Kinetex biphenyl

Fractioning was performed with an HPLC Shimadzu LC-20AT coupled with a DAD SPD-20A detector. The semi-preparative column was in a Kinetex biphenyl stationary phase (300 mm * 4.6 mm, 5µ particle of 100 A, Phenomenex, Torrance, CA, USA). Analysis was performed in gradient mode at a flow rate of 1 mL/min. The gradient started from 0% to 100% methanol during 55 min. The column was washed for 25 min with 100% water. All fractioning were realized four times with 100 µL of 10 mg/mL extracts.

Quantification measurements were performed on Agilent 6495B Triple-Quad LC/MS coupled to 1260 Infinity II LC system (Agilent Technologies, Inc., Santa Clara, CA, USA). The resulting data were quantified and processed using MassHunter Workstation Software ver. B.09.00 (Agilent Technologies). The mass spectrometer was operated in “Dynamic MRM” mode, with following source parameters - gas temp 160 ^oC, gas flow 14 L/min, sheat gas temp 390 ^oC and shear gas flow 12 L/min. The capillary voltage was set to 2800 V for positive mode and 3000 V for negative mode, with nozzle voltage at constant 0 V.
The chromatographic columns, Kinetex Biphenyl (100 × 2.1 mm, 1.7 μm) and Kinetex Polar (150 × 2.1 mm, 2.6 μm), were from Phenomenex Ltd. (Phenomenex, Torrance, CA, USA). Kinetex Polar, with inlet filter, was used for all presented work. Gradient employed for all analyses takes 23 min at flow of 0.53 mL/min and consists of mobile phase A (dH₂O with 0.03% FA (v/v)) and B (acetonitrile). Its gradient time-frame was: 0 min − 99% A, 8 min – 91.5% A, 9 min – 90% A, 15 min – 88% A, 20 min – 2% A, 21 min – 99% A, 23 min – 99% A. The column compartment was heated to 53 ^oC.

Species composition was identified by LC-MS/MS according to our recent report^{26 (link)}. Briefly, UPLC separation was performed on a Kinetex Biphenyl (2.6 µm, 150 × 2.1mm) column (Phenomenex, Torrance, CA, USA) by using a binary mobile phase of water and methanol (MeOH). The elution program (flow rate 0.3 mL/min) was from 40% to 80% MeOH in 2 min, then 100% MeOH in 13 min and 100% MeOH for other 7 min. Full scan mass spectra (ESI in negative ion mode) were acquired in the m/z range 150–2000 with a resolution of 70000. MS/MS experiments on monoisotopically isolated precursor ions were performed with a normalized collision energy between 20 and 40.

For the isolation, the same HPLC system was used as described above. Column: Kinetex^® Biphenyl, 100 Å, 250–4.6 mm (5 µm, same material pre-column; Phenomenex, Torrance, CA, USA); Injection volume 10 µL; oven temp.: 25 °C; auto sampler temp.: 10 °C; detection wavelength: 340 nm; flow: 1.2 mL/min; A = H₂O + 0.1% TFA, B = acetonitrile + 0.1% TFA; gradient: 0–2 min 10% B, 2–15 min 10% B → 70% B, 15–16 min 70% B → 10% B, 16–20 min 10% B. The peaks of the metabolites (retention times: 6.85 min for IO-G1, 7.86 min for IO-G3 and 8.4 min for IO) were collected separately and subsequently purified by another solid phase extraction. Additionally, 0.15 mg of IO-G1 and 0.4 mg of IO-G3 could be observed as purified metabolites.

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.

The HPLC system consists of: Elite LaChrom with an L2200 autosampler, L2130 pump, L2350 column oven, L2444 DAD, and EZChrom Elite 3.1.7 software (Hitachi, Tokyo, Japan); column: Kinetex^® Biphenyl, 100 Å, 250 × 4.6 mm 5 µm (same material pre-column; Phenomenex, Torrance, CA, USA); injection volume 20 µL; oven temp. 25 °C; auto sampler temp.: 10 °C; detection wavelength: 340 nm; flow: 1.2 mL/min; A = H₂O + 0.1% TFA, B = acetonitrile + 0.1% TFA; gradient: 0–2 min 10% B, 2–15 min 10% B → 70% B, 15–16 min 70% B → 10% B, 16–20 min 10% B. All samples were filtered (Phenex RC Membrane 0.2 um, Phenomenex, USA) before injection.