Occurrence of glycidyl esters in infant formula products on the Canadian market between 2015 and 2019.

Glycidyl fatty acid esters (GEs) are processing contaminants formed during refining steps of vegetable oils. 'In vivo' hydrolysis of GEs releases potentially carcinogenic and genotoxic glycidol (2,3-epoxy-1-propanol). Occurrence of GEs in vegetable oils used for infant formula manufacturing may pose a potential health concern for formula-fed infants. Refined oils are commonly used as the main fat ingredient in formula manufacturing. For this study, different infant formula products (powders, concentrates and ready-to-feed formula products) were purchased and analysed in 2015 (35 samples) and 2019 (33 samples). Seven individual GEs were analysed by LC-MS/MS via direct approach by stable isotope dilution analysis, and total bound glycidol concentrations were calculated. Concentrations of bound glycidol in reconstituted formula reached maxima of 40.3 ng/g in the 2015 samples and 31.5 ng/g in the samples collected in 2019, with respective means of 8.7 ng/g and 6.7 ng/g. The analysed bound glycidol concentrations are comparable with concentration ranges from other studies, but are higher than observed in studies from the European market. Temporal trend data show a reduction of bound glycidol concentrations in 2019. GE concentrations were compared across different manufacturers.

Chemical refining methods effectively mitigate 2-MCPD esters, 3-MCPD esters, and glycidyl esters formation in refined vegetable oils.

Esters of 3-monochloro-1,2-propanediol (3-MCPDE), 2-monochloro-1,3-propanediol (2-MCPDE), and glycidyl esters (GE) are processing contaminants that can be found in refined edible fats and oils. Recently, the European Commission has implemented maximum limits for the presence of free and bound 3-MCPDE in vegetable fats and oils and in marine and fish oils. This boosted the necessity of oil producers to develop refining methods to limit the concentration of both 3-MCPDE and GE in their final products. Physical refining may lack the potential to mitigate the formation of 2- and 3-MCPDE. Therefore, in this study, the chemical refining method were explored to provide a viable mitigation strategy aimed at industrial application. Several pilot plant treatments with organic palm oil were performed. The investigated refining methods included a neutralization, a water washing process, reduced deodorization temperature, and a combination of them. The best performing chemical refining treatment achieved a final concentration of 0.42 (-49%), 0.78 (-52%), and 0.99 (-73%) mg/kg for 2-MCPDE, 3-MCPDE, and GE in organic palm oil, respectively. Results thus showed chemical refining has great potential for the simultaneous mitigation of 2-, 3-MCPDE, and GE.

Urinary non-targeted toxicokinetics and metabolic fingerprinting of exposure to 3-monochloropropane-1,2-diol and glycidol from refined edible oils.

The widespread presence of 3-monochloropropane-1,2-diol (3-MCPD) and glycidol in refined edible oils have raised food industrial and public health concerns, but their specific biomarkers of exposure and urinary metabolic pathways indicating nephrotoxicity remain largely unknown. Here, we unraveled the in vivo biotransformation of these two contaminants and revealed how they affect metabolic pathways in rats. Urine metabolomes in rats administered with glycidol or 3-MCPD were investigated using ultra-high performance liquid chromatography combined with a quadrupole-orbitrap high-resolution mass spectrometry. Compared to the currently acknowledged metabolite which is only 2,3-dihydroxypropyl mercapturic acid, we identified 8 and 4 new specific exposure biomarkers of glycidol and 3-MCPD, respectively, via mapping the glyceryl polymerization and glutathione and sulfur conjugation. The changes of metabolites in the surrounding metabolic network were investigated to further gain insight into their metabolic fates. Exposure to glycidol up-regulated citrate, isocitrate, ketoglutarate, malate, and pyruvate in the tricarboxylic acid cycle and glycolysis pathways, while 3-MCPD intake down-regulated these signal molecules in both pathways. Nonetheless, L-cysteine, proline, and arginine were significantly decreased by the effect of either glycidol or 3-MCPD. Our findings first map the urinary metabolomics of both contaminants from edible oils and advance the omics-level recognition for their observational health hazards.

Automated sample preparation and analysis by gas chromatography tandem mass spectrometry (GC-MS/MS) for the determination of 3- and 2-monochloropropanediol (MCPD) esters and glycidol esters in edible oils.

The potentially carcinogenic process contaminant 3- and 2-monochloropropanediol esters (2-MCPD and 3-MCPD esters) and glycidyl esters (GEs) are under study in refined oils and foodstuffs. Legislation set recommended total daily intake (TDI) for 3-MCPD of 0.8 µg/kg and as low as reasonably achievable (ALARA) for glycidol. Usually, the so far adopted method for the determination of these contaminants relay on numerous and time-consuming steps for sample preparation (AOCS methods) and on GC-MS detection. The obtained sensitivities and the number of processable samples are thus limited. In this optic, new reliable methods that allow for the fast and sensitive determination of these contaminants in edible oils may be considered an improvement of the overall strategy of tackling the problem. In this paper a new automated method for sample preparation and detection by GC-MS/MS is presented and validated. Data on sensitivity (LOD at 1.5, 2.2 and 3 ng/g for 3-MCPD, 2-MCPD, 3-MBPD (deriving from glycidol), respectively), linearity across low and high calibration ranges and precision showed to be fit-for-purposes. Finally, the methodology was applied to ten extra virgin oil samples and one sample of sunflower seeds oil.

Puerarin inhibited 3-chloropropane-1,2-diol fatty acid esters formation by reacting with glycidol and glycidyl esters.

The inhibitory effects of seven polyphenols on 3-chloropropane-1,2-diol fatty acid esters (3-MCPDE) formation were investigated in palm oil models. Results showed that there was not a positive significant correlation between the free-radical scavenging activities of the tested compounds and their 3-MCPDE-formation inhibitory activities; puerarin, with weak antioxidant activity, showed the highest inhibitory capacity. Moreover, puerarin reduced the content of glycidol and glycidyl esters (GEs), two key intermediates of 3-MCPDE formation in the oil models; and a puerarin-adduct was discovered in the oil fortified with glycidol or GEs, with its structure elucidated by LC-MS/MS and comparison with newly synthesized ones. Based on its chemical structure, we proposed that puerarin, at least in part, reacted with glycidol and GEs to inhibit 3-MCPDE formation. In addition, the formed compound, puerarin-7-O-propanediol was identified in the potato chips frying system, further confirming reacting with glycidol/GEs as a key mechanism of puerarin to inhibit 3-MCPDE formation.

Urinary Excretion of 2/3-Monochloropropanediol (2/3-MCPD) and 2,3-Dihydroxypropylmercapturic Acid (DHPMA) after a Single High dose of Fatty Acid Esters of 2/3-MCPD and Glycidol: A Controlled Exposure Study in Humans.

SCOPE: 2- and 3-monochloropropanediol (2/3-MCPD) and glycidol are absorbed in the intestine after lipase-catalyzed hydrolysis of their fatty acid esters. METHODS AND RESULTS: In an exposure study with 12 non-smoking participants, the complete urinary excretion of the metabolite 2,3-dihydroxypropylmercapturic acid (DHPMA) and of 2/3-MCPD is measured on four consecutive days before and after consumption of 50 g glycidyl ester-rich palm fat or 12 g 2/3-MCPD ester-rich hazelnut oil. After controlled exposure, urinary excretion rates of 2/3-MCPD per hour strongly increase, followed by a decrease with average half-lives of 5.8 h (2-MCPD) and 3.6 h (3-MCPD). After consumption of hazelnut oil, mean excretion rates are 14.3% (2-MCPD) and 3.7% (3-MCPD) of the study doses. The latter rate is significantly higher (4.6%) after consumption of palm fat, indicating partial conversion (about 5%) of glycidol to 3-MCPD under the acidic conditions in the stomach. The average daily "background" exposure is estimated to be 0.12 and 0.32 µg per kg body weight (BW) for 2-MCPD and 3-MCPD, respectively. The relatively high and constant urinary excretion of DHPMA does not reflect the controlled exposure. CONCLUSION: Urinary excretion of 2- and 3-MCPD is suitable as biomarker for the external exposure to the respective fatty acid esters.

[Alkaline transesterification CG-MS/MS method of monochlorpropanediol and glycidyl esters' determination in some edible fats, oils and fat blends on Russian market].

Monochlorpropanediol fatty acid esters (MCPDE) and glycidyl fatty acid esters (GE) are mainly considered to be processing contaminants and their concentration can rise during high temperature refining and deodorization of edible oils. Free forms formed during digestive hydrolysis of esters such as 3-monochloropropane-1,2-diol (3-MCPD), 2-MCPD and glycidol can provoke a negative effect on human health. Therefore the quantitative determination of MCPDE and GE in edible oils, fats and fat blends is needed. The aim - this manuscript deals with MCPDE and GE concentration measured as free 3-MCPD, 2-MCPD and glycidol in different edible oils, fats and fat blends of Russian market. Material and methods. 55 edible oil and fat samples sold on Russian market including refined and non-refined oils and fat blends such as spreads, dairy fat replacers, and margarines have been analyzed. Slow alkaline transesterification method with GC-MS/MS was used. Results. According to the data obtained, the highest concentrations of the contaminants were detected in fat blends: <0.10-5.03 mg/kg for 3-MCPD, <0.10-2.50 mg/kg for 2-MCPD and 0.1 5-11.17 mg/kg for glycidol. In palm oils and its fractions concentration of 3-MCPD was <0.10-6.61 mg/kg, 2-MCPD - <0.10-2.69 mg/kg and glycidol - <0.10-6.29 mg/kg. The content of glycidol in sunflower oils fluctuated in the range <0.10-1.19 mg/kg, 3-MCPD was <0.10-2.47 mg/kg, and 2-MCPD <0.10-0.67 mg/kg. Non-refined edible oils and olive oils had no or little MCPDE or GE. Conclusion. In this work we indicate high importance of monitoring MCPDE and GE in edible oils and fats both as ready-to-eat products and as ingredients prior to the Russian market release. There is strong need in mitigation of these process contaminants during fat blends manufacturing.

Verification and evaluation of monochloropropanediol (MCPD) esters and glycidyl esters in palm oil products of different regions in Malaysia.

This study was conducted to investigate on the effect of different sampling regions of palm-refined oils and fats on the 2- and 3-monochloropropanediol fatty acid esters (MCPDE) and glycidol fatty acid esters (GE) levels. The American Oil Chemists' Society (AOCS) Official Method Cd 29a-13 on the determination of MCPDE and GE in edible oils and fats by acid transesterification was successfully verified and optimised, with slight modification using 7890A Agilent GC system equipped with 5975C quadrupole detector. The determined limits of detection (LOD) for MCPDE were 0.02 mg kg-1 and 0.05 mg kg-1 for GE. The method performance has showed good recovery between 80% and 120% for all pertinent compounds with seven replicates assayed in three separate days. Round robin test with two European laboratories, i.e. Eurofins and SGS, has shown compliance results with those of the present study. Among the sampling regions, only one refinery located in the central region of Malaysia showed a significant increment of the MCPDE and GE levels after refining process. The GE level averaging at 2.5 mg kg-1 was slightly higher than that of 3-MCPDE averaging at 1.3 mg kg-1. Both esters were preferentially partitioned into the liquid phase rather than the solid phase after fractionation. However, the overall results exhibited no direct correlation between the esters content and the different sampling locations of the palm oil products in Malaysia. Analysis of total chlorine content also displayed significant variations between sampling locations which clearly show its effect on the chlorine content in the CPO samples.

Washing bleached palm oil to reduce monochloropropanediols and glycidyl esters.

The formation of toxic compounds, potentially carcinogenic, during food processing has been considered an important food safety issue. Among them, particular attention has been given to 3-monochloropropane-1,2-diol esters (3-MCPDE), 2-monochloropropane-1,3-diol esters (2-MCPDE) and glycidyl esters (GE), which can be formed during vegetable oil refining, especially palm oil. These substances may pose a health risk to humans due to their toxicity and carcinogenicity. The aim of this study was to investigate the effect of washing bleached palm oil (BPO) with different solvents, and evaluate the reduction of 3-MCPDE, 2-MCPDE and GE as well as assess the quality parameters of the final product. For this purpose, we used two types of washing with different solvents. A single washing was carried out in one step and a double washing in two steps using a solvent gradient. Single washing had a limited reduction in the levels of 3-MCPDE and 2-MCPDE and resulted in an increased level of GE, whereas double washing slightly reduced 3-MCPDE and 2-MCPDE and resulted in a significant reduction of GE levels. The reduction achieved in this study was up to 17.1% for 3-MCPDE, 56.4% for 2-MCPDE and 76.9% for GE levels. The reduction of 3-MCPDE and 2-MCPDE might be due to the removal of part of the ethanol-soluble chlorinated precursors from the oil which suggests that highly lipophilic forms of these substances are present in BPO. The substantial reduction on GE levels might be associated with the removal of the precursors present in the oil such as diacylglycerols. Thus, the washing treatment could be used as a supplementary strategy to reduce processing contaminants from palm oil, especially GEs.

The hemoglobin adduct N-(2,3-dihydroxypropyl)-valine as biomarker of dietary exposure to glycidyl esters: a controlled exposure study in humans.

Fatty acid esters of glycidol (glycidyl esters) are heat-induced food contaminants predominantly formed during industrial deodorization of vegetable oils and fats. After consumption, the esters are digested in the gastrointestinal tract, leading to a systemic exposure to the reactive epoxide glycidol. The compound is carcinogenic, genotoxic and teratogenic in rodents, and rated as probably carcinogenic to humans (IARC group 2A). Assessment of exposure from occurrence and consumption data is difficult, as lots of different foods containing refined oils and fats may contribute to human exposure. Therefore, assessment of the internal exposure using the hemoglobin adduct of glycidol, N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val), may be promising, but a proof-of-principle study is needed to interpret adduct levels with respect to the underlying external exposure. A controlled exposure study was conducted with 11 healthy participants consuming a daily portion of about 36 g commercially available palm fat with a relatively high content of ester-bound glycidol (8.7 mg glycidol/kg) over 4 weeks (total amount 1 kg fat, individual doses between 2.7 and 5.2 µg/kg body weight per day). Frequent blood sampling was performed to monitor the 2,3-diHOPr-Val adduct levels during formation and the following removal over 15 weeks, using a modified Edman degradation and ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results demonstrated for the first time that the relatively high exposure during the intervention period was reflected in corresponding distinct increases of 2,3-diHOPr-Val levels in all participants, following the expected slope for hemoglobin adduct formation and removal over time. The mean adduct level increased from 4.0 to 12.2 pmol 2,3-diHOPr-Val/g hemoglobin. By using a nonlinear mixed model, values for the adduct level/dose ratio (k, mean 0.082 pmol 2,3-diHOPr-Val/g hemoglobin per µg glycidol/kg body weight) and the adduct lifetime (τ, mean 104 days, likely the lifetime of the erythrocytes) were determined. Interindividual variability was generally low. 2,3-DiHOPr-Val was therefore proven to be a biomarker of the external dietary exposure to fatty acid esters of glycidol. From the background adduct levels observed in our study, a mean external glycidol exposure of 0.94 µg/kg body weight was estimated. This value is considerably higher than current estimates for adults using occurrence and consumption data of food. Possible reasons for this discrepancy are discussed (other oral or inhalational glycidol sources, endogenous formation, exposure to other chemicals also forming the adduct 2,3-diHOPr-Val). Further research is necessary to clarify the issue.

Discrimination of processing grades of olive oil and other vegetable oils by monochloropropanediol esters and glycidyl esters.

In this study, the processing derived contaminants 2- and 3-monochloropropanediol (2- and 3-MCPD) esters and glycidyl esters (GEs) were analysed in 84 oil samples by GC-MS/MS for the discrimination of processing grades of olive oils as a potential authentication tool. Concentrations of 2- and 3-MCPD esters and GEs varied in the ranges 0-6 mg/kg, 0-1.5 mg/kg, and 0-1 mg/kg oil, respectively. The concentrations of the three compounds in lower grade olive oils were significantly higher (P < .001) than that in EVOO. A similar difference was observed for other refined and cold-pressed vegetable oils. The limit of fraud detection of lower grade oils in EVOO was 2% when using 3-MCPD esters, 5% for 2-MCPD esters, and 13-14% for GEs based on calculations of virtual mixtures of the current sample set. Especially the MCPD esters appear very specific and promising for the detection of lower processing grade oils in EVOO.

An Improved Enzymatic Indirect Method for Simultaneous Determinations of 3-MCPD Esters and Glycidyl Esters in Fish Oils.

The enzymatic indirect method for simultaneous determinations of 3-chloro-1, 2-propanediol fatty acid esters (3-MCPD-Es) and glycidyl fatty acid esters (Gly-Es) make use of lipase from Candida cylindracea (previously referred to as C. rugosa). Because of low substrate specificity of the lipase for esters of polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), fish oils high in PUFAs are currently excluded from the range of application of the method. The objective of this study was to make the enzymatic indirect method applicable to fats and oils containing PUFAs. By using a Burkholderia cepacia lipase, and by removing sodium bromide from hydrolysis step and adding it after completion of the hydrolysis step, satisfactory recovery rates of 91-109% for 3-MCPD, and 91-110% for glycidol (Gly) were obtained from an EPA and DHA concentrated sardine oil, three DHA concentrated tuna oils, two fish oils, and five fish-oil based dietary supplements spiked with DHA-esters or oleic acid-esters of 3-MCPD and Gly at 20 mg/kg. Further, results from unspiked samples of seven fish oil based dietary supplements and five DHA concentrated tuna oils analyzed by the improved enzymatic indirect method were compared with the results analyzed by AOCS Cd 29a. For all 3-MCPD, 2-MCPD and Gly, the 95% confidence intervals determined by the weighted Deming regression for slopes and intercepts contained the value of 1 and 0, respectively. It was therefore concluded that the results from the two methods were not statistically different. These results suggest that fish oils high in PUFAs may be included in the range of application for the improved enzymatic indirect method for simultaneous determinations of 3-MCPD and Gly esters in fats and oils.

Effects of temperature and NaCl on the formation of 3-MCPD esters and glycidyl esters in refined, bleached and deodorized palm olein during deep-fat frying of potato chips.

The effects of frying duration, frying temperature and concentration of sodium chloride on the formation of 3-monochloropropane-1,2-diol (3-MCPD) esters and glycidyl esters (GEs) of refined, bleached and deodorized (RBD) palm olein during deep-fat frying (at 160°C and 180°C) of potato chips (0%, 1%, 3% and 5% NaCl) for 100min/d for five consecutive days in eight systems were compared in this study. All oil samples collected after each frying cycle were analyzed for 3-MCPD esters, GEs, free fatty acid (FFA) contents, specific extinction at 232 and 268 nm (K232 and K268), p-anisidine value (pAV), and fatty acid composition. The 3-MCPD ester trend was decreasing when the frying duration increased, whereas the trend was increasing when frying temperature and concentration of NaCl increased. The GEs trend was increasing when the frying temperature, frying duration and concentration of NaCl increased. All of the oil qualities were within the safety limit.

Direct analysis of intact glycidyl fatty acid esters in edible oils using gas chromatography-mass spectrometry.

Glycidyl esters (GE), fatty acid esters of glycidol, are process contaminants formed during edible oil processing. A novel direct method for the determination of intact GE in oils and fats based on gas chromatography-mass spectrometry (GC-MS) is presented. The method consists of a simple extraction step of GE from the lipid matrix, purification of the extract and isolation of GE by normal phase liquid chromatography (NPLC). Individual GE in the final fraction are separated and quantified by standard GC-MS operated in selected ion monitoring (SIM) mode. The setup and conditions of the GC-MS were optimized in such a way that thermal degradation of GE and artifact formation were prevented. The method exhibits very good performance parameters: the limit of detection was approximately 0.01 mg/kg for the individual GE (corresponding to 0.002-0.003 mg/kg of free glycidol), the repeatability was in the range of 5-12% for individual GE at levels above 0.1mg/kg, and recovery values ranged from 85 to 115% depending on the level and the chain identity of the GE. The comparison of experimental values with spiked levels and with the results obtained by other methods confirmed a good trueness. Over a period of several months of extensive use the method was found to be very reliable and rugged.