Xevo tq xs

The sample extraction was done using methanol:water (2:1 v/v %), 100 mg FW/mL final sample ratio. Ultra-performance liquid chromatography with tandem mass spectrometry (Waters Acquity I class UPLC system coupled to a Waters Xevo TQ-XS instrument equipped with a UniSpray (US) ion source operated in timed MRM mode) analyses were carried out according to Vrhovsek et al. [76 (link)] with slight modifications as described in detail by Pál et al. [32 (link)]. Separation was achieved on a Waters Acquity HSS T3 column (1.8 μm, 100 mm × 2.1 mm), kept at 40 °C. Mobile phases both contained 0.1 v/v % formic acid while a water and acetonitrile gradient was used. For quantitation the transition exhibiting the highest S/N ratio was utilized (Table S1). Data processing was performed using Waters MassLynx 4.2 and TargetLynx softwares.

The analyses were performed using a Waters Xevo G2-XS quadrupole-time
of flight mass spectrometer for compound characterization in HR (Supporting
Information, section “Supplementary Results,” MS And MS/MS Characterization) and a Waters Xevo TQ-XS
(triple quadrupole) mass spectrometer both equipped with Z-spray (electrospray)
ionization and step-wave source optimization, and controlled under
MassLynx v4.2 (Waters) as fully described previously.^{13 (link)} The Waters Xevo G2-XS was used for the compounds characterization
in HR, in full-scan, and in quadrupole selection mode without or with
increasing collision energy up to 35 V for fragment generation. The
Waters Xevo TQ-XS was used for the implementation of the MRM method
as detailed in Supporting Information, section “Supplementary Results” and Table S1. The final
MRM transitions for O6-m2dGO and 2dGO analyses are given in Table 2 with expected LC
retention times of each compound and selected MRM transitions for
LC method optimization.
MassLynx and TargetLynx v.4.2 (Waters) were used for
rapid chromatogram
and spectra evaluation and for the construction of calibration curves
and the computing of quantification data, respectively. As only 60
μL out of 65 μL was taken from each sample for analysis
and CALs were prepared as 60 μL-samples, calculated concentrations
were corrected by a factor 1.083 to retrieve real concentrations in
the samples.

Targeted metabolomic analysis was performed as previously described [15 (link), 16 (link), 21 (link), 22 (link)], using an ACQUITY UPLC connected to a triple-quadrupole mass spectrometer (Xevo TQ-XS, Waters Corporation, Milford, MA, USA), and a MxP^® Quant 500 kit (Biocrates Life Science AG). Plasma samples of 10 µL were used, and sample preparation and measurements were performed according to the MxP^® Quant 500 kit manual. Metabolite concentrations were calculated using exported raw data files with the MetIDQ™ version Oxygen software (Biocrates Life Science AG).

Yeast cultures were pelleted by centrifugation at 3500× g for 5 min at 12 °C, and 150 μL aliquots of supernatant were removed for analysis. Metabolites were analyzed by LC-M/MS using a Waters Acquity UPLC and Waters Xevo TQ-XS with the mass spectrometer. Chromatography was performed using an Acquity UPLC BEHC18 column (2.1 mm × 100 mm, 1.7 μm; Waters) with 0.1% (v/v) formic acid in water as mobile phase solvent A and 0.1% (v/v) formic acid in acetonitrile as solvent B. The column was operated with a constant flow rate of 0.3 mL/min at 30 °C and a sample injection volume of 5 μL. Chromatographic separation was performed using the following gradient: 0.00–0.5 min, 5% B; 0.50–3.00 min, 5%–24% B; 3.00–4.50 min, 24%–95% B; 4.50–7.00 min, 95% B; 7.01–10.00 min, 5% B. The LC eluent was directed to the MS from 0.01–10.00 min operating with ESI in positive mode, Desolvation temperature of 500 °C, gas flow rate of 11 L/min, and nebulizer pressure of 40 psi.

Sample supernatants (5 µL) were injected using a Waters FTN Sample manager onto an ACQUITY UPLC BEH Shield RP18 column (Waters; particle size: 1.7 µm, length: 50 mm, internal diameter: 2.1 mm). The initial solvent was 81% A (solvent A: 100% water with 5 mmol/L ammonium acetate and 0.01% formic acid; solvent B: 100% methanol with 5 mmol/L ammonium acetate and 0.01% formic acid) at a flow rate of 0.4 mL/min at 45 °C. Gradient elution (Table 8) was performed using a Waters ACQUITY UPLC I-class pump. Mass spectrometric detection employed a Xevo TQ-XS (Waters) with Unispray ionization in negative ion mode. Individual bile salts were monitored in MRM windows, either as pseudo-transitions (for unconjugated species) or as glycine/taurine daughter ions.

Multiple reaction monitoring (MRM) of the studied substances was conducted using an XevoTQ-XS (Waters, Milford, MA, USA) mass spectrometer equipped with an electrospray ionization (ESI) source, which operated in the positive ion mode (ESI (+)) and negative ion mode (ESI (−)). The desolvation gas flow was set at 800 L/h at 600 °C and the source temperature was 150 °C. The applied capillary voltage was 3.0 kV. In turn, the collision gas flow was set at 0.20 mL/min.
Traced MRMs and their corresponding MS settings are listed in Table 5.

Phenolic compounds were identified and quantified by a UPLC-MS/MS analysis using an Acquity UPLC HSS T3 1.8 µm column, with a gradient elution consisting of water containing 0.1% (v/v) acetic acid (solvent A) and acetonitrile (solvent B) for 25 min at a flow rate of 0.4 mL/min. The solvent gradient was: at 0 min 5% B, 15–15.10 min 95% B, and 15.10–25 min 5% B (re-equilibration step). Phenolic compounds were identified in a Waters SYNAPT G2 HDMS Q-TOF high-resolution spectrometer by comparing the retention times of peaks and the fragmentation data in samples to those of 25 standards (Section 2.1). Quantification was performed using a Waters ACQUITY I CLASS model chromatograph instrument (Waters, Mississauga, ON, Canada) equipped with a Waters XEVO TQ-XS. Ionization was performed by UniSpray (US). Individual compounds were quantified by constructing calibration curves with commercial standards in a concentration of 1–1000 µg/L.