Acquity uplc hss t3 chromatographic column

After cells were separated from the medium, cells were freeze-dried in vacuo for 48 h, growth of them determined as DW (g). 65 mg of cells were weighed and then soaked in 1.5 mL 80% (v/v) methanol for 12 h, followed by 40 kHz ultrasonication at 25 °C for 1 h. The methanol extracts were centrifuged at 13,000 rpm for 10 min and the supernatant were filtered through a 0.22 μm microporous membrane. UPLC (1290 Infinity II, Agilent) with an ACQUITY UPLC^® HSS T3 chromatographic column (1.8 μm, 2.1 mm × 100 mm, Waters) was used for the analysis. Column temperature was 40 °C and the flow rate was 0.4 mL min⁻¹. 100% acetonitrile (mobile phase A) and 0.1% (v/v) formic acid in water (mobile phase B) with a linear gradient was used: (0 min: 70% B, 5 min: 65% B, 15 min: 30% B, 21 min: 10% B). A UV detector monitored at 219.4 nm, 260.0 and 426.0 nm, the sample injection volume was 5 μL. For determining the extracellular triptolide, celastrol and triptophenolide concentration, medium (25 mL) was extracted 3 times with the same volume of ethyl acetate. The combined ethyl acetate fraction was subsequently concentrated and then redissolved in 1 mL 80% methanol and was filtered through a 0.22 μm microporous membrane before UPLC analysis as previously described.

Analysis was undertaken using UPLC/Q-TOF MS (SYNAPT G2-Si, Waters, USA) with an ACQUITY UPLC^® HSS T3 chromatographic column (1.8 μm, 2.1 mm × 100 mm, Waters, USA) kept at 40 °C. The method was based on the methods described previously [40 (link)]. Briefly, samples (3 μL) were separated using 0.1% (v/v) formic acid (mobile phase A) and 100% acetonitrile (mobile phase B): 0 min at 30% B, 6 min at 45% B, 18 min at 60% B, 23 min at 90% B, at 0.5 mL min⁻¹ flow rate. Mass detection was conducted by electrospray ionization (ESI) in the positive ion mode. The QTOF-MS conditions were as follows: sample cone, 40 V; source temperature, 120 °C; desolvation temperature, 450 °C; cone gas flow, 50 L h⁻¹; and desolvation gas flow, 800 L/h; capillary voltage, 1000 V. The ramp collision energy was set as 20–65 eV for the high-energy scans. Data analysis was performed using the MassLynx V4.1, Progenesis QI 3.0.3 and EZinfo 3.0 software (Waters). Online database included KEGG, MassBank, Nature Communications, Nature Chemistry, Nature Chemical Biology, and ChemSpider. Variable importance parameter (VIP) value > 1.

The serum samples were thawed at room temperature and 100-μL aliquots were transferred into centrifuge tubes (1.5 mL). All serum samples were extracted using 300 μL of methanol and mixed with 10 μL of an internal standard (3.1 mg/mL, DL-o-chlorophenylalanine). The samples were then vortexed for 30 s and centrifuged at 12000 rpm and 4°C for 15 min. ACQUITY^TM UPLC -QTOF analysis system and Waters ACQUITY UPLC HSS T3 chromatographic column (2.1 mm × 100 mm, 1.8 covering m) were used for LC-MS detection. The following chromatographic separation conditions were applied: column temperature, 40°C; mobile phase A, water+0.1% formic acid; mobile phase B: acetonitrile+0.1% formic acid; flow rate: 0.35 mL/min; injection volume: 6 μL. The data were first transformed to CDF files using CDF bridge and imported into XCMS software for peak picking, peak alignment, peak filtering and peak filling. The data, including the retention time (RT), MZ, observations (samples) and peak intensity, were normalized using Excel 2007.

An ACQUITY I-class UPLC system equipped with a Xevo G2-XS Q-TOF MS instrument (Waters Corporation, Milford, MA, USA) was used to analyze the Qingpi samples. Compounds were separated using a Waters ACQUITY UPLC HSS T3 chromatographic column (2.1 mm × 100 mm, 1.8 μm; MA, USA). The injection volume was 4 μL and the flow rate was 0.4 mL/min. The temperature of column was set at 40 °C. The mobile phase was composed of water containing 0.1% (v/v) formic acid (A) and acetonitrile containing 0.1% (v/v) formic acid (B). The gradient elution program was as follows: 0–15 min for 99–1% A, 15–17 min for 1% A, 17–17.10 min for 1–99% A, and 17.10–20 min for 99% A.
For the MS conditions, the positive mode of electrospray ionization (ESI) source was used, the mass range was m/z 50 to 1200, and the scanning time was 0.25 s. The voltage was set at +2.5 KV for capillary, 40 V for cone hole voltage, 80 V for ion source compensation, and +3.0 KV for spray voltage. The cone hole gas flow rate was 50 L/h. The desolvation gas temperature was 450 °C and its flow rate was 800 L/h. The low collision energy was set as 6 eV and the high collision energy increased from 15 to 50 eV. The MS^E data acquisition mode was used to collect data in real time.