Screening of 152 Veterinary Drug Residues in Animal Source Foods by LC-MS/MS, Multilaboratory Validation Study: Final Action 2020.04.

BACKGROUND: The presence of veterinary drug residues in food-producing animals and animal products is regulated through the enforcement of maximum residue limits (MRLs). To answer the need of the food sector to monitor these substances in a wide range of food commodities, stakeholders at AOAC identified the need for a reliable confirmatory screening method. Such qualitative approach is required for compliance checking and to support product release in manufacturing. OBJECTIVE: Data were collected from 5 independent laboratories that applied the AOAC Official First Action Method AOAC 2020.04 to demonstrate adequate performance under reproducibility conditions. Probability of Detection (POD) was calculated in blank test samples and test samples spiked at the Screening Target Concentration (STC) level, with the objective to achieve PODs ≤ 10% and ≥ 90%, respectively. Additionally, the effectiveness of the screening method was assessed through participation to 92 proficiency test samples. METHODS: Four streams were optimized to screen for 152 veterinary drug residues by LC-MS/MS in a wide variety of food commodities including milk-based ingredients and related products (e.g., milk fractions, infant formula, infant cereals and baby foods), meat- and fish-based ingredients and related products (fresh, powdered, cooked, infant cereals and baby foods) and other ingredients such as eggs, animal fat and animal byproducts. The four streams covered 105 antibiotic residues, anti-inflammatory and antiparasitic agents (Stream A); 23 Beta-lactams (Stream B); 14 Aminoglycosides (Stream C) and 10 Tetracyclines (Stream D). RESULTS: The multi-laboratory validation led to PODs at the STC ≥ 94% and PODs in the blank ≤ 9%. Further application of the multi-laboratory validated method to 92 proficiency tests provided more than 99% satisfactory submitted results (n = 784). CONCLUSION: The inter-laboratory reproducibility determined for this method met the acceptance criteria defined in AOAC SMPR 2018.010. HIGHLIGHTS: AOAC has approved the method for Final Action Status.

Deciphering the origin of total estrogenic activity of complex mixtures.

Introduction: Identifying compounds with endocrine properties in food is getting increasingly important. Current chemical analysis methodology is mainly focused on the identification of known substances without bringing insight for biological activity. Recently, the application of bioassays has been promoted for their potential to detect unknown bioactive substances and to provide information on possible interactions between molecules. From the toxicological perspective, measuring endocrine activity cannot inform on endocrine disruption and/or health risks without sufficient knowledge on the nature of the responsible factors. Methods: The present study addresses a promising approach using High Performance Thin-Layer Chromatography (HPTLC) coupled to bioassays were analyzed using the Liquid Chromatography Mass-Spectrometry (LC-MS). The estrogen receptor activation was assessed using the transcription activation Estrogen Receptor Alpha Chemical Activated LUciferase gene eXpression assay (ERα- CALUX) and the HPTLC coupled to the Estrogen Screen Yeast assay (p-YES). Results: Seven isoflavones were identified in the soy isolates. Estrogen receptor activation was assessed for both, the identified isoflavones and the soy isolates with ERα-CALUX test. Correlation between the soy isolates extracts and the identified isoflavones was shown. Moreover, p-YES revealed the presence of an estrogenic bioactive zone. Analysis of the bioactive zone through LCHRMS highlighted signals corresponding to several isoflavones already detected in the isolates as well as two additional ones. For all detected isoflavones, an estrogenic activity dose-response was established in both bioassays. Conclusion: Finally, genistein, daidzein, and naringenin were found as the most active substances. A concordance analysis integrating the analytical and bioassay data indicated that genistein and daidzein were the drivers of the estrogenic activity of these soy protein isolates. Altogether, these data suggest that the integration of HPTLC-bioassay together with chemical analysis is a powerful approach to characterize the endocrine activity of complex mixtures.

Confirmation of the full conversion of ethylene oxide to 2-chloroethanol in fumigated foodstuffs: possible implications for risk assessment.

This study describes the extension of a gas chromatography mass spectrometry (GC-MS) method, initially devoted to the analysis of ethylene oxide (EO) in ice cream, to a larger range of food items including herbs, spices, vegetables, inorganic salts, food supplements, thickeners, etc. Results are reported as EOTotal according to EC 2015/868 definition (expressed as EO equivalents as the sum of native EO and 2-chloroethanol (2-CE) after acidic hydrolysis) with a limit of quantification at 0.01 mg/kg regardless of the food item. Its ruggedness was demonstrated through fortification experiments on hundreds of samples. Re-analysis of 146 positive food samples without hydrolysis demonstrated that not EO but 2-CE is the predominant analyte detected in the different processed ingredients suspected to have been previously treated with EO. A series of eight contaminated dried herbs and spices were also re-analysed by four ISO 17025 accredited commercial laboratories making use of different analytical strategies for EO determination in foods. Each laboratory reported EOTotal levels within the same concentration range, but the resulting reproducibility ranged from 23% to 41% depending on the sample. Additionally, we show that results of free EO from methods based on conversion to 2-iodoethanol may lead to artefactual detection of native EO (false positive). An official method of analysis applicable for different food matrices would be useful to avoid discrepancies of results. Altogether, these data re-enforce the fact that in absence of native EO in food items, risk assessment of EO in foodstuffs should consider the predominance of 2-CE. A toxicological risk assessment using the food additive xanthan gum as a case study is discussed.

Rapid and Reliable Data Treatment for the Control of Food Chemical Contaminants by LC-HRMS.

Liquid chromatography-high resolution mass spectrometry (LC-HRMS) is considered an unavoidable extension of low-resolution LC-MS/MS that stretches the capabilities of multi-residue analysis of chemical contaminants in food. However, LC-HRMS acquisitions generate a massive amount of information available for data processing with supplier software that still miss critical calculation features and adapted reporting tools. Consequently, routine laboratories are still reluctant to switch from LC-MS/MS to LC-HRMS, the latter is still perceived as a cumbersome and demanding technology. In that context, we propose a four-step LC-HRMS workflow to speed-up data processing in situations of multi-residue multi-matrix analysis with the goal to maximize the time spent on data interpretation rather than on data formatting. The first three steps of the workflow imply specific settings on the Orbitrap HRMS associated software (TraceFinderTM) while the fourth step is the novelty i.e. a newly coded R-script capable to translate a raw export file into a comprehensive .xlsx report file in a few seconds. As recommended by various international guidelines and in some official methods, standard addition-based applications are fully embedded in this reporting tool whilst still being the main bottleneck of supplier's software. The reporting tool also allows appropriate data formatting, filtering, and color-coding options to provide a clear picture of compounds being detected or not, and those requiring specific attention due to unmet quality control criteria as required by European legislation (European Commission SANTE 11312/2021). It is hoped that additional functionalities compatible with R scripts will be soon fully embedded in the supplier's software for easier data interpretation and reporting.

Analysis of ethylene oxide in ice creams manufactured with contaminated carob bean gum (E410).

Residues of ethylene oxide (EO), a banned fumigant in the EU, were found at amounts above the maximum residue limit (MRL) in carob (locust) bean gum (additive E410). The pesticide entered the food chain via stabiliser blends that are used as minor ingredients in the manufacture of ice cream. Consequently, all products that contained the non-compliant ingredient were withdrawn or recalled in several countries across the EU, in most cases irrespective of whether the pesticide residue was detectable or not in the final product. This is the first report of a reliable method to determine EO and its metabolite/marker compound 2-chloroethanol (2-CE), either together or independently in ice cream, with a limit of quantification at 0.01 mg EO/kg and recovery in the range of 87-104% across the levels investigated (0.01, 0.02 and 0.06 mg EO/kg). The method applies QuEChERS extraction and isotope dilution gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). High resolution mass spectrometry (HRMS) confirmed the specificity of low mass ions. Data on the stability of EO and 2-CE under thermal conditions revealed that 2-CE is relatively stable in an ice cream matrix (ca. 80% recovery of spiked material). Importantly, this study also demonstrates that not EO, but 2-CE is the predominant analyte detected in the contaminated samples, which is new information of significance in terms of the overall risk assessment of EO in foodstuffs.

Limitations of currently available in vitro oestrogenicity bioassays for effect-based testing of whole foods as the basis for decision making.

The idea that previously unknown hazards can be readily revealed in complex mixtures such as foods is a seductive one, giving rise to the hope that data from effect-based assays of food products collected in market surveys is of suitable quality to be the basis for data-driven decision-making. To study this, we undertook a comparative study of the oestrogenicity of blinded cereal samples, both in a number of external testing laboratories and in our own facility. The results clearly showed little variance in the activities of 9 samples when using a single method, but great differences between the activities from each method. Further exploration of these findings suggest that the oestrogenic activity is likely an inherent part of the natural food matrix which the varying sample preparation methods are able to release and extract to differing degrees. These issues indicate the current poor suitability of these types of datasets to be used as the basis for consumer advice or food decision-making. Data quality must be improved before such testing is used in practice.

High resolution mass spectrometry workflow for the analysis of food contaminants: Application to plant toxins, mycotoxins and phytoestrogens in plant-based ingredients.

An analytical workflow including mass spectral library, generic sample preparation, chromatographic separation, and analysis by high-resolution mass spectrometry (HRMS) was developed to gain insight into the occurrence of plant toxins, mycotoxins and phytoestrogens in plant-based food. This workflow was applied to 156 compounds including 90 plant toxins (pyrrolizidine alkaloids, tropane alkaloids, glycoalkaloids, isoquinoline alkaloids and aristolochic acids), 54 mycotoxins (including ergot alkaloids and Alternaria toxins) and 12 phytoestrogens (including isoflavones, lignans and coumestan) in plant-based protein ingredients, cereal and pseudo-cereal products. A mass spectral library was built based on fragmentation spectra collected at 10 different collision energies in both positive and negative ionisation modes for each toxin. Emphasis was put on a generic QuEChERS-like sample preparation followed by ultra-high-pressure liquid chromatography using alkaline mobile phase allowing the separation of more than 50 toxic pyrrolizidine alkaloids. HRMS acquisition comprised a full-scan event for toxins detection followed by data-dependent MS2 for toxin identification against mass spectrum. Method performance was evaluated using fortified samples in terms of sensitivity, repeatability, reproducibility and recovery. All toxins were positively identified at levels ranging from 1 µg kg-1 to 100 µg kg-1. Quantitative results obtained by a standard addition approach met SANTE/12682/2019 criteria for 132 out of 156 toxins. Such a workflow using generic, sensitive and selective multi-residue method allows a better insight into the occurrence of regulated and non-regulated toxins in plant-based foods and to conduct safety evaluation and risk assessments when needed.

Screening of 154 Veterinary Drug Residues in Foods of Animal Origin Using LC-MS/MS: First Action 2020.04.

BACKGROUND: Veterinary drug residues in food are substances (>200 compounds) exhibiting potential health risks for consumers, thus being regulated in national legislations and the Codex Alimentarius. Most of the compounds are regulated based upon a maximum residue limit (MRL) while a few of them are banned in food for humans. The food sector needs a reliable and consensus analytical platform able to monitor these substances in a wide range of food commodities. OBJECTIVE: Several confirmatory methods based on liquid chromatography-mass spectrometry are available in the literature for either screening or quantification of veterinary drug residues in food, but usually applicable to limited scope of matrices. The current work describes the single-laboratory validation (SLV) of a method for screening 154 veterinary drug residues in several food categories. METHODS: This work describes a streamlined platform making use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for screening 105 antibiotics, 41 antiparasitics, 5anti-inflammatory agents, and 3 tranquilizers in foods of animal origin. For the best performance across the commodities (dairy-, meat-, fish-, and egg-based materials), four method streams were established. As a screening tool, probabilities of detection (PODs) were assessed at the screening target concentration (STC < MRL) and the blank. RESULTS: The SLV led to PODs at the STC >94% and PODs in the blank < 4%. CONCLUSION: Performance is in agreement with the acceptance criteria defined in SMPR 2018.010. HIGHLIGHTS: The Expert Review Panel approved the present method as AOAC Official First Action 2020.04.

Levels of Alternaria Toxins in Selected Food Commodities Including Green Coffee.

Alternaria toxins are emerging mycotoxins, candidates for regulation by European Authorities. Therefore, highly sensitive, confirmatory, and reliable analytical methodologies are required for their monitoring in food. In that context, an isotope dilution LC-MS/MS method was developed for the analysis of five Alternaria toxins (Altenuene, Alternariol, Alternariol monomethylether, Tentoxin, and Tenuazonic Acid) in a broad range of commodities including cereals and cereal-based products, tomato-based products, tree nuts, vegetable oils, dried fruits, cocoa, green coffee, spices, herbs, and tea. Validation data collected in two different laboratories demonstrated the robustness of the method. Underestimation of Tenuazonic Acid level in dry samples such as cereals was reported when inappropriate extraction solvent mixtures were used as currently done in several published methodologies. An investigation survey performed on 216 food items evidenced large variations of Alternaria toxins levels, in line with data reported in the last EFSA safety assessment. The analysis of 78 green coffee samples collected from 21 producing countries demonstrated that coffee is a negligible source of exposure to Alternaria toxins. Its wide scope of application, adequate sample throughput, and high sensitivity make this method fit for purpose for the regular monitoring of Alternaria toxins in foods.

Application of a streamlined LC-MS/MS methodology for the determination of atropine and scopolamine in cereals from Asian and African countries.

Tropane alkaloids are toxic secondary metabolites produced by a wide variety of plants that can be present in edible materials or animal feed. Several human poisoning cases through consumption of cereals were reported over the last years and highlighted the need for reliable and robust analytical methodologies for safety control. To rationalize analyses in high-throughput laboratory environments dealing with shorter and shorter turn-around-around time, the scope of our multi mycotoxins method was extended to the analysis of two regulated tropane alkaloids, namely atropine and scopolamine. Extraction procedure is based on the QuEChERS (Quick, Easy, Cheap, Efficient, Rugged, and Safe) approach followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) detection. Quantification is performed by the isotopic dilution approach using labelled isotopomers as internal standard. The procedure was validated at two fortification levels (0.5 µg/kg and 10 µg/kg) on different cereal-based products according to the European SANTE/12682/2019 document and performance parameters such as precision (RSD(r) ≤ 6%, RSD(iR) ≤ 6%) and recovery (82-114%) fulfilled its requirements. The limit of quantification (0.5 µg/kg) is low enough to ensure compliance with existing regulations. The method was further applied on 95 cereals and cereal-based products collected from Asian and African countries. All samples were found free of the two targeted TAs, with the exception of a rice-based product in which both atropine and scopolamine were quantified at 9.6 µg/kg and 2.6 µg/kg, respectively. A total of 29 cereals samples, shown to be free of both atropine and scopolamine were also analysed for mycotoxins. Aflatoxins, fumonisins, and deoxynivalenol were sporadically detected at levels below the maximum levels defined by the European Union legislation often considered as the most stringent regulation.

Multiple Mycotoxins Determination in Food by LC-MS/MS: An International Collaborative Study.

An intercollaborative study was organized to evaluate the performance characteristics of a liquid chromatography tandem mass spectrometry procedure for the simultaneous determination of 12 mycotoxins in food, which were ochratoxin A, aflatoxins B1, B2, G1, G2, and M1, deoxynivalenol, zearalenone, fumonisins B1 and B2, and T-2 and HT-2 toxins. The method combined the simplicity of the QuEChERS (Quick, Easy, Cheap, Efficient, Rugged and Safe) approach with the efficiency of immunoaffinity column cleanup (the step used to enhance sensitivity and sample cleanup for some matrices only). Twenty-three entities were enrolled and were European reference laboratories for mycotoxin analysis, U.S. and European service laboratories, and Nestlé laboratories. Each participant analyzed 28 incurred and/or spiked blind samples composed of spices, nuts, milk powder, dried fruits, cereals, and baby food using the protocol given. Method performances were assessed according to ISO 5725-2. Relative standard deviations of repeatability and reproducibility and trueness values for each of the 115 mycotoxin/sample combinations ranged from 5% to 23%, 7% to 26%, and 85% to 129%, respectively, in line with requirements defined in EC 401/2006. The overall set of data gathered demonstrated that the method offered a unique platform to ensure compliance with EC 1881/2006 and EC 165/2013 regulations setting maximum limits for mycotoxins in food samples, even at low regulated levels for foods intended for infants and young children. The method was applicable regardless of the food, the regulated mycotoxin, and the concentration level, and thus is an excellent candidate for future standardization.

A new high-throughput screening method to determine multiple dyes in herbs and spices.

The unauthorised addition of colours to herbs and spices is a recurrent issue affecting food business operators. Such a practice aims at improving food visual attractiveness, masking poor product quality, and/or compensating for natural colour variation with the ultimate goal to increase profits. To detect this fraud, a new LC-MS/MS method was developed for screening 58 dyes in both herbs and spices. This extended list of targets was established based on requirements from international spices organisations, past issues identified by web scouting and by notifications from the European Rapid Alert System for Food and Feed (RASFF). The method is intended to quickly detect fraudulent addition of dyes with Screening Target Concentrations ranging from 0.1 to 2.5 mg/kg. Validation was performed according to the European Community Reference Laboratories Residues Guidelines 20/1/2010. False positive and false negative rates were below 5% for all analytes and applicability of the method was further demonstrated by analysing 117 samples collected worldwide. None of the surveyed dyes was found in herbs (n = 28, 16 varieties) whereas 6% of spice samples (n = 89, 21 varieties) was found contaminated with one or two dyes at levels ranging from 0.12 to 255 mg/kg. Four out of the nine detected compounds have never been reported in the RASFF, thus demonstrating the usefulness of this analytical approach.

Screening of veterinary drug residues in food by LC-MS/MS. Background and challenges.

Regulatory agencies and government authorities have established maximum residue limits (MRL) in various food matrices of animal origin for supporting governments and food operators in the monitoring of veterinary drug residues in the food chain, and ultimately in the consumer's plate. Today, about 200 veterinary drug residues from several families, mainly with antibiotic, antiparasitic or antiinflammatory activities, are regulated in a variety of food matrices such as milk, meat or egg. This article provides a review of the regulatory framework in milk and muscle including data from Codex Alimentarius, Europe, the U.S.A., Canada and China for about 220 veterinary drugs. The article also provides a comprehensive overview of the challenge for food control, and emphasizes the pivotal role of liquid chromatography-mass spectrometry (LC-MS), either in tandem with quadrupoles (LC-MS/MS) or high resolution MS (LC-HRMS), for ensuring an adequate consumer protection combined with an affordable cost. The capability of a streamlined LC-MS/MS platform for screening 152 veterinary drug residues in a broad range of raw materials and finished products is highlighted in a production line perspective. The rationale for a suite of four methods intended to achieve appropriate performance in terms of scope and sensitivity is presented. Overall, the platform encompasses one stream for the determination of 105 compounds in a run (based on acidic QuEChERS-like), plus two streams for 23 ß-lactams (alkaline QuEChERS-like) and 10 tetracyclines (low-temperature partitioning), respectively, and a dedicated stream for 14 aminoglycosides (molecularly-imprinted polymer).

Screening of 23 ß-lactams in foodstuffs by LC-MS/MS using an alkaline QuEChERS-like extraction.

A fast and robust high performance LC-MS/MS screening method was developed for the analysis of ß-lactam antibiotics in foods of animal origin: eggs, raw milk, processed dairy ingredients, infant formula, and meat- and fish-based products including baby foods. QuEChERS extraction with some adaptations enabled 23 drugs to be simultaneously monitored. Screening target concentrations were set at levels adequate to ensure compliance with current European, Chinese, US and Canadian regulations. The method was fully validated according to the European Community Reference Laboratories Residues Guidelines using 93 food samples of different composition. False-negative and false-positive rates were below 5% for all analytes. The method is adequate for use in high-routine laboratories. A 1-year study was additionally conducted to assess the stability of the 23 analytes in the working standard solution.

Determination of 105 antibiotic, anti-inflammatory, antiparasitic agents and tranquilizers by LC-MS/MS based on an acidic QuEChERS-like extraction.

A procedure for screening 105 veterinary drugs in foods by liquid chromatography tandem mass-spectrometry (LC-MS/MS) is presented. Its scope encompasses raw materials of animal origin (milk, meat, fish, egg and fat) but also related processed ingredients and finished products commonly used and manufactured by food business operators. Due to the complexity of the matrices considered and to efficiently deal with losses during extraction and matrix effects during MS source ionisation, each sample was analysed twice, that is 'unspiked' and 'spiked at the screening target concentration' using a QuEChERS-like extraction. The entire procedure was validated according to the European Community Reference Laboratories Residues Guidelines. False-negative and false-positive rates were below 5% for all veterinary drugs whatever the food matrix. Effectiveness of the procedure was further demonstrated through participation to five proficiency tests and its ruggedness demonstrated in quality control operations by a second laboratory.

Stability study of veterinary drugs in standard solutions for LC-MS/MS screening in food.

A study on stability of veterinary drugs in standard solutions stored at -80°C and at -20°C was conducted over 1 year. Data were acquired on 152 individual stock standard solutions and also on 15 family mixes and 2 working standard solutions. All solutions were prepared, stored and compared 1 year later against freshly prepared ones by LC-MS/MS. A statistical analysis was performed to set the acceptability criteria, taking into account the variability of standard preparations. In individual stock standard solutions stored at -80°C (12 months) and -20°C (9 months), stability was demonstrated for 141 and 140 out of 152 compounds, i.e. for 92% and 93% of compounds, respectively. Drugs were even more stable when solubilised in either diluted family mixes or working standard solutions, with more than 99% and 94% of compounds found unaltered when stored at -80°C and at -20°C, respectively. In mixes, beta-lactams from the cephalosporin (cefadroxil and cephalexin) and penicillin (amoxicillin and ampicillin) families were found to be the least stable compounds when stored at -20°C (6 months), necessitating storage at -80°C to achieve a 1-year shelf life. The study also evidenced solubility issues for two sulfonamides (sulfadiazine and sulfamerazine) in methanol-based solutions. An independent stability study conducted by a second laboratory confirmed the 1-year stability of 3 family mixes-quinolones, sulfonamides and tetracyclines.

Development of a quantitative multi-compound method for the detection of 14 nitrogen-rich adulterants by LC-MS/MS in food materials.

The increasing number of food frauds using exogenous nitrogen-rich adulterants to artificially raise the protein content for economically motivated adulteration has demonstrated the need for a robust analytical methodology. This method should be applicable for quality control in operations covering a wide range of analyte concentrations to be able to analyse high levels as usually found in adulteration, as well as low levels due to contamination. The paper describes a LC-MS/MS method covering 14 nitrogen-rich adulterants using a simple and fast sample preparation based on dilution and clean-up by dispersive SPE. Quantification is carried out by isotopic dilution reaching LOQs of 0.05-0.20 mg/kg in a broad range of food matrices (infant formula, liquid milk, dairy ingredient, high protein meal, cereal, infant cereal, and meat/fish powder). Validation of seven commodity groups was performed according to SANCO 12571/2013, giving satisfactory results demonstrating the method's fitness for purpose at the validated range at contamination level. Method ruggedness was further assessed by transferring the developed method into another laboratory devoted to routine testing for quality control. Next to the method description, emphasis is placed on challenges and problems appearing during method development as well as validation. They are discussed in detail and solutions are provided.

Heat-induced formation of mepiquat by decarboxylation of pipecolic acid and its betaine derivative. Part 1: Model system studies.

This study describes, for the first time, the role of pipecolic acid betaine and pipecolic acid, naturally present in some foods, in the formation of the plant growth regulator N,N-dimethylpiperidinium (mepiquat) under dry thermal conditions. The formation of mepiquat and intermediate compounds was investigated in model systems using high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry. Mepiquat is released with a yield of up to 0.66mol% after thermal treatment (>150°C) of pipecolic acid betaine. Similar conversion rates are attained with the congener piperidine-2-carboxylic acid (dl-pipecolic acid), albeit in the presence of alkylating agents, such as choline, glycine betaine or trigonelline, that are fairly widespread in food crops. These new pathways to mepiquat indicate that the occurrence of low levels of this thermally induced compound is probably more widespread in processed foods than initially suspected (see Part 2 of this study on the occurrence of mepiquat in selected foodstuffs).

Process-induced formation of imidazoles in selected foods.

The presence of 4-methylimidazole (4-MEI), 2-methylimidazole (2-MEI) and 2-acetyl-4-tetrahydroxybutylimidazole (THI) in some foods may result from the usage of caramel colorants E150c and E150d as food additives. This study demonstrates that alkylimidazoles are also byproducts formed from natural constituents in foods during thermal processes. A range of heat-processed foods that are known not to contain caramel colorants were analyzed by isotope dilution LC-MS/MS to determine the contamination levels. Highest 4-MEI concentrations (up to 466µg/kg) were observed in roasted barley, roasted malt and cocoa powders, with the concomitant presence of 2-MEI and/or THI in some cases, albeit at significantly lower levels. Low amounts of 4-MEI (<20µg/kg) were also detected in cereal-based foods such as breakfast cereals and bread toasted to a brown color (medium toasted). The occurrence of 4-MEI in certain processed foods is therefore not a reliable indicator of the presence of the additives E150c or E150d.

Heat-induced formation of mepiquat by decarboxylation of pipecolic acid and its betaine derivative. Part 2: Natural formation in cooked vegetables and selected food products.

Mepiquat (N,N-dimethylpiperidinium) is a plant growth regulator registered for use as its chloride salt in many countries on cereals and other crops. Recent model system studies have shown that natural chemicals present in crop plants, such as pipecolic acid and pipecolic acid betaine, may furnish mepiquat through different chemical pathways, when subjected to temperatures in the range of 200°C. In this study, we cooked raw vegetables that did not contain mepiquat to a palatable state using different traditional cooking methods, and detected mepiquat in 9 out of 11 oven-cooked vegetables, reaching up to 189µg/kg dry wt in oven-cooked broccoli. Commercial oven potato fries generated mepiquat during cooking, typically in the range of 20-60µg/kg. Only traces of mepiquat (<5µg/kg) were found in commercial potato crisps. This work demonstrates that mepiquat occurs at µg/kg levels in a variety of cooked vegetables, including potatoes.