Direct Determination of MCPD Fatty Acid Esters and Glycidyl Fatty Acid Esters in Vegetable Oils by LC–TOFMS

Analysis of MCPD esters and glycidyl esters in vegetable oils using the indirect method proposed by the DGF gave inconsistent results when salting out conditions were varied. Subsequent investigation showed that the method was destroying and reforming MCPD during the analysis. An LC time of flight MS method was developed for direct analysis of both MCPD esters and glycidyl esters in vegetable oils. The results of the LC–TOFMS method were compared with the DGF method. The DGF method consistently gave results that were greater than the LC–TOFMS method. The levels of MCPD esters and glycidyl esters found in a variety of vegetable oils are reported. MCPD monoesters were not found in any oil samples. MCPD diesters were found only in samples containing palm oil, and were not present in all palm oil samples. Glycidyl esters were found in a wide variety of oils. Some processing conditions that influence the concentration of MCPD esters and glycidyl esters are discussed.     

Influence of precursors on the formation of 3‐MCPD and glycidyl esters in a model oil under simulated deodorization conditions

To find new ways for reducing the potential of palm oil to form 3‐monochloropropane‐1,2‐diol (3‐MCPD) and glycidyl esters during refining it is helpful to know more about the influence of different precursors like diacylglycerols (DAGs) and monoacylglycerols (MAGs), lecithin, and chlorine containing compounds. After adding increasing amounts of the different precursors to a model oil obtained by removal of polar compounds from crude palm oil and heating the mixture under standardized conditions to 240°C for 2 h the contents of 3‐MCPD and glycidyl esters were analyzed according to the standard procedure of DGF C‐VI 18 (10). DAGs and MAGs were found to increase the potential of palm oil to form 3‐MCPD and glycidyl esters, but refined lecithin showed no influence. Sodium chloride as well as tetra‐n‐butylammoniumchloride (TBAC) led to higher contents of the esters. Whereas the addition of TBAC raised the amount of glycidyl esters as well as 3‐MCPD esters, sodium chloride largely raised the amount of 3‐MCPD esters. An addition of 5 mmol of sodium carbonate/kg model oil spiked with sodium chloride reduced the amount of glycidyl esters almost completely; the 3‐MCPD esters were reduced by 50%. About 1 mmol sodium hydrogen carbonate/kg oil reduced both 3‐MCPD and glycidyl esters almost completely. Practical applications : For the mitigation of the formation of 3‐MCPD esters and related compounds in refined edible oils, it is helpful to know more about the effect of different possible precursors. Using a broader data basis, it is possible to adopt the oil processing but especially the choice of the raw material to the demands of the market for lower contents of the esters in the refined products.     

Mitigation of 3‐MCPD and glycidyl esters within the production chain of vegetable oils especially palm oil

In 2007 the oil producing industry and the downstream food processing industry were worried by the announcement that fatty acid esters of 3‐MCPD and later in 2008 that glycidyl esters have been found in different types of vegetable oils after processing. The reason for the concern was that the German Federal Institute for Risk Assessment assumed in a first statement the complete degradation of the esters to free 3‐MCPD and glycidol both classified by the International Agency for Research on Cancer (IARC) as possible and probably, respectively, carcinogenic to humans. In the meantime a lot of research has been done on these heat‐induced compounds to mitigate their formation during oil processing and today the content in vegetable oils could be remarkably lower if all approaches were implemented. This paper summarizes the different mitigation strategies and shows their effect on lowering the amount of 3‐MCPD and glycidyl esters in refined vegetable oils.     

Generation of 3‐monochloro‐1,2‐propanediol and related materials from tri‐, di‐, and monoolein at deodorization temperature

Heating tests of pure tri‐, di‐, and monoolein (TO, DO, and MO, respectively) with and without the addition of tetrabutylammonium chloride as a chloride source at 240°C revealed the characteristic reactions that generate 3‐monochloro‐1,2‐propanediol‐related materials (3‐MCPD‐RM) in each acylglycerol. 3‐MCPD‐RM were formed mainly from DO and MO, with only a small amount from TO. Glycidyl ester was the predominant class of 3‐MCPD‐RM generated from both DO and MO, and was increased continuously throughout the heating period with comparable rates in both DO and MO, which also generated 3‐MCPD esters with chloride in a short completion time with an achieved level that was fourfold higher for MO than for DO. The production of free glycidol and 3‐MCPD was confirmed only in heated MO, but not from TO and DO, in a closed heating system, although these compounds were never detected in oils heated under simulated distillation conditions using a gas stream. In a closed system, both free glycidol and 3‐MCPD were increased throughout the heating period, which differed from the esters. Since an interesterification reaction, which produced free glycerol, was observed only in heated MO, free glycerol might be one of the precursors for those free forms. For clarification, further investigation is required.     

Determination of 3‐MCPD esters in edible oil – methods of analysis and comparability of results

The discovery of fatty acid esters of 3‐chloropropane‐1,2‐diol (3‐MCPD) in edible oil products initiated food monitoring campaigns in many EU Member States. As the determination of 3‐MCPD esters was new to most laboratories, questions on the reliability of the produced analysis data were raised. In response to this, the Institute for Reference Materials and Measurements (IRMM) of the European Commission's Joint Research Centre (JRC) organised a proficiency test on the determination of 3‐chloropropane‐1,2‐diol esters (3‐MCPD esters) in edible oils. The aim of this proficiency test was to scrutinise the capabilities of official food control laboratories, private food control laboratories as well as laboratories from food industry to determine the 3‐MCPD esters content of edible oils. The study was carried out in accordance with “The International Harmonised Protocol for the Proficiency Testing of Analytical Chemistry Laboratories” and ISO Guide 43. The test materials dispatched to the participants were: refined palm oil, extra virgin olive oil spiked with 3‐chloropropane‐1,2‐dioleate and 3‐MCPD standard solution in sodium chloride. Altogether 41 laboratories from 11 EU Member States, Switzerland and Macedonia subscribed for participation in the study. The analysis task was to determine the 3‐MCPD esters content as total 3‐MCPD content of the test samples. Participants were free to choose their analysis methods. In total, 34 laboratories reported results to the organisers of the study. The performance of laboratories in the determination of 3‐MCPD esters in edible oils was expressed by z‐scores. About 56% of the participants performed satisfactorily in the determination of 3‐MCPD esters in palm oil and 85% for the spiked extra virgin olive oil test sample. The study revealed that the direct transesterification of the sample without the prior removal of glycidol esters might lead to strong positive bias.     

Determination of total 3‐chloropropane‐1,2‐diol (3‐MCPD) in edible oils by cleavage of MCPD esters with sodium methoxide

A method for the determination of total 3‐chloropropane‐1,2‐diol (3‐MCPD) in edible fats and oils was presented. 3‐MCPD was released from 3‐MCPD fatty acid esters by transesterification with NaOCH₃/methanol. After derivatization with phenylboronic acid, 3‐MCPD was determined by GC‐MS. Deuterium‐labeled 3‐MCPD was used as internal standard. In a model experiment, it was shown that acidic hydrolysis with methanol/sulfuric acid, which is normally used for the release of 3‐MCPD from its esters, can cause problems because under acidic conditions additional 3‐MCPD can be formed. No additional 3‐MCPD was formed using NaOCH₃/methanol for transesterification. Eleven samples of cold‐pressed and refined safflower oils were analyzed with this method. Levels of total 3‐MCPD were in the range from <100 up to 3200 µg/kg.     

Biotransformation of glycidol by the unspecific wax ester synthase/acyl‐CoA:diacylglycerol acyltransferase of Acinetobacter baylyi ADP1

Glycidol was biologically derivatized by the unspecific wax ester synthase/acyl coenzyme A (acyl‐CoA): diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter baylyi ADP1 into glycidyl acyl ester. Catalysis of in vitro conversion of glycidol to glycidyl acyl ester by the WS/DGAT from A. baylyi was verified by (i) a radiometric assay, (ii) thin‐layer chromatography and (iii) also by ESI‐MS. A specific activity of 50 nmol·mg^–1·min^–1 was obtained when 10 mM glycidol and 5 µM palmitoyl‐CoA were used. In vivo synthesized glycidyl acyl esters in recombinant E. coli were detected and quantified by staining with the epoxide‐specific reagent 4‐(4‐nitrobenzyl)‐pyridine. Of glycidyl acyl esters, 1.5 mg/L was obtained from the culture in the presence of 10 mM glycidol and 10 mM oleate.     

Glycidols Esters, 2-Chloropropane-1,3-Diols,and 3-Chloropropane-1,2-Diols Contents in Real Olive Oil Samples and their Relation with Diacylglycerols

A series of authentic virgin, refined, and mixtures of olive oils was analyzed for their content of 2-and 3-chloropropanediol (MCPD) esters expressed as 2−/3-MCPD, glycidol (and related glycidyl esters) (GE), and diglycerides (DAG). High concentrations of MCPD and GE were found, above all, in pomace oils, which come from the poorer starting raw materials, while virgin olive oils, as expected, do not contain any process contaminant. On the other hand, DAGs are present in all samples, demonstrating that their involvement in the formation of such contaminants only occurs when temperatures are higher than that used during the refining steps. The lack of correlation between the amounts of MCPD and GE can be ascribed to their completely different chemical stability as the epoxy ring of the GE opens easily, leading to both short-chain derivatives and/or MCPD itself. This finding can also explain the data about the absence of 2-MCPD in all the analyzed oil samples: other than the statistical probability and the steric effect of the SN2 formation mechanism, both in favor of the 3- derivative, we have also to consider the MCPD formation pathway involving glycidol that, under opportune conditions of refining, can increase the whole amount of 3-MCPD (under thermodynamic control, 3-MCPD is more stable).     

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

Distilled monoglycerides (DMG) and mono-di-glycerides (MDG) are commonly used as emulsifiers in various processed foods, e.g., bread, margarine, and ice cream. DMG and MDG are derived from triacylglyceride (TAG) oils, and thus it is speculated that the harmful substances glycidol and 3-monochloropropane-1,2-diol (MCPD) or their esters can also appear in DMG and MDG as they do in edible oil. However, present analysis methods, often developed for MCPD esters and glycidyl esters in TAG oils are not suitable for many DMG and MDG products. Dissolution of the DMG and MDG is the main problem. In this work a method for quantifying content of glycidol esters (GE) and 3-MCPD using an indirect method quantifying the compounds with GC-MS has been tested. The development has been developed based on the American Oil Chemists' Society (AOCS) Cd 29c method including the modifications implemented by Axel Semrau in the automated procedure for triglycerides. With the proposed procedure it is shown that the new analysis method produces valid results in the range 1–10 ppm, and acceptable results at 0.5 ppm for both 3-MCPD and glycidol. The method is an important part of providing safe and healthy emulsifier improved food products to consumers in European Commission and the rest of the world. Practical Applications: The European Commission (EU) has requested the emulsifier industry to supply data for GE and MCPD content as a first step in making legislation for maximum content as in edible oils and fats. However, there is no analysis method for emulsifiers (e.g., mono- and diglycerides). This work is a contribution to development of such an analysis method.     

Commercially available alternatives to palm oil

Since several years there has been a demand for food products free of palm oil, noticeable in the Western European market. Alternatives based on liquid oils, fully hydrogenated fats, and exotic fats like shea and sal etc., have been developed by the research groups of several specialty oils and fats suppliers. This article describes the advantages and disadvantages of those products and compares them to similar products based on palm oil. It is also discussed how reasonable the replacement of palm products would be, since sustainable and 3‐MCPD/glycidolester‐reduced palm based specialty oils are also available on the market.     

Influence of Enzymatic Remediation on Compositional and Thermal Properties of Palm Oil and Palm Oleins from Dry Fractionation

Characteristics of crude palm oil are high FFA and DAG contents. High DAG content may affect throughput and yield during fractionation: high‐grade specialty fats such as hard palm mid fraction require premium crude palm oil to secure adequate crystallization properties. Moreover, DAGs are generally considered the main precursors for the formation of glycidyl esters during high‐temperature deodorization. The purpose of this study was to investigate the effect of enzymatic remediation on the reduction of FFA and DAG in crude palm oil. In practice, series of process parameters (vacuum and reaction time) were investigated, and the quality of enzymatically remediated crude palm oils was examined in terms of FFA and DAG reduction, TAG composition, and SFC and DSC melting profiles. Fully refined enzymatically remediated palm oils were then dry fractionated. The quality of the oleins derived from the enzymatically remediated palm oil was compared to that of regular RBD palm oleins.     

Application of Indirect Enzymatic Method for Determinations of 2-/3-MCPD-Es and Gly-Es in Foods Containing fats and Oils

Because of the potential health risks, fatty acid esters of 3‐chloro‐1,2‐propanediol (3‐MCPD‐Es), 2‐chloro‐1,3‐propanediol (2‐MCPD‐Es) and glycidol (Gly‐Es) in foods are drawing the attention of public health authorities. To assess applicability of the rapid indirect method developed earlier by using a Candida rugosa lipase for the analyses of refined fats and oils was applied to the analyses of various foods. Mayonnaise, vegetable oil margarine and fat spread could be analyzed with the hydrolysis condition of 30 min at room temperature. Analyses of 3‐MCPD‐Es in margarines and fat spreads containing milk fat could be analyzed by increasing the hydrolysis temperature to 40 °C. The results in a mayonnaise, four fat spreads and five margarines analyzed by the enzymatic method were 0.10–0.98 mg/kg for 3‐MCPD, 0.05–0.41 mg/kg for 2‐MCPD and 0.15–0.59 mg/kg for Gly, and correlated well with the results obtained by AOCS Cd 29a with Cd 30–15 with slopes of 0.99–1.13, and R²s of 0.87–0.99. Further, by adding a simple fat extraction step using a solvent mix at 60 °C, foods high in protein and carbohydrate, such as infant formulas, could also be successfully analyzed with >90 % recovery in 1 day. Because the enzymatic method requires only 30 min for hydrolysis, the method is considered suitable for routine analyses of 2‐/3‐MCPD‐Es and Gly‐Es in foods.     

A comparison of the indirect and direct quantification of glycidol ester by kinetic analysis

In a comparison of the German society's official indirect method and the direct LC‐MS method to determine the levels of glycidol fatty acid esters (GEs) in edible oils, the indirect method showed lower GE levels in cases of a high level of GEs and/or containing partial acylglycerols (PGs). The present study used kinetic analysis to compare the scope of both methods. A kinetic model combination of reversible decomposition of GEs and 3‐monochloro‐1,2‐propanediol forming substances (MCPD‐FS) generated from PGs accurately predicted a persistent level of underestimation of commercial vegetable oils when the indirect method was used. The results of the kinetic prediction showed that the underestimation in the indirect method was proportional to the PG and GE levels in oils. Although most conventional cooking oils are low in GEs and PGs, significant error may occur in oils such as palm oil, which are reported to have a higher content of GEs and DAGs. The direct method was affected by neither the GEs nor the PGs, and proved to be a truer and more accurate determinant of GE levels in a wide range of edible oil products.     

Ester content evaluation in biodiesel from animal fats and lauric oils

A modified method for the determination of ester contents of biodiesel based on EN 14103 has been developed. The method includes natural contents of heptadecanoic acid ester, which are found in animal fats and interfere with the standard method, into the calculation of ester content values. As a result, biodiesel samples prepared from waste animal fats and oils showed an increase in ester content between 2 and 7 wt‐% compared to values measured according to EN 14103. Furthermore, modifications of the GC temperature program made it possible to include also short‐chain fatty acid esters C₈–C₁₂, which can be found in coconut and palm kernel oil, into the calculation. Measurements showed that the ester content of such biodiesel differs by more than 40 wt‐% compared to EN 14103 determinations. However, also the stability of the internal standard solution methyl heptadecanoate influences the values of ester content. It can be demonstrated that after a period of 7 days, an ester content decrease of about 2 wt‐% can be observed. Therefore, the use of almost freshly prepared standard solutions should be recommended.     

A Novel Method for Simultaneous Monitoring of 2-MCPD, 3-MCPD and Glycidyl Esters in Oils and Fats

The availability of a reliable methodology for the quantification of fatty acid esters of monochloropropropanediol (MCPD) and glycidol is essential for understanding the mechanism of formation of these process contaminants and for developing effective mitigation strategies. While several analytical methods for the determination of MCPD esters have already been developed and evaluated, only very few procedures are currently available for the analysis of glycidyl esters. This work presents a new indirect method for the simultaneous quantification of fatty acid esters of 2-MCPD, 3-MCPD and glycidol. The method is based on the acid-catalyzed conversion of glycidyl esters into 3-monobromopropanediol (3-MBPD) monoesters which, owing to the structural similarity to MCPD esters, are quantified by using the procedure we previously optimized for the analysis of MCPD esters. The critical step of the method, which is the conversion of glycidyl esters, was optimized by testing different reagent concentrations and varying other condition settings. The novel method showed good repeatability (RSD <2.5 %) and between-day reproducibility (RSD ≤5 %). The limit of detection was 0.04 mg/kg for bound 2-MCPD and 3-MCPD and 0.06 mg/kg for bound glycidol. The trueness of the method was evaluated by the analysis of spiked samples and by interlaboratory comparison.     

Factors Impacting the Formation of 3-MCPD Esters and Glycidyl Esters During Deep Fat Frying of Chicken Breast Meat

The effect of the frying temperature, frying duration and the addition of NaCl on the formation of 3‐monochloropropane‐1,2‐diol (3‐MCPD) esters and glycidyl esters (GE) in palm olein after deep frying was examined in this study. The eight frying systems were deep‐fat frying (at 160 and 180 °C) of chicken breast meat (CBM) (with 0, 1, 3 and 5% sodium chloride, NaCl) for 100 min/day for five consecutive days. All oil samples collected after each day were analyzed for 3‐MCPD ester, GE, and free fatty acid (FFA) contents, specific extinctions at 232 and 268 nm (K₂₃₂ and K₂₆₈), p‐anisidine value (pA), and fatty acid composition. There was a significant (p < 0.05) decrease in the 3‐MCPD esters and a significant (p < 0.05) decrease in the GE with the increasing of the frying duration. There were significant (p < 0.05) increases in the 3‐MCPD esters formed when the concentration of NaCl increased from 0 to 5%. The addition of NaCl to the CBM during deep frying had no significant effect on the GE generation. The FFA contents, K₂₃₂ and K₂₆₈ and pA showed that all the frying oils were within the safety limit.     

Wax ester fraction of edible oils: Analysis by on‐line LC‐GC‐MS and GC×GC‐FID

The wax ester fraction of various plant oils was isolated by normal‐phase HPLC (NPLC) on‐line coupled to GC via the on‐column interface and applying concurrent eluent evaporation. The esters were analyzed by on‐line NPLC‐GC‐MS and by comprehensive two‐dimensional GC with flame ionization detection (GC×GC‐FID) off‐line combined with NPLC‐GC. GC×GC‐FID enables to group the various classes of wax esters, in particular the phytol esters, geranylgeraniol esters and the straight‐chain esters of palmitic acids and the unsaturated C₁₈ acids. Optimization of the GC×GC columns and the conditions must take into account the limited thermostability of the diterpene esters. Chromatograms are shown for a range of oils, with particular focus on the various classes of wax esters in olive oil and the geranylgeraniol esters 22:0 and 24:0 in a variety of oils.     

The Effect of Type of Oil and Degree of Degradation on Glycidyl Esters Content During the Frying of French Fries

The aim of the study was to determine the effect of oil degradation on the content of glycidyl esters (GEs) in oils used for the frying of French fries. As frying media, refined oils such as rapeseed, palm, palm olein and blend were used. French fries were fried for 40 h in oils heated to 180 °C in 30‐min cycles. After every 8 h of frying, fresh oil and samples were analyzed for acid and anisidine values, color, refractive index, fatty acid composition, and content and composition of the polar fraction. GEs were determined by LC–MS. Hydrolysis and polymerization occurred most intensively in palm olein, while oxidation was reported for rapeseed oil. The degradation of oil caused increased changes in the RI of frying oils. Losses of mono‐ and polyunsaturated fatty acids were observed in all samples, with the largest share in blend. The highest content of GE found in fresh oil was in palm olein (25 mg kg^?1) and the lowest content of GE was found in rapeseed oil (0.8 mg kg^?1). The palm oil, palm olein and blend were dominated by GEs of palmitic and oleic acids, while rapeseed oil was dominated by GE of oleic acid. With increasing frying time, the content of GEs decreased with losses from 47 % in rapeseed oil to 78 % in palm oil after finishing frying.     

Enzymatic removal of 3‐monochloro‐1,2‐propanediol (3‐MCPD) and its esters from oils

3‐Monochloro‐1,2‐propanediol (3‐MCPD) is a contaminant in processed food well known for about 30 years. More recently, this compound has observed attendance due to its occurrence as fatty acid esters in edible oils and products derived from them. In this study, the first enzymatic approach to remove 3‐MCPD and its esters from aqueous and biphasic systems by converting it into glycerol is described. First, 3‐MCPD was converted in an aqueous system by an enzyme cascade consisting of a halohydrin dehalogenase from Arthrobacter sp. AD2 and an epoxide hydrolase from Agrobacterium radiobacter AD1 with complete conversion to glycerol. Next, it could also be shown, that the corresponding oleic acid monoester of 3‐monochloropropanediol‐1‐monooleic‐ester (3‐MCPD‐ester) was converted in a biphasic system in the presence of an edible oil by Candida antarctica lipase A to yield free 3‐MCPD and the corresponding fatty acid. Hence, also 3‐MCPD‐esters can be converted by an enzyme cascade into the harmless product glycerol. Practical applications: Since several reports have been recently published on the contamination of foods with 3‐MCPD and its fatty acid esters, there is a great demand to remove these compounds and an urgency to find useful methods for this. In this contribution, we present an easy enzymatic way to remove 3‐MCPD and its esters from the reaction media (i.e., plant oil) by converting it to the nontoxic glycerol. The method requires neither high temperature nor organic solvents.