For measurement of nucleotides and deoxy-nucleotides prior to mass spectrometry analysis, the dried extract was suspended in 50 µL of methanol: water (50:50) containing 0.1% formic acid. Samples were delivered to the MS via reverse phase chromatography using a RRHD SB-CN column (1.8 µm, 3.0 × 100 mm, Agilent Technologies) at 300 µl/min. Gradient spanning 2% B to 98% B over a 15-minute period followed by 98% B to 2% B for a 1-minute period. Then gradient is continued for a 4-minute time period to re-equilibrate the column. Buffers A and B were comprised of 0.1% formic acid in water and acetonitrile, respectively.
Ten microliters were injected and analyzed using a 6495 QQQ triple quadrupole mass spectrometer (Agilent Technologies) coupled to a 1290 series HPLC system via Selected Reaction Monitoring (SRM). Metabolites were measured using positive ionization mode with an ESI voltage of +4000 ev, respectively. Approximately 9–12 data points were acquired per detected metabolite.
Ten microliters were injected and analyzed using a 6495 QQQ triple quadrupole mass spectrometer (Agilent Technologies) coupled to a 1290 series HPLC system via Selected Reaction Monitoring (SRM). Metabolites were measured using positive ionization mode with an ESI voltage of +4000 ev, respectively. Approximately 9–12 data points were acquired per detected metabolite.