The metabolite standards, instrumentation and CE-TOF-MS condition were used in this study as previously described (Soga et al. 2006 (link)), with slight modifications in the lock mass system setting. All chemical standards were of analytical or reagent grade and were obtained from commercial sources. They were dissolved in Milli-Q water (Millipore, Bedford, MA, USA), 0.1 mol/l HCl or 0.1 mol/l NaOH to obtain 1, 10 or 100 mmol/l stock solutions. The working solution was prepared prior to use by diluting with Milli-Q water to the appropriate concentration.
All CE-MS experiments were performed using an Agilent CE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany), an Agilent G3250AA LC/MSD TOF system (Agilent Technologies, Palo Alto, CA, USA), an Agilent 1100 series binary HPLC pump, and the G1603A Agilent CE-MS adapter and G1607A Agilent CE-ESI-MS sprayer kit. System control and data acquisition were done with G2201AA Agilent Chemstation software for CE and Analyst QS software for TOF-MS (ver. 1.1).
All samples were measured in single mode (see below); separation was done in fused-silica capillaries (50 μm i.d. × 100 cm total length) filled with 1 M formic acid as the background electrolyte. Sample solutions were injected at 50 mbar for 3 s and a voltage of 30 kV was applied. The capillary temperature was maintained at 20°C and the temperature of the sample tray was kept below 5°C using an external thermostatic cooler. The sheath liquid, comprising methanol/water (50% v/v) and 0.5 μM reserpine, was delivered at 10 μl/min. ESI-TOF-MS was conducted in the positive ion mode. The capillary voltage was set at 4 kV; the flow rate of nitrogen gas (heater temperature 300°C) was set at 10 psig. In TOF-MS, the fragmentor, skimmer and OCT RFV voltage were set at 75, 50 and 125 V, respectively. In the present study, we used a methanol dimer adduct ion ([2MeOH + H]+, m/z 65.059706) and hexakis phosphazene ([M + H]+, m/z 622.028963) to provide the lock mass for exact mass measurements. Exact mass data were acquired at the rate of 1.5 cycles/s over a 50–1000 m/z range.
All CE-MS experiments were performed using an Agilent CE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany), an Agilent G3250AA LC/MSD TOF system (Agilent Technologies, Palo Alto, CA, USA), an Agilent 1100 series binary HPLC pump, and the G1603A Agilent CE-MS adapter and G1607A Agilent CE-ESI-MS sprayer kit. System control and data acquisition were done with G2201AA Agilent Chemstation software for CE and Analyst QS software for TOF-MS (ver. 1.1).
All samples were measured in single mode (see below); separation was done in fused-silica capillaries (50 μm i.d. × 100 cm total length) filled with 1 M formic acid as the background electrolyte. Sample solutions were injected at 50 mbar for 3 s and a voltage of 30 kV was applied. The capillary temperature was maintained at 20°C and the temperature of the sample tray was kept below 5°C using an external thermostatic cooler. The sheath liquid, comprising methanol/water (50% v/v) and 0.5 μM reserpine, was delivered at 10 μl/min. ESI-TOF-MS was conducted in the positive ion mode. The capillary voltage was set at 4 kV; the flow rate of nitrogen gas (heater temperature 300°C) was set at 10 psig. In TOF-MS, the fragmentor, skimmer and OCT RFV voltage were set at 75, 50 and 125 V, respectively. In the present study, we used a methanol dimer adduct ion ([2MeOH + H]+, m/z 65.059706) and hexakis phosphazene ([M + H]+, m/z 622.028963) to provide the lock mass for exact mass measurements. Exact mass data were acquired at the rate of 1.5 cycles/s over a 50–1000 m/z range.
