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SPE purification<br />
SPE columns w … SPE purification<br />
SPE columns were conditioned with adequate amounts of methanol and then water. A standard mixture of organic and inorganic compounds was dissolved in 1% formic acid (FA); thereafter, 500 mL of the mixture was loaded on to each of the columns. The columns were then washed and the organic metabolites were eluted with the solvents listed in Table 1. The solvent volume used for washing and elution was 20 times the volume of the solid-phase packing. The eluted solution was dried by centrifugal concentration and then resuspended in 20 mL of water. Five microliters of the solution was used for CE-MS analysis. The conditions used in CE-MS analysis have been described in a previous report.<br />
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CE-MS analysis<br />
All CE-TOFMS 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, a G1603A Agilent CE-MS adapter, and a G1607A Agilent CE-ESI-MS sprayer kit. G2201AA Agilent ChemStation and Analyst QS software were used for CE and TOFMS, respectively. Separations were carried out using a fused silica capillary (50 mm id x 100 cm total length) filled with 1M FA or 20mM ammonium formate (pH 10.0) as the electrolyte for cation and anion analyses, respectively. The capillary temperature was maintained at 201C. Fifteen nanoliters of the sample solutions was injected at 50 mbar for 15 s. The sample tray was cooled below 41C. Prior to each run, the capillary was flushed with the electrolyte for 5 min. The voltage used for the separation was set at 30 kV. Methanol/water (50%, v/v) containing 0.5 mM reserpine was delivered as the sheath liquid at 10 mL/min. ESI-TOFMS was conducted in the positive-ion mode for cation analyses and in the negative-ion mode for anion analyses, and the capillary voltage was set at 4 kV. The flow rate of heated dry nitrogen gas (heating temperature 3001C) was maintained at 10 psig. The fragmentor, skimmer, and Oct RFV voltage were set at 110, 50, and 160 V, respectively, for cation analysis and at 120, 60, and 220 V, respectively, for anion analysis. Automatic recalibration of each acquired spectrum was performed using reference masses of reference standards. The methanol dimer ion ([2M+H]+, m/z=65.0597) and reserpine ([M+H]+, m/z=609.2806) for cation analyses or the FA dimer ion ([2M–H]-,m/z=91.0037) and reserpine ([M–H]-, m/z=607.2661) for anion analyses provided the lock mass for exact mass measurements. Exact mass data were acquired at a rate of 1.5 cycles/s over a 50–1000 m/z range. of 1.5 cycles/s over a 50–1000 m/z range.
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