E2695 separations module

Ginsenoside contents in MGE and FGE were analyzed using a HPLC system (e2695 Separations Module) equipped with a HPLC pump, an autosampler, a column oven, and a diode array UV/Vis detector (2998 PDA detector; Waters Corp., Milford, MA, USA) with a C₁₈ column (4.6 × 250 mm, 5 μm; YMC Co., Ltd., Kyoto, Japan). The ginsenosides were eluted in a gradient system composed of solvent A (0.2% phosphoric acid) and solvent B (acetonitrile). The gradient was 80-80-77-70-60-50-15-15-80-80% of solvent A at the gradient time t G = 0-5-20-25-30-35-60-62-65-70 min, respectively. The temperature of the column oven was 30°C, and the flow rate was 1.0 ml/min; an injection volume of 10 μl was applied. The UV/Vis detector was set at the wavelength range of 203 nm. The ginsenoside standards and the sample solution (0.1 mg/ml) were dissolved and diluted in methanol. The relative standard deviation of the measured concentrations was used to assess precision. A comparison of the mean measured concentration versus the corresponding nominal concentration was used to assess accuracy.

Analysis was performed on the High-performance liquid chromatography (HPLC) system (Waters Corporation, Milford, MA, USA). The HPLC system was equipped with a Waters Alliance e2695 Separations Module and UV/VIS detector (Waters, 2489). The output signal of the detector was recorded using a Empower® 3 software. The separation was executed on a YMC Hydrosphere C₁₈ column (12 nm, 150 × 4.6 mm, 5 μm). The mobile phase was composed of 0.1% formic acid and acetonitrile with the gradient elution system at a flow rate of 0.4 ml/min. The gradient flows were as follows: 10–35% B at 0–8 min, 35–45% B at 8–20 min, 45–70% B at 20–25 min, 70–90% B at 25–35 min, 90% B at 35–40 min. The injection volume was 10 μl and the detection wavelength was set at 205 nm.

The extraction methods for active components (CA, LAB, RA, TI, TIIA, DT, CT) and HPLC were carried out in accordance with the general method used previously in our laboratory [52 (link)]. Dry sample powder (20 mg) was dissolved in 2 mL of 70% methanol and soaked for 12 h. The mixture was sonicated for 45 min and centrifuged at 8000 rpm for 10 min. The supernatant was filtered through a 0.22 um microporous membrane (F) for HPLC analysis using a Waters e2695 Separations Module and 2998 PDA Detector. Chromatography separation was performed with a C18 column (Waters), the temperature of column was 30 °C. The detected wavelength was 288 nm for phenolic acids and 270 nm for tanshinones. The mobile phase was acetonitrile (C) and 0.026% phosphoric acid solution (D) with the flow rate of 1.00 mL/min. The elution gradient was set as follows: t = 0–10 min, 5–20% C; t = 10–15 min, 20–25% C; t = 15–20 min, 25% C; t = 20–25 min, 25–20% C; t = 25–28 min, 20–30% C; t = 28–40 min, 30% C; t = 40–45 min, 45% C; t = 45–58 min, 45–58% C; t = 58–67 min, 58–50% C; t = 67–70 min, 50–60% C; t = 70–80 min, 60–65% C; t = 80–85 min, 65–95 C; t = 85–95 min, 95 C; t = 95–96 min, 5% C. Compounds were identified by comparison with the standard substances, which were purchased from the National Institutes for Food and Drug Control (Beijing, China).

A Waters system (Waters e2695 Separations Module and Waters 2998 PDA Detector, Milford, MA, USA) with an A Sepax GP-C18 column (4.6 × 250 mm, 5 µm) was used for the HPLC analysis. The injection volume was 10 µL, and the column temperature was maintained at 30 °C.
Small molecular components of toad venom. Fresh venom from toads from different areas was extracted with methanol and detected by HPLC according to the methods recommended by Cao et al. and the “Pharmacopoeia of the People’s Republic of China” [2 (link)].
Total proteins from the soluble fraction of toad venom. Samples were fractionated with a linear gradient of 5–90% acetonitrile acidified with 0.5% (v/v) trifluoroacetic acid for 105 min at a flow rate of 1.0 mL/min. The detection wavelength was set at 280 nm [13 (link)].

Cellular nucleotides were extracted according to published procedures^{75 (link)}. Briefly, 1 × 10⁶ cells were washed with phosphate-buffered saline and quenched with liquid nitrogen, and then vigorously mixed with methanol and acetonitrile (1:1, v:v). After incubation on ice for 15 min, the samples were centrifuged at 12,000× g at 4 °C for 10 min. Cellular nucleotides were separated and quantified using a C18 column (Agilent Eclipse XDB-C18, 4.6 × 250 mm, average particle size 5 μm) assembled on the Waters Alliance e2695 Separations Module (Milford, MA, USA). Acetonitrile (5%) and 50 mM KH₂PO₄ (pH 6.5) containing 10 mM tetrabutylammonium bromide were used as mobile phase A, and acetonitrile was used as mobile phase B. All samples were separated in the mobile phase at a flow rate of 1 mL/min for 30 min at 22 °C. No degradation of individual nucleotides or changes in the ratios of nucleotide mixtures was detected during the experiments. The GTP concentration in the samples was calculated based on the slope of the calibration curves generated using pooled authentic samples (to mimic the matrix), and guanosine-5′-triphosphoric acid disodium salt was used as a standard.

The main components responsible for the pharmacological actions of S. baicalensis are flavonoids, including baicalin, baicalein, and wogonin. The contents of baicalin, baicalein, and wogonin in S. baicalensis extract were quantified using a Waters e2695 separations module (MA, USA) equipped with a Waters 2707 autosampler, a Waters 1525 pump, and a Waters 2998 photodiode array detector. The separation was achieved using a SunFire™ C18 column (5 μm particle size, 250 × 4.6 mm i.d.; Waters) maintained at 30°C with a flow rate of 1 mL/min. The mobile phase consisted of 1% (v/v) phosphoric acid in distilled water (A) and acetonitrile (B). The gradient elution was set as follows: 5%–50% B at 0–60 min, 50%–70% B at 60–61 min, 70%–5% B at 61–63 min, and 5%–5% B at 63–68 min. The injection volume was 10 μL. The detector wavelength for quantification was set at 254 nm, and a 3D chromatogram of UV absorption was acquired.