Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism

Qi Liu; Amita Gupta; Ayse Okesli-Armlovich; Wenjie Qiao; Curt R. Fischer; Mark Smith; Jan E. Carette; Michael C. Bassik; Chaitan Khosla

LC-MS/MS was performed on an Agilent 1290 infinity II LC system tandem with 6470 triple quad mass spectrometer using ion pairing chromatography. LC separation was performed at 40 ºC with a solvent flow rate of 0.25 mL/min on a Zorbax RRHD Extended-C18 column (2.1 × 150 mm, 1.8 µM). Buffer A contained 97% H2O, 3% MeOH, 5 mM tributylamine, 5.5 mM acetic acid and 1 µM medronic acid at pH=5.0;Buffer B contained ca. 100% MeOH, 5 mM tributylamine, 5.5 mM acetic acid and 10 µM medronic acid at pH=7.0 The initial mobile phase composition was 100% solvent A, and the gradient (%B) after sample injection was as follows: 0% at 2.5 min, 20% at 7.5 min, 45% at 14 min, 99% at 20 to 23 min, and 0% from 23.1 to 27.1 min. The autosampler temperature was set to 4 °C and 5 µL of sample was injected. Samples were measured in the negative ESI mode with capillary voltage at −3.5kV. Further source settings were as follows: gas temperature, 250 °C; gas flow, 13 L/min; nebulizer, 35 psi; sheath gas temperature, 325 ºC; sheath gas flow, 12 L/min; nozzle voltage, 500 V; and delta EMV, -200. Acquisition was performed in dynamic multiple reaction monitoring (dMRM) mode with fragmentation pattern and retention time setting as Table SI .