Optimization of an Indirect Enzymatic Method for the Simultaneous Analysis of 3-MCPD, 2-MCPD, and Glycidyl Esters in Edible Oils

Abstract

We developed a novel, indirect enzymatic method for the analysis of fatty acid esters of 3-monochloro-1,2-propanediol (3-MCPD), 2-monochloro-1,3-propanediol (2-MCPD), and glycidol (Gly) in edible oils and fats. Using this method, the ester analytes were rapidly cleavaged by Candida rugosa lipase at room temperature for 0.5 h. As a result of the simultaneous hydrolysis and bromination steps, 3-MCPD esters, 2-MCPD esters, and glycidyl esters were converted to free 3-MCPD, 2-MCPD, and 3-monobromo-1,2-propanediol (3-MBPD), respectively. After the addition of internal standards, the mixtures were washed with hexane, derivatized with phenylboronic acid, and analyzed by gas chromatography-mass spectrometer (GC-MS). The analytical method was evaluated in preliminary and feasibility studies performed by 13 laboratories. The preliminary study from 4 laboratories showed the reproducibility (RSD R ) of < 10% and recoveries in the range of 102-111% for the spiked 3-MCPD and 2-MCPD in extra virgin olive (EVO) oil, semi-solid palm oil, and solid palm oil. However, the RSD R and recoveries of Gly in the palm oil samples were not satisfactory. The Gly content of refrigerated palm oil samples decreased whereas the samples at room temperature were stable for three months, and this may be due to the depletion of Gly during cold storage. The feasibility studies performed by all 13 laboratories were conducted based on modifications of the shaking conditions for ester cleavage, the conditions of Gly bromination, and the removal of gel formed by residual lipase. Satisfactory RSD R were obtained for EVO oil samples spiked with standard esters (4.4% for 3-MCPD, 11.2% for 2-MCPD, and 6.6% for Gly).

Collaborative Study of an Indirect Enzymatic Method for the Simultaneous Analysis of 3-MCPD, 2-MCPD, and Glycidyl Esters in Edible Oils

“…The method developed by Miyazaki et al 21 and modified following the feasibility study by JOCS 22 was used in this study Fig. 1 .…”

“…Using reference standards in the ester form necessitates long hydrolysis times, as needed for the sample being analyzed, to construct calibration curves. An indirect method using a lipase for ester cleavage was developed by Miyazaki et al 21 and was improved through preliminary and feasibility studies conducted by the MCPD subcommittee organized within JOCS for method validation 22 . In this method, ester hydrolysis is completed in 0.5 hour at room temperature.…”

Enzymatic hydrolysis and detection of 3‐monochloropropane‐1,2‐diol esters

“…At present, there are more and more studies on the determination of 3‐MCPD esters by indirect enzymatic hydrolysis method. An indirect method using a lipase for ester cleavage was developed by Miyazaki et al (2012) and was improved through preliminary and feasibility studies conducted by the MCPD subcommittee organized within JOCS for method validation (Koyama et al, 2015). In this method, ester hydrolysis is completed in 0.5 h at room temperature.…”

Glycidol and 3‐MCPD Analysis Methods for Mono‐ and Diglycerides

“…Variations of the method have been made to improve special purposes of the methods. [ 6–14 ] Some methods are two sets of procedures; one where both GE and MCPDs are detected and quantified after chlorination (assay A), and one where only MCPDs are detected and quantified after fast bromation, the fast brumation result in unreliable direct quantification of glycidol (Assay B). GE content is then calculated as the difference between the two results.…”