2998 diode array detector

ESIMS was performed using an Agilent 1100 series LC/MSD ion trap mass spectrometer (Agilent, Santa Clara, CA, USA). The 1D- and 2D-NMR spectra were obtained in CD₃OD and DMSO with TMS as the internal standard on Varian 500 MHz and Bruker AV500-III spetrometers (Bruker Corporation, Billerica, MA, USA). Column chromatography was performed over silica gel (160–200 mesh, Qingdao Marin Chemical, Inc., Qingdao, China), RP-18 reverse phase silica gel (43–60 μm), cyanopropyl silica gel (43–60 μm) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). LPLC experiments were performed with Combiflash with a UV-detector (ISCO Companion, Lincoln, NE, USA). HPLC experiments were performed using a Waters 2545 system with 2998 diode array detector (Waters Corporation, Milford, MA, USA), using the Waters Sunfire and X-Bridge (250 mm × 10 mm i.d.) preparative columns packed with C18 (5 μm) (Alltech Associates, Inc., Bannockburn, IL, USA). TLC was carried out with glass precoated silica gel GF254 glass plates. Spots were visualized under UV light and by spraying with 8% H₂SO₄ in 95% EtOH followed by heating.

A. oryzae transformant strains were analysed for production of metabolites through HPLC-MS. Each strain was grown in 100 ml of CMP medium (35 g l⁻¹ Czapek-Dox liquid, 20 g l⁻¹ maltose, 10 g l⁻¹ peptone) at 28 °C for 10 days prior to performing metabolite extractions with ethyl acetate, followed by concentration of the crude extract in vacuo methanol. Crude extracts were dissolved in methanol and analysed by HPLC-MS including a Waters 2767 HPLC system with a Waters 2545 pump, a Phenomenex LUNA column (2.6 μ, C18, 100 Å, 4.6 × 100 mm) and a Phenomenex Security Guard precolumn (Luna C5 300 Å) for reverse-phase chromatography. UV absorbance was detected between 200 and 400 nm through a Waters 2998 diode array detector while a mass range between 150 and 800 Da in positive and negative ion mode was scanned by the Waters Quattro Micro spectrometer. Chromatography was achieved using a gradient of solvents ((A) HPLC grade H₂O containing 0.05% formic acid; (B) HPLC grade CH₃CN containing 0.045% formic acid) with the following programme: 0 minutes, 20% B; 15 minutes, 90% B; 16 minutes 95% B; 17 minutes 95% B; 18 minutes 10% B, 20 minutes 10% B. The flow rate was set at 1 ml min⁻¹.

Sample solutions (20 μl) were analyzed using a Waters LCMS system comprising of a Phenomenex KINETEX column (2.6 μ, C18, 100 Å, 4.6 × 100 mm) equipped with a Phenomenex Security Guard precolumn (Luna C5 300 Å) eluted at 1 ml/min. Detection was by Waters 2998 Diode Array detector between 200 and 600 nm; Waters 2424 ELSD and Waters SQD-2 mass detector operating simultaneously in ES⁺ and ES⁻ modes between 100 and 800 m/z. Solvent A was HPLC grade water containing 0.05% formic acid, and solvent B was HPLC grade acetonitrile containing 0.05% formic acid. Gradient program was as follows: 5–95% of B over 15 min. 0 min, 5% B; 1 min, 5% B; 2 min, 40% B; 15 min, 95% B; 17 min, 95% B; 18 min, 5% B; 20 min, 5% B; flow rate 1 ml/min.

A Waters E2695 HPLC coupled with a 2998 diode array detector (Waters, Milford, MA, USA) was used to analyze the phenolic compounds in the ‘Hangbaiju’ samples. A Phenomenex Luna C₁₈ column (250 mm × 4.6 mm, 5 µm, Torrance, CA, USA) was used to separate the phenolic compounds under a 0.8 mL/min flow rate. The mobile phase consisted of (A) acetonitrile and (B) 0.1% phosphoric acid in water (v/v). The sample injection volume was 10 µL, and the column was maintained at 35 °C during the elution program. The gradient was programed as follows, 0 to 11 min, 10% A to 18% A; 11 to 32 min, 18% A isocratic; 32 to 40 min, 18% A to 30% A; 40 to 48 min, 30% A to 35% A; 48 to 50 min, 35% A to 40% A; 50 to 55 min, 40% A isocratic; 55 to 60 min, 40% A to 70% A; and 60 to 70 min, 70% A to 10% A. Phenolic compounds were identified by comparing their retention time with corresponding external standards. The quantification was also conducted using the standards.

Sample analysis was performed on a liquid chromatograph Alliance 2695, equipped with a 2998 Diode Array Detector (Waters, Milford, MA, USA) and Software Empower 3. The separation was carried out on a 5 micron (100 Å, 250 × 4.6 mm) C-18 reverse phase Kromasil column (Ale, Bohus, Sweden) at room temperature. The mobile phase was phosphoric acid at 5 mM (solvent A) and MeOH (solvent B), at a flow rate of 1 mL/min. The elution gradient was as follows: a linear gradient of 85–80% solvent B (0–5 min), 60% B (6–10 min), 70% B (11–20 min), 80% B (21–25 min) and, finally, 85% B (26–30 min). The injection volume was 20 µL, and the 5CQA was detected at a wavelength of 325 nm. This method was adapted from Fujioka and Shibamoto (2008) [60 (link)]. Sample chromatograms were compared with those of the 5CQA standard for identification. The measurements were carried out in triplicate. Instrumental calibration: Eight different levels of concentration were employed for 5CQA. The Pearson correlation coefficient (r) was calculated to estimate the type of adjustment of the experimental points in the calibration curve, and subsequently, statistical analyses with Student’s t-test [61 ] and variance analysis were performed, to verify its significance.

Structural characterization of flavonols in bayberry leaves was conducted by HPLC, LC-MS, and Nuclear Magnetic Resonance (NMR) Spectroscopy. The operated HPLC system was equipped with a e2695 pump, a 2998 diode array detector, and a SunFire C18 analytical column (5 μm, 4.5 × 250 mm) (Waters, USA). The mobile phase solution was the same as that of pre-HPLC. Gradient program was as follows: 0-40 min, 10%-38% of B; 40-60 min, 38%-48% of B; 60-70 min, 48%-100% of B; 70-75 min, 100%-10% of B; and 75-80 min, 10% of B. Flow rate was set at 1 mL/min, and injection value was 10 μL.
LC-MS identification was carried out as our previous report [11 (link)]. Chromatographic separations were performed under the same gradient procedure as HPLC using an Agilent 1290 Infinity system (Agilent Technologies, USA) equipped with an X-Bridge C18 analytical column (4.6 × 250 mm). MS analysis was conducted by an Agilent 6460 triple quadrupole mass spectrometer coupled to an electrospray ionization source (Agilent Technologies, Santa Clara, CA, USA) and operated in the negative ionization mode.
The ¹³C-NMR spectra were measured in DMSO-d6 at 25°C on an Agilent 600 MHz instrument. Solvent residual peak δ 39.52 was used to calibrate all ¹³C chemical shifts in the present study. The chemical shifts of NMR were calculated and extracted by MestReNova (version 6.1.0).