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.