E2695 hplc system

The HPLC system was comprised of a Waters e2695 HPLC system equipped with a Waters SunFire^TM C₁₈ column (250 mm × 4.6 mm, 5 μm, Waters, Milford, MA, USA) as well as a Diode Array Detector (DAD, Waters 2998, Milford, MA, USA). The mobile phase consisted of 0.1% formic acid aqueous solution (v/v, solution A) and acetonitrile solution (solution B) in the gradient elution at 0.8 mL/min with time-course increasing of solution B to 15% B for 0–5 min, 15–20% B for 5–10 min, 20–25% B for 10–20 min, 25–35% B for 20–30 min, 35–50% B for 30–40 min, 80% B for 40–50 min, and 15% B for 50–55 min. The column temperature was set at 30 °C. The detected wavelength was set at 280 nm. Before HPLC analysis, the samples were filtered through a 0.25-μm membrane filter (Millipore, Billerica, MA, USA). Accurate amounts of standard phenolics were added to GL extracts after enzymatic hydrolysis, and they were extracted as described in Section 4.3, Section 4.4 and Section 4.5. As calculated according to the amount found and amount added, the recovery rate of these phenolics ranged from 95.31% to 101.07% (Table 3). The contents of individual phenolics were expressed as milligram per 100 g DM of GL samples.

Sixteen ginsenosides 20(S)-ginsengoside Rg2, 20 (S)-ginsengoside Rh1, 20 (R)-ginsenoside Rh1, F1, Rg6, F4, Rk3, Rh4, 20 (S)-sinsenoside Rg3, 20 (R)-ginsenoside Rg3, CK, Rk1, Rg5, 20 (S)-ginsenoside Rh2, 20 (R)-ginsenoside Rh2, and PPD standards were purchased from the Hongjiu Biotech Co., Ltd. (Jilin, China). The purity of all these standards was over 98% as indicated by the manufacturer. HPLC-grade acetonitrile was purchased from Merck Co. (Merck, Darmstadt, Germany). Wahaha purified water was purchased from Hangzhou Wahaha Group Co., LTD. Ultraviolet spectrophotometer (UV) was purchased from Shanghai Metash instruments Co., LTD. (UV-9000, Shanghai, China). Other chemicals were of reagent grade. Waters e 2695 HPLC system (Palo Alto, CA, United States).

Photosynthetic pigment content was quantified as described previously (Arnon, 1949 (link); Wei et al., 2012 (link)). The normal green tea cultivar C. sinensis (L.) O. Kuntze cv. Rougui was used as a control for pigment and ultra-structural analyses. For Chl analysis, we first performed a quick measurement of the relative amount of Chl content using a SPAD-502PLUS Chl meter (Spectrum Technologies, Konica Minolta, Japan) according to a previous report (Akita et al., 2013 (link)). Then, total Chl and carotenoids were extracted and quantified using a TU-1810PC spectrophotometer (Purkinje General Instrument Co. Ltd., Beijing, China). Lutein content was measured according to a previous report (Pan et al., 2007 (link)), and further confirmed by comparison with an authentic compound (Sigma-Aldrich, St. Louis, MO, USA) using an E2695 HPLC system (Waters, USA). The assays were conducted in triplicate.
Samples for ultra-structural observation were prepared as described previously (Du et al., 2008 ). Fresh tea leaves were cut into small pieces (1.0 × 2.0 mm) with a scalpel and fixed in glutaraldehyde solution (2.5%, v/v) overnight at 4°C. Ultrathin sections (70–90 nm) were prepared using a LKB Nova Ultra-microtome (LKB, Bromma, Sweden), examined and imaged using a JEM2100HC transmission electron microscope (JEOL, Tokyo, Japan).

The plasma concentrations of enrofloxacin were detected through Agilent 1200 high-performance liquid chromatography (HPLC) system as described previously with minor modification [13] (link), [20] (link). Briefly, the blood samples thawed out at room temperature and centrifuged at 2 000 g for 5 min, the supernatant (0.5 ml) was applied to acetonitrile and the organic and water phases were separated by centrifugation. The organic phase was evaporated to dryness under a nitrogen stream and the residue was re-suspended with mobile phase solution. Twenty microliters of the mixture was injected into the HPLC column. The composition of the mobile phase was 0.1 M phosphoric acid (adjust pH to 3.0 with triethylamine)/acetonitrile (84:16, v/v). Enrofloxacin plasma concentration was determined using a Waters e2695 HPLC system (Waters, Japan). HPLC analysis was performed on Kromasil C₁₈ HPLC Columns (5 µm, 25 cm×4.6 mm). The flow rate of the mobile phase was set to 0.85 ml/min. UV absorbance was measured at 278 nm.

Waters e2695 HPLC system (Waters, Milford, USA) was used for the chromatographic analysis. The analytical column was an XTERRA MS C18 column (4.6 mm × 250 mm, 5 µm) (Waters, Milford, USA). The mobile phase was 0.1% formic acid solution (A)—acetonitrile (B) with gradient elution (0–10 min, 10–25% B; 10–37 min, 10–25% B; 37–41 min, 35–100% B; 41–45 min, 100–10% B). The flow rate was 1.0 mL/min and column temperature was maintained at 25 °C. The detection wavelength was set at 360 nm. The injection volume was 10 μL with needle wash.

trans-resveratrol levels in berry skins and leaves were measured using HPLC method. The standard for trans-resveratrol was purchased from Sigma-Aldrich (USA). Mocked leaves and VdMYB overexpressed leaves inoculated with E. necator for 0 and 24 h. One gram samples were ground to powder using a porcelain mortar and pestle in liquid nitrogen, extracted by 15 mL extraction solution (methanol: ethyl acetate, 1:1 v/v) for 24 h at room temperature in the dark. After centrifugation at 20000 g at 4 °C for 10 min, the supernatant was evaporated at 40 °C until the solvent was volatilized completely and then dissolved in 2 ml methanol. The extract was filtered through a 0.22 μm PTFE membrane filter before resveratrol analysis. Extractable amounts of resveratrol were analyzed using a Waters e2695 HPLC system (USA). Elution was carried out with a mobile phase delivered using a Waters C18 HPLC pump at a flow rate 0.8 mL·min^− 1. A Waters 2996 UV detector was used at 306 nm. Mean values and standard deviations were obtained from three biological replicates.