Peptides were purified by RP-HPLC on a Waters Prep LC system incorporating a Waters 486 tuneable absorbance detector set at 214 nm and a Phenomenex Luna C8(2) column (250 × 21.2 mm ID, 100 Å, 10 micron). Peptides were eluted with a gradient of 100% Buffer A (0.1% TFA/water) to 60% Buffer B (0.1%TFA/acetonitrile) over 60 min at a flow rate of 15 mL/min. Fractions collected were analysed by LC/MS on a Shimadzu 2020 LCMS system. LC analysis was carried out at 214 nm using a Phenomenex Luna C8(2) column (100 × 2.0 mm ID, 100 Å, 3 micron), eluting with a gradient of 100% Buffer A (0.05% TFA/water) to 60% Buffer B (0.05%TFA/acetonitrile) over 10 min at a flow rate of 0.2 mL/min. Mass spectra were acquired in positive ion mode with a scan range of 200–2000 m/z.
Luna c8 2 column
Manufactured by Phenomenex
Sourced in United States, United Kingdom, Germany
The Luna C8(2) column is a reversed-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. The column features a octylsilane (C8) stationary phase, which provides a moderate level of hydrophobicity for the retention of moderately polar to nonpolar analytes.
Automatically generated - may contain errors
Lab products found in correlation
Analyst software v1, by AB Sciex (2 mentions)
Securityguard c18 column, by Phenomenex (2 mentions)
Api4000 triple quadrupole lc ms ms, by Thermo Fisher Scientific (2 mentions)
Series 200 lc, by PerkinElmer (2 mentions)
Xcalibur software 2, by Thermo Fisher Scientific (1 mentions)
717 plus autosampler, by Waters Corporation (1 mentions)
2487 dual λ absorbance detector, by Waters Corporation (1 mentions)
2020 lc ms system, by Shimadzu (1 mentions)
Prep lc system, by Waters Corporation (1 mentions)
Esi it tof mass spectrometer, by Shimadzu (1 mentions)
E2695, by Waters Corporation (1 mentions)
Libra s11 spectrophotometer, by Harvard Bioscience (1 mentions)
9 protocols using luna c8 2 column
1
Preparative HPLC Purification of Peptides
2
UHPLC Determination of Coumarin Derivatives
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Determination and quantification of the components were performed using a Dionex Ultimate 3000 UHPLC system equipped with a diode array detector (DAD) (ThermoFisher Scientific, Basel, Switzerland). The elution was performed at 40 °C on a LunaC8(2) column (100 × 4.6 mm) with a 3.0 μm particle size (Phenomenex, Torrance, CA, USA). The mobile phase consisted of (A) 0.1% aqueous formic acid solution and (B) 0.1% acetonitrile formic acid solution, which was applied in the following gradient elution: 5% B in the first min, 5–95% B for 1.0–14.0 min, from 95% to 5% B at 14.0 min and 5% B until 20.4 min. The flow rate was set to 0.5 mL min−1 and the detection wavelengths to 254 and 280 nm. The injection volume was 10 μL. Samples were filtered through a 0.45 mL membrane filter before being analyzed.
Xcalibur software 2.2 (ThermoFisher Scientific, Bremen, Germany) was used for the instrument control. The compounds were quantitated by a set peak detection algorithm and direct plot ICIS.
A stock solution of each ingredient was prepared in DMSO (4-hydroxycoumarin 0.75 mg/1 mL; 3-acetyl-hydroxycoumarin 0.65 mg/1 mL;L1 1.8 mg/1 mL; L2 1.86/1 mL; L3 2 mg/1 mL; L4 1.75 mg/1 mL; and L5 1.86/1 mL;). Dilution of the stock solution with methanol was always done 10 times.
Xcalibur software 2.2 (ThermoFisher Scientific, Bremen, Germany) was used for the instrument control. The compounds were quantitated by a set peak detection algorithm and direct plot ICIS.
A stock solution of each ingredient was prepared in DMSO (4-hydroxycoumarin 0.75 mg/1 mL; 3-acetyl-hydroxycoumarin 0.65 mg/1 mL;
3
HPLC Analysis of Pyridostigmine Bromide
4
Quantitative Analysis of Testosterone
5
Peptide Purification by RP-HPLC
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For purification, the peptides were re-dissolved in H2O. To peptides WAW7 and WAW8 a 10-fold excess of dithiothreitol was added, and the mixtures were incubated in an ultrasonic bath at 60°C for 30 min. Each peptide was purified by RP-HPLC with UV-Vis detection (SHIMADZU), using a semi-preparative Luna C8(2) column (250 × 21.2 mm, 5 μm, 100 Å) (Phenomenex). Purification was carried out using a gradient of 5%–50% B in A over 180 min (mobile phase A—H2O with 0.1% TFA; mobile phase B—80% acetonitrile in H2O with 0.08% TFA). The purity of the peptides was checked by RP-UHPLC with PDA and ELSD-LT detectors (SHIMADZU) using a Kromasil C8 analytical column Kinetex C8 (100 × 2.1 mm, 2.6 μm, 100 Å) and a linear gradient of 5%–100% B in A over 15 min, with detection using an ESI-IT-TOF mass spectrometer (SHIMADZU).
6
HPLC Analysis of Acetylglycoside Standards
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Briefly, 16.63 mg of AGS was dissolved in 1 mL of 100% ethanol, and filtered through a syringe filter. Three standards were prepared as stock solutions in 30% acetone in methanol. HPLC analysis was performed on a chromatographic system equipped with a separation module (e2695), a 2424 evaporative light-scattering detector (ELSD; Waters Co., Milford, MA, USA), and nitrogen gas generator (Genius, Peak Scientific Instruments Ltd., Scotland, UK). Confirmations of peaks in AGS chromatograms were processed using the Empower 3 program (Waters Co., Milford, MA, USA). Liquid chromatography separations were performed on a Luna C8(2) column (5 μm, 4.6 × 250 mm, Phenomenex Inc. (Torrance, CA, USA)) with 10 μL injected volume and 0.9 mL/min flow rate. The mobile phase consisted of 0.05% formic acid in distilled water (A) and acetonitrile (B), and the linear gradient program was as follows: 100% A→80% A (5 min), 50% A→45% A (15 min), 45% A→30% A (17 min), 30% A→28% A (20 min), 28% A→7% A (40 min), 7% A→0% A (42 min), 0% A isocratic mode for 47 min. ELSD conditions were 50 of detector gain, 40 psi of nitrogen gas, and 50 °C of drift tube. All standard compounds were confirmed by comparing the retention time and λmax of each compounds.
7
Quantitation of Testosterone by LC-MS/MS
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Quantitation of testosterone was performed in selected reaction monitoring (SRM) mode. Mass transitions and optimized MS/MS parameters are given in Table S5 . Analyst® software v1.4.1 (AB SCIEX) was used for SRM, peak integration, and analyte quantitation. Standard curves were prepared for testosterone in both tissue culture media and water in the range 0–20 ng/ml and the limit of detection (LOD) and lower limit of quantitation (LLOQ) were established in both matrices. The concentration of testosterone was measured in FM, SDM and APSCE media. Peak areas for these samples were quantified against the external calibration curves of testosterone (Table S6 ). Reverse phase chromatographic separation of testosterone was achieved using a Perkin Elmer Series 200 LC (Beaconsfield, UK) equipped with a Luna C8(2) column (3 μm; 20 × 4 mm i.d.) and SecurityGuard C18 column (4 × 3 mm) (Phenomenex, UK) maintained at 30 °C and a flow rate of 0.5 ml min−1 using the conditions in Table S7 . An API4000 triple quadrupole LC/MS/MS (Applied Biosystems, USA) was used for analysis with electrospray ionization (ESI) performed in positive ion mode using nitrogen gas with source parameters found in Table S8 .
8
Optimized HPLC Analysis of Compounds
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All analytical experiments were performed on an Agilent 1100 HPLC (Waldbronn, Germany) using a Luna C8(2) column (150 mm × 2.0 mm, 3 µm particle size; Phenomenex, Torrance, CA, USA) as the stationary phase and a mobile phase comprising 0.05% trifluoroacetic acid in water (A) and acetonitrile (B).
Elution started at 2% B. This proportion was rapidly increased to 20% B in only 0.1 min, followed by an increase of up to 50% B in the first 15 min and to 70% in another 20 min. In the next 5 min elution was increased to 98% B and this composition was maintained for additional 10 min. The column was then re-equilibrated for 10 min prior to the next run. The DAD-wavelengths were adjusted to 210, 280, and 310 nm, whereas flow rate, sample injection volume, and column temperature were set at 0.25 mL/min, 5 μL, and 30 °C, respectively.
Elution started at 2% B. This proportion was rapidly increased to 20% B in only 0.1 min, followed by an increase of up to 50% B in the first 15 min and to 70% in another 20 min. In the next 5 min elution was increased to 98% B and this composition was maintained for additional 10 min. The column was then re-equilibrated for 10 min prior to the next run. The DAD-wavelengths were adjusted to 210, 280, and 310 nm, whereas flow rate, sample injection volume, and column temperature were set at 0.25 mL/min, 5 μL, and 30 °C, respectively.
9
Analysis of DAME Oxygenation and Transamination
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Analysis of DAME and its oxygenation and transamination products was carried out via gas chromatography using a Thermo Scientific Ultra TM Chromatograph (Waltham, MA) and reversed phase high pressure liquid chromatography using a VWR Hitachi LaChrome Elite HPLC system (Darmstadt, Germany) equipped with a Luna C8(2) column (4.6 x 150 mm, 5 µm, 100Å, Phenomenex, Aschaffenburg, Germany) and connected to a charged aerosol Corona detector (ESA Biosciences Inc, Chelmsford, MA) as described before (Schrewe et al., 2013b) .
Cell concentrations were determined via measurement of the optical density at 450 nm (OD450) (Libra S11 spectrophotometer; Biochrom Ltd., Cambridge, United Kingdom) with an OD450 of 1 corresponding to 0.166 gCDW L -1 (Blank et al., 2008) (link). SDS-PAGE analysis was performed according to the protocol of (Laemmli, 1970) (link).
Cell concentrations were determined via measurement of the optical density at 450 nm (OD450) (Libra S11 spectrophotometer; Biochrom Ltd., Cambridge, United Kingdom) with an OD450 of 1 corresponding to 0.166 gCDW L -1 (Blank et al., 2008) (link). SDS-PAGE analysis was performed according to the protocol of (Laemmli, 1970) (link).
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