Kinetex xb c18 column

Analysis was performed using an ultra-high-performance liquid chromatography coupled with tandem mass spectrometry method (UHPLC─QqQ─MS/MS). Chromatographic separation was carried out with the use of a Kinetex^® XB-C18 column (150 × 2.1 mm i.d., particle size 2.6 μm; Phenomenex, Torrance, CA, USA). The mobile phase consisted of 0.1% formic acid and 10 mM ammonium formate in water (A) and 0.1% formic acid in acetonitrile (B). The gradient (at a constant flow of 0.4 mL/min) applied was as follows: 0 min, 5% B; 12 min, 98% B; 14 min, 98% B; and 15 min, 5% B. Return to the initial gradient compositions was performed for 5 min. The injection volume was 2.0 μL. Detection was achieved using a triple quadrupole mass spectrometer (Shimadzu 8050, Kyoto, Japan) equipped with an electrospray ionization (ESI) source. Determination of the substances was carried out in the multiple reaction monitoring (MRM) mode. The following MS parameters were fixed: nebulizing gas flow, 3 L/min; heating gas flow, 10 L/min; interface temperature, 250 °C; desolvation line temperature, 200 °C; heat block temperature, 350 °C; and drying gas flow, 10 L/min. A summary of precursor, product ions, collision energies, Q1–Q3 pre bias voltages and retention time for each compound are presented in Table 1.

The standards and ginger extracts were analyzed on a Shimadzu UPLC system (Nexera X2, Shimadzu, Kyoto, Japan) using the specifications of [30 (link)]: injection volume, 2 μL; flow rate, 0.3 mL/min; retention time, 30 min; wavelength, 280 nm; and eluents, 0.1% acetic acid in water (A) and 0.1% acetic acid in acetonitrile (B). The gradient elution had the following profile: 0–0.5 min, 90% A; 0.5–2.5 min, 60% A; 2.5–4.5 min, 45% A; 4.5–6.0 min, 40% A; 6.0–11.5 min, 35% A; 11.5–13.0 min, 30% A; 13.0–14.5 min, 25% A; 14.5–16.0 min, 20% A; 16.0–17.5 min, 15% A; 17.5–25.0 min, 10% A; 25.0–30.0 min, 90% A. In the UPLC analysis, a Kinetex XB.C18 column (1.7 μm, 150 × 2.1 mm, Phenomenex, Torrnace, CA, USA) was used and the column temperature was maintained at 30 °C. The concentrations in each sample were calculated by comparing their response with the corresponding standard curve. The calibration curve for this method has the six calibration standards solutions (6G, 8G, 10G, 6S, 8S, and 10S) each covering the range of 10–1000 μg/mL (calculation by the equations (R2 coefficients): 6G; y = 942.59x + 16,123 (0.9993), 8G; y = 781.68x + 9155 (0.9994), 10G; y = 752.09x + 10,667 (0.9994), 6S; y = 1103.3x + 13,039 (0.9994), 8S; y = 944.76x + 16,414 (0.9994), 10S; y = 558.24x + 6124 (0.9995)).

The assessment of chemical composition of each extract (RE, LBE) was carried out on a 1260 Infinity chromatograph (Agilent, Santa Clara, CA, USA) consisting of binary pump, a column oven, and photo-diode array (PDA) detector over 55 min period. The separation was performed using a Kinetex XB C18 column (150 × 3 mm, 2.6 µm) (Phenomenex, Torrance, CA, USA). The mobile phase was 0.1% (v/v) FA in water (A) and 0.1% FA in acetonitrile (B). The separation was achieved by a gradient of 0–2 min 1% B; 2–20 min 1–20% B; 20–40 min 20–75% B; 40–45 min 75–95% B; 45–48 min 95% B; 48–49 min 95–1% B; 49–55 min 1% B. The flow rate was 0.5 mL/min and the column temperature was maintained at 25 ± 0.8 °C. The UV–Vis spectra was recorded from 190 to 540 nm with selective wavelength monitoring at 280 nm. Mass spectrometry (MS) detection was carried out on a 6230 MS/TOF mass spectrometer (Agilent, Santa Clara, CA, USA) equipped with an electrospray ionization source with Agilent Jet Stream thermal focusing. The parameters used for ionization source were set as follows: drying and sheath gas flow: 12 L/min; nebulizer: 35 psi; source temperature 350 °C; ion spray voltage 4500 V for the positive mode analysis. The data were collected in 115–1900 m/z range and processing was performed using Mass Hunter qualitative analysis software.

NMR spectra were recorded either in CDCl₃ or in DMSO-d₆ on a Bruker AVANCE III HD 400 (resonance frequency 400.17 MHz for ¹H). Optical rotation was determined using a PolAAr31 polarimeter (Optical Activity Ltd., Ramsey, UK). RP-HPLC separations were performed using an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector (PAD). Analytical chromatographic separations were carried out either on a Kinetex XB-C18 column (4.6 × 250 mm, 5 μm total particle size; Phenomenex, Torrance, CA, USA; nonpolar compounds) or on a Zorbax Eclipse XDB-C18 column (4.6 × 150 mm; Agilent Technologies, USA; phenolic compounds). Semipreparative RP-HPLC was conducted on a Vertex Plus column (Eurospher II 100-5 C18, 8 × 250 mm; Knauer GmbH, Berlin, Germany), with an isocratic elution, using MeOH-H₂O mixtures of different polarities, at a flow rate of 1.0–2.0 mL min⁻¹. The column was coupled to a Knauer P4.1S pump and a dual wavelength UV/VIS detector operating at 210 and 260 nm. Conventional column chromatography was carried out on Silica gel 60 (0.063–0.2 mm, Merck, Germany) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). TLC separations were performed using precoated plates (Silica gel 60 without fluorescence indicator, Art. No 5553, Merck, Germany).

HPLC separations were conducted on an Agilent 1100 HPLC system with a Kinetex XB-C18 column (4.60 × 150 mm, 5 μm in particle size and 100 Å in pore size; Phenomenex Inc., Torrance, CA, USA). For the purification of ODNs, a triethylammonium acetate buffer (50 mM, pH 6.8, Solution A) and a mixture of solution A and acetonitrile (70/30, v/v, Solution B) were employed as mobile phases. The flow rate was 0.8 ml/min and the gradient profile was 5–25% B in 5 min followed by 25–55% B in 60 min. The HPLC traces for the purifications of the 12-mer lesion-containing ODNs are shown in Supplementary Figure S19 and the electrospray ionization-mass spectrometry (ESI-MS) and tandem MS (MS/MS) of the purified lesion-containing ODNs are displayed in Supplementary Figures S20–S26.