Zorbax eclipse xbd c18 column

Defatted samples (250 mg) of fermented and dried beans were subjected to extraction of methylxanthines (theobromine, theophylline and caffeine) and monomeric phenolic compounds (catechin and epicatechin), according to previous studies [14 (link),50 (link),51 (link)]. Aliquots of the extracts (20 µL) obtained were injected into a chromatograph equipped with a diode array detector (HPLC-DAD) at 280 nm (1260 Infinity Agilent Technologies, La Jolla, CA, USA) with Zorbax Eclipse XBD-C18 column (4.6 × 150 mm, 5 µm, Agilent Technologies, La Jolla, CA, USA) at 25 °C, at flow rate of 1.2 mL/min of mobile phases (A) water/acetonitrile (99.8: 0.2, v/v) and (B) methanol, in gradient mode according Chagas Junior et al. [14 (link)].
The tentative identification of the compounds was based on the retention time of each analyzed standard and quantified through the external analytical curves: theobromine (from 3.125to 50 µg/mL, R² ≥ 0.99, LOQ = 0.64 mg/g), theophylline (from 3.125to 100 µg/mL, R² ≥ 0.99, LOQ = 0.02 mg/g), caffeine (from 3.125 to 100 µg/mL, R² ≥ 0.99, LOQ = 0.04 mg/g), catechin (from 3.125 to 50 µg/mL, R² ≥ 0.99, LOQ = 0.31 mg/g) and epicatechin (from 3.125to 100 µg/mL, R² ≥ 0.99, LOQ = 0.11 mg/g) [11 (link)]. All experiments were performed in duplicate.

Ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3, Rd was added to methanol and dissolved to make mixed control solutions with concentrations of 101 μg/ml, 103 μg/ml, 102 μg/ml, 103 μg/ml, 101 μg/ml, 98 μg/ml, 98 μg/ml, and 99 μg/ml. The solution was shaken well, then filtered through a 0.22 μm microporous membrane and set aside. Next, 2 g each of ginseng core and ginseng bark powder were weighed to prepare a lyophilized powder of the n-butanol layer in a 2 ml volumetric flask. Methanol was added and dissolved to fix the volume to the scale. The solution was then shaken well and filtered through 0.22 μm microporous membrane. The test solution was obtained from continued filtration.
An Agilent ZORBAX Eclipse XBD-C18 column (4.6 mm × 250 mm, 5 μm) was used; acetonitrile was used as mobile phase A and water as mobile phase B for elution (0 min–30 min, 19% A; 30 min–35 min, 19%–20% A; 35 min–50 min, 20%–26% A; 50 min–55 min, 26%–28% A; 55 min–110 min, 28%–29% A; 110 min–140 min, 29%–33% A). The flow rate was 0.8 ml/min; the detection wavelength was 203 nm; the column temperature was 40°C, and the injection volume was 10 μl.