Waters 2707

The Prep-HPLC analysis was performed as described in our recent studies [9 (link),10 (link)]. The extract sample (10 mg/mL) was dissolved in ultrapure water (analytical grade, Merck KGaA, Darmstadt, Germany), centrifugated at 8000× g for 20 min, and then passed through 0.22 μm membrane (Shanghai Sangon Biological Engineeing Technology and Service Co., Ltd., Shanghai, China). The filtration was subjected to Prep-HPLC using a Waters 2707 (Waters, Milford, MA, USA) linked with a UPLC Sunfire C18 column (5 μm, 10 × 250 mm) (Waters, Milford, MA, USA) with the parameters described recently by Liu et al. [9 (link)]. After the Prep-HPLC, the isolated fragments were individually collected, concentrated, and evaporated using the rotary evaporator (IKA, Staufen, Germany), and then redissolved in ultrapure water for further analyses.

The mice were placed in a prone position, and the neck was bent at a 45‐degree angle. The occipital bone to the atlas muscle was cut with iris scissors to expose the white dura, and it was punctured at 2 mm between the occipital bone and the atlas cone with a needle. A micropipette was used to extract 2.5 μl of cerebrospinal fluid (CSF). The collected CSF was centrifuged at 250 g for 10 min and was frozen at −80°C. The glutamate level was determined using a Waters high‐performance liquid chromatography (HPLC) system (Waters Breeze 1525) equipped with a UV–Visible detector (Waters 2489), an autosampler (Waters 2707), and an isocratic pump (Waters 1515). Trigeminal ganglions (TGs) from the unilateral surface were dissected immediately and frozen at −80°C for further assays.

Aliquots (10 mg/mL) of freeze-dried samples resolved in H₂O (Analytical grade, Merck KGaA, Darmstadt, Germany) were centrifuged at 12,000 rpm for 20 min. The supernatant was filtered through 0.22 µm membrane (Sangon, Shanghai, China), and the filtration was collected for further analysis. Prep-HPLC was run using Waters 2707 (Waters, Milford, Massachusetts, USA) linked with UPLC Sunfire C18 column (5 μm, 10 × 250 mm) (Waters, Massachusetts, USA) at the following parameters: column temperature, 40 °C; injection volume, 100 μL; and mobile phase of methanol (eluent A) and water (eluent B) at a flow rate of 4 mL/min (isocratic elution: 0–15 min, 20% eluent A and 80% eluent B). Photo-diode array (PDA) spectra were measured in the wavelength ranging from 200 to 600 nm.

Five milligrams of lyophilized skin secretion were dissolved in 1 ml of deionized water containing 0.05% (v/v) trifluoroacetic acid (TFA, obtained from Sigma-Aldrich, St. Louis, MO, USA). The sample was subsequently centrifuged and the supernatant was further filtered through a 0.45 μm RC membrane (Phenomenex, Macclesfield, UK). The filtrate was injected into a reverse-phase HPLC system (UV Detector: Waters 2489; Binary HPLC Pump: Waters 1525; Autosampler: Waters 2707 (Waters Ireland, Dublin, Ireland); Column: Phenomenex Aeris Peptide, C18, 250 × 10.0 mm (Phenomenex, Macclesfield, UK), followed by elution with a gradient program from 0.05/99.95 (v/v) TFA/water (0 min) to 0.05/19.95/80.0 (v/v/v) TFA/water/acetonitrile in 240 min. The column effluent was monitored by UV absorbance at 214 nm, and fractions were collected automatically at 1 min intervals.

For HPLC analysis, the soluble-free, soluble-conjugated and insoluble-bound phenolic extracts were filtered through 0.45 μm membrane filters before analysis. Then, 10 μl of each sample extract was fractionated using a waters HPLC system equipped with a waters 2707 autosampler, a Waters 2489 UV/Visible detector and a Waters 1525 binary pump on a 250 mm × 4.6 mm i.d., 5 μm, Inertsil C18 analytical column. Gradient elution was performed with a mobile phase consisting of A (0.1% acetic acid in water) and B (0.1% acetic acid in methanol). The flow rate was 1 ml min⁻¹, and the gradient elution was set according to the method reported by Shao et al. as follows: 0 to 11 min, 9% to 14% B; 11 to 14 min, 14% to 15% B; 14 to 17 min, 15% B; 17 to 24 min, 15% to 16.5% B; 24 to 28 min, 16.5% to 19% B; 28 to 30 min, 19% to 25% B; 30 to 36 min, 25% to 26% B; 36 to 38 min, 26% to 28% B; 38 to 41 min, 28% to 35% B; 41 to 46 min, 35% to 40% B; 46 to 48 min, 40% to 48% B; 48 to 53 min, 48% to 53% B; 53 to 70 min, 53% to 70% B; 70 to 72 min, 9% B; 72 to 75 min, 9% B.^{15 (link)} The column was warmed to 40 °C. The phenolic acids were detected at a wavelength of 280 nm, and the phenolic acid contents were quantified using the external calibration curves.