2.4. Enzymatic hydrolysis of 3-MCPD esters standard
1 mg of 3-MCPD esters standard was weighed and dissolved in a 10 mL test
tube with ethyl acetate. After mixing, 20 uL of the above solution was
put into a 5 mL test tube. After adding 3 mL of the 30% sodium bromide
aqueous solution containing 150 U/mL lipase, the mixture was shaken by a
high-speed shaker for 30 min at room temperature to hydrolyze the
esters. The mixture was then heated in a water bath at 80℃ for 10 min.
The mixture was cooled to room temperature, and 50 μL of 2.0 μg/mL
internal standard mix was added. Next, 3 mL of hexane was added and the
tube was vortexed for 10 s. The aqueous layer was transferred to a new
test tube, to which another 3 mL of hexane was added and the tube was
vortexed again for 10 sec. After the hexane layer was removed, 3-MCPD in
the aqueous layer and the deuterated free form internal standards were
derivatized by adding 100 μL of PBA solution and 3 mL of hexane, and
shaking at room temperature for 5-10 min. The organic layer was then
transferred to a new test tube containing sodium sulfate and
concentrated to approximately 0.5-0.8 mL under a stream of nitrogen.
After filtration through a 0.2 μm membrane filter, the sample was
subjected to GC-MS analysis.
2.5. Procedures for analysis of3-MCPD esters
In a 10 mL test tube, 0.1 g of oil sample was dissolved in 200–500 μL
of isooctane. After adding 3 mL of 30% NaBr aqueous solution containing
150 U/mL lipase, the mixture was shaken by a high-speed shaker for 30
min at room temperature. The test tube was heated for 10 min in an 80℃
water bath. The test tube was cooled to room temperature, and 50 μL of
2.0 μg/mL internal standard mixed solution was added. Next, 3 mL of
hexane was added and shaken for 10 min. The resulting aqueous layer was
transferred to a new test tube. Another 3 mL of hexane was added and the
tube was shaken for 10 sec; the resulting hexane layer was subsequently
removed. A 100 μL PBA solution was added to the aqueous layer, and the
mixture was agitated with a vortex mixer for 10 sec. Next, 3 mL of
hexane was added and shaken for 10 sec. The resulting organic layer was
transferred to a new test tube and concentrated to approximately
0.5–0.8 mL using a stream of nitrogen. After filtration through a 0.2
μm membrane filter, the samples were subjected to GC-MS analysis.
2.6. GC-MS Analysis
A GC capillary column was used with a (5%-phenyl)-methylpolysiloxane
liquid phase, 30 m length, 0.25 mm internal diameter, and 0.25 μm film
thickness. The carrier gas was helium at a fixed flow rate of 1.2
mL/min. The sample was injected in splitless mode at 250℃. The column
oven temperature was held at 60℃ for 1 min, then raised to 150℃ at
10℃/min, 180℃ at 3℃/min, and finally 300℃ at 30℃/min, before being held
at 300℃ for 8 min (total run=32 min). MS was performed in positive
electron ionization mode with an ion source temperature of 230℃.
Quantitative and qualitative analyses were performed by selective ion
monitoring (SIM) using ions at m/z 147 and 196 for the 3-MCPD
derivative, 150 and 201 for the 3-MCPD-d5 derivative.
Results and Discussions