2.3. GC-MS Analysis

The dried samples were chemically derivatized via the methyl chloroformate (MCF) method in accordance with the protocol published by Smart et al. [10] The dried extracts were resuspended in the order of following solutions: 200 μL of sodium hydroxide (1 M), 167 μL of methanol, and 34 μL of pyridine. To commence the MCF derivatization, 20 μL of MCF was added and mixed for 30 seconds twice. To stop derivatization, 400 μL chloroform and 400 μL of bicarbonate (50 mM) were added and mixed 10 s each time. The mixture was then centrifuged for 2 min at 2000 rpm to isolate the organic layer from the aqueous layer. Lastly, the aqueous layer was removed and added anhydrous sodium sulphate to eliminate the remaining water. The organic layer containing derivatized metabolites was transferred into GC vials readily for GC-MS analysis. For GC-MS, a ZB-1701 gas-phase capillary column (30 m × 320 μm × 0.25 μm; Phenomenex, Torrance, CA, USA), inlet for a splitter/splitless inlet (Agilent Technologies, Santa Clara, CA, USA) was used, with an inlet temperature of 250°C, a flame ionization detector (FID) detector temperature of 250°C, a nitrogen flow rate of 40 mL/min, a hydrogen flow rate of 40 mL/min, an air flow rate of 450 mL/min, a split ratio of 20 : 1, and a sample injection volume of 1 μl. In addition, the temperature protocol was as follows: an initial temperature of 80°C, with an increase from 25°C/min to 200°C, and then with an increase from 3°C/min to 215°C. Finally, the metabolites were analyzed at an increase from 2°C/min to 230°C.