HPLC analyses of the described above the final supernatants obtained after electrochemical, enzymatic and cellular transformations were performed using a Nexera-I LC-2040C 3D HPLC system and the LabSolution software package (Shimadzu Corp., Kyoto, Japan). Samples were separated using a reversed-phase 5 μm Suplex pKb-100 analytical column (0.46 × 25 cm, C18) (Supelco, Bellefonte, PA, USA) warmed to 25 °C. The analyses were performed at a flow rate of 1 mL/min with the following mobile-phase system: a linear gradient from 15% to 80% methanol in ammonium formate buffer (0.05 M, pH 3.4) for 25 min, followed by linear gradient from 80% to 100% methanol in ammonium formate buffer for 3 min. The column was then re-equilibrated at initial conditions for 10 min between runs. The elution of each metabolite was monitored at 430 nm.
Suplex pkb 100 analytical column
Manufactured by Merck Group
Sourced in United States
The Suplex pKb-100 analytical column is a laboratory equipment product designed for chromatographic analysis. It is a component used in analytical processes to separate and identify chemical compounds within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Labsolution software package, by Shimadzu (1 mentions)
Nexera 1 lc 2040c 3d hplc system, by Shimadzu (1 mentions)
2487 dual, by Waters Corporation (1 mentions)
717 plus, by Waters Corporation (1 mentions)
1525 binary pump, by Waters Corporation (1 mentions)
7725i rheodyne injector, by Waters Corporation (1 mentions)
Breeze software, by Waters Corporation (1 mentions)
Analyst 1, by Thermo Fisher Scientific (1 mentions)
Agilent 1100 series, by Agilent Technologies (1 mentions)
Chemstation software, by Agilent Technologies (1 mentions)
4 protocols using suplex pkb 100 analytical column
1
HPLC Analysis of Electrochemical, Enzymatic, and Cellular Transformations
2
Quantitative HPLC Analysis of Metabolic Transformations
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All metabolic transformations were analyzed using RP-HPLC with UV-vis detection with 5 mm Suplex pKb-100 analytical column (0.46 cm × 25 cm, C18) (Supelco, Bellefonte, PA, USA) with Waters HPLC system equipped with 1525 binary pump, 7725i Rheodyne injector, 2487 dual λ absorbance detector and 717 plus autosampler controlled with Breeze software (Waters Co., Milford, MA, USA). Aliquots of 200 µL of the prepared samples were analyzed by HPLC-UV-vis at a flow rate of 1 mL/min in 50 mM ammonium formate buffer, pH = 3.2, with a linear gradient from 15% to 80% methanol for 25 min, followed by a linear gradient from 80% to 100% for 3 min. The column was then re-equilibrated at initial conditions for 20 min between runs. The elution of sample components was monitored at 254 nm (testosterone transformation), 330 nm (7-OH-TFC glucuronidation) or 420 nm (C-1305 and C-1311 metabolism).
3
Quantitative HPLC-MS/MS Analysis
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All experiments were performed on an Agilent 1100 high-performance liquid chromatography (HPLC) (Palo Alto, CA, USA) with an Applied Biosystems 3200 Q Trap mass spectrometermass spectrometer system (Concord, ON, Canada) equipped with a turbospray ionization source. The liquid chromatograph used was an Agilent 1100 Series with a quaternary pump, a vacuum degasser and an autosampler. A SuplexpKb 100 analytical column (25 cm × 2.1 mm) protected by a 5-μm SuplexpKb 100 precolumn (2 cm × 2.1 mm) (Supelco, Bellefonte, PA, USA) was used. The liquid chromatography/mass spectrometry (LC/MS-MS) was controlled through the Analyst 1.4 software (Applied Biosystems) run on a Dell Precision T3400 computer under the Microsoft Windows XP operating system.
4
HPLC-UV Analysis of NI-12a and NI-ST-05 Metabolites
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HPLC–UV
analysis
of the supernatants was carried out on an HP1050 LC system equipped
with a variable wavelength UV–DAD detector. Instrument control
and data collection was accomplished using Agilent ChemStation software.
The samples were separated on a reversed-phase 5 μm Suplex pKb-100
analytical column (0.46 cm × 25 cm, C18) (Supelco, USA) maintained
at 25 °C. HPLC analyses were carried out at a flow rate of 1
mL/min with the following elution system: a linear gradient from 15
to 80% methanol in ammonium formate buffer (0.05 M, pH 3.4) for 25
min followed by a linear gradient from 80 to 100% methanol in ammonium
formate for 3 min. The column was then re-equilibrated to initial
conditions for 10 min between runs. The elution of each metabolite
of NI-12a was monitored at 335 nm, whereas the elution of the metabolite
of NI-ST-05 was monitored at 325 nm.
analysis
of the supernatants was carried out on an HP1050 LC system equipped
with a variable wavelength UV–DAD detector. Instrument control
and data collection was accomplished using Agilent ChemStation software.
The samples were separated on a reversed-phase 5 μm Suplex pKb-100
analytical column (0.46 cm × 25 cm, C18) (Supelco, USA) maintained
at 25 °C. HPLC analyses were carried out at a flow rate of 1
mL/min with the following elution system: a linear gradient from 15
to 80% methanol in ammonium formate buffer (0.05 M, pH 3.4) for 25
min followed by a linear gradient from 80 to 100% methanol in ammonium
formate for 3 min. The column was then re-equilibrated to initial
conditions for 10 min between runs. The elution of each metabolite
of NI-12a was monitored at 335 nm, whereas the elution of the metabolite
of NI-ST-05 was monitored at 325 nm.
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