Gemini c18 column

Alkaloid extraction was performed according to the methods of Wang et al. [31 (link)]. Alkaloid extracts were dissolved in 1.0 mL of methanol and analyzed by HPLC and ion-pair extraction-spectrophotometry as described by Yang et al. [33 ].
The contents of vindoline, catharanthine, and ajmalicine were determined at 25 °C by HPLC analysis using an Agilent 1260 series system (Agilent Technologies, Santa Clara, CA, USA) and a Phenomenex Gemini C18 column (250 mm × 4.6 mm, 5 μm) (Phenomenex Inc., Torrance, CA, USA). The mobile phase consisted of methanol/acetonitrile/10 mM ammonium acetate (15:40:45, v/v/v) at a flow rate of 1.0 mL/min. The detection wavelength was 280 nm, and the injection volume was 10 μL. Before injection, all samples were filtered with 0.45-μm nylon membrane filters (Jinteng Corp., Tianjin, China). Alkaloids were identified and quantified by comparing retention time and UV absorbance spectra with the standards. In a citric acid-phosphate buffer of pH 3.0, an ion-pair complex was formed between all alkaloids in the sample and the color reagent bromophenol blue upon 5 min of reaction at 30 °C. The complex was extracted with CHCl₃ in which the absorbance of total alkaloids was measured at the wavelength of 413 nm. Vindoline was used as a reference standard in the preparation of the calibration curve. Each sample was analyzed in triplicate.

HPLC-grade acetonitrile, methanol, dichloromethane, n-hexane and lycopene analytical standard were purchased from Sigma (St. Louis, MO, USA). The assay was performed according to the procedure described by Kozukue and Friedman (2003) slightly modified [27 (link)]. HPLC analysis was performed on a Jasco Extrema LC-4000 system (Jasco Inc., Easton, MD, USA) equipped with photo diode-array detector and autosampler. Data were acquired and analyses were performed using JASCO ChromNAV (version 2.02.04). Samples were analyzed on a Gemini C18 column (250 × 4.6 mm, 5 µm, Phenomenex) and lycopene was detected at 450 nm. The column was eluted at a flow rate of 1 mL/min with a two-solvent system, namely, (A) acetonitrile, (B) n-hexane/dichloromethane/methanol (1:1:1) with 82–76% A for the first 10 min, then in 2 min 58% A, in 6 min 40% A, and finally returned to 82% A in 5 min. This was followed by isocratic elution for 2 min. Calibration curve was performed with lycopene samples prepared freshly on a daily basis. The retention time of lycopene was 15 min. Each analysis was performed in triplicate. Lycopene standard was diluted to obtain the following ppm concentrations: 1, 5, 25, 50 and 100. A good linear fit range was found, the regression equation and correlation coefficient were respectively: y = 11660x + 776.75, R = 0.9998.

The BPW extracts’ phenolic profiles were analyzed by high-performance liquid chromatography (HPLC) with photodiode array (PDA) detection. The analysis was conducted using an HPLC system (Shimadzu Corporation, Kyoto, Japan) with a 250 mm × 4.6 mm Gemini C₁₈ column from Phenomenex (Madrid, Spain) and a guard column with the same characteristics that was kept at 25 °C, according to the method described by Pinto et al. [26 (link)]. The mobile phase was composed of methanol (A) and water (B), both acidified with 0.1% formic acid, and the elution gradient was carried out at a flow rate of 1.0 mL/min. A PDA at 280, 320 and 360 nm was used to detect and quantify the individual phenolic compounds. The extracts were prepared in methanol: water 20:80 v/v and filtered through a 0.22 μm-pore-sized nylon filter before injection. Standards were prepared in a 50:50 v/v methanol/water mixture in a linearity range of 1 to 200 mg/L. Each extract was analyzed three times, and the results were expressed as mg of compound/g of dw.

The monosaccharide compositions of crud LJLP were measured by HPLC with procedure of previous report (Liu et al., 2017), which had a few modifications. 10 mg of LJLP was treated with 3 mol/L trifluoroacetic acid (TFA) at 100°C for 6 hr. After removal of TFA, other impurities were stripped from the residue by washing with methanol for 3 times and then redissolved in 2 ml water. After adding 0.2 ml 0.5 mol/L 1‐phenyl‐3‐methyl‐5‐pyrazolone (PMP) methanol solution, 0.2 ml 0.3 mol/L NaOH solution was mixed with the hydrolysate and incubated for 45 min at 65°C. The next step is the acid neutralization treatment of the mixture, followed by adding 1 ml trichloromethane and extracting for 3 times. The aqueous phase was collected to be determined by HPLC.
The determination process was carried out with an Agilent 1,260 HPLC system. The chromatographic conditions were as follows: Phenomenex Gemini C₁₈ column (250 mm × 4.6 mm, 5 μm) with the temperature of 35°C; flow rate 0.8 ml/min; and detector wavelength 245 nm. As for the eluting solvents, it was a mixture of acetonitrile (82:18, v/v) and 0.05 M phosphate buffer (pH 6.8). The injection volume was 10 μl.

As rutin and quercetin were reported to be the major constituents of the Euonymus alatus leaf, the quantities of those compounds in the EAE were determined by HPLC analysis. HPLC analysis was performed on the reversed-phase Gemini C18 column supplied by Phenomenex (250 × 4.6 mm, 5 μm, Torrance, CA, USA) with 1% acetic acid (40:15:45) MeOH:ACN:DW as the mobile phase and a flow rate of 1 mL/min. The column temperature was maintained at 30 °C throughout the analysis. The chromatograms were procured at a wavelength of 360 nm, and the injection volume was 10 μL. EAE was injected three times each, and the averages of the peak areas on the chromatograms were obtained and extrapolated to the standard curve of known concentrations of rutin and quercetin for quantification.