Challenges and future directions in LC-MS-based multiclass method development for the quantification of food contaminants.

Monitoring of food contaminants and residues has undergone a significant improvement in recent years and is now performed in an intensive manner. Achievements in the area of chromatography-mass spectrometry coupling techniques enabled the development of quantitative multi-target approaches covering several hundred analytes. Although the majority of methods are focusing on the analysis of one specific group of substances, such as pesticides, mycotoxins, or veterinary drugs, current trends are going towards the simultaneous determination of multiclass compounds from several families of contaminants and residues. This work provides an overview of relevant multiclass concepts based on LC-MS/MS and LC-HRMS instruments. Merits and shortcomings will be critically discussed based on current performance characteristics of the EU legislation system. In addition, the discussion of a recently developed multiclass approach covering >1000 substances is presented as a case study to illustrate the current developments in this area.

Advanced LC-MS-based methods to study the co-occurrence and metabolization of multiple mycotoxins in cereals and cereal-based food.

Liquid chromatography (LC) coupled with mass spectrometry (MS) is widely used for the determination of mycotoxins in cereals and cereal-based products. In addition to the regulated mycotoxins, for which official control is required, LC-MS is often used for the screening of a large range of mycotoxins and/or for the identification and characterization of novel metabolites. This review provides insight into the LC-MS methods used for the determination of co-occurring mycotoxins with special emphasis on multiple-analyte applications. The first part of the review is focused on targeted LC-MS approaches using cleanup methods such as solid-phase extraction and immunoaffinity chromatography, as well as on methods based on minimum cleanup (quick, easy, cheap, effective, rugged, and safe; QuEChERS) and dilute and shoot. The second part of the review deals with the untargeted determination of mycotoxins by LC coupled with high-resolution MS, which includes also metabolomics techniques to study the fate of mycotoxins in plants.

Determinants and Expansion of Specificity in a Trichothecene UDP-Glucosyltransferase from Oryza sativa.

Family 1 UDP-glycosyltransferases (UGTs) in plants primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, which is a plant disease of global significance that leads to reduced crop yields and contamination with trichothecene mycotoxins such as deoxynivalenol (DON), T-2 toxin, and many other structural variants. The UGT Os79 from rice has emerged as a promising candidate for inactivation of mycotoxins because of its ability to glycosylate DON, nivalenol, and hydrolyzed T-2 toxin (HT-2). However, Os79 is unable to modify T-2 toxin (T-2), produced by pathogens such as Fusarium sporotrichioides and Fusarium langsethii. Activity toward T-2 is desirable because it would allow a single UGT to inactivate co-occurring mycotoxins. Here, the structure of Os79 in complex with the products UDP and deoxynivalenol 3-O-glucoside is reported together with a kinetic analysis of a broad range of trichothecene mycotoxins. Residues associated with the trichothecene binding pocket were examined by site-directed mutagenesis that revealed that trichothecenes substituted at the C4 position, which are not glycosylated by wild-type Os79, can be accommodated in the binding pocket by increasing its volume. The H122A/L123A/Q202L triple mutation, which increases the volume of the active site and attenuates polar contacts, led to strong and equivalent activity toward trichothecenes with C4 acetyl groups. This mutant enzyme provides the broad specificity required to control multiple toxins produced by different Fusarium species and chemotypes.

A barley UDP-glucosyltransferase inactivates nivalenol and provides Fusarium Head Blight resistance in transgenic wheat.

Fusarium Head Blight is a disease of cereal crops that causes severe yield losses and mycotoxin contamination of grain. The main causal pathogen, Fusarium graminearum, produces the trichothecene toxins deoxynivalenol or nivalenol as virulence factors. Nivalenol-producing isolates are most prevalent in Asia but co-exist with deoxynivalenol producers in lower frequency in North America and Europe. Previous studies identified a barley UDP-glucosyltransferase, HvUGT13248, that efficiently detoxifies deoxynivalenol, and when expressed in transgenic wheat results in high levels of type II resistance against deoxynivalenol-producing F. graminearum. Here we show that HvUGT13248 is also capable of converting nivalenol into the non-toxic nivalenol-3-O-ß-d-glucoside. We describe the enzymatic preparation of a nivalenol-glucoside standard and its use in development of an analytical method to detect the nivalenol-glucoside conjugate. Recombinant Escherichia coli expressing HvUGT13248 glycosylates nivalenol more efficiently than deoxynivalenol. Overexpression in yeast, Arabidopsis thaliana, and wheat leads to increased nivalenol resistance. Increased ability to convert nivalenol to nivalenol-glucoside was observed in transgenic wheat, which also exhibits type II resistance to a nivalenol-producing F. graminearum strain. Our results demonstrate the HvUGT13248 can act to detoxify deoxynivalenol and nivalenol and provide resistance to deoxynivalenol- and nivalenol-producing Fusarium.

In vivo contribution of deoxynivalenol-3-ß-D-glucoside to deoxynivalenol exposure in broiler chickens and pigs: oral bioavailability, hydrolysis and toxicokinetics.

Crossover animal trials were performed with intravenous and oral administration of deoxynivalenol-3-ß-D-glucoside (DON3G) and deoxynivalenol (DON) to broiler chickens and pigs. Systemic plasma concentrations of DON, DON3G and de-epoxy-DON were quantified using liquid chromatography-tandem mass spectrometry. Liquid chromatography coupled to high-resolution mass spectrometry was used to unravel phase II metabolism of DON. Additionally for pigs, portal plasma was analysed to study presystemic hydrolysis and metabolism. Data were processed via tailor-made compartmental toxicokinetic models. The results in broiler chickens indicate that DON3G is not hydrolysed to DON in vivo. Furthermore, the absolute oral bioavailability of DON3G in broiler chickens was low (3.79 ± 2.68 %) and comparable to that of DON (5.56 ± 2.05 %). After PO DON3G administration to pigs, only DON was detected in plasma, indicating a complete presystemic hydrolysis of the absorbed fraction of DON3G. However, the absorbed fraction of DON3G, recovered as DON, was approximately 5 times lower than after PO DON administration, 16.1 ± 5.4 compared with 81.3 ± 17.4 %. Analysis of phase II metabolites revealed that biotransformation of DON and DON3G in pigs mainly consists of glucuronidation, whereas in chickens predominantly conjugation with sulphate occurred. The extent of phase II metabolism is notably higher for chickens than for pigs, which might explain the differences in sensitivity of these species to DON. Although in vitro studies demonstrate a decreased toxicity of DON3G compared with DON, the species-dependent toxicokinetic data and in vivo hydrolysis to DON illustrate the toxicological relevance and consequently the need for further research to establish a tolerable daily intake.

A Versatile Family 3 Glycoside Hydrolase from Bifidobacterium adolescentis Hydrolyzes ß-Glucosides of the Fusarium Mycotoxins Deoxynivalenol, Nivalenol, and HT-2 Toxin in Cereal Matrices.

Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-ß-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of ß-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial ß-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 ß-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-ß-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A Km of 5.4 mM and a Vmax of 16 µmol min(-1) mg(-1) were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-ß-glucosides in cereal samples.

Critical evaluation of indirect methods for the determination of deoxynivalenol and its conjugated forms in cereals.

A critical assessment of three previously published indirect methods based on acidic hydrolysis using superacids for the determination of "free" and "total" deoxynivalenol (DON) was carried out. The modified mycotoxins DON-3-glucoside (D3G), 3-acetyl-DON (3ADON), and 15-acetyl-DON (15ADON) were chosen as model analytes. The initial experiments focused on the stability/degradation of DON under hydrolytic conditions and the ability to release DON from the modified forms. Acidic conditions that were capable of cleaving D3G, 3ADON, and 15ADON to DON were not found, raising doubts over the efficacy of previously published indirect methods for total DON determination. Validation of these indirect methods for wheat, maize, and barley using UHPLC-MS/MS was performed in order to test the accuracy of the generated results. Validation data for DON, D3G, 3ADON, and 15ADON in nonhydrolyzed and hydrolyzed matrices were obtained. Under the tested conditions, DON was not released from D3G, 3ADON, or 15ADON after hydrolysis and thus none of the published methods were able to cleave the modified forms of DON. In addition to acids, alkaline hydrolysis with KOH for an extended time and at elevated temperatures was also tested. 3ADON and 15ADON were cleaved under the alkaline pH caused by the addition of KOH or aqueous K2CO3 to "neutralize" the acidic sample extracts in the published studies. The published additional DON increase after hydrolysis may have been caused by huge differences in matrix effects and the recovery of DON in nonhydrolyzed and hydrolyzed matrices as well as by the alkaline cleavage of 3ADON or 15ADON after the neutralization of hydrolyzed extracts.

Simultaneous determination of major type A and B trichothecenes, zearalenone and certain modified metabolites in Finnish cereal grains with a novel liquid chromatography-tandem mass spectrometric method.

A reliable and sensitive liquid chromatography-tandem mass spectrometric method was developed for the simultaneous quantitative determination in cereals of the Fusarium mycotoxins HT-2 toxin, T-2 toxin, deoxynivalenol, nivalenol and zearalenone, as well as the modified metabolites 3-acetyl-deoxynivalenol, α-zearalenol, ß-zearalenol, deoxynivalenol-3-glucoside, HT-2-3-glucoside, nivalenol-3-glucoside, zearalenone-14-glucoside, zearalenone-14-sulphate, zearalenone-16-glucoside, α-zearalenol-14-glucoside and ß-zearalenol-14-glucoside. The 'dilute and shoot' approach was used for sample preparation after extraction with acetonitrile:water:acetic acid (79:20:1, v/v/v). Separation was carried out using reversed-phase liquid chromatography, and detection was performed using tandem mass spectrometry in the selected reaction monitoring mode. The method was in-house validated according to performance characteristics, established in Commission Regulation EC No 401/2006 and Commission Decision EC No 657/2002, prior to its application in a nationwide survey for the analysis of barley, oat and wheat samples (n = 95) harvested in Finland during 2013. Deoxynivalenol and its glucosylated form were the most abundant of the analytes, being detected in 93 and 81 % of the samples, respectively. Concentrations of deoxynivalenol were unusually high in 2013, especially in oats, with some cases exceeding the maximum legislative limits for unprocessed oats placed on the market for first-stage processing. All modified mycotoxins analysed were detected, and the natural occurrence of some of these compounds (e.g. zearalenone-16-glucoside and nivalenol-3-glucoside) in barley, oats and/or wheat was documented for the first time.

Tracing the metabolism of HT-2 toxin and T-2 toxin in barley by isotope-assisted untargeted screening and quantitative LC-HRMS analysis.

An extensive study of the metabolism of the type A trichothecene mycotoxins HT-2 toxin and T-2 toxin in barley using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) is reported. A recently developed untargeted approach based on stable isotopic labelling, LC-Orbitrap-MS analysis with fast polarity switching and data processing by MetExtract software was combined with targeted LC-Q-TOF-MS(/MS) analysis for metabolite structure elucidation and quantification. In total, 9 HT-2 toxin and 13 T-2 toxin metabolites plus tentative isomers were detected, which were successfully annotated by calculation of elemental formulas and further LC-HRMS/MS measurements as well as partly identified with authentic standards. As a result, glucosylated forms of the toxins, malonylglucosides, and acetyl and feruloyl conjugates were elucidated. Additionally, time courses of metabolite formation and mass balances were established. For absolute quantification of those compounds for which standards were available, the method was validated by determining apparent recovery, signal suppression, or enhancement and extraction recovery. Most importantly, T-2 toxin was rapidly metabolised to HT-2 toxin and for both parent toxins HT-2 toxin-3-O-ß-glucoside was identified (confirmed by authentic standard) as the main metabolite, which reached its maximum already 1 day after toxin treatment. Graphical Abstract Isotope-assisted untargeted screening of HT-2 toxin and T-2 toxin metabolites in barley.

Mycotoxin contamination in moldy slices of bread is mostly limited to the immediate vicinity of the visible infestation.

Bread is an important staple food that is susceptible to spoilage, making it one of the most wasted foods. To determine the safety of partially moldy bread, five types of bread were inoculated with common mold species. After incubation, the metabolite profile was determined in and under the inoculation spot, as well as at a lateral distance of 3 cm from the moldy spot. The result showed that the metabolites were exclusively concentrated in the inoculation area and directly below the inoculation area. The only exception was citrinin, a mycotoxin produced by Penicillia such as Penicillium citrinum, which was detected in almost all tested bread areas when inoculated with the corresponding strains. The results of our study suggest that the removal of moldy parts may be a solution to reduce food waste if the remaining bread is to be used, for example for insect farming to produce animal feed.

Extention and interlaboratory comparison of an LC-MS/MS multi-class method for the determination of 15 different classes of veterinary drug residues in milk and poultry feed.

An HPLC-MS/MS multi-class method for quantitation of 15 different classes of veterinary drug residues (>140 analytes) in milk and poultry feed was developed and validated. Accuracy criteria for routine laboratories were met for the majority of analytes, > 83 % in milk and between 50 and 60 % in chicken feed, with an apparent recovery of 60-140 %. Extraction efficiency criteria were met for >95 % of the analytes for milk and > 80 % for chicken feed. Intermediate precision meets the SANTE criterion of RSD < 20 % for 80-90 % of the analytes in both matrices. For all analytes with an existing MRL in milk, the LOQ was below the related MRL. Twenty-nine samples of commercial milk and chicken feed were analyzed within the interlaboratory comparison. No residues of veterinary drugs were found in the milk samples. However, the feed samples exhibited high levels of nicarbazin, salinomycin, and decoquinate.

Introduction to This Special Issue of Toxins: Reduction and Control of Mycotoxins along Entire Food and Feed Chain.

Contamination of food and feed by mycotoxins is considered a significant issue in food and feed safety worldwide [...].

An Interlaboratory Comparison Study of Regulated and Emerging Mycotoxins Using Liquid Chromatography Mass Spectrometry: Challenges and Future Directions of Routine Multi-Mycotoxin Analysis including Emerging Mycotoxins.

The present interlaboratory comparison study involved nine laboratories located throughout the world that tested for 24 regulated and non-regulated mycotoxins by applying their in-house LC-MS/MS multi-toxin method to 10 individual lots of 4 matrix commodities, including complex chicken and swine feed, soy and corn gluten. In total, more than 6000 data points were collected and analyzed statistically by calculating a consensus value in combination with a target standard deviation following a modified Horwitz equation. The performance of each participant was evaluated by a z-score assessment with a satisfying range of ±2, leading to an overall success rate of 70% for all tested compounds. Equal performance for both regulated and emerging mycotoxins indicates that participating routine laboratories have successfully expanded their analytical portfolio in view of potentially new regulations. In addition, the study design proved to be fit for the purpose of providing future certified reference materials, which surpass current analyte matrix combinations and exceed the typical scope of the regulatory framework.

Fusarium metabolites in maize from regions of Northern Serbia in 2016-2017.

The main objective of this study was to determine the presence of Fusarium metabolites in maize samples collected from different regions of Northern Serbia (Backa, Banat and Srem) during a period of two years (2016-2017). A total of 458 maize samples were analysed by liquid chromatography-tandem mass spectrometry. A total of 40 metabolites were detected, where 94% of the samples contained at least 5 metabolites. Fumonisins (including B1, B2, B3 and B4), moniliformin and bikaverin were the most frequent (80-98%) Fusarium metabolites in both years. Furthermore, in samples from 2016, fumonisin A1 and A2, deoxynivalenol, deoxynivalenol-3-glucoside, zearalenone, culmorin, 15-hydroxyculmorin, fusapyron, fusaproliferin and aurofusarin were detected with frequencies of 58-80%. Levels of certain Fusarium metabolites in 2016 were higher on average due to increased humidity when compared to 2017, which was characterised by warm and dry conditions.

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%.

Fusarium mycotoxins in various barley cultivars and their transfer into malt.

BACKGROUND: Fusarium toxins, secondary metabolites of toxinogenic Fusarium species, are found in a range of cereal grains. In this study the occurrence of the most commonest Fusarium toxins, namely nivalenol (NIV), deoxynivalenol (DON), deoxynivalenol-3-glucoside, fusarenon-X, 3- and 15-acetyldeoxynivalenol, HT-2 and T-2 toxins and zearalenone, in various barley cultivars harvested in 2005-2008 was monitored. The impact of weather, locality, fungicide treatment and barley cultivar (hulless or covered) on contamination was evaluated. The transfer of these mycotoxins into malt was assessed. RESULTS: The most prevalent toxin was DON, which was found in 83% of samples (maximum level 180 µg kg(-1)), while HT-2 was detected in 62% of samples (maximum level 716 µg kg(-1)). Using analysis of covariance, weather was found to be the key factor in all years (P < 0.001). A relationship between cultivar and contamination was confirmed only for HT-2 (P < 0.001) and T-2 (P = 0.037), with higher levels of these toxins being observed in hulless cultivars. With the exception of NIV (P = 0.008), no significant relationship was found between fungicide treatment and contamination. No distinct trend regarding DON levels in malt was found, with both decreases and increases occurring. CONCLUSION: The results show an inter-annual variation in mycotoxin occurrence in barley cultivars as well as differences in contamination of malt produced from fungicide-treated and untreated barley.

Realizing the simultaneous liquid chromatography-tandem mass spectrometry based quantification of >1200 biotoxins, pesticides and veterinary drugs in complex feed.

The first quantitative multiclass approach enabling the accurate quantification of >1200 biotoxins, pesticides and veterinary drugs in complex feed using liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed. Optimization of HPLC/UHPLC (chromatographic column, flow rate and injection volume) and MS/MS conditions (dwell time and cycle time) were carried out in order to allow the combination of five major substance classes and the high number of target analytes with different physico-chemical properties. Cycle times and retention windows were carefully optimized and ensured appropriate dwell times reducing the overall measurement error. Validation was carried out in two compound feed matrices according to the EU SANTE validation guideline. Apparent recoveries matching the acceptable range of 60-140% accounted 60% and 79% for all analytes in cattle and chicken feed, respectively. High extraction efficiencies were obtained for all analyte/matrix combinations and revealed matrix effects as the main source for deviation of the targeted performance criteria. Concerning the methods repeatability 99% of all analytes in chicken and 96% in cattle feed complied with the acceptable RSD ≤ 20% criterion. Limits of quantification were between 1-10 µg/kg for the vast majority of compounds. Finally, the methods applicability was tested in >130 real compound feed samples and provides first insights into co-exposure of agro-contaminants in animal feed.

Evaluation of Matrix Effects and Extraction Efficiencies of LC-MS/MS Methods as the Essential Part for Proper Validation of Multiclass Contaminants in Complex Feed.

This work provides a proposal for proper determination of matrix effects and extraction efficiencies as an integral part of full validation of liquid chromatography coupled to tandem mass spectrometry-based multiclass methods for complex feedstuff. Analytical performance data have been determined for 100 selected analytes in three compound feed matrices and twelve single feed ingredients using seven individual samples per matrix type. Apparent recoveries ranged from 60-140% for 52-89% of all compounds in single feed materials and 51-72% in complex compound feed. Regarding extraction efficiencies, 84-97% of all analytes ranged within 70-120% in all tested feed materials, implying that signal suppression due to matrix effects is the main source for the deviation from 100% of the expected target deriving from external calibration. However, the comparison between compound feed and single feed materials shows great variances regarding the apparent recoveries and matrix effects. Therefore, model compound feed formulas for cattle, pig, and chicken were prepared in-house in order to circumvent the issue of the lack of a true blank sample material and to simulate compositional uncertainties. The results of this work highlight that compound feed modeling enables a more realistic estimation of the method performance and therefore should be implemented in future validation guidelines.

Mycotoxins in maize harvested in Serbia in the period 2012-2015. Part 2: Non-regulated mycotoxins and other fungal metabolites.

The main objective of this study was to screen, for the first time, the natural occurrence of non-regulated fungal metabolites in 204 maize samples harvested in Serbia in maize growing seasons with extreme drought (2012), extreme precipitation and flood (2014) and moderate drought conditions (2013 and 2015). In total, 109 non-regulated fungal metabolites were detected in examined samples, whereby each sample was contaminated between 13 and 55 non-regulated fungal metabolites. Moniliformin and beauvericin occurred in all samples collected from each year. In samples from year 2012, oxaline, questiomycin A, cyclo (l-Pro-l-Val), cyclo (l-Pro-l-Tyr), bikaverin, kojic acid and 3-nitropropionic acid were the most predominant (98.0-100%). All samples from 2014 were contaminated with 7-hydroxypestalotin, 15-hydroxyculmorin, culmorin, butenolid and aurofusarin. Bikaverin and oxaline were quantified in 100% samples from 2013 and 2015, while 3-nitropropionic acid additionally occurred in 100% samples from 2015.

Mycotoxins in maize harvested in Republic of Serbia in the period 2012-2015. Part 1: Regulated mycotoxins and its derivatives.

The main objective of this study was to apply a liquid chromatography-tandem mass spectrometric method to investigate the presence of 20 mycotoxins in 204 maize samples harvested in Northern Serbia in the period 2012-2015, including seasons with extreme drought (2012), hot and dry conditions (2013 and 2015) and extreme precipitation (2014). Between 2 and 20 mycotoxins contaminated examined samples. In samples collected from each year, all of six examined fumonisins were detected with very high prevalence (from 76% to 100%). Aflatoxin B1 was detected in 94% and 90% maize samples from 2012 and 2015, respectively. In samples from year 2014, deoxynivalenol, zearalenone and its derivatives were detected in 100% of samples. Furthermore, ochratoxin A (25%) was the most predominant in samples from 2012. The obtained results indicate that changes in weather conditions, recorded in the period of four years, had significant influence on the occurrence of examined mycotoxins in maize.