Xevo g2 q tof ms

A 200 μl aliquot of the organic layer was dried and resuspended in 100 μl of methanol and toluene (1:4, v/v). Single injections of 3 μl of extract were made on an Acquity UPLC system (Waters Corporation) in discrete, randomized blocks. The pooled QC was injected after every 10 sample injections.
Separation was performed with an Acquity UPLC CSH Phenyl Hexyl column (1.7 μM, 1.0 × 100 mm; Waters Corporation), using a gradient from solvent A (water, 0.1% formic acid) to solvent B (acetonitrile, 0.1% formic acid). Injections were made in 100% A, held at 100% A for 1 min, ramped to 98% B over 12 min, held at 98% B for 3 min, and then returned to starting conditions over 0.05 min and allowed to re‐equilibrate for 3.95 min, with a 200 μl/min constant flow rate. The column and samples were held at 65°C and 6°C, respectively. The column eluent was infused into a Xevo G2 Q‐TOF‐MS (Waters Corporation) with an electrospray source in positive mode, scanning 50–2,000 m/z at 0.2 s per scan, alternating between MS (6 V collision energy) and MSE mode (15–30 V ramp). Calibration was performed using sodium iodide with 1 ppm mass accuracy. The capillary voltage was held at 2,200 V, source temp at 150°C, and N desolvation temp at 350°C with a flow rate of 800 L/hr.

The UPLC analysis was performed on a Waters ACQUITY Ultra Performance LC system (Waters, USA) equipped with a Waters Xevo G2 QTof MS. Chromatographic separation was carried out at 45°C on an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm). The mobile phase consisted of water (A) and acetonitrile (B), each containing 0.1% formic acid. The optimized UPLC elution conditions were 0–0.5 min, 1% B; 0.5–12.0 min, 1–30% B; 12.0–15.0 min, 30–99% B; 15.0–16.0 min, 99% B; 16.0–20.0 min, 99.0–1.0% B. The flow rate was 0.45 mL/min. The autosampler was maintained at 4°C. Every 2 μL sample solution was injected for each run.

Laboratory procedures for metabolomics have been previously described [21] , [27] (link) and are detailed in the S1 Text. Briefly, metabolomic profiling was performed on Acquity UPLC coupled to a Xevo G2 Q-TOFMS (Waters Corporation, Milford, USA) with an atmospheric electrospray interface operating in positive ion mode. Non-consecutive duplicate sample aliquots of 1 µL were injected onto a Acquity UPLC BEH C8 analytical column. Mass analysis was performed in the full scan mode (m/z 50–1200).
Genotyping arrays used in each study are described in the S1 Text. All the samples underwent the same quality control (QC) and imputation of polymorphic 1000 genome CEU SNPs (Phase I, version 3) performed using IMPUTE2.
Methods for measuring the 21 biological markers and imaging features in PIVUS have been previously described [28] (link)–[30] (link).

Commercially available reagents were purchased from commercial sources and used as received without further purification. If no further details are given, the reaction was performed under ambient atmosphere and temperature. Analytical thin layer chromatography (TLC) was performed on silica gel-coated plates (Merck, 60 F254) with the indicated solvent mixture, and visualization was performed using ultraviolet (UV) irradiation (λ = 254 nm) and/or staining with aqueous KMnO₄. If not specially mentioned, flash column chromatography used silica gel (200–300 mesh) supplied by Tsingtao Haiyang Chemicals (Qingdao, China).
¹H NMR spectra were recorded on a Bruker Avance III 400 (400 MHz) spectrometer. TMS (δH 0.00) were used as the internal reference. ¹³C NMR spectra were recorded on a Bruker Advance III 400 (100 MHz) spectrometer in CDCl₃ (δC 77.16) using their central resonance as the internal reference. All ¹³C NMR spectra were proton decoupled. High-resolution mass spectra (HRMS) were recorded on a Waters Xevo G2 QTOF MS. A commercially available UV lamp (model: Philips TUV 25W/G25 T8, emission wave-length range: 200–280 nm; λmax: 254 nm) was used as light resource.

A quantity of 3 μL of extract was injected into a Waters Acquity UPLC system and separated using a Waters Acquity UPLC HSS T3 (1.8 µM, 1.0 × 100 mm), using a gradient from solvent A (water, 0.1% formic acid) to solvent B (acetonitrile, 0.1% formic acid). Injections were made in 100% A, held at 100% A for 1 min, ramped to 98% B over 12 min, held at 95% B for 3 min, and then returned to starting conditions over 0.05 min and allowed to re-equilibrate for 3.95 min, with a 200 µL/min constant flow rate. The column and samples were held at 40 °C and 6 °C, respectively. The column eluent was infused into a Waters Xevo G2 Q-TOF-MS with an electrospray source in negative mode, scanning 50–1200 m/z at 0.2 seconds per scan, alternating between MS (6 V collision energy) and MSE mode (15–30 V ramp). Calibration was performed using sodium iodide with 1 ppm mass accuracy. The capillary voltage was held at 2200 V, source temp at 150 °C, and nitrogen desolvation temp at 350 °C with a flow rate of 800 L/hr.