The supernatant was loaded into the

The supernatant was loaded into the Chem Elut column
after 4 min of equilibration. EC was eluted with 160 mL of
methylene chloride. The eluent was concentrated to about
2~3 mL using a rotary evaporator, transferred into v-vial,
and further concentrated to 1 mL under a gentle stream of
nitrogen.
A 7820A GC-5977E MS (Agilent Technologies, Santa
Clara, CA, USA) was used for the quantification and identification
of EC. The GC conditions were as follows: capillary
column 30 m length × 0.25 mm i.d., 0.25 μm film
thickness DB-WAX (J&W, Folsom, CA, USA), helium carrier
gas at 1 mL/min, injection volume with 2 μL in splitless
mode, and injector 210oC. Oven temperature: 60oC,
10oC/min to 90oC, 2oC/min to 130oC held for 5 min, 20oC/
min to 220oC, and then held for 3 min. The MS was operated
in the selected ion monitoring (SIM) with electron
impact ionization (70 eV). The MS transfer line and ion
source were kept at 240oC and 230oC, respectively. Mass to
charge (m/z) 62 and 64 were major fragment ions of EC and
d5-EC. EC was quantified using calibration curves made
from peak area ratios of EC/d5-EC (m/z 62 vs m/z 64). EC
peak was identified by comparing the area ratios of m/z 62
vs m/z 74 that were major fragment ions of EC.