1525 hplc system

Plant fresh material (0.05 g) was boiled in 2 mL of milliQ water for 10 min before being filtered using 0.22 µm filters. The soluble sugar fraction was analyzed using a Waters 1525 HPLC system coupled to a 2424 evaporative light scattering detector (ELSD), as previously described [52 (link)]. The source parameters of ELSD were the following: gain 75, data rate one point per second, nebulizer heating 60%, drift tube 50 °C, and gas pressure 2.8 Kg/cm². Samples of 20 µL were injected into a Prontosil 120-3-amino column (4.6 × 125 mm; 3 µm particle size) and were maintained at room temperature with a Waters 717 auto-sampler. An isocratic flux (1 mL/min) of 85% acetonitrile (J.T. Baker) was applied in each run for 25 min. Glucose, fructose, and sucrose standards were employed to identify peaks by co-injection. Sugars were quantified with peak integration using the Waters Empower software, and comparisons were made with the glucose, fructose, and sucrose standard calibration curves.

The phenolic compounds were identified through HPLC-PAD-ESI-microTOF/MS as explained in Aramburu et al. (13 (link)) with an Agilent 1200 HPLC system (Agilent Technologies, Wilmington, USA) provided with a binary pump (model G1312B), an autosampler (model G1367D), a degasser (model G1379B), and a PAD (model G1315C). Spectral data from all peaks were accumulated in the 200–800 nm range. The HPLC equipment was coupled with a high-resolution mass spectrometer Bruker microTOF-QII (Bruker Daltonics, Billerica, MA, USA) with an electrospray ionization source (ESI).
The polyphenols identified were quantified with a Waters 1525 HPLC system (Milford, MA, USA) equipped with a binary pump and photodiode array detector (PAD). A C18 column (250 × 4.0 mm, 5 μm particle size; Luna, Phenomenex, USA) was used. The mobile phase was composed of water (1% formic acid) (A) and methanol (B), and the flow rate was 0.8 mL/min and 20 μL injection volume. A linear gradient starting with 10% of B until reaching 30% at 20 min was used. Spectral data from all peaks were accumulated in the 200–400 nm range. The content of each phenolic compound in the SP was expressed as mg/100 g of SP.

In order to analyze THSG content, different groups of transgenic hairy roots were sub-cultured in a new liquid MS medium (pH=5.7) in the dark at 25°C and 130 rpm for 45 d prior to compounds contents assay. The stilbenes were then extracted as described by Zhu et al. (2013). First, the samples (0.2000 g each) were finely ground under liquid nitrogen and dried at 45°C. They were then soaked with 25 mL of diluted ethanol (52.9:47.1, V: V) for 12 h, and were resuspended every 2 h during this period. These extracts were centrifuged at 5,500 rpm for 20 min to remove impurities, and the supernatant was filtered through a 0.45 μm nylon filter (Jinlong, China) as test solution. Analysis of the test solution was performed on a Waters 1525 HPLC system equipped with a PDA detector, and separation was achieved using a Phenomenex 00G-4252-E0 (250 mm × 4.6 mm I.D., 0.45 μm) with the gradient program set to: acetonitrile (A): water (B) (25: 75, V: V) for 20 min. The flow rate was 1.0 mL/min, and the injection volume was 20 μL, with a detection wavelength of 320 nm. Finally, we dissolved THSG standard in diluted ethanol and diluted it to 8 gradient concentrations (700.00, 350.00, 175.00, 87.50, 43.75, 21.88, 10.94 and 5.47 μg/mL). The standard curve was then plotted with the concentrations and peak areas as the abscissa and ordinate, respectively.

The galantamine hydrobromide analysis was studied by using Waters 1525 HPLC system with Waters 2489 UV/VIS detector. The isocratic HPLC system consists of Agilent Eclipse XDB RF C18 column (5 μm, 4.6 × 150 mm), with trifluoroacetic acid/water/acetonitrile (0.01/85/15 v/v) as the mobile phase. The mobile phase was delivered in the flow rate of 1 mL/min and the wavelength chosen for galantamine hydrobromide detection was 290 nm. The standard calibration curve was plotted in concentration range of 100 ppm to 1000 ppm. The calibration curve showed linear relationship between the peak area and the concentration of galantamine hydrobromide solution, with R² > 0.99.

The water-soluble sugar fraction was analyzed using a Waters 1,525 HPLC system coupled to a 2,424 evaporative light scattering detector (ELSD). The source parameters of ELSD were the following: gain 75, data rate 1 point per second, nebulizer heating 60%, drift tube 50°C, and gas pressure 2.8 Kg/cm². Plant dry material (50 mg) was boiled in 10 ml MilliQ water for 10 min and then filtered through 0.22 μm nylon filters. Twenty microliters of aliquots were injected into a Prontosil 120-3-amino column (4.6 × 125 mm; 3 μm particle size) maintained at room temperature, using a Waters 717 autosampler. In each run, an isocratic flux of 85% acetonitrile was applied for 25 min at 1 mL/min. Fructose (Fru) and sucrose (Suc) were identified as the major carbohydrates in the samples and were quantified by peak integration using the Waters Empower software and comparison with the corresponding purified standards.

The molecular weight of BSP was determined by high-performance gel permeation chromatography (HPGPC). Briefly, BSP was dissolved in 0.1 M NaNO₃ solution of which 20 μl was loaded into a Waters 1525 HPLC system (Milford, MA, United States) equipped with two columns connected in series Waters Ultrahydrogel^™ linear column (7.8 mm × 300 mm), a 2410 refractive index detector (Waters Corp., Milford, MA, United States). The eluent was 0.1 M NaNO₃ at a flow rate of 0.8 ml/min. The temperature of the column was maintained at 30°C. Molecular weights were determined relative to dextran standards (dextran T-20000, M_W = 2,000,000; dextranT-150, M_W = 133,800; dextranT-40, M_W = 36,800; dextran T-10, M_W = 9,700; dextran T-5, M_W = 2,700). All dextran reagents were purchased from Sigma-Aldrich.