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Extraction from sample<br />
A 10 m … Extraction from sample<br />
A 10 mg sample of each dried root was weighed and homogenized in a 2-mL Eppendorf tube and 1,000 μL of a single-phase mixture of methanol/water/chloroform (5:2:2, v/v/v) containing an internal standard (ribitol, 0.4 mg/mL H2O) was added for metabolite extraction.The extraction was carried out at room temperature using mixer mills (MM 301, Verder Scientific Co. Ltd., Haan, German) at 20 Hz for 5 min.After centrifugation at 16000 ×g for 3 min at 4°C, 800 μL of the supernatant was transferred to a new 1.5-mL Eppendorf tube, to which 400 μL of distilled water was added and vortexed for 10 s. After centrifugation at 16000 ×g for 3 min at 4 °C, 400 μL of the upper layer containing hydrophilic metabolites was transferred to a new 1.5-mL Eppendorf tube, and then freeze-dried (Taitec model VD-800F freeze dryer, Taitec Co. Ltd., Saitama, Japan). For quality control samples, 10 mg of each sample was added into a 15-mL tube (total 360 mg; six biological samples and six biological replicates) and then divided into six 2-mL Eppendorf tubes such that they ontained 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, and 60 mg, respectively. An empty tube was also prepared, labeled as 0 mg QC, for monitoring background effects. Metabolite extraction of these dilution series was performed sing the procedure described above.<br />
For oximation, 50 μL of methoxyamine hydrochloride in pyridine (20 mg/mL) was added and incubated at 30 °C for 90 min. For silylation, 50 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was added and ncubated at 37 °C for 30 min. The mixture was centrifuged at 16000 ×g for 5 min at 20 °C and a 50-μL aliquot of the resultant supernatant was transferred into GC–MS vial. Each sample (1 μL) was injected into a GCMS-QP2010 Ultra instrument (Shimadzu Co., Kyoto, Japan) in split mode (25/1, v/v). Each diluted QC sample was analyzed four times from the same GC–MS vial. Analytical conditions were optimized as described in a previous study.14 The column was a fused silica capillary column (30 m × 0.25 mm i.d.) coated with 0.25 μm CP-SIL 8 CB low bleed/MS (Agilent Technologies Japan, Ltd., California, USA). The front inlet temperaturewas 230 °C, the helium gas flow rate was 1 mL/min, and the column temperature was held at 80 °C for 2 min isothermally, then raised by 15 °C/min to 330 °C, and finally held for 6 min isothermally. The transfer line and ion source temperatures were 250 °C and 200 °C, respectively. Twenty scans per second were recorded over the mass range 85–500 m/z. e recorded over the mass range 85–500 m/z.
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