Methanol for hplc

Methanol and brutanol were obtained from Duksan Pure Chemicals Co. Ltd., Ansan, South Korea, for use in extracting saponin. Methanol for HPLC was obtained from Sigma-Aldrich, St Louis, MO, USA. Among the various ginsenoide types, the RG, Rb1 and Rc (Ace EMzyme Co., Anseong-si, Korea) were obtained as standards for ginsenoside HPLC analysis. Additionally, carbazole was obtained from Samchun Pure Chemicals Co., Seoul, Korea, for use in determining acidic polysaccharide content.

Microfiltered and ultrapure water was used for the preparation of the solutions via the Merck Millipore ZRQS0P3FR Direct system. All solvents and reagents used in the experiments were of a high degree of purity. Ethanol ≥99.9% ACS for analysis, methanol for HPLC, anhydrous 95% n-hexane, chloroform for chromatography, 1-butanol ACS reagent ≥99.5%, hydrochloric acid 37% RPE for analysis, potassium bicarbonate, anhydrous sodium carbonate for analysis, gallic acid ACS for analysis and Sudan IV reagent were purchased from Sigma-Aldrich Chemical Company (Milan, Italy). Folin-Ciocălteu reagent and hypergrade acetonitrile for LC-MS were purchased from Merck Millipore GmbH (Milan, Italy). 2,2-diphenyl-1-picrylhydrazyl (DPPH·) was obtained from Alfa Aesar (from Thermo Fisher Scientific companies in Rodano, Milan, Italy). Polyphenol standards (malvidin-3-O-glucoside, naringin, catechin, quercetin, gallic acid, vanillic acid, caffeic acid, ferulic acid), amino acid standard mixture, methanol, acetic acid, gallic acid, were purchased from Merck (Darmstadt, Germany); 2-propanol and acetonitrile (ACN) from Honeywell (Charlotte, USA), formic acid by J.T. Baker (Rodano, Italy).

The following standards and chemicals were used: chlorogenic acid (Dr Ehrenstorfer GmbH, Augsburg, Germany), caffeine (Sigma-Aldrich, Prague, Czech Republic), methanol for HPLC (Sigma-Aldrich, Prague, Czech Republic), and acetic acid 100% for HPLC (Sigma-Aldrich, Prague, Czech Republic).
The sample of ground coffee (7 g) was added into a beaker (250 mL), and the caffeine and CQA were coextracted for 5 min with 100 mL of hot (90 °C) demineralised water. Then the sample was filtered through the paper filter. Coffee extracts were analysed by the HPLC method described in Yoe-Ray et al. [21 (link)] with the following modifications. caffeine and CQA were separated on the column Kinetex, Biphenyl, 150 × 4.6 mm, particle size 5 µm (Phenomenex, Torrance, CA, USA) with the guard column (SecurityGuard ULTRA Cartridge, UHPLC Biphenyl, for 4.6 mm ID columns, Phenomenex) using a liquid chromatograph Agilent 1260 Infinity II (Agilent Technologies, Santa Clara, CA, USA) with quaternary pump and diode array detector. The column was thermostated at 35 °C. Methanol/acetic acid 1% (20:80 v/v) was used as a mobile phase. The flow rate of the mobile phase was 1 mL min⁻¹. The separated CQA and caffeine, respectively, were detected at 273 nm. The calibration curve was used for the quantitative determination; the calibration curve points ranged from 10 to 100 µg mL⁻¹.