Influence of regional and yearly weather patterns on multi-mycotoxin occurrence in Austrian wheat: a liquid chromatographic-tandem mass spectrometric and multivariate statistics approach.

BACKGROUND: Mycotoxin surveys play an essential role in our food safety system. The obtained occurrence data form the basis for the assessment of the exposure of humans and animals to these toxic fungal secondary metabolites. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become the gold standard for mycotoxin determination because it enables selective and sensitive multi-toxin analysis. Simultaneous determination of several hundreds of secondary fungal metabolites is feasible using this technique. In this study, we combined a targeted dilute-and-shoot LC-MS/MS-based multi-analyte approach with multivariate statistics for the analysis of Austrian wheat from two different years and different geographical origins. RESULTS: We quantified 47 secondary fungal metabolites, including regulated emerging and masked mycotoxins. The resulting multi-mycotoxin occurrence data were further analyzed using both multivariate and univariate statistics. Principal component analysis (PCA) and analysis of variance (ANOVA) simultaneous component analysis (ASCA) were employed to identify regional and yearly trends within the dataset and to quantify the variance in metabolite occurrence attributed to the different effects. In addition, secondary fungal metabolites significantly impacted by these factors were selected via ANOVA. Of the 47 secondary metabolites identified, 39 were affected by the year, region or a combined effect. Moreover, our findings show that 43 of the secondary fungal metabolites were significantly influenced by the weather conditions. CONCLUSION: The results presented in this study underline the added value of combining targeted LC-MS/MS with multivariate statistics for monitoring a broad spectrum of secondary fungal metabolites in food crops. Through multivariate statistics, trends associated with the year or region can be readily studied. The approach presented could pave the way for a better understanding of the impact of climate change on plant pathogenic fungi and its implications for food safety. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The potential and applicability of infrared spectroscopic methods for the rapid screening and routine analysis of mycotoxins in food crops.

Infrared (IR) spectroscopy is increasingly being used to analyze food crops for quality and safety purposes in a rapid, nondestructive, and eco-friendly manner. The lack of sensitivity and the overlapping absorption characteristics of major sample matrix components, however, often prevent the direct determination of food contaminants at trace levels. By measuring fungal-induced matrix changes with near IR and mid IR spectroscopy as well as hyperspectral imaging, the indirect determination of mycotoxins in food crops has been realized. Recent studies underline that such IR spectroscopic platforms have great potential for the rapid analysis of mycotoxins along the food and feed supply chain. However, there are no published reports on the validation of IR methods according to official regulations, and those publications that demonstrate their applicability in a routine analytical set-up are scarce. Therefore, the purpose of this review is to discuss the current state-of-the-art and the potential of IR spectroscopic methods for the rapid determination of mycotoxins in food crops. The study critically reflects on the applicability and limitations of IR spectroscopy in routine analysis and provides guidance to non-spectroscopists from the food and feed sector considering implementation of IR spectroscopy for rapid mycotoxin screening. Finally, an outlook on trends, possible fields of applications, and different ways of implementation in the food and feed safety area are discussed.

3D Printing for Low-Cost and Versatile Attenuated Total Reflection Infrared Spectroscopy.

In this work, we present a fully 3D-printed module for attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy ready for use in commercial FTIR spectrometers. The developed setup stands out in terms of robustness and ease of sample application. Rapid prototyping paired with theoretical considerations were employed to design and fabricate the module. Cost-efficient commercial available silicon and germanium ATR crystals prepared from double-sided polished wafers were mounted in the setup. While low-noise levels and stability bear comparison with commercial systems, the multibounce ATR crystal's long interaction pathlengths as well as their interchangeability turns the presented ATR module into an even more sophisticated tool. The versatility of the proposed setup is demonstrated for various spectroscopic challenges: Curing of a cyanoacrylate and a two-component epoxy based adhesive was monitored by tracking polymerization processes at room and high temperatures. To emphasize potential applications of the disposable ATR module in life science studies exploring potential biohazardous samples, mid-IR spectra of Escherichia coli and bovine serum albumin were recorded. The total printing time of the ATR module is 10.5 h, enabling overnight fabrication at a total cost ranging from 150 to 613 €, making the high versatility of ATR spectroscopy accessible to a broader audience. This proves the potential of 3D printing to generate optical instruments tailored to the needs of individual analytical problems.

An Acoustic Trap for Bead Injection Attenuated Total Reflection Infrared Spectroscopy.

In this work, we introduce a system combining an acoustic trap for bead injection with attenuated total reflection (ATR) infrared (IR) spectroscopy. By mounting an acoustofluidic cell hosting an ultrasound source on top of a custom-built ATR fixture we were able to trap beads labeled with the enzyme alkaline phosphatase without requiring any mechanical retention elements. Sequential injection analysis was employed for reproducible sample handling and bead injection into the acoustic trap. To showcase potential applications of the presented setup for kinetic studies, we monitored the conversion of p-nitrophenylphosphate into p-nitrophenol and phosphate via beads carrying the immobilized enzyme using ATR-IR spectroscopy. Retaining the labeled beads via ultrasound particle manipulation resulted in excellent experimental reproducibility (relative standard deviation, 3.91%). It was demonstrated that trapped beads remained stably restrained with up to eight cell volumes of liquid passing through the acoustofluidic cell. Beads could be discarded in a straightforward manner by switching off the ultrasound, in contrast to systems containing mechanical retention elements, which require backflushing. Multiple experiments were performed by employing different substrate concentrations with the same batch of trapped beads as well as varying the amount of enzyme present in the cell, enabling enzyme kinetic studies and emphasizing the application of the proposed setup in studies where enzymatic reuse is desired. This proves the potential of the acoustic trap combined with ATR-IR spectroscopy to monitor the activity of immobilized enzymes and its ability to perform complex bead-based assays.

Beyond Fourier Transform Infrared Spectroscopy: External Cavity Quantum Cascade Laser-Based Mid-infrared Transmission Spectroscopy of Proteins in the Amide I and Amide II Region.

In this work, we present a setup for mid-IR measurements of the protein amide I and amide II bands in aqueous solution. Employing a latest generation external cavity-quantum cascade laser (EC-QCL) at room temperature in pulsed operation mode allowed implementing a high optical path length of 31 µm that ensures robust sample handling. By application of a data processing routine, which removes occasionally deviating EC-QCL scans, the noise level could be lowered by a factor of 4. The thereby accomplished signal-to-noise ratio is better by a factor of approximately 2 compared to research-grade Fourier transform infrared (FT-IR) spectrometers at equal acquisition times. Employing this setup, characteristic spectral features of three representative proteins with different secondary structures could be measured at concentrations as low as 1 mg mL-1. Mathematical evaluation of the spectral overlap confirms excellent agreement of the quantum cascade laser infrared spectroscropy (QCL-IR) transmission measurements with protein spectra acquired by FT-IR spectroscopy. The presented setup combines performance surpassing FT-IR spectroscopy with large applicable optical paths and coverage of the relevant spectral range for protein analysis. This holds high potential for future EC-QCL-based protein studies, including the investigation of dynamic secondary structure changes and chemometrics-based protein quantification in complex matrices.

WaterSpy: A High Sensitivity, Portable Photonic Device for Pervasive Water Quality Analysis.

In this paper, we present WaterSpy, a project developing an innovative, compact, cost-effective photonic device for pervasive water quality sensing, operating in the mid-IR spectral range. The approach combines the use of advanced Quantum Cascade Lasers (QCLs) employing the Vernier effect, used as light source, with novel, fibre-coupled, fast and sensitive Higher Operation Temperature (HOT) photodetectors, used as sensors. These will be complemented by optimised laser driving and detector electronics, laser modulation and signal conditioning technologies. The paper presents the WaterSpy concept, the requirements elicited, the preliminary architecture design of the device, the use cases in which it will be validated, while highlighting the innovative technologies that contribute to the advancement of the current state of the art.

Advancing Mycotoxin Detection in Food and Feed: Novel Insights from Surface-Enhanced Raman Spectroscopy (SERS).

The implementation of low-cost and rapid technologies for the on-site detection of mycotoxin-contaminated crops is a promising solution to address the growing concerns of the agri-food industry. Recently, there have been significant developments in surface-enhanced Raman spectroscopy (SERS) for the direct detection of mycotoxins in food and feed. This review provides an overview of the most recent advancements in the utilization of SERS through the successful fabrication of novel nanostructured materials. Various bottom-up and top-down approaches have demonstrated their potential in improving sensitivity, while many applications exploit the immobilization of recognition elements and molecular imprinted polymers (MIPs) to enhance specificity and reproducibility in complex matrices. Therefore, the design and fabrication of nanomaterials is of utmost importance and are presented herein. This paper uncovers that limited studies establish detection limits or conduct validation using naturally contaminated samples. One decade on, SERS is still lacking significant progress and there is a disconnect between the technology, the European regulatory limits, and the intended end-user. Ongoing challenges and potential solutions are discussed including nanofabrication, molecular binders, and data analytics. Recommendations to assay design, portability, and substrate stability are made to help improve the potential and feasibility of SERS for future on-site agri-food applications.

Interconnectable 3D-printed sample processing modules for portable mycotoxin screening of intact wheat.

BACKGROUND: The increasing demand for food and feed products is stretching the capacity of the food value chain to its limits. A key step for ensuring food safety is checking for mycotoxin contamination of wheat. However, this analysis is typically performed by rather complex and expensive chromatographic methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). These costly methods require extensive sample preparation that is not easily carried out at different points along the food supply chain. To overcome such challenges in sample processing, an inexpensive and portable sample preparation device was needed, that required low skill, for rapid sample-to-result mycotoxin screening. RESULTS: We describe 3D-printed and interconnectable modules for simple, integrated and on-site sample preparation, including grinding of wheat kernels, and solvent-based extraction. We characterized these 3D-printed modules for mycotoxin screening and benchmarked them against a laboratory mill using commercial lateral flow device(s) (LFD) and in-house validated LC-MS/MS analysis. Different integrated sieve configurations were compared based on grinding efficiency, and we selected a sieve size of 2 mm allowing grinding of 10 g of wheat within 5 min. Moreover, 10 first time-users were able to operate the grinder module with minimal instructions. Screening for deoxynivalenol (DON) in naturally contaminated samples at the regulatory/legal limit (1.25 mg kg-1) was demonstrated using the developed 3D-printed prototype. The whole process only takes 15 min, from sample preparation to screening result. The results showed a clear correlation (R2 = 0.96) between the LFD and LC-MS/MS. SIGNIFICANCE: Our findings demonstrate the potential of 3D-printed sample handling equipment as a valuable extension of existing analytical procedures, facilitating the on-site implementation of rapid methods for the determination of mycotoxins in grains. The presented prototype is inexpensive with material costs of 2.5€, relies on biodegradable 3D printing filament and can be produced with consumer-grade printers, making the prototype readily available. As a future perspective, the modular character of our developed tool kit will allow for adaptation to other hard food commodities beyond the determination of DON in wheat.

Prediction of Deoxynivalenol Contamination in Wheat via Infrared Attenuated Total Reflection Spectroscopy and Multivariate Data Analysis.

The climate crisis further exacerbates the challenges for food production. For instance, the increasingly unpredictable growth of fungal species in the field can lead to an unprecedented high prevalence of several mycotoxins, including the most important toxic secondary metabolite produced by Fusarium spp., i.e., deoxynivalenol (DON). The presence of DON in crops may cause health problems in the population and livestock. Hence, there is a demand for advanced strategies facilitating the detection of DON contamination in cereal-based products. To address this need, we introduce infrared attenuated total reflection (IR-ATR) spectroscopy combined with advanced data modeling routines and optimized sample preparation protocols. In this study, we address the limited exploration of wheat commodities to date via IR-ATR spectroscopy. The focus of this study was optimizing the extraction protocol for wheat by testing various solvents aligned with a greener and more sustainable analytical approach. The employed chemometric method, i.e., sparse partial least-squares discriminant analysis, not only facilitated establishing robust classification models capable of discriminating between high vs low DON-contaminated samples adhering to the EU regulatory limit of 1250 µg/kg but also provided valuable insights into the relevant parameters shaping these models.

A Portable Infrared Attenuated Total Reflection Spectrometer for Food Analysis.

The analytical performance of a compact infrared attenuated total reflection spectrometer using a pyroelectric detector array has been evaluated and compared to a conventional laboratory Fourier transform infrared system for applications in food analysis. Analytical characteristics including sensitivity, repeatability, linearity of the calibration functions, signal-to-noise ratio, and spectral resolution have been derived for both approaches. Representative analytes of relevance in food industries (i.e., organic solvents, fatty acids, and mycotoxins) have been used for the assessment of the performance of the device and to discuss the potential of this technology in food and feed analysis.

An Automatic Immunoaffinity Pretreatment of Deoxynivalenol Coupled with UPLC-UV Analysis.

An immunoaffinity magnetic beads (IMBs) based automatic pretreatment method was developed for the quantitative analysis of deoxynivalenol (DON) by ultra-performance liquid chromatography and ultraviolet detector (UPLC-UV). First, N-hydroxysuccinimide-terminated magnetic beads (NHS-MBs) with good magnetic responsivity and dispersibility were synthesized and characterized by optical microscopy, scanning electron microscopy (SEM), and laser diffraction-based particle size analyzer. Then, the amino groups of anti-DON monoclonal antibody (mAb) and the NHS groups of NHS-MBs were linked by covalent bonds to prepare IMB, without any activation reagent. The essential factors affecting the binding and elution of DON were meticulously tuned. Under optimal conditions, DON could be extracted from a real sample and eluted from IMB by water, enabling environmentally friendly and green analysis. Hence, there was no need for dilution or evaporation prior to UPLC-UV analysis. DON in 20 samples could be purified and concentrated within 30 min by the mycotoxin automated purification instrument (MAPI), allowing for automated, green, high-throughput and simple clean-up. Recoveries at four distinct spiking levels in corn and wheat ranged from 92.0% to 109.5% with good relative standard deviations (RSD, 2.1-7.0%). Comparing the test results of IAC and IMB in commercial samples demonstrated the reliability and superiority of IMB for quantitatively analyzing massive samples.

Optimizing extraction solvents for deoxynivalenol analysis in maize via infrared attenuated total reflection spectroscopy and chemometric methods.

Farmers, cereal suppliers and processors demand rapid techniques for the assessment of mould-associated contamination. Deoxynivalenol (DON) is among the most important Fusarium toxins and related to human and animal diseases besides causing significant economic losses. Routine analytical techniques for the analysis of DON are either based on chromatographic or immunoanalytical techniques, which are time-consuming and frequently rely on hazardous consumables. The present study evaluates the feasibility of infrared attenuated total reflection spectroscopy (IR-ATR) for the analysis of maize extracts via different solvents optimized for the determination of DON contamination along the regulatory requirements by the European Union (EU) for unprocessed maize (1750 µg kg-1). Reference analysis was done by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The studied maize samples were either naturally infected or had been artificially inoculated in the field with Fusarium graminearum, Fusarium culmorum or Fusarium verticillioides. Principal component analysis demonstrated that water and methanol-water (70 : 30% v) were optimum solvents for differentiating DON contamination levels. Supervised partial least squares discriminant analysis resulted in excellent classification accuracies of 86.7% and 90.8% for water and methanol-water extracts, respectively. The IR spectra of samples with fungal infection and high DON contamination had distinct spectral features, which could be related to carbohydrates, proteins and lipid content within the investigated extracts.

Polarimetric Balanced Detection: Background-Free Mid-IR Evanescent Field Laser Spectroscopy for Low-Noise, Long-term Stable Chemical Sensing.

In this work, we introduce polarimetric balanced detection as a new attenuated total reflection (ATR) infrared (IR) sensing scheme, leveraging unequal effective thicknesses achieved with laser light of different polarizations. We combined a monolithic widely tunable Vernier quantum cascade laser (QCL-XT) and a multibounce ATR IR spectroscopy setup for analysis of liquids in a process analytical setting. Polarimetric balanced detection enables simultaneous recording of background and sample spectra, significantly reducing long-term drifts. The root-mean-square noise could be improved by a factor of 10 in a long-term experiment, compared to conventional absorbance measurements obtained via the single-ended optical channel. The sensing performance of the device was further evaluated by on-site measurements of ethanol in water, leading to an improved limit of detection (LOD) achieved with polarimetric balanced detection. Sequential injection analysis was employed for automated injection of samples into a custom-built ATR flow cell mounted above a zinc sulfide multibounce ATR element. The QCL-XT posed to be suitable for mid-IR-based sensing in liquids due to its wide tunability. Polarimetric balanced detection proved to enhance the robustness and long-term stability of the sensing device, along with improving the LOD by a factor of 5. This demonstrates the potential for new polarimetric QCL-based ATR mid-IR sensing schemes for in-field measurements or process monitoring usually prone to a multitude of interferences.

A thermoelectrically stabilized aluminium acoustic trap combined with attenuated total reflection infrared spectroscopy for detection of Escherichia coli in water.

Acoustic trapping is a non-contact particle manipulation method that holds great potential for performing automated assays. We demonstrate an aluminium acoustic trap in combination with attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) for detection of E. coli in water. The thermal conductivity of aluminium was exploited to thermo-electrically heat and hold the acoustic trap at the desired assay temperature of 37 °C. Systematic characterisation and optimisation of the acoustic trap allowed high flow rates while maintaining high acoustic trapping performance. The ATR element serves not only as a reflector for ultrasound standing wave generation but also as a sensing interface. The enzyme conversion induced by alkaline phosphatase-labelled bacteria was directly monitored in the acoustic trap using ATR-FTIR spectroscopy. Sequential injection analysis allowed automated liquid handling, including non-contact bacteria retention, washing and enzyme-substrate exchange within the acoustic trap. The presented method was able to detect E. coli concentrations as low as 1.95 × 106 bacteria per mL in 197 min. The demonstrated ultrasound assisted assay paves the way to fully automated bacteria detection devices based on acoustic trapping combined with ATR-FTIR spectroscopy.