Towards a consensus LC-MS/MS method for the determination of acrylamide in food that prevents overestimation due to interferences.

Acrylamide is prone to misquantification, and critical steps in the analytical procedures need to be identified and controlled to ensure a reliable determination. Four methods were considered to illustrate misquantification issues with acrylamide. For two methods varying by the extent of their sample preparations, cases of overestimation in cocoa samples reaching up to a 20-fold factor are shown. A second example, applied to a variety of food products, includes two other methods varying by their chromatographic conditions. As a follow up of a study conducted in 2020 about the identification of N-acetyl-ß-alanine as an interference of acrylamide in coffee, the extent of this interference was evaluated in a selection of coffee samples, cereal-based products and baby foods. The ultimate objective of this manuscript was to resolve such cases of misquantification and validate a wide scope and robust method allowing an interference free acrylamide analysis. To do so, an extraction procedure based on the EN 16618:2015 standard with water extraction and two consecutive solid phase extraction (SPE) steps was applied with modified liquid chromatographic conditions. The method was validated in coffee, cereals, baby foods, cocoa and pet foods with excellent performance in terms of recovery (97-108%) and precision (RSDr and RSDiR <12 %). The breath of scope was further proved through trueness determination in quality control materials and reference materials including French fries, potato crisps, vegetable crisps, instant coffee, infant food and biscuit (cookie), with trueness values found within a 94-107% range.

Quantification of furan and 5 alkylfurans with complete separation of 2-ethylfuran and 2,5-dimethylfuran isomers in cereals, coffee and Infant products by GC-MS.

Alkylfurans have been found concurrently to furan in thermally processed food and might add to the overall exposure, thereby increase the health concern. The analytical methods developed for these compounds are based on gas chromatography separation coupled to mass spectrometry. Two of those alkylfurans, 2,5-dimethylfuran and 2-ethylfuran, are isomers, for which accurate quantification require either complete chromatographic separation or tandem mass spectrometry (MS/MS) selectivity. A new chromatographic method is reported using the Supelco Equity-1 column, demonstrating complete baseline separation of these two isomers, with a shorter runtime when using single mass spectrometry. Full validation was performed successfully for furan, 2- and 3-methylfuran, 2-ethylfuran, 2,5-dimethylfuran and 2-pentylfuran on different food matrices with recovery rates in the range of 80-110 %, repeatability below 14%, intermediate reproducibility below 22% as well as expanded uncertainty under 50%.

Fate of acrylamide during coffee roasting and in vitro digestion assessed with carbon 14- and carbon 13-labeled materials.

Acrylamide (AA) formation during coffee roasting happens rapidly, reaching a peak value within the first minutes of roasting followed by a fast decrease to reach an asymptote at approximately 200 µg/kg. Today, the mechanisms by which AA is reduced during roasting remain unclear. In this research, the fate of AA during roasting followed by drip brewed-like extraction was studied using 14C-radiolabeled (14C-AA) and 13C-labeled (13C3-AA) materials. Results showed that 28% of the spiked 14C-AA was lost during the roasting process, presumably by degradation to volatile compounds and 25% was non-extractable; therefore, appeared bound to the matrix. About 50% of initial AA went into the water extract, either unchanged or transformed by conjugation/binding. The release of bound acrylamide was further evidenced by increasing levels of 13C3-AA over prolonged roasting times. In addition, the absence of 14C activity in the hexane extracts suggested acrylamide not to bind to any lipophilic material.

Sources of overestimation in the analysis of acrylamide-in coffee by liquid chromatography mass spectrometry.

Analysis of acrylamide in coffee by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is prone to interferences. According to our study, unknown background ions can entail an overestimation by up to 40% in case of coelution with acrylamide. In order to develop a precise and accurate quantification method for acrylamide, identification and removal of these interfering ions is requested. We thus studied potential isobaric impurities of acrylamide using liquid chromatography-high resolution mass spectrometry (LC-HRMS). An in-source fragment of N-Acetyl-ß-alanine, a substance not yet reported in coffee, was identified as the main interfering ion. The characterization of the interference further triggered modification of the mobile phase-pH to alter the retention of N-Acetyl-ß-alanine and achieve an interference free acrylamide determination. Two other compounds closely related to acrylamide namely 3-aminopropanamide and lactamide were also susceptible to in-source fragmentation, highlighting the pivotal role of chromatographic conditions to ensure a reliable quantification of acrylamide.

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.

N,N-dimethylpiperidinium (mepiquat) Part 2. Formation in roasted coffee and barley during thermal processing.

Previous work in model systems has demonstrated that mepiquat can be formed under typical roasting conditions from the amino acid lysine via the Maillard reaction and trigonelline, the latter alkaloid serving as a methyl donor. This study shows for the first time that mepiquat is formed in low mg kg(-1) amounts during the coffee roasting process and consequently can be detected in roast and ground as well as soluble coffee up to levels of 1.4 mg kg(-1). Darker roast coffees contain relatively higher amounts of mepiquat versus light roasted beans, with an excellent correlation of mepiquat formation to roast colour (r(2) = 0.99) in robusta beans. A survey of 20 of the major green coffee origins (robusta and arabica coffees) showed the absence of mepiquat (<0.005 mg kg(-1)). Preliminary studies indicate that mepiquat is not formed during processing (thermal treatment) in most of the cereal-based foods such as pizza and ready-to-eat cereals, but was detected in barley after roasting (0.64 mg kg(-1)). Mepiquat can therefore be considered a process-induced compound formed from natural constituents during the roasting process. Even considering a high intake of seven cups per day of soluble coffee containing 1.4 mg kg(-1) mepiquat in the coffee powder (the highest amount measured in this study), the resulting intake would exhaust less than 0.2% of the ADI of mepiquat.

Instant coffee with high chlorogenic acid levels protects humans against oxidative damage of macromolecules.

SCOPE: Coffee is among the most frequently consumed beverages. Its consumption is inversely associated to the incidence of diseases related to reactive oxygen species; the phenomenon may be due to its antioxidant properties. Our primary objective was to investigate the impact of consumption of a coffee containing high levels of chlorogenic acids on the oxidation of proteins, DNA and membrane lipids; additionally, other redox biomarkers were monitored in an intervention trial. METHODS AND RESULTS: The treatment group (n=36) consumed instant coffee co-extracted from green and roasted beans, whereas the control consumed water (800 mL/P/day, 5 days). A global statistical analysis of four main biomarkers selected as primary outcomes showed that the overall changes are significant. 8-Isoprostaglandin F2α in urine declined by 15.3%, 3-nitrotyrosine was decreased by 16.1%, DNA migration due to oxidized purines and pyrimidines was (not significantly) reduced in lymphocytes by 12.5 and 14.1%. Other markers such as the total antioxidant capacity were moderately increased; e.g. LDL and malondialdehyde were shifted towards a non-significant reduction. CONCLUSION: The oxidation of DNA, lipids and proteins associated with the incidence of various diseases and the protection against their oxidative damage may be indicative for beneficial health effects of coffee.

In silico methods for physiologically based biokinetic models describing bioactivation and detoxification of coumarin and estragole: implications for risk assessment.

In chemical safety assessment, information on adverse effects after chronic exposure to low levels of hazardous compounds is essential for estimating human risks. Results from in vitro studies are often not directly applicable to the in vivo situation, and in vivo animal studies often have to be performed at unrealistic high levels of exposure. Physiologically based biokinetic (PBBK) modeling can be used as a platform for integrating in vitro metabolic data to predict dose- and species-dependent in vivo effects on biokinetics, and can provide a method to obtain a better mechanistic basis for extrapolations of data obtained in experimental animal studies to the human situation. Recently, we have developed PBBK models for the bioactivation of the alkenylbenzene estragole to its DNA binding ultimate carcinogenic metabolite 1'-sulfooxyestragole in both rat and human, as well as rat and human PBBK models for the bioactivation of coumarin to its hepatotoxic o-hydroxyphenylacetaldehyde metabolite. This article presents an overview of the results obtained so far with these in silico methods for PBBK modeling, focusing on the possible implications for risk assessment, and some additional considerations and future perspectives.

Basil extract inhibits the sulfotransferase mediated formation of DNA adducts of the procarcinogen 1'-hydroxyestragole by rat and human liver S9 homogenates and in HepG2 human hepatoma cells.

The effects of a basil extract on the sulfation and concomitant DNA adduct formation of the proximate carcinogen 1'-hydroxyestragole were studied using rat and human liver S9 homogenates and the human hepatoma cell line HepG2. Basil was chosen since it contains the procarcinogen estragole that can be metabolized to 1'-hydroxyestragole by cytochrome P450 enzymes. Basil extract addition to incubations of rat and human liver S9 homogenates with 1'-hydroxyestragole, the sulfotransferase cofactor PAPS, and 2'-deoxyguanosine resulted in a dose-dependent inhibition of N2-(trans-isoestragol-3'-yl)-2'-deoxyguanosine formation. Because the inhibition resembled the inhibition by the sulfotransferase inhibitor pentachlorophenol and since the inhibition was not observed in incubations with the direct electrophile 1'-acetoxyestragole it is concluded that the inhibition occurs at the level of the sulfotransferase mediated bioactivation step. Additional experiments in HepG2 cells revealed the same protective effect of basil extract in intact cells, demonstrating that the inhibitors are able to enter the cells. The results of this study suggest that bioactivation and subsequent adverse effects of 1'-hydroxyestragole might be lower in a matrix of other basil ingredients than what would be expected on the basis of experiments using 1'-hydroxyestragole as a single compound.

Improved sample preparation to determine acrylamide in difficult matrixes such as chocolate powder, cocoa, and coffee by liquid chromatography tandem mass spectroscopy.

An improved sample preparation (extraction and cleanup) is presented that enables the quantification of low levels of acrylamide in difficult matrixes, including soluble chocolate powder, cocoa, coffee, and coffee surrogate. Final analysis is done by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) using d3-acrylamide as internal standard. Sample pretreatment essentially encompasses (a) protein precipitation with Carrez I and II solutions, (b) extraction of the analyte into ethyl acetate, and (c) solid-phase extraction on a Multimode cartridge. The stability of acrylamide in final extracts and in certain commercial foods and beverages is also reported. This approach provided good performance in terms of linearity, accuracy and precision. Full validation was conducted in soluble chocolate powder, achieving a decision limit (CCalpha) and detection capability (CCbeta) of 9.2 and 12.5 microg/kg, respectively. The method was extended to the analysis of acrylamide in various foodstuffs such as mashed potatoes, crisp bread, and butter biscuit and cookies. Furthermore, the accuracy of the method is demonstrated by the results obtained in three inter-laboratory proficiency tests.

Artifactual nitration controlled measurement of protein-bound 3-nitro-L-tyrosine in biological fluids and tissues by isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry.

A sensitive and selective method is presented to accurately determine the level of protein-bound 3-nitro-L-tyrosine (NTyr) in rat plasma and kidney samples. This assay is based on isotope dilution liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The sample preparation entails protein precipitation, acid hydrolysis with 6 N HCl, and solid-phase extraction (using reverse and aminopropyl phase cartridges) prior to the determinative step. For kidney samples, NTyr is converted into its butyl ester to improve sensitivity. The potential formation of artifactual NTyr during the acid hydrolysis step was carefully followed and determined by supplementation of the samples with (13)C-labeled L-tyrosine (Tyr) prior to protein digestion. Hence, the concomitant measurement of formation of (13)C-enriched NTyr enabled the accurate determination of artifactual NTyr. This approach was employed to measure the basal level of protein-bound NTyr in rat plasma and kidney samples, revealing levels in the range of 4-18 micromol/mol of Tyr and 50-68 micromol/mol of Tyr, respectively. No artifactual nitration of Tyr was observed in kidney proteins, whereas in the case of plasma the contribution of the artifactual response ranged from 16 to 40%. This method allows the analysis of protein-bound NTyr with a full control of the artifactual nitration of tyrosine during the proteolysis and/or sample preparation. Reliable detection of NTyr in proteins may allow insight into the role of nitric oxide-derived oxidants under various pathological conditions.