Delipidation. The leaf samples were extracted by isopropanol at 85 oC for 10 min and room temperature overnight, followed by isopropanol, chloroform/methanol (2:1, v/v), chloroform/methanol (1:2, v/v), and methanol extraction at room temperature 24 h for each. The samples were dried under a gentle stream of nitrogen and afterward were dried in vacuum dessicator for two days.
Depolymerization. Each delipidated sample (10-100 mg) with 25 μg of methyl heptadecanoate (internal standard) reacted with 2 ml of freshly made medium which contained 1.2 ml of methanol, 0.3 ml of methyl acetate and 0.5 ml of 25% sodium methoxide in methanol (Sigma) at 60 oC for 2 h. In each tube, 4ml of dichloromethane, 0.7 ml of glacial acetic acid, and 1 ml of 0.9% NaCl (w/v) Tris 100 mM pH 8.0 were added. The samples were well shaken and phases were separated by centrifugation for 5 min at 3000 rpm. The lower organic phase was collected, washed with 2 ml of 0.9% NaCl (w/v) Tris 100 mM pH 8.0, and dry over anhydrous sodium sulfate, followed by evaporation to dryness under a gentle nitrogen stream.
Derivatization. The dried sample was derivatized with 100 μl of BSTFA and 100 μl of anhydrous pyridine at 80 oC for 1h. The derivatized sample was dried under light stream of nitrogen and resolved in 200 μl of chloroform for GC-MS/FID analysis.
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