 2–4 min, 20–25% B; 4–9 min, 25–60% B; 9–14 min, 60–100% B; 14–16 min, 100% B; 16–16.1 min, 100–5% B; 16.1–18.1 min, 5% B. The column temperature was maintained at 40 °C with a flow rate of 0.35 mL/min, and the injection volume was 5 μL.
The mass spectrometry scan parameters were set as follows: S-lens RF level, 50; mass range, 100 to 1200 m/z; full MS resolution, 70,000; MS/MS resolution, 17,500; NCE/stepped NCE was set at 10, 20, and 40 eV. The ESI ion source parameters were set as follows: spray voltage, 3800 V; sheath gas flow rate, 40 for ESI+ and 5 for ESI−; capillary temperature, 320 °C; probe heater temperature, 350 °C; aux gas flow rate, 10. During the mass spectrometry operation, one QC sample was inserted for every six formal samples. The QC samples were used to evaluate the stability of the mass spectrometry platform throughout the experimental process.

Take 1 mL of the filtrate diluted 10-fold, filter it through a 0.22 μm membrane, and transfer it to a liquid-phase bottle. The sample separation was performed using the ACQUITY ultra-high-performance liquid chromatography system (Waters, Milford, MA, USA) and the Waters ACQUITY HSS C18 chromatographic column (2.1 × 100 mm, 1.8 μm). The mobile phase A is 0.1% formic acid, and mobile phase B is pure methanol. The injection volume is 3.0 μL, and the column temperature is 35 °C. The eluate from the chromatographic system was ionized using the Agilent 5600 time-of-flight high-resolution mass spectrometry equipped with an electrospray ion source in negative ion mode to obtain mass spectrometry data. The operating parameters are as follows: the ion source temperature is 500 °C; the capillary voltage is 4500 V; the nebulizer gas pressure is 50 psi; collision voltage is −10 eV; the mass scan range is 50–1000 m/z.

A Waters ACQUITY ultra-high-performance liquid chromatography system (Waters, Milford, MA, USA) equipped with a Quantiva triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA, USA) was used to analyze the isothiazolinone biocides. An Agilent ZORBAX Eclipse Plus C18 column (2.1 × 50 mm, 1.8 µm) was installed in the instrument. The analyses were carried out using 0.1% formic acid in ultra-pure water as the “A” mobile phase and methanol in the “B” mobile phase at a flow rate of 0.3 mL/min. The column temperature was set to 40 °C, and the injection volume was 1 μL. The mobile phase gradient was started at 30% B, and a linear gradient was applied to increase eluent B to 100% over 10 min, where it was held for 3 min.
An electrospray ion source (ESI) was used to ionize the isothiazolinone biocides. The ion source temperature was set at 350 °C, cone gas flow, 20 psi, heated probe temperature, 250 °C; probe gas flow, 45 psi, and nebulizer gas flow, 55 psi. The spray voltage was set to 4500 V. Xcalibur 4.1.50 (Thermo Scientific, San Jose, CA, USA) software was used for data processing.
The BIT, MIT, and OIT were monitored using MRM (multiple reaction monitoring) with mass transition set for BIT as 152.0 m/z → 133.8 m/z (22.0 V), MIT: 115.8 m/z → 101.1 m/z (22.0 V) and OIT: 214.1 m/z → 102.1 m/z (11.0 V). Representative chromatograms are shown in Figure 3.

Carotenoids, ergosterol and ubiquinone (Coenzyme Q9) extracted by the method described previously, were separated and identified by Liquid Chromatography with photodiode array detection. An Acquity ultra high performance liquid chromatography system (Waters) was used with an Ethylene Bridged Hybrid (BEH C18) column (2.1 × 100 mm, 1.7 mm) with a BEH C18 VanGuard precolumn (2.1 × 50 mm, 1.7 mm). The mobile phase used was A, methanol/water (50/50), and B, acetonitrile (ACN)/ethyl acetate (75:25) and the flow rate was 0.5 ml min⁻¹. All solvents used were HPLC grade and filtered prior to use through a 0.2-mm filter. The gradient was 30% A:70% B for 0.5 min and then stepped to 0.1% A:99.9% B for 5.5 min and then to 30% A:70% B for the last 2 min. Column temperature was maintained at 30 °C and the temperature of samples at 8 °C. Online scanning across the UV/visible range was performed in a continuous manner from 250 to 600 nm, using an extended wavelength photo diode array detector (Waters, Watford, UK).