Ultimate 3000 high performance liquid chromatography system

The cells of logarithmic HCCLM3 were subcultured and treated with the proteasome inhibitor MG132 for 2 h when the cell convergence reached 60–70%. Cells were cultured for 24 h after replacement of the medium with 100 μM Sal B. Then the cell protein was collected for immunoprecipitation. A total of 100 μL sterilized double-distilled water were added to the precipitate of the control group and the treatment group, and then each mixture was boiled in a protein dry thermostat at 100 °C for 10 min to precipitate the denatured protein, and the tested substance was separated from the protein and dissolved in water for UHPLC/MS analysis. Sal B standard as the positive control, sterile double-distilled water as the control, and PBS and the cell lysate as the samples were to be tested.
The UHPLC/MS analysis used a Thermo Fisher Scientific q active four-stage rod-electrostatic field orbital trap high resolution mass spectrometry system and Dionex UltiMate 3000 high-performance liquid chromatography system. Thermo Fisher Scientific Xcaliber and Thermo Fisher Scientific Pathfinder were used for data collection and processing, respectively.

Freeze-dried soil (2 g) was added to a centrifuge tube and supplemented with 5 mL of methanol. The tube was securely closed, agitated for 2 min on a vortex mixer, and extracted for 20 min with ultrasonication, then centrifuged for 10 min at 4000 rpm. Soil was sequentially extracted a further two times, the supernatants pooled and passed through a 0.22-μm PTFE filter membrane. All final samples were stored at −20 °C prior to instrumental analysis. Instrumental analysis was performed on a Ultimate 3000 high-performance liquid chromatography system (Dionex, Sunnyvale CA, USA). The chromatographic column was a C18 reversed phase column (4.6 × 150 mm, 5 μm) with 80% methanol in water and water as the mobile phases B and A, respectively, and the system was programmed (with respect to mobile phase B) for 0–15 min, at a flow rate of 0.8 mL/min. The injection volume was 10 μL and the column temperature was 30 °C. Quality control for PPCPs extraction tests was assessed by the use of a soil spiked with 4 mL of PPCPs (1000 mg/L in methanol), and duplicate samples. The recovery rates for TCS and CBZ in two soils varied from 87% to 97%.