2.1. Reagents
As the ester-form standard reagent,
3-MCPD dioleate (purity 99.8%) was
purchased from Wako Pure Chemical (Osaka, Japan). As the free-form
standard reagents, 3-MCPD (purity 98%)
was purchased from Macklin
Biochemical Co. Ltd (Shanghai,
China, www.macklin.cn),3-MCPD-d5 (purity 98.0%) from Larodan AB (Sweden,www.larodan.com). Phenylboronic
acid (PBA) (purity 95%) was purchased from Sigma-Aldrich (USA,www.sigmaaldrich.cn).Lipase
from Candida rugosa was purchased from Sigma-Aldrich. Lipase from
Candida sp. and lipase from
Thermophila sparsiformis was purchased from Shanghai Aladdin Biochemical
Technology Co. Ltd (Shanghai, China,www.aladdin.cn).
A 30% (w/v) sodium bromide aqueous solution was prepared and adjusted
pH with citric acid and disodium hydrogen phosphate aqueous solutions.
The lipase was dissolved with this solution to prepare an aqueous
solution of sodium bromide containing 150 U/mL lipase. A PBA solution
was prepared by dissolving 0.25 g PBA in 10 mL water/acetone mixed
solution (1:19, v/v).
1 mg of 3-MCPD esters standard was
weighed, dissolved in ethyl acetate, transferred to a 10 mL volumetric
flask, volumetric to calibration with ethyl acetatel. For the standard
stock solutions, 300 μg/mL ethanol solutions were prepared for 3-MCPD
and 200 μg/mL ethanol solutions were prepared for
3-MCPD-d5 as internal standard stock solutions. These
stock solutions could be stored at ≤ 20℃ for at least three months. On
the day of the study, the standard stock solution was diluted with the
30% sodium bromide aqueous solution to prepare 1.5 μg/mL standard
solutions. Additionally, 2.0 μg/mL internal standard solutions was
prepared by combining the internal standard stock solutions and diluting
it with the 30% sodium bromide aqueous solution.