Agilent 1120 compact lc

A quantitative analysis of phenolic compounds was performed by using high-performance liquid chromatography (HPLC) in accordance with Cichova et al.^{35 (link)}. The analytical system used was an Agilent 1120 Compact LC (Agilent Technologies, Germany) with a UV 280 nm detector and equipped with a 4.6 × 250 mm column Luna C18, 100 Å, 5 µm (Phenomenex, USA). The flow rate was 1 mL/min, the temperature of the column oven was 25 °C, and the injection volume was 20 µL. The mobile phase consisted of water–formic acid (95:5, v/v) as solvent A and acetonitrile–solvent A (60:40, v/v) as solvent B. A gradient program was carried out as follows: 0 min, 100% A; 10 min, 85% A; 20 min, 82% A; 50 min, 0% A; 65 min, 100% A; 70 min, 100% A.
The analytical standards were prepared at a stock concentration of 200 μg/mL. The preparation of the sample before running the HPLC auto-sampler consisted of injecting 5 mL of methanol and 10 mL of water in a Sep-Pak C18 cartridge (Phenomenex, USA); then, 5 mL of orange peel extract, adjusted to pH 1.6, was passed through the cartridge. Phenolic compounds were collected with 12 mL of acetone, which was then evaporated with nitrogen and dissolved with 1 mL of HCl 0.6 M/aqueous methanol 75%. The results are expressed in mg per 100 g of orange peel.

Polyamines except for thermospermine were purchased from Nakalai Tesque (Tokyo, Japan). Thermospermine was kindly provided by Dr. Masaru Niitsu. For HPLC, polyamines were extracted from seedlings in 3% perchloric acid and benzoylated according to Naka et al [36 (link)]. The resulting samples were injected into a reverse-phase column (TSK-gel ODS-100V, 5 μm, 4.6 × 150 mm, Tosoh, Tokyo, Japan) and eluted with 42% (v/v) acetonitrile at a flow-rate of 0.2 mL/min for 30 min using the Agilent 1120 Compact LC. The benzoyl polyamines were detected at 254 nm.

Mycelia from 5 mL culture broth were harvested by filtration on filter paper, dried overnight at 100°C and weighed. Filtered medium was passed through a 0.2 μm PVDF membrane (Acrodisc® LC; Pall, Life Sciences) and injected into an AQUASIL C18 140×4.6 mm column (Thermo Scientific) connected to an HPLC device (Agilent 1120 Compact LC) to determine extracellular nucleoside concentrations by monitoring absorbance at 260 nm. The separation of nucleosides was achieved by using an isocratic flow of phosphate buffer, pH 5.5, plus 0.5% of acetonitrile. Quantification was carried out using a calibration curve prepared with pure standards of inosine and guanosine (Sigma-Aldrich). All analyses were performed using three biological replicates.
Metabolism quenching and metabolite extraction were carried out as described [28 (link),29 (link)], using the mycelia obtained from 7 mL of culture broth. Briefly, samples were quenched by the addition of methanol at −40°C, followed by boiling ethanol, and extracted upon the addition of acetonitrile by mechanical homogenization and subsequent chloroform/chloromethane organic extraction steps. The intracellular concentrations of the metabolites in the purine pathway were determined following previously published methodologies [30 (link)].