Hss c18 column

UPLC-HRMS analyses of samples were conducted using a Waters ACQUITY H-class LC system coupled with an LTQ-Orbitrap Velos pro mass spectrometer (Thermo Fisher Scientific, MA. USA). Saliva metabolites were separated with a 17-min gradient using a Waters HSS C18 column (3.0× 100 mm, 1.7 μm) at a flow rate of 0.3 mL/min. Mobile phase A was 0.1% formic acid in H₂O, and mobile phase B was acetonitrile. Saliva metabolites were separated with a gradient as follows: 0–2 min, 2% solvent B; 2–5 min, 2–55% solvent B; 5–15 min, 55–100% solvent B; 15–20 min, 100% solvent B; 20–20.1 min, 100–2% solvent B; and 20.1–29 min, 2% solvent B. The column temperature was set at 45°C. Full MS acquisition was performed from 100 to 1000 m/z at a resolution of 60 K. The automatic gain control (AGC) target was 1× 10⁶, and the maximum injection time (IT) was 500 ms. UPLC targeted-MS/MS data were acquired at a resolution of 15 K with an AGC target of 5× 10⁴, a maximum IT of 250 ms, and an isolation window of 3 m/z. The collision energy was optimized as 20, 40, 60 or 80 for each target with higher-energy collisional dissociation (HCD) fragmentation. The injection order of the saliva samples was randomized to reduce experimental bias.

Ethanolic extract from F. formosa leaves was dissolved in methanol and filtered through a 0.22 µm microfilter. The UPLC ACQUITY system (Waters) was used, coupled with a DAD detector—scanning from 220 to 400 nm—and a mass spectrometry detector (MS), with an HSS C18 column (1.7 μm particles, 50 × 3 mm i.d.), operating at a flow rate of 0.3 mL/min and maintaining the column oven at 40 °C. A gradient of H₂O (0.1% formic acid) and ACN (0.1% formic acid) was applied, involving a long linear elution period (5–95% H₂O over 10 min), followed by a short isocratic elution period (95% ACN for 1 min). In obtaining the mass spectra, an Electrospray ionization (ESI) system was used, with a capillary temperature of 250 °C, the spray voltage set at 3.5 kV, capillary voltage at + 3 V and − 47 V for positive and negative polarities respectively, and a tube lens offset of 0 V and − 25 V for positive and negative polarities respectively. Nitrogen was used as the collision gas with a flow rate of 50 arbitrary units. Mass analysis was conducted in full scan mode from 100 to 1500 Da in negative mode.

The Waters ACQUITY H-class LC system coupled with an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, MA. USA) was launched to perform the ultra-performance LC-MS analyses of urine samples. We separated urinary metabolites with a 17 min gradient on a Waters HSS C18 column (3.0× 100 mm, 1.7 μm), and the flow speed was 0.5 ml/min. Mobile phases A and B were 0.1% formic acid in H₂O and acetonitrile, respectively. The gradient was described as follows: 0–1 min, 2% solvent B; 1–3 min, 2–15% solvent B; 3–6 min, 15–50% solvent B; 6–9 min, 50–95% solvent B; 9–9.1 min, 95–100% solvent B; 9.1–12 min, 100% solvent B; 12–12.1 min, 100–2% solvent B; and 12–17 min, 2% solvent B. The temperature of the process was 50 °C. Scans from 100 to 1000 m/z at a resolution of 60 K were used to acquire the Full MS. The automatic gain control (AGC) target was 1× 10⁶, and the maximum injection time (IT) was 100 ms. Then, UPLC targeted-MS/MS analyses of the QC sample were conducted to identify the differential metabolites. A resolution of 15 K with an AGC target of 5× 10⁵, a maximum IT of 50 ms, and an isolation window of 3 m/z was obtained. In terms of every target with higher-energy collisional dissociation (HCD) fragmentation, 20, 40 and 60 were set as the optimal collision energies.

A Waters ACQUITY H-class LC system coupled with an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, MA, USA) was utilized to analyze the urine samples. To separate urine metabolites, a 17-minute gradient was applied to a Waters HSS C18 column (3.0x100 mm, 1.7 um) at a flow rate of 0.5 ml/min. Mobile phase B consisted of acetonitrile, whereas mobile phase A involved 0.1% formic acid in water. Followed were the gradient’s setting:0-2 min,2% solvent B;2-5 min, 2-55% solvent B;5-15 min,55-100% solvent B;15-20 min,100% solvent B;20-20.1 min,100-2% solvent B; and 20.1-29 min,2% solvent B. The column’s temperature was kept at 50°C throughout the procedure. The mass scan was in the 100–1,000 m/z range. The MS1 and MS2 analyses were run at resolutions of 60K and 15K, respectively. The MS1 automatic gain objective was set to 1 x 106, and the maximum injection time (IT) was 100 milliseconds. The maximum IT was set to 50ms, and the MS2 automatic gain control target was 5x105. The ideal collision energy of 20, 40, 60, or 80 was employed to dissociate the various metabolites using the higher-energy collisional dissociation (HCD) fragmentation mode. As a result, the features in MetaAnalyst 4.0 (http://www.meta-boanalyst.ca) may be more comparable.

HRLC-MS was selected for urinary metabolite detection due to its high sensitivity and reproducibility. Urine metabolite separation and analysis were conducted using a Waters ACQUITY H-class LC system coupled with an LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific, MA). The following 18-min gradient on a Waters HSS C18 column (3.0 × 100 mm, 1.7 μm) at a flow rate of 0.5 mL/min was used: 0–1 min, 2% solvent B (mobile phase A: 0.1% formic acid in H₂O; mobile phase B: acetonitrile); 1–3 min, 2%–55% solvent B; 3–8 min, 55%–100% solvent B; 8–13 min, 100% solvent B; 13–13.1 min, 100%–2% solvent B; 13.1–18 min, 2% solvent B. The column temperature was set at 45 °C.
All samples were fully scanned from 100 to 1000 m/z at a resolution of 60 K. The automatic gain control (AGC) target was 1 × 10⁶, and the maximum injection time (IT) was 100 ms. The extracted MS features were divided into several targeted lists and imported to the MS2 method for targeted data-dependent analysis. MS/MS fragment acquisition was performed at a resolution of 15 K with an AGC target of 5 × 10⁵. Collision energy was optimised as 20, 40, or 60 for each targeted list with higher-energy collisional dissociation (HCD) fragmentation. The injection order of urine samples was randomised to reduce any experimental bias. The QC sample was injected regularly to monitor system stability.

The analysis of probe metabolites from CYP‐specific marker reactions was conducted with a LC/MS/MS method modified from earlier works (Turpeinen et al., 2005; Tolonen et al., 2007) and also reported by (Showande et al., 2013). Briefly, a Waters Acquity UPLC system (Waters Corp., Milford, MA) was used together with a Waters HSS C18 column (2.1 mm × 50 mm; 1.8 μm particle size) and an online filter at 35°C. The injection volume was 4 μl, and UPLC eluents were aqueous 0.1% acetic acid (pH 3.2, A) and acetonitrile (B). The gradient elution from 2%–65%–95% B was applied in 0–2.5–3.5 min, followed by column equilibration, giving a total time of 4.5 min/injection. The eluent flow rate was 0.5 ml/min. Data were acquired using a Thermo TSQ Endura triple quadrupole MS. Multiple reaction monitoring (MRM) mode using positive ion mode. For all compounds, the spray voltage was 4500 V, vaporizer temperature and transfer tube temperature were 400°C and 350°C, respectively. The CID argon pressure was set to 2.0 mTorr. The MRM transitions were as previously described (Tolonen et al., 2007; Turpeinen et al., 2005). For acetaminophen, hydroxyrepaglinide and 4‐hydroxydiclofenac MRM's were m/z 152 >  m/z 110, m/z 469 >  m/z 246, m/z 312 >  m/z 231, respectively. The instruments were controlled using Thermo Xcalibur 3.0.63 software.