Uplc hss t3 column

Chromatographic separation was performed using an Acquity UPLC HSS-T3 column (100 × 2.1 mm, dp: 1.8 μm) in combination with a guard column of the same type (Waters NV/SA, Asse, Belgium). The gradient elution programme consisted of two mobile phases (A and B). Mobile phase A and B were 0.1% formic acid in ultrapure H₂O and 0.1% formic acid in methanol, respectively. The following program was applied: 0 min (60% A, 40% B), 0–2 min (linear gradient to 100% B), 2–4 min (100% B), 4–4.1 min (linear gradient to 60% A, 40% B) and 4.1–8 min (60% A, 40% B). Flow rate was set at 0.4 mL/min. The LC eluent was interfaced to a Xevo TQ-XS triple quadrupole mass spectrometer (Waters NV/SA, Asse, Belgium) with ion source heated electrospray ionization operating in positive ionization mode. Acquisition was performed in selected reaction monitoring mode. For CBD and internal standard, the following two most intense product ions were followed: CBD: mass-to-charge ratio 315.08 > 193.00/122.96 and CBD-d3: mass-to-charge ratio 318.12 > 196.03/122.96. The LC-MS/MS analytical methods were validated using matrix-matched calibrator and quality control samples, based on blank plasma of untreated dogs. The limit of quantification (LOQ) was 1 ng/mL. The LC-MS/MS analyses were conducted in accordance with the international guidelines (33 –35 ).

UPLC-MS/MS was used to analyze the components of the JPQCHSD formula. The collision voltages of the positive and negative ion sources were 3.0 and 2.2 kV, respectively; the temperature of the heated vaporizer was 300 °C; and nitrogen was used as the sheath, auxiliary, and collision gas. Waters Masslynx 4.1 and UNIFI Scientific Information Systems (Milford, MA) were used to collect and analyze the data. The Waters Acquity UPLC H-Class system was used to analyze the components of JPQCHSD with a chromatographic column (UPLC HSS T3 column (2.1 × 100 mm, 1.8 mm). The mobile phase consisted of A (water: 0.1% formic acid, v/v) and B (acetonitrile) and was run at a flow rate of 0.4 mL/min with a column temperature of 55 °C.

Blood was collected in EDTA-coated tubes, and plasma was separated by centrifuging blood at 2000× g for 10 min at 20 °C. Plasma EGCG and EGC levels were analyzed using a previously validated ultra-high performance liquid chromatography (UPLC) method for analysis of tea catechins [33 (link)]. The Acquity UPLC system was interfaced with a photodiode array detector and a quaternary solvent manager (Waters Corporation, Milford, MA, USA) and the instrument was fitted with an Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 um) protected with a 0.2 um in-line filter. Reference standards included EGCG (97%) and EGC (95%) purchased from Sigma (St. Louis, MO, USA). Standard stock solutions were prepared in 3% acetonitrile and further diluted with 3% acetonitrile to obtain different concentration levels for preparation of standard curves.

The AB ExionLC system (AB SCIEX, Framingham, MA, USA) coupled to the AB SCIEX API 6500 Qtrap+ system (AB SCIEX) was used to analyze targeted phenolic metabolite profiles. Specifically, the positive and negative electrospray ionization modes and the AB SCIEX OS workstation (version 1.7.1) were used. A Waters UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm) was used in the positive and negative ion modes. The binary gradient elution system consisted of (A) water (containing 0.1% formic acid, v/v) and (B) acetonitrile. The injection volume was 5 μL. Using an electrospray ion source, the analyte was analyzed in the multiple reaction monitoring (MRM) mode for both positive and negative ion scanning, resulting in increased sensitivity. Mass spectrometry parameters, such as the declustering potential and collision energy, were optimized to enable the rapid screening of target compound ion pairs. The optimized mass spectrometry conditions were as follows: positive mode—collision gas: 35, ion spray voltage: 5500 V, ion spray temperature: 600 °C, ion source gas 1: 60, and ion source gas 2: 50; negative mode—collision gas: 35, ion spray voltage: −4500 V, ion spray temperature: 600 °C, ion source gas 1: 60, and ion source gas 2: 50.

The metabolite analysis was based on literature reports [13 (link),14 (link)] in sample preparation and metabolite analysis. Briefly, an Ultra-high performance liquid phase tandem Thermo Q Exactive Orbitrap mass spectrometer, which was equipped with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) (Waters Corporation, Shanghai, China), and the control software (Xcalibur, version: 4.0.27, Thermo Scientific (Waltham, MA, USA)) was used as the liquid-mass system for primary and secondary mass spectrometry data acquisition. The mobile phase under a positive ion mode was composed of A: 0.1% formic acid aqueous solution and B: acetonitrile. For negative ion mode, mobile phase A was 5 mmol/L ammonium acetate aqueous solution and mobile phase B was acetonitrile. MS-DIAL [15 (link)] software was used to identify the metabolites based on the open-access database of MetaboLights (https://www.ebi.ac.uk/metabolights (accessed on 9 October 2021)).