Hyperclone c18 ods column

Dried extracts were dissolved in 70 μl 5 mM sodium phosphate buffer (Na₂HPO₄ and NaH₂PO₄) pH 6.2. The samples were centrifuged at 20,000 g for 30 min at 4°C. Amino acids were measured by precolumn derivatisation with ortho-phthaldialdehyde in combination with fluorescence detection (excitation wavelength 330 nm; emission wavelength 450 nm) (Lindroth and Mopper, 1979 (link); Rajendra, 1987 (link); Watanabe et al., 2013 (link)). Elution was achieved on a Hyperclone C18 ODS column (Phenomenex) connected to a HPLC system (Dionex), applying a solvent gradient with increasing hydrophobicity (buffer A: 0.2% [v/v] tetrahydrofolate, 8.5 mM sodium phosphate buffer, pH 6.8; buffer B: 32.5% [v/v] methanol, 20.5% [v/v] acetonitrile, and 18.5 mM sodium phosphate buffer, pH 6.8; flow: 1.0 mL/min; 0–3 min: 100% A, 5 min: 93% A, 7% B; 14 min: 60% A, 40% B; 18 min: 55% A, 45% B; 28–31 min 100% B, 31.3–34 min: 100% A).

The ADP and ATP content was measured by High Performance Liquid Chromatography (HPLC) with 20 mg of fresh weight plant materials (Zhang et al., 2020 (link)). Briefly, 0.2 ml of 0.1 M HCL was mixed with the plant materials on ice. 15 μl extract/standard (different concentration) was mixed with 77 μl CP Buffer [62 mM, citric acid monohydrate and 76 mM (Na)₂HPO₄ × 2H₂O] and 8 μl 45% chloroacetaldehyde and incubated at 80°C for 10 min. The mixture was then centrifuged at 16,000×g at 20°C for 30 min. 90 μl of the supernatant was then measured by the HPLC [Hyperclone C18 (ODS) column (Phenomenex)]. The result was calculated using the standard ADP and ATP gradient.

HPLC analyses were carried out using a Shimadzu UFLC XR device at 40°C, a flow rate of 1.0 mL min⁻¹, injection volume was 10 μL, detection wavelength 254 nm. The stationary phase was a Phenomenex® HyperClone ODS (C18) column, 120 Å, 5 μm, 150 × 4.60 mm; mobile phase A: H₂O + 0.9% AA + 0.1% FA; mobile phase B: CH₃CN + 0.9% AA + 0.1% FA; 0 min: 90% A to 30 min: 2% A, 30-35 min: 2% A.

Ethyl acetate honey extracts in methanol solution were diluted fivefold with water, filtered (0.45 µm) and analyzed by HPLC (high-performance liquid chromatography) (Agilent 1100 Series HPLC-MSD, Agilent Technology) connected to a diode array detector (DAD) as described earlier [21] . Analyses were performed on a HyperClone ODS (C18) column (2.0 mm, 200 mm, 5 µm, Phenomenex) using the following gradient run (0.2 ml/min) with methanol (A) and 0.3% formic acid (B): 90–75% B from 0 to 5 min, 75–69% B from 5 to 20 min and 69–40% B from 20 to 40 min. The run was stopped at 65 min and post time was 25 min. Phenolic acids were quantified at 260 nm or 320 nm, and 5- (hydroxymethyl)-2-furaldehyde (HMF) at 280 nm. The authentic chemical compounds used for identification and quantification of phenolic acids were protocatechuic, benzoic vanillic, syringic, sinapic, trans-cinnamic, p-coumaric, caffeic, p-hydroxybenzoic, gallic, ferulic and chlorogenic acid.