Acquity uplc hss t3 c18 1.8 μm

Briefly, adding buffer S1 nuclease (Takara Biotechnology, Dalian, Liaoning, China), Alkaline Phosphatase (Takara Biotechnology) and Phosphodiesterase I (Sigma‐Aldrich) into 1 μg RNA, then the mixture was incubated at 37°C. After the RNA was digested into nucleosides completely, the mixture was extracted with chloroform (Sigma‐Aldrich). The resulting aqueous layer was collected for analysis with LC‐MS/MS. The sample extracts were analyzed using an UltraPerformanceLiquid (UPLC)‐MS/MS system (UPLC, ExionLC™ AD, Applied Biosystems 6500 Triple Quadrupole; oster City, CA, USA). The analytical conditions were as follows, LC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 μm; Waters, Milford, MA, USA); solvent system, water (2 mmol/L NH₄HCO₃): methanol (2 mmol/L NH₄HCO₃); gradient program, 95:5V/V at 0 min, 95:5V/V at 1 min, 5:95 V/V at 9 min, 5:95 V/V at 11 min, 95:5 V/V at 11.1 min, 95:5 V/V at 14 min; flow rate, 0.30 mL/min; temperature, 40°C; injection volume: 10 μL. The effluent was alternatively connected to an Electrospray Ionization‐triple quadrupole‐linear ion trap. A specific set of multiple reaction monitoring transitions were monitored for each period according to the metabolites eluted within this period.