Kornél Nagy

Formation mechanisms of Monochloropropanediol (MCPD) fatty acid diesters in refined palm (Elaeis guineensis) oil and related fractions

Monochloropropanediol (MCPD) fatty acid esters are process contaminants generated during the deodorisation of edible oils. In particular, MCPD diesters are found in higher abundance in refined palm oil than other edible oils. In the present study, a series of model reactions mimicking palm oil deodorisation has been conducted with pure acylglycerols in the presence or absence of either organic or inorganic chlorine-containing compounds. Results showed that the bulk of MCPD diesters are formed above 200°C through the reaction of organochlorines with triacylglycerols (TAG). Additional experiments confirmed that this reaction can be initiated during palm oil deodorisation by hydrogen chloride (HCl) gas evolved through the thermal degradation of organochlorines present in the oil. Therein, the majority of the ultimately produced MCPD diesters are the result of HCl reacting with TAG, via protonation, followed by the elimination of a fatty acid residue. Two possible MCPD diester formation mechanisms are highlighted, both of which involve acyloxonium ion reactive intermediates. Investigations with pure TAG regio-isomers showed that MCPD ester formation is regioselective and the sn-1(3) position of the glycerol backbone is favoured.

Mass-defect filtering of isotope signatures to reveal the source of chlorinated palm oil contaminants

This paper reports new insights at the molecular level into the route of a worldwide problem of the food industry: the occurrence of monochloro-propanediol (MCPD) esters. The application of mass defect-driven workflows is described to generate a hypothesis on the identity and occurrence of those thermally labile, chlorinated contaminant precursors that may act as chlorine donors during the formation of MCPD esters. For the first time, holistic mass-defect filtering of isotope signatures is used to pinpoint completely unknown and unexpected chlorine-containing substances naturally present in various extracts of palm fruit and partially and fully refined oils. Supervised multivariate analysis showed the effective classification of samples from various stages of industrial processing, suggesting that these steps strongly impact a complex and dynamic pool of chlorinated substances. In-vitro experiments confirmed that several of these naturally occurring chlorinated plant constituents decompose upon heat treatment, thus potentially being a source of chlorine for further reactions with palm oil lipids in a subsequent chlorination cascade. It is hypothesised that during oil refining the organochlorines naturally present in palm fruits act as a ‘chlorine source’ for the generation MCPD diesters. This discovery implies that industrial efforts targeting the mitigation of chlorinated substances must intervene at the earliest possible production stage or preferably even prior to oil processing. Current performance and limitations of mass-defect filtering are discussed and future developments are outlined.

Glycidyl esters in refined palm (Elaeis guineensis) oil and related fractions. Part II: Practical recommendations for effective mitigation.

In a previous work, it was shown that at high temperatures (up to 280°C) glycidyl esters (GE) are formed from diacylglycerols (DAG) via elimination of free fatty acid (FFA). In the present study, the impact of DAG content and temperature on the formation of GE using a model vacuum system mimicking industrial edible oil deodorization is investigated. These deodorization experiments confirmed that the formation of GE from DAG is extensive at temperatures above 230-240°C, and therefore, this value should be considered as an upper limit for refining operations. Furthermore, experimental data suggest that the formation of GE accelerates in particular when the DAG levels in refined oils exceed 3-4% of total lipids. Analysis of the lipid composition of crude palm oil (CPO) samples allowed the estimation that this critical DAG content corresponds to about 1.9-2.5% of FFA, which is the conventional quality marker of CPO. Moreover, high levels (>100ppm) of GE were also found in palm fatty acid distillate samples, which may indicate that the level of GE in fully refined palm oils also depends on the elimination rate of GE into the fatty acid distillate.

Factors impacting the formation of Monochloropropanediol (MCPD) fatty acid diesters during palm (Elaeis guineensis) oil production

Recently, organic and inorganic chlorinated compounds were detected in crude and commercially refined palm oils. Further, the predominant formation mechanism of monochloropropanediol (MCPD) diesters at high temperatures (>170–180°C) was revealed. The present study involved the development and comparison of solutions to mitigate MCPD diester levels in oils from various stages of palm oil production. Partially refined palm oil samples and oil extracted from fresh palm fruits were submitted to bench-top deodorisation experiments. Application of glycerol and ethanol as refining aids during the deodorisation of refined-bleached palm oil proved to be moderately effective; about 25%–35% reduction of MCPD diester levels was achieved. Washing crude palm oil with ethanol–water (1:1) prior to deodorisation was also an effective strategy yielding an ∼30% reduction of MCPD diester contents. Washing palm fruit pulp before oil extraction, however, was most impactful, resulting in a 95% reduction of MCPD diesters when compared to the deodorised control oil. This suggests that intervention upstream in the process chain is most efficient in reducing levels of these contaminants in refined oils. Following the study, a root-cause analysis was performed in order to map the parameters potentially responsible for the occurrence of MCPD diesters in refined palm oil and related fractions.

First identification of dimethoxycinnamic acids in human plasma after coffee intake by liquid chromatography-mass spectrometry.

There is a substantial amount of published literature on the bioavailability of various coffee components including the most abundant metabolites, caffeic and ferulic acids. Surprisingly, to date, the appearance of dimethoxycinnamic acid derivatives in humans has not been reported despite the fact that methylated form of catechol-type polyphenols could help maintain, modify or even improve their biological activities. This study reports an LC-MS method for the detection of dimethoxycinnamic acid in human plasma after treatment with an esterase. Liquid chromatography, including the combination of methanol and acetonitrile as organic eluent, was optimized to resolve all interferences and enable reliable detection and identification of 3,4-dimethoxycinnamic and 3,4-dimethoxy-dihydrocinnamic acids. In addition to the good mass accuracy achieved (better than 5 ppm), tandem mass spectrometric and co-chromatography experiments further confirmed the identity of the compounds. The optimized method was applied to analyze samples obtained immediately, 1 and 10 h after coffee ingestion. The results show that in particular 3,4-dimethoxycinnamic acid appears in high abundance (∼380 nM at 60 min) in plasma upon coffee intake, indicating that it is important to consider these derivatives in future bioavailability and bioefficacy studies.

Hydrolysis of Rosmarinic Acid from Rosemary Extract with Esterases and Lactobacillus johnsonii in Vitro and in a Gastrointestinal Model

Rosmarinic acid (RA) was identified as one of the main components of rosemary extracts and has been ascribed to a number of health benefits. Several studies suggested that after ingestion, RA is metabolized by gut microflora into caffeic acid and derivatives. However, only limited information on the microorganisms and enzymes involved in this biotransformation is available. In this study, we investigated the hydrolysis of RA from rosemary extract with enzymes and a probiotic bacterium Lactobacillus johnsonii NCC 533. Chlorogenate esterase from Aspergillus japonicus (0.02 U/mg) hydrolyzed 90% of RA (5 mg/mL) after 2 h at pH 7.0 and 40 degrees C. Complete hydrolysis of RA (5 mg/mL) was achieved with a preparation of L. johnsonii (25 mg/mL, 3.3 E9 cfu/g) after 2 h of incubation at pH 7.0 and 37 degrees C. No hydrolysis of RA was observed after the passage of rosemary extract through the gastrointestinal tract model (GI model). Thus, RA is hydrolyzed neither chemically under the conditions of the GI model (temperature, pH, and bile salts) nor by secreted enzymatic activity (lipase and pancreatic enzymes). The addition of L. johnsonii cells to rosemary extract in the GI model resulted in substantial hydrolysis of RA (up to 99%).

Non-covalent binding of proteins to polyphenols correlates with their amino acid sequence

The present paper describes the assessment of non-covalent binding (NCB) between milk proteins and polyphenols and its correlation with the physicochemical parameters of proteins. A method based on ultrafiltration and liquid chromatography-tandem mass spectrometry was used to analyse free and non-covalently bound polyphenols (ligands) in mixtures with major milk proteins. Binding strength values of individual polyphenols were normalised to those obtained with quercitrin (quercetin-3-O-rhamnoside), used as a reference compound. NCB data acquired by experiments at pH 6.6 without any preliminary protein denaturation were correlated with the physicochemical parameters of ligands and proteins. Unsupervised multivariate analysis revealed that NCB of proteins clustered according to their family (caseins separated from albumins). Based on this model, a predictive relationship was observed between protein-polyphenol binding strength and primary/secondary structure parameters of the proteins e.g. number of charges, proline residues and extended strand. These results confirm that, under the investigated experimental conditions, the NCB between polyphenols and protein mixtures can be predicted and optimised based on the molecular structures.

Identification of monoacylglycerol regio-isomers by gas chromatography–mass spectrometry

Monoacylglycerols (MAGs) are lipids found in trace amounts in plants and animal tissues. While they are widely used in various industrial applications, accurate determination of the regio-specific distribution is hindered by the lack of stable, commercially available standards. Indeed, unsaturated beta-MAG (or Sn-2 MAG) readily undergoes isomerization into alpha-MAG (acyl chain is attached to the Sn-1 or the Sn-3 position). In the present study, we describe structural elucidation of alpha- and beta-regio-isomers of monopalmitoyl-glycerol (MAG C16:0) as model compounds in their silylated forms using gas chromatography-mass spectrometry (GC-MS) with electronic impact (EI) ionization. MS fragmentation of alpha-MAG C16:0 is characterized by the loss of methylene(trimethylsilyl)oxonium (103 amu) and the consecutive loss of acyl chain yielding a fragment ion at m/z 205. The fragmentation pattern of beta-MAG C16:0 shows a series of diagnostic fragments at m/z 218, 203, 191 and 103 that are not formed from the alpha-isomer and hereby enable reliable distinction of these regio-isomers. Possible fragmentation scenarios are postulated to explain the formation of these marker ions, which were also applied to characterize the regio-isomer composition of a complex mixture of MAG sample containing n-3 long-chain polyunsaturated fatty acids.

Quantification of alkylresorcinols in human plasma by liquid chromatography/tandem mass spectrometry

Alkylresorcinols (AR) are of interest as biomarkers of wholegrain wheat and rye intake in epidemiological studies and are currently mainly measured by gas chromatography/mass spectrometry (GC/MS) after labour-intensive sample preparation including liquid-liquid extraction, solid-phase extraction (SPE) and chemical derivatization. This manuscript describes and validates an alternative approach based on normal-phase liquid chromatography/tandem mass spectrometry for the quantification of alkylresorcinols in human plasma. The method requires neither SPE nor chemical derivatization and has a shortened run time compared to GC/MS. Normal- and reversed-phase columns and various mobile phases were evaluated with and without previous SPE of the samples. Normal-phase chromatography allowed separation of AR from the interfering triacylglycerols, diacylglycerols and sterols and enabled detection of AR even without SPE of the samples. The described method has instrumental lower limits of detection in the 25-75 pg range, and lower limits of quantification in the 75-250 pg range. Pooled human plasma and (2)H(4)-nonadecylresorcinol (internal standard) was applied to calibrate the method in the 20-12 000 nM range. The overall method showed intra-batch precision of 8.6% and an averaged accuracy of 100.2%. Applications for diverse human plasma samples are presented and are compared with the results determined by GC/MS. Based on the presented data; this method requiring less sample preparation is suggested for further evaluation as an alternative to GC/MS for analysis of biomarkers of wholegrain wheat and rye intake in epidemiological studies.

Quantification of flavan-3-ols and phenolic acids in milk-based food products by reversed-phase liquid chromatography-tandem mass spectrometry

This article reports the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the comprehensive quantification of flavan-3-ol and phenolic acid constituents of milk-based food products. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with the stable isotope labelled internal standards and ultrafiltration to preserve overall polyphenol composition, but to eliminate milk proteins in order to comply with LC. Reversed-phase liquid chromatography was optimized to achieve separation of 22 analytes in 8 min in order to reduce suppression effects, achieve a wide dynamic range and, most importantly, to resolve isomeric compounds. Negative-ion electrospray mass spectrometric detection and fragmentation of analytes was optimized, final transitions were selected for maximized selectivity, reliable quantification and reduction of false positives. The quantitative performance of the method was validated, the main features include: (1) range of lower limits of detection 5-15 ng/mL for flavan-3-ols, 60-100 ng/mL for procyanidins, 1-60 ng/mL for other phenolic acids, (2) lower limits of quantification 15-45 ng/mL for flavan-3-ols, 200-300 ng/mL for procyanidins, 3-200 ng/mL for other phenolic acids, (3) averaged intra-day precision 9.5%, (4) calibrated range 60-300,000 ng/mL for flavan-3-ols, 900-900,000 ng/mL for procyanidins, 9-225,000 ng/mL for other phenolic acids, (5) averaged accuracy 99.5%. Applications for yoghurt and ice-cream products are given. The presented data suggest that this method will help to better characterize the polyphenol composition of milk-based food products for quality control, assessment of dietary intake and for polyphenol bioavailability/bioefficacy studies.