Tsq quantum access

Cucumber leaves treated with H₂O and aged 7, 14, 21, 28, 35, and 42 days were taken and immediately frozen in liquid nitrogen. The frozen leaves were dried in a vacuum freeze dryer and ground into frozen powder under liquid nitrogen freezing, and 0.1 g of powder was weighed for MT extraction [27 (link)]. A 10 μL extract was then tested on an HPLC–MS system (Thermo Fisher Scientific, TSQ Quantum Access, Waltham, MA, USA) with the test conditions set according to Bian [73 ].

Samples (100 μl; Blank, Calibration or test samples) were added to individual injection vials containing Internal Standard-2u (50 μl; 2 μg/ml AcetylCholine-D4, Cat#D-1555, CDN Isotopes, Canada) and DS1 (900 μl, contains 0.1% Formic Acid in 50% HPLC grade Acetonitrile), vortexed, and injected into the LCMS system. DS1 was run between high and low concentration samples and between sets of the test sample. Calibration ranges were 10–200 μg/ml for Creatine (Cat# BS-9561E, BioServ UK Limited) and 0.10–35.0 μg/ml for Choline (Cat# 67-48-1, Sigma Aldrich, UK). Samples were analysed on a Thermo Accela Pump and CTC Auto sampler coupled to a Thermo TSQ Quantum Access. LC Solvent A (0.1% Formic Acid in water); LC Solvent B (0.1% Formic Acid in Acetonitrile). LCMS parameters are detailed in Table 2.

Optical rotations were measured using AA-55 polarimeter (Optical Activity, Ramsey, United Kingdom) in CH₂Cl₂. The UV spectra were measured in MeCN on a DU800 UV-vis spectrophotometer (Beckman Coulter, Brea, CA, USA). ¹H- and ¹³C-NMR spectra were recorded at 400 and 100 MHz, respectively, on a WM-400 spectrometer (Bruker, Billerica, MA, USA) in CDCl₃, unless otherwise noted. The same solvent was used as the internal standard. COSY, ROESY, DEPT, HMBC and HMQC experiments [35 ] were recorded using Bruker microprograms. ESI-MS were obtained on a TSQ Quantum Access™ mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Analytical and PTLC were performed on silica gel plates (Kieselgel 60, F254, Merck, Darmstadt, Germany, 0.25 and 0.5 mm, respectively). The spots were visualized exposure to UV radiation (254 nm). Column chromatography was performed on a silica gel (Kieselgel 60, 0.063−0.200 mm, Merck) Reverse phase column chromatography was carried out using a C-18ec Chromabond 10-g cartridge (Macherey-Nagel, Düren, Germany). Medium pressure liquid chromatography (MPLC) was performed with a Sepacore system (Büchi, Flawil, Switzerland) equipped with a UV-detector. Fractionation was performed with a PuriFlash SiHC cartridge (SiOH, 40 g, Interchim, Montluçon, France).

Reduced (GSH) and oxidized glutathione (GSSG) levels were measured in the blood by means of an LC-MS/MS method previously developed by us [18 (link)]. The analysis was performed by means of an Accela chromatographic system coupled with a triple quadrupole mass spectrometer TSQ Quantum Access (Thermo Fisher Scientific, Rodano, Milan, Italy) with an electrospray ionization source in positive ion mode. The transitions monitored were m/z 308.1 → m/z 76.2 + 84.2 + 161.9 for GSH and m/z 613.2 → m/z 230.5 + 234.6 + 354.8 for GSSG. The data were obtained by comparison with calibration curves using GSH and GSSG standard solutions (Sigma-Aldrich, Merck Life Science S.r.l., Milan, Italy). The intra- and inter-day CVs % were <5%, and the limits of detection were 0.031 µmol/L and 0.008 µmol/L for GSH and GSSG, respectively. The levels of GSH and GSSG were corrected for hemoglobin (Hb) and expressed as µmol/g Hb.

Reduced (GSH) and oxidized glutathione (GSSG) quantitation was performed as previously described.²⁸ Cell pellet was resuspended in 50 µl of PBS, proteins were precipitated with 50 µl of 10% trichloroacetic acid plus 1 mM EDTA and, after a further dilution 1:5 with formic acid 0.1%, samples were analysed by LC‐MS/MS. The LC‐MS/MS analysis was performed using an Accela HPLC System (Thermo Fisher Scientific, Waltham, Massachusetts, USA) coupled to a triple quadrupole mass spectrometer TSQ Quantum Access (Thermo Fisher Scientific, Waltham, Massachusetts, USA) outfitted with electrospray ionization source operating in positive mode. The chromatographic separation was conducted on a Luna PFP column (2.0 × 100 mm, particle size 3.0 µm, Phenomenex, Torrance, California, USA) maintained at 35°C. Analytes were eluted under isocratic conditions at 200 µl/min by 1% methanol in 0.75 mM ammonium formate adjusted to pH 3.5 with formic acid. The analytes were detected by multiple reaction monitoring, and the transitions monitored (precursor ion > product‐fragment ions) were m/z 308.1 → m/z 76.2, 84.2, 161.9 (GSH) and m/z 613.2 → m/z 230.5, 234.6, 354.8 (GSSG). A linear 6‐point calibration curve (range 0.25–8 µM for GSH and 0.008–0.25 µM for GSSG) was used for the quantification.

Liquid chromatography was conducted for the identification and quantification of tyrosol, hydroxytyrosol, and oleuropein, by using the isocratic HPLC Varian ProStar 210 machine equipped with UV 9050 detector, managed by Varian Workstation software (Varian, Inc). For other hydroxycinnamic acids and flavonoid analysis, the HPLC‐MS method was optimized by using a HPLC Agilent 1,200 Series (Agilent Technologies) coupled with a mass spectrometer TSQ Quantum Access (Thermo Scientific), equipped with electrospray ion source (ESI) and triple quadrupole analyzer. The Xcalibur software (Thermo Scientific) managed the instrument and collected the data.

HPLC separation was performed using a Surveyor plus HPLC system (Thermo Fisher, Singapore) equipped with a Hypersil Golden C₁₈ column (100 mm × 2.1 mm, 3 μm particles) at 20°C. The mobile phase consisted of water (component A) and methanol (component B) and the flow rate was 0.2 mL min^–1. The gradient program was: 0–2.0 min, 30% B; 2.0–5.0 min, 30–80% B; 5.0–13.0 min, 80% B; 13.0–15.0 min, 80–90% B; 15.0–25.0 min, 90% B. HESI-MS/MS data were acquired with a triple quadrupole mass spectrometer (TSQ Quantum Access, Thermo Fisher, United States) coupled with an electrospray source in positive ionization mode. The MS parameters were set as follows: vaporizer temperature 375°C, capillary temperature 300°C, sheath gas 40 (arbitrary units), aux gas 5 (arbitrary units) and spray voltage 3.5 kV. For qualitative and semi-quantitative analysis of metabolites, selected reaction monitoring (SRM) mode was used. The contents of ursolic acid 28-O-β-D-glucopyranoside were determined by calculating relative peak areas using the product prepared from IaAU1 enzymatic activity assay as standard.