Inertsil ods 3 c18 column

Freshly harvested spores (5 × 10⁴) of representative isolates of the gene knockout strains, ΔmrpigAup1, ΔmrpigAup2, ΔmrpigA, ΔmrpigO, ΔmrpigP, ΔmrpigPdown1, ΔmrpigPdown2, and the wild-type strain M. ruber M7 were spread on cellophane membranes on PDA plates, and cultivated at 28°C for 10 days. The mycelia were harvested by scraping from the membranes, freeze-dried, and ground in a mortar with a pestle under liquid nitrogen. The mycelia powder (0.05 g) was suspended in 1.5 mL methanol, incubated at 65°C for 1 h, then centrifuged at 10,000 × g for 10 min to collect the supernatant for analysis. HPLC was performed following the method described by Liu Q. et al. (2016) (link) on a Waters system fitted with an Inertsil ODS–3 C18 column (250 × 4.6 mm, 5.0 μm, GL Sciences). The mobile phases consisted of water (A), acetonitrile (B), and 0.5% phosphoric acid in water (C). The flow rate was kept at 0.8 mL/min. The system was run with the following gradient program: from 40 to 30% A for 3 min, from 30 to 5% A for 22 min, 5% A for 5 min, from 5 to 40% A for 1 min, and 40% A for 5 min. C was kept constant at 5% throughout the program. Absorbance was monitored with a 2487 UV/Vis Detector (Waters) at 190- to 700-nm wavelength. Metabolites were identified based on comparison to authentic standards (Chen et al., 2017 (link), 2019 (link)).

To determine the concentration of p-CA and caffeic acid, the samples cultured in SC drop-out medium were centrifuged at 12,000 × g for 5 min. After filtration through 0.22 μm filters, the supernatants were analyzed by high-performance liquid chromatography (HPLC). For samples fermented in MYPD medium, supernatants were extracted with ethyl acetate and dried with a centrifugal vacuum concentrator, as described previously (Li et al., 2020 (link)). The p-CA and caffeic acid were quantified using a CoMetro 6000 HPLC instrument (CoMetro, South Plainfield, NJ, United States), equipped with an Inertsil ODS-3/C18 column (250 mm × 4.6 mm, 5 μm; GL Sciences, Tokyo, Japan) and a CoMetro 6000 UV detector. The gradient program was performed with solvent A (5:95 acetonitrile/water, with 0.1% v/v TFA) and solvent B (acetonitrile/0.1% TFA) as the mobile phases, at a flow rate of 1 mL/min, a detection wavelength of 310 nm and an injection volume of 10 μL. The concentrations of p-CA and caffeic acid were calculated by comparison with standard solutions of p-CA and caffeic acid in absolute ethanol.

HPLC analysis was conducted on an LC-20A HPLC system with a diode array detector (Shimadzu, Kyoto, Japan). The separation of dihydroflavonol substrates and anthocyanidin products was accomplished on an Inertsil-ODS3 C18 column (5 μm, 250 × 4.6 mm, GL Science). The mobile phase consisted of 0.1% (v/v) formic acid (A) and acetonitrile containing 0.1% (v/v) formic acid (B). The gradient profile was optimized as follows: 0 min, 95% A/5% B; 30 min, 45% A/55% B; 45 min, 35% A/65% B; 50 min, 0% A/100% B; 52 min, 95% A/5% B; and 60 min, 95% A/5% B. The flow rate was 1 mL·min⁻¹, and the column temperature was maintained at 30 °C. The detection wavelength was 288 nm for dihydroflavonols and 520 nm for anthocyanidins.

The extracts were filtered through a 0.45-µL syringe filter before being injected into a high-performance liquid chromatograph (HPLC) with a UV detector (1260 Infinity II, Agilent Technologies, Santa Clara, CA, USA) to analyse the GSAC, SAC and 5-HMF contents. The HPLC column was an Inertsil ODS-3 (C18 column, 5 µm, 4.6 × 250 mm, GL Sciences Inc., Japan). The isocratic HPLC system was used for analysis. The mobile phase was prepared by mixing 50 mM phosphate buffer (pH 2.8) and methanol in a ratio of 85:15 v/v, respectively, all HPLC grade. The flow rate of the mobile phase was 1 mL/min, with a sample injection volume of 10 µL and column temperature of 25 °C. The detection wavelength was 205 nm for GSAC and SAC detection^{46 (link)} and 285 nm for 5-HMF detection⁴⁷ .

HPLC analysis was carried out using the Shimadzu HPLC system (Shimadzu Corp.; Kyoto, Japan), consisting of quaternary pumps (10ATVP), an online degasser (DGU-14A), an auto-injector (SIL-10ADVP), a column oven (CTO-10ASvp), a fluorescence detector (RF-10AXL), and the LC Solution software (Shimadzu, Kyoto, Japan). The excitation and emission wavelengths were 360 and 450 nm, respectively. Separation was performed on an Inertsil ODS-3 C18 Column (250 mm × 4.6 mm, 5 µm) (GL Sciences; Tokyo, Japan) equipped with Ascentis^® C18 Supelguard^™ (2 cm × 4 mm, 5 µm) (Supleco; Bellefonte, PA, USA). The column temperature was controlled at 35 °C. The injection volume was 20 µL. The chromatographic separation was performed using a mobile phase with a water-to-methanol-to-acetonitrile ration of 6:3:2 (v/v/v), containing 2% (v/v) acetic acid as solvent A and 1% (v/v) acetic acid in water as solvent B, at a flow rate of 1 mL/min. The gradient elution program was: 0 to 5 min 45%A; 5 to 7 min, 45 to 100%A; 7 to 20 min 100%A; 20 to 22 min, 100 to 45%A; and 22 to 30 min, 45%A.

The accumulation of flavonoid aglycones in the petals was analyzed as described previously [57 (link)], with some modifications. Briefly, 100 mg of ground petal sample was subjected to a 2-h acid hydrolysis in 300 μL of 50% methanol containing 2 N HCl at 90°C. After centrifugation at 15,000× g for 10 min at 4 °C, the supernatant was transferred to a new tube. The remaining pellet was rinsed with 200 μL acid hydrolysis solution, which was then combined with the first extract. A 10 μL aliquot of the extract was used in a HPLC analysis performed on an LC-20A HPLC system (Shimadzu, Kyoto, Japan) equipped with an Inertsil-ODS3 C18 column (5 μm, 250 × 4.6 mm; GL Science, Eindhoven, Netherlands). The chromatographic separation was carried out using 0.1% formic acid in water (solution A) and 0.1% formic acid in acetonitrile (solution B) with the following gradient conditions; 0–30 min, linear gradient of 5–55% (v/v) solution B; 30-45 min, linear gradient of 55–65% (v/v) solution B; and 45–50 min, linear gradient of 65–100% (v/v) solution B at a flow rate of 1 mL∙min⁻¹. The temperature of the column was maintained at 40 °C. A diode-array detector was used for compound detection. The spectra of the compounds were recorded between 210 and 800 nm, and the peak corresponding to each compound was identified by comparing the retention times and UV spectra with those of the standards.