Determination of indole-3-pyruvic acid levels in Arabidopsis thaliana by gas chromatography-selected ion monitoring-mass spectrometry.
A rapid and simple method is described for the determination of indole-3-pyruvic acid (IPA) levels in Arabidopsis thaliana by gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS). The method includes derivatization of IPA with hydroxylamine in the crude extract, followed by ethyl acetate partitioning, solid-phase extraction with C18 resin, reversed-phase high-performance liquid chromatography (HPLC), and GC-SIM-MS. Three derivatizing reagents were tested; these were pentafluorobenzylhydroxylamine, pentafluorophenylhydrazine, and hydroxylamine. Hydroxylamine proved to be the most useful, as the IPA-oxime was easiest to purify from plant extracts and was the most stable. IPA was quantified in Arabidopsis seedlings ranging in age from 5 to 12 days; levels varied from 4 to 13 ng/g, peaking at 7-9 days.
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Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003, USA.
Normanly
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J
D017360:Arabidopsis / Q000737:chemistry*; D002138:Calibration; D016166:Free Radical Scavengers / Q000032:analysis* / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D019811:Hydroxylamine / Q000737:chemistry; D007211:Indoles / Q000032:analysis* / Q000737:chemistry
D016166:Free Radical Scavengers; D007211:Indoles; D019811:Hydroxylamine; C008122:indol-3-yl pyruvic acid