Venusil mp c18 column

SAM, SAH, and the SAM:SAH ratio were determined in brain tissue samples. The brain tissue samples were homogenized using a motor-driven tissue homogenizer (PT1200E, Kinematica, Lucerne, Switzerland). Next, 100 mg extracts of the brain tissue were resuspended in 300 μL 0.4 mol/L perchloric acid. Homogenates were centrifuged at 20,000× g for 10 min at 4 °C. The supernatant was filtered through a 0.45-μm membrane filter (Millipore, Billerica, MA, USA), followed by loading into a Venusil MP-C18 column (4.6 mm × 250 mm, 5 μm particle, Agela Technologies, Wilmington, DE, USA) fitted with a matched guard column, run by HPLC system (Waters, Milford, MA, USA). Absorption of eluted compounds was monitored at λ = 254 nm with an ultraviolet detector. A two-buffer elution system was used: mobile phase A and B, both contained 4 mmol/L 1-heptanesulfonic acid (pH 4) and 10 mmol/L ammonium formate. The mobile phase B contained 50% acentonitrile by volume. Elution of SAM and SAH was achieved at a flow rate of 1 mL/min with the following parameters: 0–0.5 min, 100% A; 0.5–20 min, linear gradient to 75% A and 25% B; 20–30 min, 25% B; 30–45 min, 100% A.

As a pretreatment, HUVECs (2 × 10⁷) were washed twice with cold phosphate buffered saline (PBS). The cellular protein content was determined using the BCA protein assay kit (BosterBio, Wuhan, China). Subsequently, the HUVECs and plasma were resuspended ice-cold perchloric acid (0.4 mol/L) and centrifuged at 20,000× g for 10 min at 4 °C. The supernatants were collected and filtered through 0.45 μm membrane (Millipore, Billerica, MA, USA) prior to application to a high-performance liquid chromatography system (Waters, Milford, MA, USA) containing a Venusil MP-C18 column (250 mm × 4.6 mm, 5 μm particles; Agela Technologies, Wilmington, DE, USA) fitted with a matched guard column and an ultraviolet detector. The mobile phase contained 50 mmol/L sodium dihydrogen phosphate and 10 mmol/L sodium heptanesulfonate (pH 4). Elution of SAM and SAH was performed at a flow rate of 1 mL/min. Absorption of eluted compounds was monitored at λ = 254 nm. The SAM and SAH elution peaks were identified by comparing to SAM and SAH standards. Cellular SAM and SAH concentrations were normalized to cellular protein content.

To track the translocation of derived peptide, synthetic peptides (SBS Genetech Co., Ltd, Beijing, China) are labeled with radioactive elements. ¹²⁵I is commonly used for labelling peptides on tyrosine^{55 (link),56 (link)}. There was a tyrosine previous to the N-terminal of peptide B (H₂N-TNPVLEN-COOH) in the Bt Cry1Ac amino acid sequence. So peptide H₂N-YTNPVLEN-COOH was synthesized for iodination labelling by chloramine-T method^{39 (link)} at the State Key Laboratory of Radioactive Chemical Drugs of Beijing Normal University. An aliquot of 100 µL of chloramine-T (0.99 µg/µL) and 1.0 mCi of Na¹²⁵I (18.5 MBq) were added into 2.5 mL of peptide solution (40 μg/mL). After stirred for 5 min at room temperature, 100 µL of sodium pyrosulfite (Acfa Aesar, Beijing, China) solution (1.67 µg/µL) was added to stop the reaction. The mixture was loaded on SCL-10Avp high performance liquid chromatograph (Shimadzu, Japan), separated by a semi-preparative Venusil MP C-18 column (10 mm × 250 mm, Bonna-Agela Technologies). The radiolabeled peptide was eluted with acetonitrile at a flow rate of 2.0 mL/min and monitored by a UV detector (254 nm). The fraction at the retention time period of 7.6–8.3 min was collected. The radioactivity was determined by a Gamma Counter (Wallac, USA). The ¹²⁵I-peptide was purified for the following experiment.

GA3 was analyzed by high-performance liquid chromatography (Dionex U3000) equipped with a Venusil MPC18 column (5 μm, Agela Technologies). The pretreated samples were separated using a mobile phase composed of methanol/water/phosphoric acid (68:32:0.05) at a flow rate of 0.7 mL/min. The detection wavelength was 210 nm, and the injection volume was 10 μL. The retention time of GA3 was 24.12 min. The standard curve was prepared by diluting the 0.1 g GA3 standard with 10 ml methanol to yield solutions of 50, 100, 200, 400, 600, and 800 mg/L. Then, the standard solutions were filtered through a 0.22 μm pore-size organic membrane for liquid phase analysis. The standard curve was made, and the formula was as follows:
Y = 0.1256*X-0.2476 (R² = 0.9991),
where Y is the peak area and X represents the concentration of GA3.

The high-performance liquid chromatography (HPLC) system used was composed of a Waters 717 plus Autosampler, a Waters 600 Controller, a Waters 996 Photodiode Array Detector, and a Waters Millog workstation (Waters, Shinagawa, Tokyo, Japan). Optical rotations were measured in methanol on a PerkinElmer-341 polarimeter. The IR spectra were run on a NicoletAvatar-360FT-IR spectrometer. ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectra were measured at 25°C on a Bruker AVANCE DMX 500 NMR spectrometer with TMS as internal standard. CD spectra were measured on a JASCO J-715 (JASCO) spectropolarimeter. UV spectra were also recorded in methanol on a Shimadzu UV2550. ESIMS were recorded on an Agilent 6460 Triple Quad LCMS. Preparative HPLC was performed on a ChuangXinTongHeng system equipped with a Venusil MP-C18 column (10 mm × 250 mm, Agela Technologies, Tianjin, China). The organic solvents used in chromatographic separation were of analytical grade purchased from Sayfo Technology (Tianjin China) and chromatographic grade for HPLC analysis purchased from Tedia, United States. Deionized water was prepared by reverse osmosis Milli-Q water (18 MW; Millipore, Bedford, MA, United States) and used for all solutions and dilutions. Agar powder for plate culture and cobalt chloride was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

CCE nucleosides were analyzed by high-performance liquid chromatography (HPLC). CCE powder (0.5 g) was dissolved with 50 mL distilled water, and the solution was filtered through a 0.45-μm aqueous phase filter membrane for liquid chromatography analysis. The chromatography column was a Venusil MP C18 column (5 μm, 4.6 mm × 250 mm, Agela Technologies of America). The mobile phase was composed of ultrapure water (A) and methanol (B) with a gradient elution as follows: 0–5 min: 100% A; 5–10 min A: 95 A; 10–30 min: 70% A; 30–40 min: 95% A; 40–45 min: 100% A. The injection volume was 10 μL, the flow rate was 1.0 mL/min, and the column temperature was 40°C. The UV detection wavelength was set at 254 nm.