Zorbax stablebond analytical sb c18 column

Thermo Scientific Dionex Ultimate 3000 HPLC (Thermo, Waltham, MA, USA) coupled with a Triple Quad^TM 5500 mass spectrometer (Sciex, Framingham, CA, USA) with an ESI source (Turbo Ionspray) was used for the analysis of the oxidation product of 5hmdU and labeled product of 5fdU. The LC separation was performed on a Zorbax Stablebond Analytical SB-C18 column (2.1 mm × 100 mm, 3.5 μm, Agilent Technologies, Santa Clara, CA, USA) at 35 °C. Water containing 0.0085% FA (v/v, solvent A) and MeOH containing 0.0085% FA (v/v, solvent B) was employed as the mobile phase. A gradient of 0–5% B for 6 min, 5–80% B for 0.5 min, 80% B for 5 min and 100% B for 5 min was used. The flow rate of the mobile phase was set at 0.3 mL min⁻¹. The mass spectrometry detection was performed under the positive ESI mode. The nucleosides and labeled products were monitored using the multiple reaction monitoring (MRM) mode. The MRM parameters were optimized to achieve maximal detection sensitivity (Table S1). The values for curtain gas, collision gas, ionspray voltage, ion source gas 1, ion source gas 2, temperature, declustering potential and entrance potential were 20, 8, 5500, 55, 60, 600, 70 and 10, respectively.