Nexera uhplc system

The filtered Obeth was analyzed for the presence of polyphenols using UHPLC Nexera system (Shimadzu, MA, USA), column (C18). The solvent used in mobile phase consisted of solvent A (0.2% acetic acid) and solvent B (methanol). The flow rate of 1.0 mL/min was sustained for 21 min of running time. The gradient flow used was: 20% B (0–10 min), 55% B (10–12 min), 70% B (12–14 min), 50% B (14–15 min), 40% B (15–17 min), and 30% B (17 –21 min). For testing, the injection volume of the sample was 10 μL with column temperature maintained at 25°C. The spectra were observed at 280 nm with a photodiode array (PDA) analyzer. The peaks of the compound were identified by comparing retention time and distribution of UV-VIS spectra of samples with those of standard compounds.

The quantitative HPLC profiling of fractions from C. fistula was carried out using a Shimadzu UHPLC Nexera system (Shimadzu, MA, USA), equipped with a quaternary pump (LC-30AD) and a degasser. The injection volume of the sample was 5 μL. The analytical column used was the Enable C18 column (150 mm × 4.6 mm i.d. × 5 μm p.s.), (Shimadzu, MA, USA) that is also provided with the guard column (10 × 4 mm). For chromatographic analysis, the mobile phase elution was a continuous gradient of solvent A (0.1% acetic acid in water, pH 3) and solvent B (methanol). The setting of gradient program was as mentioned (0–10 min) 30% B, (10–14 min) 65% B, (14–16 min) 80% B, (16–17 min) 40% B, (17–17.50 min) 35% B and (17.50–21 min) 30% B. The total run time was 21 min with a constant flow rate of 1.0 mL/min. The column oven (CTO-10AS) helps to maintain a constant temperature of 25 °C. The photodiode array (PDA) detector (SPD-M20A) (Shimadzu, MA, USA) was used to monitor the chromatogram at 280 nm. The solvent delivery, detection, and data processing were performed with the help of the Labsolutions software (version 5.09, Shimadzu, MA, USA).

The analysis was performed using a Nexera UHPLC system (Shimadzu) coupled to a Q-TOF mass spectrometer (TripleTOF 6600, AB Sciex). Separation of metabolites from the spiked human plasma metabolite extracts was performed using a UPLC BEH C18 2.1 × 100, 1.7 µm analytic column (Waters Corp.). The mobile phase was 0.1% formic acid in water (eluent A) and 0.1% formic acid in ACN (eluent B). The gradient profile was 5% B from 0 to 0.5 min, 100% B at 10 min for 3 min and 5% B at 13.5 to 16 min. A volume of 5 µL of the sample was injected. As indicated above different samples were measured in DDA and DIA/SWATH. MS settings were as follows: Gas 1 55, Gas 2 65, Cur 35, Temperature 500 °C, Ion Spray Voltage 5500 V, declustering potential 80 V Information Dependent Acquisition was used for the generation of assay libraries. The IDA duty cycle was 200 ms for MS1, 80 ms for MS2. The mass range of the TOF MS and MS/MS scans were 50–2000 m/z and the collision energy was ramped from 20–50 V or 50–80 V depending on the sample. SWATH acquisition was performed with one TOF MS survey scan (240 ms) followed by 8 SWATH scans (90 ms). The fragment ion window for SWATH was from 100 to 900 m/z. Here, variable windows were used, optimized on the plasma matrix using the SWATH Variable Window Calculator (SCIEX) (Supplementary Table 1).

The methanolic extract was investigated using the NEXERA UHPLC system (Shimadzu, Tokyo, Japan) equipped with a Luna^® Omega C-18 column (50 × 2.1 mm i.d., 1.6 μm particle size). Two μL of each sample were injected. The mobile phase was constituted by water (solvent A) and acetonitrile (solvent B), both acidified with formic acid (0.1% v/v). A linear gradient was used as follows: 0–10 min, 5%→32% B; 10–28 min, 32→75% B; 28-29 min, 75%→95% B; 29–30 min, 95% B; 30–32 min, column re-equilibration. The flow rate was set at 400 μL/min. High-Resolution Mass Spectrometry (HR-MS) data were obtained by an AB SCIEX Triple TOF^® 4600 mass spectrometer (AB Sciex, Concord, ON, Canada), equipped with a DuoSpray^TM ion source (AB Sciex, Concord, ON, Canada) operating in the negative ElectroSpray (ESI) mode. A full-scan Time-Of-Flight (TOF) survey and 8 information-dependent acquisition MS/MS scans were acquired, using the following parameters: curtain gas 35 psi, nebulizer and heated gases 60 psi, ion spray voltage 4500 V, ion source temperature 600 °C, declustering potential −80 V and collision energy −40 ± 15 V. The instrument was controlled by Analyst^® TF 1.7 software (AB Sciex, Concord, ON, Canada), whereas MS data were processed by PeakView^® software version 2.2 (AB Sciex, Concord, ON, Canada).

Extracts were analyzed using a NEXERA UHPLC system (Shimadzu, Tokyo, Japan) and the Luna^® Omega C-18 column (Shimadzu, Tokyo, Japan). The linear gradient for separation purposes consisted of water (A) and acetonitrile (B), both with 0.1% formic acid. B eluent was held at 5% for 0.5 min and was allowed to reach 17.5% at 5 min; then, it was increased to 45% in 5 min and held at this % for the other 2 min. Finally, it was augmented to 95% in 1 min, and following another min at 95%, initial conditions were restored in 1 min, allowing for the system to re-equilibrate for 1 min. The flow rate was 0.5 mL/min. The injection volume was 2.0 μL. MS analysis was performed using the AB SCIEX Triple TOF^® 4600 (AB Sciex, Concord, ON, Canada), equipped with a DuoSpray^TM ion source, operating in negative ESI mode. A full scan TOF survey (170–1700 Da) and eight IDA (information-dependent acquisition) experiments were performed using the following parameters: curtain gas 35 psi; nebulizer gas 60 psi; heated gas 60 psi; ion spray voltage −4.5 kV; interface heater temperature 600 °C; declustering potential −80V; collision energy −45 ± 10 V. The instrument was controlled by Analyst^® TF 1.7.1 software. Data processing was carried out by PeakView^® software version 2.2.