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Aliquots of 50 mg each were prepared and stored for up to 3 years at –80°C until further processing. , 2000). Non-sample controls were entered into analysis at this step of the protocol. Extraction was performed in three steps: (i) the first extraction step was initiated by adding a 300 µL volume of pre-cooled polar water-miscible solvent, which contained the internal standard substances, ribitol, D(-)-isoascorbic acid for monitoring the recovery of GC-separated, oxidation-sensitive vitamin C, L(+)-ascorbic acid, and 2,3,3,3-D4 alanine, to the deep-frozen powder without removal of the steel ball, (ii) after initial incubation a 200 µL volume of chloroform was added and shortly incubated 5 min at 37°C (iii) finally, polar and lipid phases were separated by adding 400 µL of bi-distilled water 1 min vigorous mixing and 10 min centrifugation at room temperature in a microvial centrifuge set to maximum speed.
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And Hall, R. , 2004, Potential of metabolomics as a functional genomics tool, Trends in Plant Science 9(9):418-425. , 1999, The Unified Modeling Language User Guide, Reading, Massachusetts, Addison-Wesley. , 2001, Minimum information about a microarray experiment (MIAME) - toward standards for microarray data, Nature Genetics 29(4):365-371. , 2003, AnIMLs in the spectroscopic laboratory? Spectroscopy Europe 15(5):25-28. Dromey, R. , and Stefik, M. , 1976, Extraction of mass spectra free of background and neighbouring component contributions from gas chromatography/mass spectrometry data, Analytical Chemistry 48(9):1368-1375.