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.