Waters symmetry c18 column

IAA and ABA were extracted, purified, and measured by high-performance liquid chromatography (HPLC) using the method described by Yang et al. (2018) (link). Root samples (0.1 g) from each treatment were ground to a powder in liquid nitrogen, 4 mL acetonitrile was added, and the homogenate was incubated in the dark at 4°C for 12 h. The extract was centrifuged at 10800 rpm for 10 min at 4°C. The residue was extracted twice with the same solvent. The supernatant was combined and concentrated to residue at 37°C by rotatory evaporation, and re-dissolved in 8.0 mL 0.4 mol L^-1 phosphate buffer and then added 6.0 mL chloroform and to remove pigment. The pH of the aqueous phase was adjusted to pH3 using pure formic acid. The aqueous phase was extracted thrice with ethyl acetate (3.0 mL). The ethyl acetate phase was concentrated by rotary evaporation and redissolved in 1 mL of the mobile phase. Phytohormone extracts were filtered through 0.2-μm hydrophobic membranes and 10-μL aliquots were injected into a Waters Symmetry C18 column (4.6 mm × 150.0 mm; 5 μm; Waters Corp., Milford, MA, USA) using acetonitrile: methanol: 0.6% acetic acid (5:50:45, v:v:v) as the mobile phase. The flow rate was held at 0.6 mL min^-1 and the peaks were identified with a photodiode array detector (Waters 2998; Waters Corp., Milford, MA, USA) at an absorbance of 254 nm.

Plasma samples were analyzed using LC-MS/MS (Waters TQ-S) with telmisartan as the internal standard (IS) [32 (link),33 (link)]. Briefly, 400 μL of IS solution (0.25 µg/mL of IS acetonitrile solution) was added to thawed plasma samples at ambient temperature and mixed vigorously by vortexing. Then, the sample was centrifuged at 4 °C (11,000 r/min for 15 min) to remove proteins and obtain the supernatant. The supernatant was further filtered through a 0.22 μm syringe-driven filter prior to analysis.
An ACQUITY UHPLC ^TM I-Class system (Waters, Milford, CT, USA) was employed to separate the analytes on a Waters Symmetry^® C18 column (2.1 mm × 100 mm, 3.5 μm). The temperature of the column oven and sample tray were set at 40 °C and 10 °C, respectively. Methanol was used as mobile phase A and 2 mM ammonium acetate aqueous solution containing 0.1% formic acid was used as mobile phase B. A total flow rate of 0.2 mL/min was used, and the gradient program was performed as follows: 0–0.5, 95% B; 1.2–2.0 min, 5% B; 2.1–4.0 min, 95% B. The injection volume was set to 1 μL. Data acquisition and quantitative analyses were performed using a Waters TQ-S system (Waters MS Technologies, Manchester, UK). Multiple reaction monitoring (MRM) was employed to analyze the analytes and IS in positive polarity mode with electrospray ionization (ESI).

The flavonoid compounds; nicotiflorin and rutin were purchased from Extrasynthese (Genay, France) whereas clitorin and manghaslin were isolated from C. papaya leaf extract and the structural elucidation were compared to previous publish data [19 (link)–21 (link)]. Analysis of HPLC was performed to monitor these flavonoid compounds in C. papaya leaf samples on a Waters 2695 HPLC system (Milford, USA) equipped with a Waters 996 photodiode array detector and a Waters 2695 separation module with empower 2.0 software. A Waters Symmetry C18 Column, 300 Å, 5 μm, 3.9 mm X 150 mm, (Milford, USA) was used as stationary phase and the mobile phase consisted of water containing 0.01% Trifluoroacetic acid (TFA) (Solvent A) and acetonitrile containing 0.01% TFA (Solvent B). The gradient solvent system of solvent A and solvent B as follows: 90% solvent A until 5 min, followed by 90–750% solvent A over 20 min, then 75–5% A over 5 min, then going back to 90% solvent A until 5 min, and finally reconditioning the column with 90% solvent A isocratic for 5 min. The flow rate for this analysis was maintain in 1.0 ml/min and the injection volume were 20 μl. The chromatogram was monitored using a wavelength range of 210–400 nm.

For chromatographic analysis, stock solutions (200 μg/mL) of each of the investigated samples were prepared in methanol. HPLC measurements were performed utilizing Shimadzu Prominence HPLC system (Shimadzu Corporation, Japan) consisting of LC-20 AD pump, DGU-20 A5 degasser, CBM-20A interface, and SPD-20A UV–Vis detector with a 20 µL injection loop. The column utilized for studying the chromatographic behavior of the analytes was the most frequently used for lipophilicity investigations: Waters symmetry^® C₁₈ column [250 mm × 4.6 mm (i.d.), 5-μm particle size]. The flow rate was 1.2 mL/min at room temperature. The retention behaviour of the analytes was investigated as a function of mobile phase composition ranging from 60–80% methanol and 40–20% water. The HPLC analyses were carried out at room temperature under isocratic conditions. Membrane filters (0.45 μm) from Sartorius (Goettingen, Germany) were used for filtration of samples. The absorbance of the analytes during a chromatographic run was performed at 200 nm. Each experiment was run in duplicate. Chromatographic data were collected using LabSolutions CS software. H¹NMR spectra were recorded using Bruker NMR spectrometer (Bruker GmbH, Germany),