2.4. Analytical methods
For derivatization, samples containing carboxylic acid or amine functional groups, such as l-PA, were diluted to less than 10 mM, and 100 μL of the diluted samples was mixed with 400 μL of 1 N NaOH, 66 μL of pyridine, and 334 μL of ethanol by gentle shaking. Then, 80 μL of ECF was added to the mixture, followed by vortexing for 30 s. To complete the derivatization and separate the layers, 800 μL of 30 mM NaHCO3 and chloroform were added into the derivatized mixture, followed by shaking for 10 s. After settling, the bottom layer was filtered using a 0.2 μm syringe filter. All reactions were performed at room temperature (Yi et al., 2015).
The derivatized samples were analyzed by gas chromatography-mass spectrometry (GC-MS) (Perkin Elmer Clarus 680 GC system and Clarus SQ 8T MS system). Sample solution (1 μL) was injected into an Elite 5MS column (30 m × 0.25 mm × 0.25 μm film thickness) using helium as a carrier gas at a flow rate of 1 mL/min. The initial oven temperature was set at 120 °C for 5 min and increased to 200 °C in increments of 5 °C/min. When the temperature reached 200 °C, the rate was changed to 2 °C/min. After the temperature increased to 220 °C, the rate was set to 10 °C/min. It was finished when the oven temperature reached 300 °C. The temperature of the injector and ion source were set to 270 °C and 250 °C, respectively. The electron energy was 70 eV. The mass spectrometer was operated in full scan mode from 45 to 400 m/z, with a scan time of 0.2 s.