Ribonucleosides were separated using a Luna Omega Polar (150 × 2.1 mm; particle size, 2.5 μm; pore size, 100 Å; Phenomenex, Torrance, CA) on an Agilent 1290 series HPLC system equipped with a diode array detector. Mobile phase A was 5 mM NH4OAc (≥99%; HiPerSolv CHROMANORM, VWR) adjusted to pH 5.3 with glacial acetic acid (≥99%, HiPerSolv CHROMANORM, VWR), and mobile phase B was pure acetonitrile (LC-MS grade, purity ≥99.95; Roth). Gradient elution started with 98% A and increased to 10% B after 2 min, 30% B after 3 min, and 60% B after 3.5 min. Starting conditions are re-established at 4 min followed by 2 min of equilibration. The flow rate was 0.4 ml/min, and the column temperature was 30°C. The effluent from the column was directed through the DAD (diode array detector) before entering the Agilent 6490 Triple Quadrupole mass spectrometer in dynamic multiple reaction monitoring (MRM) mode. The MS was operated in positive-ion mode with the following parameters: electrospray ionization (ESI-MS, Agilent Jetstream); fragmentor voltage, (set in tunefile to) 250 V; cell accelerator voltage, 2 V; N2-gas temperature, 150°C; N2-gas flow, 15 liters/min; nebulizer, 30 psi; sheath gas (N2) temperature, 275°C; sheath gas flow, 11 liters/min; capillary, 2500 V; and nozzle voltage, 500 V. The instrument was operated in dynamic MRM mode with the method listed in table S2.
Luna omega polar
Manufactured by Phenomenex
Sourced in Japan, United States
The Luna Omega Polar is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of polar analytes. It features a proprietary stationary phase that provides enhanced selectivity and resolution for a wide range of polar compounds.
Automatically generated - may contain errors
Lab products found in correlation
Jet stream, by Agilent Technologies (2 mentions)
Chromanorm, by Avantor (1 mentions)
Oasis hlb, by Waters Corporation (1 mentions)
Strata x, by Phenomenex (1 mentions)
Oasis prime hlb, by Waters Corporation (1 mentions)
Kinetex polar c18, by Phenomenex (1 mentions)
Kinetex xb c18, by Phenomenex (1 mentions)
Zorbax bonus rp, by Agilent Technologies (1 mentions)
Zorbax eclipse plus c18, by Agilent Technologies (1 mentions)
C18 guard column, by Phenomenex (1 mentions)
4 protocols using luna omega polar
1
Quantitative HPLC-MS/MS Analysis of Ribonucleosides
2
Comprehensive Analytical Method Development
3
HPLC-MS/MS Analysis of Ribonucleosides
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Ribonucleosides were separated using a Luna Omega Polar, 150 × 2.1 mm, 2.5-µm particle size, 100 Å pore size from Phenomenex (Torrance), on an Agilent 1290 series HPLC system equipped with a diode array detector. Mobile phase A was 5 mM NH4OAc (≥99%, HiPerSolv Chromanorm, VWR) adjusted to pH 5.3 with glacial acetic acid (≥99%, HiPerSolv Chromanorm, VWR) and mobile phase B was pure acetonitrile (Roth, LC-MS grade, purity ≥99.95%). Gradient elution started with 98% A, increased to 10% B after 2 min, 30% B after 3 min, and 60% B after 3.5 min. Starting conditions are re-established at 4 min, followed by 2 min of equilibration. The flow rate was 0.4 mL/min and column temperature was 30°C. The effluent was directed through the DAD before entering the Agilent 6490 Triple Quadrupole mass spectrometer in dynamic multiple reaction monitoring (MRM) mode. The MS was operated in positive ion mode with the following parameters: electrospray ionization (ESI-MS, Agilent Jetstream), fragmentor voltage (set in tunefile to) 250 V, cell accelerator voltage 2 V, N2 gas temperature 150°C, N2 gas flow 15 L/min, nebulizer 30 psi, sheath gas (N2) temperature 275°C, sheath gas flow 11 l/min, capillary 2500 V, and nozzle voltage 500 V. The instrument was operated in dynamic MRM mode with the method listed in Supplemental Table S2a .
4
Quantitative Mass Spectrometry Analysis
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The ESI-source parameters which were used for all measurements were as follows: source temperature 350 °C, curtain gas 35 psi, gas 1 60 psi, gas 2 35 psi. The ion spray voltage was set at + / − 4000 V, collision gas pressure (nitrogen): high. These parameters were used to perform analyses in both the positive and negative ion modes. The MRM transitions, optimum declustering potentials and collision energies selected for each transition are given in Table S1 (MRM−) and Table S2 (MRM +).
The analytical column used was a Luna Omega Polar (2 × 150 mm, 3 µm, Phenomenex, Torrance, CA, USA) column equipped with a C18 guard column (2 × 4.6 mm, ID; Phenomenex, Torrance, CA, USA) kept at 35 °C. Eluent A was 95% MeOH (5% 10 mM ammonium acetate and 0.001% acetic acid in water) and eluent B was 95% 10 mM ammonium acetate and 0.001% acetic acid in water (5% MeOH). The following gradient elution programme was run: (0–1.0 min, 100% B), (1.0–2.0 min, linear gradient to 60% A), (2.0–3.0 min, linear gradient to 80% A), (3.0–4.5 min, 80% B), (4.5–6.0 min, linear gradient to 95% A), (6.0–10 min, 95% A), (10.0–10.1 min, 95% A), (10.1–15 min, 100% B). The injection volume was 5 µL, and the flow rate was 0.45 mL/min. The autosampler temperature was set at 4 °C.
The analytical column used was a Luna Omega Polar (2 × 150 mm, 3 µm, Phenomenex, Torrance, CA, USA) column equipped with a C18 guard column (2 × 4.6 mm, ID; Phenomenex, Torrance, CA, USA) kept at 35 °C. Eluent A was 95% MeOH (5% 10 mM ammonium acetate and 0.001% acetic acid in water) and eluent B was 95% 10 mM ammonium acetate and 0.001% acetic acid in water (5% MeOH). The following gradient elution programme was run: (0–1.0 min, 100% B), (1.0–2.0 min, linear gradient to 60% A), (2.0–3.0 min, linear gradient to 80% A), (3.0–4.5 min, 80% B), (4.5–6.0 min, linear gradient to 95% A), (6.0–10 min, 95% A), (10.0–10.1 min, 95% A), (10.1–15 min, 100% B). The injection volume was 5 µL, and the flow rate was 0.45 mL/min. The autosampler temperature was set at 4 °C.
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