Stable isotope labeling was accomplished by culturing cells in media containing 400 μM uniformly labeled 13C OA (Cambridge Isotope Laboratories, Tewksbury, MA). Briefly, SiHa cells were labeled for 24 hours, irradiated with 6Gy, incubated for additional 24 hours with label and then washed with PBS and LC/MS-grade water before quenching, with cold methanol and transferring, to microfuge tubes with a cell scraper. Methanol was evaporated by using a SpeedVac. Pellets were extracted with 2:2:1 methanol: acetonitrile: water. Extracts were reconstituted in 1:2 water: acetonitrile, with volume normalized to the dry mass of the cell pellet. The mixture was then sonicated, centrifuged, and the supernatant was taken for LC/MS analysis. Ultra-high performance LC (UHPLC)/MS was performed with an Agilent 1290 Infinity II LC system interfaced with an Agilent 6540 QTOF mass spectrometer (Agilent Technologies, Santa Clara, CA). For reversed-phase liquid chromatography (RPLC) separations, a Waters CORTECS C8 column (50 mm × 2.1 mm, 1.6 μm) was coupled to the MS detector. For hydrophilic interaction liquid chromatography (HILIC) separations, a ZIC-pHILIC column (100 mm × 2.1 mm, 5 μm) was coupled to the MS detector. LC/MS data were processed and analyzed with the Agilent Profinder software and a natural-abundance correction of 13C was applied.
Cortecs c8 column
Manufactured by Waters Corporation
Sourced in United States
CORTECS C8 column is a reversed-phase high performance liquid chromatography (HPLC) column designed for the separation of small molecules. It features a spherical, solid-core particle design with a C8 stationary phase. The column provides efficient and reproducible separations.
Automatically generated - may contain errors
3 protocols using cortecs c8 column
1
Stable Isotope Labeling for Metabolomic Analysis
2
Triterpene Analysis in Cyanobacteria
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Extracts from Synechocystis and R. capsulatus, dissolved in chloroform/methanol (2:1) with 0.05% BHT, were diluted 1:100 with methanol (+ 0.05% BHT) prior to injection of 10 μl sample volumes. Triterpenes were separated on a Dionex HPG 3200 HPLC system (Thermo Scientific) equipped with a 150 x 2.1 mm, 2.7 μm, Cortecs C8 column (Waters) with a binary gradient system. Mobile phase A consisted of water + 0.1% formic acid (FA) and mobile phase B consisted of methanol + 0.1% FA. The mobile phase gradient was as follows: Starting conditions were 75% mobile phase B, increased to 85% B within 4 min and then further increased to 100% B in 13.5 min, the plateau was held for another 4.5 min and the system was returned to starting conditions within 3 min. The flow rate was 0.5 mL/min. Triterpenes were analyzed by Q-TOF (quadrupole-time-of-flight) MS and MS/MS on a maXis 4G instrument (Bruker Daltonics) equipped with an ESI (electrospray ionization) source. The instrument was operated in positive-ion mode and the operating conditions were as follows: dry gas (nitrogen): 8.0 L/min, dry heater: 220 °C, nebulizer pressure: 1.8 bar, capillary voltage: 4500 V.
3
UPLC-MS/MS Quantification of Compounds
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Next, 1 µL of the extracted samples were injected into a reverse-phase CORTECS C8 Column, 90Å, 1.6 µm, 2.1 mm × 100 mm (Waters Corp. Milford, MA, USA) with a gradient mobile phase comprising 0.1% ammonium acetate with 1% formic acid (A) and acetonitrile containing 0.1% ammonium acetate with 1% formic acid (B). Each sample was resolved for 3.7 min at a flow rate of 0.5 mL/min with the linear gradient 0–1.2 min from 10 to 98% B; 1.2–2.2 min 98% B, and 10% B for 1.5 min. The column temperature was 45 °C. The eluent was introduced by electrospray ionization into the mass spectrometer (Xevo TQ-S, Waters Corp.), operating in positive ion electrospray ionization mode (ESI+). The capillary voltage was set to 3000 V and the Source Offset to 50 V. The dissolving gas flow was set to 1200 L/h and the temperature to 650 °C. The cone gas flow was 200 L/h, and the source temperature was set to 150 °C.
To establish the appropriate multiple reaction monitoring (MRM) conditions for the individual compounds, the cone voltage was optimized to maximize the intensity of the protonated molecular species [M + H]+ and the collision energy (eV) was adjusted to optimize the signal for the most abundant product ions, which were subsequently used for MRM analysis (Table 1 ).
To establish the appropriate multiple reaction monitoring (MRM) conditions for the individual compounds, the cone voltage was optimized to maximize the intensity of the protonated molecular species [M + H]+ and the collision energy (eV) was adjusted to optimize the signal for the most abundant product ions, which were subsequently used for MRM analysis (
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