Abstract
Gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) has become a promising technique for simultaneous and rapid analysis of small metabolites in complex mixtures. The aim of this work was to establish the quantitative nature of the information generated by amino acid analysis of crude leaf extracts using GC-TOF-MS. Dried aliquots of methanol/water extracts of Arabidopsis leaves were analysed in parallel by GC-TOF-MS following trimethylsilylation or high performance liquid chromatography and fluorescence detection of o-phthaldialdehyde derivatives (OPA-HPLC). Twenty amino acids could be routinely detected in leaf extracts by both methods. Because of instability of some trimethylsilylated derivatives, all GC-TOF-MS analyses were performed within a window of 2 h 30 min following derivatization. Repeatability studies showed that relative standard deviations for multiple injections of a single extract were below 20% for both techniques, though significantly smaller for OPA-HPLC. Similar between-extract variability and condition-independent biological variation were detected by OPA-HPLC and GC-TOF-MS, and both techniques detected similar environmentally induced changes in four major amino acids. Recovery of standard compounds through the extraction procedure was between 80% and 120% for OPA-HPLC but more variable when analysed by GC-TOF-MS. When quantified on the basis of tissue fresh weight according to response factors of mixed standards, the two techniques gave consistent values for a number of amino acids but divergent values for others. Taken together, the results suggest GC-TOF-MS analysis of Arabidopsis leaves with the present protocol can be used for absolute quantification of 4–7 amino acids, accurate relative quantification of 8–11 amino acids, and more limited quantification for five compounds of this class.
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