Metabolomics sample preparation analysis
850 μL methanol: water mixture (9:1, v/v) was added to 150 μL of sample. The mixture was thoroughly vortexed for 1 minute, and then centrifugaiton was performed at 15,000 rpm for 10 minutes. 400 μL supernatant was evaporated overnight at 4°C using a vacuum centrifuge. The dried samples were methoxylated and derivatized as described previously (8,9). 20 μL of methoxyamine hydrochloride (20 mg/mL in pyridine) was added, and the samples were incubated 90 minutes at 30°C. Finally, N-methyl-N-(trimethylsilyl)-trifluoroacetamide solution with 1% trimethylchlorosilane was added, and the samples were incubated for at 37°C for 30 minutes.