Milena Zachariasova

Novel approaches in analysis of Fusarium mycotoxins in cereals employing ultra performance liquid chromatography coupled with high resolution mass spectrometry.

Rapid, simple and cost-effective analytical methods with performance characteristics matching regulatory requirements are needed for effective control of occurrence of Fusarium toxins in cereals and cereal-based products to which they might be transferred during processing. Within this study, two alternative approaches enabling retrospective data analysis and identification of unknown signals in sample extracts have been implemented and validated for determination of 11 major Fusarium toxins. In both cases, ultra-high performance liquid chromatography (U-HPLC) coupled with high resolution mass spectrometry (HR MS) was employed. (13)C isotopically labeled surrogates as well as matrix-matched standards were employed for quantification. As far as time of flight mass analyzer (TOF-MS) was a detection tool, the use of modified QuEChERS (quick easy cheap effective rugged and safe) sample preparation procedure, widely employed in multi-pesticides residue analysis, was shown as an optimal approach to obtain low detection limits. The second challenging alternative, enabling direct analysis of crude extract, was the use of mass analyzer based on Orbitrap technology. In addition to demonstration of full compliance of the new methods with Commission Regulation (EC) No. 401/2006, also their potential to be used for confirmatory purposes according to Commission Decision 2002/657/EC has been critically assessed.

Transfer of Fusarium mycotoxins and ‘masked’ deoxynivalenol (deoxynivalenol-3-glucoside) from field barley through malt to beer

The fate of five Fusarium toxins — deoxynivalenol (DON), sum of 15- and 3-acetyl-deoxynivalenol (ADONs), HT-2 toxin (HT-2) representing the main trichothecenes and zearalenone (ZON) during the malting and brewing processes — was investigated. In addition to these ‘free’ mycotoxins, the occurrence of deoxynivalenol-3-glucoside (DON-3-Glc) was monitored for the first time in a beer production chain (currently, only DON and ZON are regulated). Two batches of barley, naturally infected and artificially inoculated with Fusarium spp. during the time of flowering, were used as a raw material for processing experiments. A highly sensitive procedure employing high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was validated for the analysis of ‘free’ Fusarium mycotoxins and DON-conjugate in all types of matrices. The method was also able to detect nivalenol (NIV), fusarenon-X (FUS-X) and T-2 toxin (T-2); nevertheless, none of these toxins was found in any of the samples. While steeping of barley grains (the first step in the malting process) apparently reduced Fusarium mycotoxin levels to below their quantification limits (5–10 µg kg−1), their successive accumulation occurred during germination. In malt, the content of monitored mycotoxins was higher compared with the original barley. The most significant increase was found for DON-3-Glc. During the brewing process, significant further increases in levels occurred. Concentrations of this ‘masked’ DON in final beers exceeded ‘free’ DON, while in malt grists this trichothecene was the most abundant, with the DON/DON-3-Glc ratio being approximately 5:1 in both sample series. When calculating mass balance, no significant changes were observed during brewing for ADONs. The content of DON and ZON slightly decreased by a maximum of 30%. Only traces of HT-2 were detected in some processing intermediates (wort after trub removal and green beer).

Analysis of multiple mycotoxins in cereals under ambient conditions using direct analysis in real time (DART) ionization coupled to high resolution mass spectrometry

Direct analysis in real time (DART) ionization coupled to an (ultra)high resolution mass spectrometer based on orbitrap technology (orbitrapMS) was used for rapid quantitative analysis of multiple mycotoxins isolated from wheat and maize by modified QuEChERS procedure. After initial evaluation of ionization efficiencies for major groups of mycotoxins achievable with DART technology, sample preparation procedure and instrument parameter settings were optimized to obtain sensitive and accurate determination of most intensively ionizing toxins (deoxynivalenol, nivalenol, zearalenon, actyldeoxynivalenol, deepoxy-deoxynivalenol, fusarenon-X, altenuene, alternariol, alternariolmethylether, diacetoxyscirpenol, sterigmatocystin). The lowest calibration levels (LCLs) estimated for the respective analytes ranged from 50 to 150 μg kg(-1). Quantitative analysis was performed either with the use of matrix-matched standards or by employing commercially available (13)C-labeled internal standards (available for deoxynivalenol, nivalenol and zearalenon). Good recoveries (100-108%) and repeatabilities (RSD 5.4-6.9%) were obtained at spiking level 500 μg kg(-1) with isotope dilution technique. Based on matrix-matched calibration, recoveries and repeatabilities were in the range 84-118% and 7.9-12.0% (RSD), respectively. The trueness of data obtained for deoxynivalenol and zearalenon in wheat/maize by DART-orbitrapMS was demonstrated by analysis of certified reference materials (CRMs). Good agreement of these results with data generated by validated ultra-high pressure liquid chromatography-time-of-flight mass spectrometry method was documented.

Analysis of multiple mycotoxins in beer employing (ultra)-high-resolution mass spectrometry

The objective of the presented study was to develop and optimize a simple, high-throughput method for the control of 32 mycotoxins (Fusarium and Alternaria toxins, aflatoxins, ergot alkaloids, ochratoxins, and sterigmatocystin) in beer. Due to the broad range of their physicochemical properties, the sample preparation step was simplified as much as possible to avoid analyte losses. The addition of acetonitrile to beer samples enabled precipitation of abundant matrix components. The clean-up efficiency was controlled by ambient mass spectrometry employing a direct analysis in real time (DART) ion source. For determination of analytes, ultra-high-performance liquid chromatography hyphenated with high-resolution mass spectrometry utilizing an orbitrap (U-HPLC-orbitrapMS) or time-of-flight (TOFMS) technology was used. Because of significantly better detection capabilities of the orbitrap technology, the U-HPLC-orbitrapMS method was chosen as a determinative step and fully validated. To compensate matrix effects, matrix-matched calibration was employed. The lowest calibration levels for most of the target mycotoxins ranged from 1 to 8  µg  L(-1) beer and the recoveries of analytes were in range from 86 to 124%.

Deoxynivalenol and its conjugates in beer: A critical assessment of data obtained by enzyme-linked immunosorbent assay and liquid chromatography coupled to tandem mass spectrometry

Enzyme-linked immunosorbent assays (ELISAs) are often employed for the control of deoxynivalenol (DON) in barley and other intermediates involved in beer production chain. Because of the occurrence of high levels of DON-3-glucoside (DON-3-Glc) in malt and beer that have been reported for the first time in our earlier study, research focused on the accuracy of DON determination by immunoassays in cereal-based matrices has been initiated. DON-3-Glc was strongly cross-reacting in all examined commercial ELISA test kits (Ridascreen) DON (R-Biopharm), Veratox 5/5 DON) (Neogen Corporation), Deoxynivalenol EIA (Euro-Diagnostica), and AgraQuant) DON Assay 0.25/5.0 Test Kit (Romer Labs). The highest overestimation in beer analysis, up to 1000%, when taking the DON content determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) as a reference method, was obtained by AgraQuant assay. Besides of DON-3-Glc and 3- and 15-acetyldeoxynivalenol (ADONs), also other, not known yet, matrix components contributed to false positive results. Similar phenomenon, although in a lesser extent due to lower content of these substances, was observed for using ELISA in the analysis of wheat. The relationship between a way of sample handling and DON overestimation was demonstrated; higher ELISA response was measured in an aqueous extract compared to that prepared by acetonitrile-water (84:16, v/v). Most of cross-reacting co-extracts were removed by MycoSep# 226 cartridge, what leads us to the hypothesis on the presence of currently unknown cross-reactive species.

Analysis of mycotoxins in barley using ultra high liquid chromatography high resolution mass spectrometry: comparison of efficiency and efficacy of different extraction procedures.

The effectiveness of four extraction methods (modified QuEChERS, matrix solid-phase dispersion (MSPD), solid-liquid extraction (SLE) and solid-phase extraction (SPE) clean-up) were evaluated for simultaneous determination of 32 mycotoxins produced by the genus Fusarium, Claviceps, Aspergillus, Penicillium and Alternaria in barley by ultra high pressure liquid chromatography coupled to ultra-high resolution mass spectrometry (UHPLC-Orbitrap(®) MS). The efficiency and efficacy of extraction methods were evaluated and compared in number of extracted mycotoxins and obtained recoveries. From the one point of view, QuEChERS procedure was fast and easy, as well as it was able to successfully extract all selected mycotoxins. On the other hand, SLE method, MSPD and SPE clean-up method did not extract adequately all selected mycotoxins and recoveries were not suitable enough. Thereby, method employing QuEChERS extraction connected with UHPLC-Orbitrap(®) MS was developed to quantify 32 mycotoxins in barley within this study. Analytical method was validated and recoveries ranged from 72% to 101% for selected mycotoxins with only one exception nivalenol (NIV) and deoxynivalenol-3-glucoside (D3G), which were lower than 67%. Relative standard deviations (RSD) were lower than 17.4% for all target mycotoxins. The lowest calibration levels (LCLs) ranged from 1 to 100 μg/kg. Validated method was finally used for monitoring mycotoxins in a total of 15 Czech barley samples, when only Fusarium toxins representatives were detected in 53% of samples and the mycotoxins with the highest incidence were enniatins.

'Emerging' mycotoxins in cereals processing chains: changes of enniatins during beer and bread making.

Enniatins represent an emerging food safety issue because of their extensive incidence, documented in recent decades, in various small grain cereals. This study was concerned with the fate of these Fusarium mycotoxins within malting, brewing, milling and baking, when employed for the processing of contaminated barley and wheat. Besides enniatins A, A1, B and B1, also deoxynivalenol and its conjugated form (deoxynivalenol-3-glucoside) were determined in almost all tested cereal-based samples. Significant decline of enniatins occurred within all technologies, with the largest drop in their concentrations observed in the brewing process. While enniatins were not detectable in final beers, they were almost quantitatively transferred to spent grains, probably because of their limited water solubility. Regarding bread baking, levels of enniatins decreased down to 30% of their concentration in the initial flour used for baking. In this case, degradation at higher temperatures might be assumed.

The study of deoxynivalenol and its masked metabolites fate during the brewing process realised by UPLC-TOFMS method.

The co-occurrence of deoxynivalenol (DON) and deoxynivalenol-3-glucoside (DON-3-Glc) has been recently reported in malt and beer. In this study, the concentration changes were monitored within the brewing process of four beer brands: light, dark tap and two lagers, produced from ground malt mixtures differing in composition, and also mycotoxins content. A simple and rapid method employing DON-dedicated immunoaffinity columns (IAC) for the selective pre-concentration, followed by ultra-performance liquid chromatography coupled to a time-of-flight mass spectrometer (UPLC-TOFMS) system for the reliable quantification at (ultra)trace levels, was validated for all experimental matrices. The results document the key role of the malt contamination nature. While in the first monitoring period a significant increase of both DON and DON-3-Glc occurred (up to 250% and 450%, respectively), fairly different trends were observed when new malts were used for identical technological processing (in some beers a decrease of DON and only a small increase of DON-3-Glc occurred). Worth noticing, that the outcome of the brewing process was surprisingly reproducible for a particular malt mixture. In the final phase, a small monitoring study comparing Czech and Austrian alcohol-free and conventional beers was carried out.

Advances in high-resolution mass spectrometry based on metabolomics studies for food – a review

Food authenticity becomes a necessity for global food policies, since food placed in the market without fail has to be authentic. It has always been a challenge, since in the past minor components, called also markers, have been mainly monitored by chromatographic methods in order to authenticate the food. Nevertheless, nowadays, advanced analytical methods have allowed food fingerprints to be achieved. At the same time they have been also combined with chemometrics, which uses statistical methods in order to verify food and to provide maximum information by analysing chemical data. These sophisticated methods based on different separation techniques or stand alone have been recently coupled to high-resolution mass spectrometry (HRMS) in order to verify the authenticity of food. The new generation of HRMS detectors have experienced significant advances in resolving power, sensitivity, robustness, extended dynamic range, easier mass calibration and tandem mass capabilities, making HRMS more attractive and useful to the food metabolomics community, therefore becoming a reliable tool for food authenticity. The purpose of this review is to summarise and describe the most recent metabolomics approaches in the area of food metabolomics, and to discuss the strengths and drawbacks of the HRMS analytical platforms combined with chemometrics. Graphical Abstract

Saffron authentication based on liquid chromatography high resolution tandem mass spectrometry and multivariate data analysis

Saffron is one of the oldest and most expensive spices, which is often target of fraudulent activities. In this research, a new strategy of saffron authentication based on metabolic fingerprinting was developed. In the first phase, a solid liquid extraction procedure was optimized, the main aim was to isolate as maximal representation of small molecules contained in saffron as possible. In the second step, a detection method based on liquid chromatography coupled with high-resolution mass spectrometry was developed. Initially, principal component analysis (PCA) revealed clear differences between saffron cultivated and packaged in Spain, protected designation of origin (PDO), and saffron packaged in Spain of unknown origin, labeled Spanish saffron. Afterwards, orthogonal partial least square discriminant analysis (OPLS-DA) was favorably used to discriminate between Spanish saffron. The tentative identification of markers showed glycerophospholipids and their oxidized lipids were significant markers according to their origin.