Agilent zorbax eclipse xdb c18

Using Agilent 1260 Infinity Quaternary LC, equipped with Agilent 1260 Infinity; Quaternary Pump (G1311B), and Diode Array Detector (G1315D, VL) coupled to Agilent Open LAB Chem Station B.04.03 software, chromatographic analysis was carried out. Analytes were separated on Agilent Zorbax Eclipse XDB-C18 (250 mm × 4.6 mm i.d., 5_mparticle diameter) protected with Agilent Zorbax XDB-C18 pre-column (Agilent Technologies, Palo Alto, CA, USA). The flow rate of 1.0 mL/min and the UV detector was set at 280 nm. Separation was carried out with acetonitrile (A) and 0.1% trifluoroacetic acid in water (B) and gradient elution program at a flow rate of 1 ml/min starting with 20% A in B for 5 min, 20–30% A/B in 5 min, 30–50% A in B in 5 min, 50–100% A in B in 10 min followed by washing with 100% A for 5 min.

LC-MS/ESI-MS analysis was performed with an Agilent 1290 Infinity LC system (Agilent Technologies, Santa Clara, CA, USA) coupled with an Agilent 6520 accurate-mass Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with dual ESI sources operated in positive-ion mode. The MS was operated with the electrospray voltage set to 4000 V, a sheath gas flow of 10 L/min, fragmented voltage of 125 V, gas temperature of 300 °C and nebulizer gas at 45 psig. Chromatographic separation of metabolites was achieved using an Agilent Zorbax Eclipse XDB-C18 (Agilent Technologies, Santa Clara, CA, USA) narrow-bore 2.1 × 150 mm, 3.5 micron (particle size) operated at 25 °C. The column was eluted at a flow rate of 0.5 mL/min with aqueous solvent A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile. The chemical structure of all the identified bioactive compounds were visualized using ChemDraw JS Sample Page (version 19.0.0-CDJS-19.0.x+da9bec968, PerkinElmer, Waltham, MA, USA).

The lyophilized fractions of the CWE-EA, HWE-EA, and ME-EA were reconstituted in 1 mL of 20% aqueous methanol (v/v) and passed through a 0.2 μm nylon filter. Eleven standards were included for flavonoids (quercetin and kaempferol), triterpenoids (arjunic acid and euscaphic acid), polyphenols (caffeic acid, chlorogenic acid, ferulic acid, gallic acid, tannic acid, and coumarin), and kojic acid. Chromatographic separations were performed on an Agilent Zorbax Eclipse XDB-C18 (Agilent, Santa Clara, CA, USA) column (4.6 mm × 150 mm, 5 μm). Samples (10 μL) were injected into the HPLC instrument (Shimadzu Prominence LC 20A series HPLC system, Shimadzu Corp, Kyoto, Japan) with a PDA detector. The mobile phase for CWE-EA, HWE-EA, and ME-EA consisted of 0.1% phosphoric acid in water (solvent A) and 0.1% phosphoric acid in acetonitrile (solvent B). Elution from the column was achieved with the following gradient: 0–5 min, 97% A and 3% B; 15–20 min, 90% A and 10% B; 30–40 min, 50% A and 50% B; 40.1–50 min, 97% A and 3% B. The preparative system was run for 40 min of the total running time at a constant flow rate of 0.8 mL/min at ambient temperature, and the spectrum was monitored at 272 nm. The identification of each compound was based on a combination of the retention time and UV spectral matching.

The methanol extract of N. biserrata was analyzed using an Agilent 1290 Infinity LC system coupled with an Agilent 6520 QTOF mass spectrometry system. A 2.0 µl sample was injected into a reversed-phase column, namely Agilent Zorbax Eclipse XDB-C18 (narrow bore, 2.1 mm × 150 mm × 3.5 µm; Agilent Technologies, Santa Clara, CA, USA). The column was maintained at 25 °C at a flow rate of 500µL/min during analysis. The mobile phases were composed of solvent A (H₂O—0.1% HCOOH) and solvent B (acetonitrile—0.1% HCOOH). The gradient elution program was initiated at 5% of solvent B for 5 min, then from 5 to 100% solvent B in 15 min. and kept for 5 min. Later, the column was conditioned as initial for 5 min. prior to next injection.
The mass spectrometry (MS) signals were acquired as described by Ling et al. (2018)^{38 (link)} with minor modifications. Briefly, voltage for positive electrospray ionization was changed to 4 kV, while the remaining settings for ion source were maintained. The MS was calibrated with Tuning Mix (Agilent Technologies, Santa Clara, CA, USA) before each batch analysis. Internal mass calibration standards, purine (m/z 121.0508) and hexakis-(1H, 1H, 3H-tetrafluoropropoxy)-phosphazine (m/z 922.0097), were introduced throughout the runs for automated mass correction^{39 (link)}.

The bioactive compound analysis was carried out using the Agilent 1290 Infinity and 6520 iFunnel Q-ToF-LCMS (Agilent Technology, Santa Clara, Calif.) fitted with an electrospray interface and operating in positive ion mode, as reported by Saleem et al. (2019) with minor modifications [21 (link)]. About 20 µg of SCE-2 was made by dissolving it in 200 µL of methanol. The column (2.1 × 150 mm, 3.5 µm Agilent Zorbax Eclipse XDB-C18) was kept at 25 °C, while the auto-sampler was kept at 4 °C. Fresh 0.1% formic acid in water and 0.1% formic acid in acetonitrile mixtures were prepared for the mobile phase A and B, respectively. The flow rate at 0.5 mL/min, injection volume at 1.0 μL, the run time was 25 min, and the recovery period was 5 min. Using an electrospray ion source in positive mode, full scan MS analysis was done over the m/z 100–1000 range. The experiment was conducted using a capillary voltage of 3500 V. The data were processed using Agilent Mass Hunter Qualitative Analysis B.05.00 (Method: Metabolomics-2017-00004.m). The compounds were discovered by comparisons and searching the METLIN database.

Analysis was conducted using a Hewlett-Packard series 1100 HPLC system Agilent Technologies Inc., formerly Hewlett-Packard Co., Palo Alto, CA, USA) equipped with a degasser, a quaternary pump, an autosampler, a column oven, and a diode-array detector; data were collected and integrated using the Agilent ChemStation software. Chromatographic conditions were as follows: column, Agilent Zorbax Eclipse XDB-C18 (3.0 × 150 mm, 5 μm; Agilent Technologies Inc.); mobile phase, 10 mM ammonium bicarbonate, pH 6.8/acetonitrile (90:10 v/v; solvent A) and acetonitrile (solvent B); elution program, isocratic elution with 100% solvent A for 2 min, linear gradient from 0 to 70% solvent B in 8 min, followed by isocratic elution with 70% solvent B for 5 min; post-run time, 7 min; flow rate, 0.4 mL/min; injection volume, 30 μL; column temperature, 28 °C; the diode-array detector was set to monitor at 433 nm, and the online UV-visible spectra were recorded in the scanning range of 200–600 nm. Under the above conditions, the retention times of compound 1 and authentic compound 3 were 13.4 and 9.7 min, respectively.

Phenolics in KRPBE were quantitatively analyzed using a reversed-phase HPLC system (Agilent 1200; Agilent Technologies, Santa Clara, CA, USA) equipped with an autosampler, a diode array detector, and a degasser. A reversed-phase column (250 × 4.6 mm, 5 μm; Agilent Zorbax Eclipse XDB-C18; Agilent Technologies) was used. The injection volume was 5 μL. The flow rate was 0.8 mL/min. The two mobile phases were water with 0.1% (v/v) formic acid (solvent A) and acetonitrile with 0.1 (v/v) formic acid (solvent B). The gradient elution profile was as follows: 95% A/5% B at 0 min, 85% A/15% B at 25 min, 65% A/35% B at 45 min, 30% A/70% B at 50 min, 20% A/80% B at 58 min, 95% A/5% B at 60 min, and 95% A/5% B at 65 min. The wavelengths for detection were set at 320 nm for caffeic acid and 280 nm for protocatechuic acid, procyanidin B1, catechin, vanillin, and taxifolin. Phenolics in KRPBE were identified by comparison of UV–visible spectra, retention times, and spiked inputs with commercial standards. Phenolics were quantified using calibration curves that relate different concentrations of authentic standards to the areas of their corresponding peaks.