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Preparation and analysis of polar metabolites
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Polar metabolites were methoximated
and trimethylsilylated to improve chromatographic behavior.
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120µL
of 15mg/mL methoxyamine hydrochloride in pyridine was added, incubated at 50°C,
and sonicated until crystallized metabolites went into solution.
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120µL
MSTFA + 1%TMCS was subsequently added and the solution incubated for 30 min
at 50°C.
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1.0 µL
of the solution was injected at 15:1 split ratio onto a HP 6890 GC equipped
with a 60M DB-5-MS column
coupled to a HP 5973 MS.
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The injection port and transfer arm
were held at 280°C,
and the oven ramped from 80°C
(2 min) to 315°C
(6 min) at 5°C/min.
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The MS source was held at 250°C
and the quadrupole at 150°C
and scanned from 50-650.
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