Agilent 1290 UPLC (ultra-high pressure liquid chromatography) system combined with an Agilent 6460 triple quadrupole mass spectrometer via an electrospray ionization interface, was used for analysis. The chromatographic separation was performed using a ZORBAX Eclipse Plus C18 analytical column (2.1X100mm; id: 1.8 µm). Samples were eluted using mobile phase A, consisting of 20% acetonitrile and 10 mM ammonium acetate in water, and mobile phase B, which consisted of 80% acetonitrile and 10 mM ammonium acetate in water, at a flow rate of 0.4 mL/min. The gradient profile details used for the LC pump are described in Table S3 . The injection volume of the samples was 5 µl. The column temperature was set at 45 °C and the sample tray temperature was maintained at 9 °C. MS/MS spectra were produced using the negative ionization mode.
Eclipse plus c18 analytical column
Manufactured by Agilent Technologies
Sourced in United States
The Eclipse Plus C18 analytical column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a silica-based stationary phase with a C18 bonded ligand, providing excellent retention and selectivity for a variety of analytes.
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Lab products found in correlation
6460 triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
1290 uplc, by Agilent Technologies (1 mentions)
Lc 20 ad prominence liquid chromatograph system, by Shimadzu (1 mentions)
Spd m20a prominence diode array detector, by Shimadzu (1 mentions)
Dgu 20a3 prominence degasser, by Shimadzu (1 mentions)
Q exactive, by Thermo Fisher Scientific (1 mentions)
Agilent 1260 high performance liquid chromatography, by Agilent Technologies (1 mentions)
6 protocols using eclipse plus c18 analytical column
1
UPLC-MS/MS Analysis of Metabolites
2
HPLC Analysis of Medicinal Plant Phytochemicals
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The stock solution of MPSEs was freshly prepared at 1 mg/mL volume in purified water and was filtrated with a 0.45 μm syringe filter (Whatman's). HPLC profiles of MPSEs were performed using a Shimadzu LC-20AD Prominence Liquid Chromatograph system equipped with a SPD-M20A Prominence Diode Array Detector and with a DGU-20A3 Prominence Degasser (Shimadzu, Japan). The wavelength scanning was done between 190 and 800 nm, while any wavelength occurring at 270 nm was carefully monitored. Notably, 20 μL of each extract (300 μg/mL) was injected into the ZORBAX Eclipse Plus C18 Analytical column (250 × 4.6 mm i.d., 5 μm particle) with an Eclipse Plus-C18 Analytical Guard Column (12.5 × 4.6 mm i.d., 5 μm particle. Successful separation of MPSEs phytochemicals was accomplished using the following mobile phase; mobile A: 0.1% aqueous trifluoroacetic acid and mobile B: 100% acetonitrile. The time program for gradient elution was 0–5 minutes, 5% B; 7–12 minutes, 10% B; 14–19 minutes, 15% B; 21–26 minutes, 20% B; 30–35 minutes, 25% B; 37–45 minutes, 30% B; 50–55 minutes, and 100%B; 60–65 minutes, 5% B.
3
UHPLC-QQQ-MS/MS for Compound Quantification
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UHPLC-QQQ-MS/MS was carried out via a UHPLC/MS system, with an Eclipse Plus C18 analytical column (2.1 mm × 100 mm, 1.8 µm), obtained from Agilent Technology Co., Ltd. (Santa Clara, CA, USA), at a 0.35 mL/min flow rate. A total of 2 µL of water + 0.1% formic acid (v/v) (A) and acetonitrile + 0.1% formic acid mobile phase (v/v) (B) were used for the mobile phase, respectively. The gradient elution process was as follows: 5% B at the beginning; followed by 5% B at the 1st min; 50% B in the 2nd min; 50% B in the 5th min; 100% B at minute 5.1; and 100% B at the 6th min. Post run time was set as 2 min. The ingredients were examined through a multiple reaction monitoring (MRM) system. The parameters for ESI source were as follows: 3.5 kV capillary voltage, 380 V fragmentor voltage, 200 °C drying gas (with a 15 L/min flow rate), 330 °C sheath gas (flow rate of 12 L/min), and 45 psi nebulizer pressure. The related compounds ionization parameters are listed in Table S1 .
4
Fractionation and Characterization of Spider Venom
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All reagents and chemicals that were used in the study were analytical grade and
bought from RCL Labscan, Thailand; Mym Biological Technology Company, Hi-Media
Laboratories, India; Sigma-Aldrich Life Sciences; LaserBio Labs, France; Macron
Fine Chemicals, Cayman Chemical Company, USA and Molecular Probes Invitrogen,
USA. On the other hand, Waters Alliance e2695 RP-HPLC system equipped with w2489
UV-VIS spectrophotometer and Agilent Eclipse PlusC18 analytical column (5 µm,
5.6 x 150 mm, pore size-100 Å) were used for the fractionation of whole spider
venom. Lyophilization was done using Sim International FD5-series freeze dryer.
Aside from this, ThermoFisher Multiskan microplate spectrophotometer and Promega
GloMax Explorer multimode microplate reader was used for the colorimetric
enzymatic assays. Moreover, Shimadzu Axima Confidence Linear/Reflectron
MALDI-TOF-MS system was used to determine the masses from the spider venom
fractions.
bought from RCL Labscan, Thailand; Mym Biological Technology Company, Hi-Media
Laboratories, India; Sigma-Aldrich Life Sciences; LaserBio Labs, France; Macron
Fine Chemicals, Cayman Chemical Company, USA and Molecular Probes Invitrogen,
USA. On the other hand, Waters Alliance e2695 RP-HPLC system equipped with w2489
UV-VIS spectrophotometer and Agilent Eclipse PlusC18 analytical column (5 µm,
5.6 x 150 mm, pore size-100 Å) were used for the fractionation of whole spider
venom. Lyophilization was done using Sim International FD5-series freeze dryer.
Aside from this, ThermoFisher Multiskan microplate spectrophotometer and Promega
GloMax Explorer multimode microplate reader was used for the colorimetric
enzymatic assays. Moreover, Shimadzu Axima Confidence Linear/Reflectron
MALDI-TOF-MS system was used to determine the masses from the spider venom
fractions.
5
Polyphenol Quantification by LC-MS
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LC-MS analyses were carried out using a Dinonex Ultimate 3000 UHPLC system (Ultimate 3000—Thermo Scientific, Waltham, MA, USA), coupled with a quadrupole-orbitrap hybrid mass analyzer (Q-Exactive, Thermo Scientific). The chromatographic separation of the polyphenol extract was achieved on an Eclipse Plus C18 analytical column (250 mm × 4.6 m, 2.6 µm, ZORBAX, Agilent, Palo Alto, CA, USA). The column temperature was set at 30 °C. The mobile phase was composed of (A) water with 0.2% formic acid and (B) acetonitrile with 0.2% formic acid. Elution was accomplished with the following solvents gradient: 0-3 min 10% B, 18% B at 13 min and kept unchanged until 16 min, and 30% B at 25 min and kept unchanged until 30 min. Finally, the system returned to 10% B in 2 min. The flow rate and the injection volume were 0.6 mL/min and 10 μL, respectively. The acquisition was carried out in negative ionization mode (ESI-). The ESI temperature was set at 300 °C, the capillary temperature at 320 °C, and the electrospray voltage at 2.8 kV. Sheath and auxiliary gas were 30 and 5 arbitrary units, respectively. The acquisition was performed in full scan/ddMS 2 modes. The parameters were optimized as follows: (i) full scan acquisition: resolution 70,000 FWHM (at m/z 200); (ii) dd-MS 2: resolution 17,500 FWHM (at m/z 200). The normalised collision energy (NCE) was set at 30.
6
Quantitative Analysis of COR and CGR
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1.0 g COR and CGR powder was accurately weighed respectively, accurately added 50 mL of methanol, weighed it, heated to re ux for 2 h. The mixture was cooled down to room temperature and weighed it again, methanol were added to make up the lost weight, shook and ltered through lter paper. Took 20 mL of the ltrate and evaporated to dryness, dissolved the residue with methanol, transferred to a 10 mL volumetric ask, then added methanol to the mark to obtain the sample solution. The solution was ltered through a 0.22 µm membrane lter before injection into the HPLC system.
The sample solution of COR and CGR were analyzed by Agilent 1260 High Performance Liquid Chromatography (Agilent Technologies, CA, USA) equipped with a Zorbax Eclipse Plus C18 analytical column (4.6 mm × 150 mm, 5 µm) and a guard column. The temperature was set at 30 ℃, the injection volume was 10 µL, and the detection wavelength was set to 285 nm. A binary elution at a ow rate of 1.0 mL/min was employed using an aqueous phase of 0.1% formic acid as solvent A and acetonitrile as solvent B, the gradient elution procedure was A:B = 21:79, detection time was 18 minutes.
The sample solution of COR and CGR were analyzed by Agilent 1260 High Performance Liquid Chromatography (Agilent Technologies, CA, USA) equipped with a Zorbax Eclipse Plus C18 analytical column (4.6 mm × 150 mm, 5 µm) and a guard column. The temperature was set at 30 ℃, the injection volume was 10 µL, and the detection wavelength was set to 285 nm. A binary elution at a ow rate of 1.0 mL/min was employed using an aqueous phase of 0.1% formic acid as solvent A and acetonitrile as solvent B, the gradient elution procedure was A:B = 21:79, detection time was 18 minutes.
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