Wageningen Academic World Mycotoxin Journal, 2021; 14 (1): 3-26 Publishers

Developments in mycotoxin analysis: an update for 2019-2020

S.A. Tittlemier1*, J. Brunkhorst2, B. Cramer3, M.C. DeRosa4, V.M.T. Lattanzio5, R. Malone2, C. Maragos6, M. Stranska7 and M.W. Sumarah8

1Canadian Grain Commission, Grain Research Laboratory, 1404-303 Main St, Winnipeg, MB, R3C 3G8, Canada; 2Trilogy Analytical Laboratory, 870 Vossbrink Dr, Washington, MO 63090, USA; 3University of Münster, Institute of Food Chemistry, Corrensstr. 45, 48149 Münster, Germany; 4Department of Chemistry, Carleton University, Ottawa, ON, K1S 5B6, Canada; 5National Research Council of Italy, Institute of Sciences of Food Production, via Amendola 122/O, 70126 Bari, Italy; 6United States Department of Agriculture, ARS National Center for Agricultural Utilization Research, Peoria, IL 61604, USA; 7Department of Food Analysis and Nutrition, Faculty of Food and Biochemical Technology, University of Chemistry and Technology, Technicka 5, Prague, 166 28, Czech Republic; 8Agriculture and Agri-Food Canada, London Research and Development Centre, 1391 Sandford Street, London, ON, N5V 4T3, Canada; [email protected]

Received: 18 November 2020 / Accepted: 16 December 2020 © 2021 Wageningen Academic Publishers OPEN ACCESS REVIEW ARTICLE Abstract

This review summarises developments on the analysis of various matrices for mycotoxins published in the period from mid-2019 to mid-2020. Notable developments in all aspects of mycotoxin analysis, from sampling and quality assurance/ quality control of analytical results, to the various detection and quantitation technologies ranging from single mycotoxin biosensors to comprehensive instrumental methods are presented and discussed. Aside from sampling and quality control, discussion of this past year’s developments is organised by detection and quantitation technology and covers chromatography with targeted or non-targeted high resolution mass spectrometry, tandem mass spectrometry, detection other than mass spectrometry, biosensors, as well as assays that use alternatives to antibodies. This critical review aims to briefly present the most important recent developments and trends in mycotoxin determination as well as to address limitations of the presented methodologies.

Keywords: sampling, multi-mycotoxin analysis, quality control, multiplex, biosensor, chromatography, mass spectrometry, aptamer, ELISA, molecularly imprinted polymer, high resolution mass spectrometry

1. Introduction and quantitation technologies available to the analyst. Our hope is that this reorganisation allows readers to more easily This article is the latest instalment in a series of annual access new information relevant to the analytical tools that reviews highlighting analytical method developments for they have at hand in order to improve their mycotoxin mycotoxin determination, continuing from the previous analyses. review covering the mid-2018 to mid-2019 period (Tittlemier et al., 2020a). As with the previous reviews Specific topics included in this review are sampling in this series, our primary purpose is to raise awareness (Section 2), quality control of mycotoxin analyses (Section of the developments and advances in analytical methods 3), chromatography with tandem mass spectrometry for mycotoxins published between mid-2019 to mid-2020. (MS/MS; Section 4), chromatography with targeted high resolution mass spectrometry (HRMS; Section 5), Globally, this past year has been full of changes. Our annual chromatography with non-targeted HRMS (Section 6), review has also changed. We have reorganised the review chromatography with non-mass spectrometric (MS) into (mostly) new sections that try to cover all aspects of detection (Section 7), multiplex biosensors (Section 8), mycotoxin analysis, from sampling and quality assurance/ single mycotoxin or single mycotoxin family biosensors quality control of analytical results, to the various detection (Section 9), and assays using antibody analogues (Section

ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2020.2664 3 S.A. Tittlemier et al.

10). A list of commonly used abbreviations in the article monitoring as a more intensive sampling plan was needed is provided in the Appendix. to minimise the probability of accepting a non-compliant batch to less than 5%. With this reorganisation we have still tried to maintain the annual review as a discussion of the most novel and Fischer et al. (2019) examined the main contributing relevant advances in analytical methodology as selected by factors to variance in the analysis of aflatoxins (AFs) in the experts in the broad field of ‘mycotoxin analysis’. As with truckloads of bagged maize delivering to Kenyan mills. past years, this review is not meant to be an exhaustive list of They also estimated the number of samples that would publications on mycotoxin analytical methods, nor a list of provide sufficient power to their sampling plan to assess incremental improvements of more ‘mature’ methodology. AFs concentration in a truckload of bagged maize. For the Critical comments on the selected methods, their validation experimental design, at each of three different commercial parameters, or unique applications are included to guide mills in Kenya three trucks were selected and 10 bags of readers in assessing the impact of these developments. This maize were each sampled twice from each of the three review should therefore appeal to all readers. trucks. Sampling of maize was performed manually, and the specific trier used to sample grain from bags was described. 2. Sampling Unfortunately, the procedures for bag sampling, bag selection, and sub-sampling of maize were not described Research on sampling and sample processing published in the paper. Concentrations of AFs varied greatly between over the past year covers a wide range of topics ranging bags sampled, and ranged from less than the detection from modelling to evaluate the costs of sampling plans limit to 1,100 μg/kg. The distribution of AFs in samples (Focker et al., 2019), grinding and dividing grain samples taken from truckloads of bagged maize differed from the in preparation for analysis (Tittlemier et al., 2020b), and the distribution observed in bulk maize transported by truck characterisation of mycotoxin (and variance of mycotoxin in Texas, emphasising that mycotoxin distributions in analysis) in fields (Cowger et al., 2020), stored grain (Kerry commodities depend on the handling and history of a et al., 2019), and transported grain (Fischer et al., 2019). commodity lot. Variability of AF concentrations in maize between bags (61% of total variance) and variability of AF Focker et al. (2019) expanded upon their previous work and concentrations in maize within a bag (27%) were the top two used optimisation modelling to assess sampling plans for factors contributing to the variance of AFs concentration monitoring aflatoxin B1 (AFB1) in maize. They evaluated observed in the study, followed by variability due to analysis six different scenarios of AFB1 production and monitoring (6%). The authors stated that the low variance from the along the multi-stage maize supply chain from the Black analysis step does not indicate ‘low risk from testing’ but Sea maize growing region to processors in the Netherlands. that the variance from the analysis step can be mitigated The six scenarios were set with AFB1 production and by good quality control procedures, such as routine use of monitoring at certain points of a simplified maize supply reference materials and control charting. The experimental chain. Production of AFB1 in the field was limited to occur data were also used to estimate that a minimum of 20 at three concentrations (1, 4, and 10 μg/kg) and production bags per truck need to be sampled in order to have a false during shipping (100 μg/kg in 5% of the maize transported). positive rate of 5% and a false negative rate of 20%. The Monitoring points were limited to storage bins used after authors outlined the assumptions used in their work, and harvest, ship compartments for sea transport, barges for noted that the concentrations of AFs were relatively low in domestic transport, and processing plants. The assumptions the year of their study but did not elaborate on how higher of the model were constraining and do not reflect the wide concentrations may affect the factors contributing to total variety of conditions encountered in real life, but they are variance nor affect the minimum number of bags to be thoroughly described by the authors. The model also sampled. Despite this limitation, the study provides good included the need to replace non-compliant batches of insight into how risk from AFs in maize can be managed at maize (i.e. AFB1 concentrations at the end of the supply a commercial mill, and demonstrates that modifications to chain greater than an industry specification of 2.5 μg/kg) the Kenyan sampling recommendation decreases the risk to meet grain volumes required by the processors. The of mischaracterising the AFs content of maize. optimisation model minimised the total cost of monitoring and replacing batches of non-compliant grain. As expected, Tittlemier et al. (2020b) evaluated grinders and dividing model outputs for the six scenarios showed that the optimal equipment used in the preparation of whole oats for number of maize batches to analyse, and the optimal mycotoxin analysis. The analysis of hulls from naturally monitoring point in the supply chain, depended upon the infected whole oats demonstrated that a variety of concentration of AFB1 in maize and whether the AFB1 Fusarium-produced mycotoxins, as well as ochratoxin A was produced in field or during shipping. The scenarios (OTA), are predominantly present in hulls as compared where the AFB1 concentration was closest to 2.5 μg/kg at to groats. The challenge in comminuting whole oats lies the end of the supply chain incurred the greatest costs for with the hulls. The burr mill type grinders evaluated

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generated lower amounts of smaller particles than rotor appropriate scale in order to detect the ‘minority’ species beater type grinders; the remaining larger particles were and their mycotoxins. visually identified as pieces of hull. Therefore a rotor beater type grinder with a 500 μm screen was chosen for 3. Quality control of mycotoxin analyses the preparation of whole oats for mycotoxin analysis. This system provided good balance between maximising the The accuracy of analytical results describing mycotoxin amount of smallest particles produced, and minimising concentrations is essential for comparability of measurements the time needed to comminute samples and clean the between laboratories. Besides the arrangement of the grinder. No practical differences were observed amongst analytical method itself, including extraction/purification and the four methods evaluated for the division of ground whole separation/detection, other key factors influencing results oats. Even though hulls contained the majority of many are the analytical standards (native or isotopically labelled) mycotoxins naturally present in the oats sample, the small and reference materials used. The analytical standards can differences in particle size fraction distributions observed differ between manufacturers and there are variances in in the test portions divided by the four methods were not uncertainties of reference values and purity of standards, expected to impact analytical results. However, the authors even between various batches. Differences between matrix cautioned that the division method could impact results if reference materials lie with the nature of mycotoxins differences in mycotoxin concentrations between particle present, i.e. single- or multi-mycotoxin, and whether or size fractions were greater than those observed in this not these analytes are incurred or fortified. particular study. When considering liquid chromatography mass spectro­ Kerry et al. (2019) re-analysed two existing data sets using metry (LC-MS) as the mainstream technique for analysis additional statistical spatial analysis tools with the aim of of mycotoxins in foods, the majority of problems with locating where the most contaminated grain, or the highest accurate quantification are associated with matrix effects potential of mycotoxin formation during storage of grain, and how to compensate for these effects. The first, most would occur so that action, such as grain removal or drying, common option to compensate matrix effects is use of could be taken to mitigate risks to health. The outcomes external matrix-matched calibration curves. The second, of such research provide information on the distributional and increasingly more popular method to mitigate impacts heterogeneity of mycotoxins in bulk commodities and allow on quantification is the stable isotope dilution technique. analysts to ensure that sampling plans and schemes can Several groups published methods for quantification of adequately account for such heterogeneity. For the first some, mainly regulated, mycotoxins in different matrices dataset of OTA and deoxynivalenol (DON) in a truckload by this approach [e.g. DON, zearalenone (ZEN), HT-2 of bulk wheat first described by Biselli et al. (2008), the re- toxin (HT-2), T-2 toxin (T-2), fumonisin B (FBs), AFs analysis showed spatial structure for OTA, which occurred and OTA in maize, peanut, rice, cereal mixture (Li et al., in tighter clusters than DON, as well as co-location of 2019a), and edible oils (Zhang and Xu, 2019), and DON, OTA and DON. The authors suggested that this may ZEN, HT-2, T-2, AFs and OTA in legumes (Kunz et al., indicate that OTA is developing from points of high DON 2020)]. In general, it can be said that the stable isotope concentration. The second dataset (Rivas Casado et al., dilution approach is very suitable and advantageous in 2010) showed spatial similarities between points of low cases when analysed matrices are chemically complex and fumonisin concentrations clustering at the top of maize non-uniform, and when signals of particular mycotoxins and points of high fumonisin concentrations clustering of interest are impacted by matrix effects. However, the in the moister locations at the base and outer layers of the significant drawback of this approach is the relative cost stored maize. The authors claim the clustering of low and of isotopically labelled mycotoxins, as well as the limited high fumonisin concentrations was due to the production portfolio of these analogues on the market. When matrix of fumonisins in the moister, more oxygen rich locations at effects on two closely eluting mycotoxins vary significantly, the base and outer layers of stored maize, but this research and the isotopically labelled standards are not available for did not monitor concentrations over time. both of them, simple use of closely eluting labelled standard may not be appropriate (Li et al., 2019a). On the other Another recent publication provided some information on hand, in the study of Debevere et al. dealing with LC-MS the spatial variation of mycotoxins within a field. While determination of DON, de-epoxydeoxynivalenol (DOM- the focus of Cowger et al.’s work (2020) was on Fusarium 1), nivalenol (NIV), enniatin B (ENN B), mycophenolic species present in 59 wheat fields in North Carolina, USA, acid (MPA), roquefortine C (ROC), ZEN, zearalanone variation in DON (0.77 to 153 mg/kg) and moniliformin (ZAN), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), (MON; <1 to 39 mg/kg) was reported. This variation in α-zearalanol (α-ZAL), β-zearalanol (β-ZAL) in rumen fluids, mycotoxin concentrations amongst the fields sampled, as use of structurally related isotopically labelled internal 13 well as the variation in Fusarium species present even in standards, in particular C15-DON for quantification of 13 nearby fields, also emphasise that sampling must be at an DOM-1 and NIV in addition to DON, and C18-ZEN for

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quantification of ZAN, α-ZOL, β-ZOL, α-ZAL, β-ZAL in The matrix reference material has been commercialised addition to ZEN, was shown to be advantageous, as they as ‘SRM 1565 Mycotoxins in Corn’ (Phillips et al., have similar physicochemical properties (Debevere et al., 2019). The authors’ thorough workflow, comprising all 2019). While the use of structurally related, as opposed to the important steps including particle size evaluation, 13 stable isotope labelled, internal standards (e.g. C15-DON homogeneity and stability testing, etc., can be further for quantification of DOM-1 and NIV) may mitigate the used for future development of multi-mycotoxin matrix effects of losses during sample extraction and clean up, reference materials in different matrices. or variations in sample volume, matrix effects during LC- MS analysis could still differ. Even though structures are Another study of De Santis et al. (2020) dealing with similar, the internal standard and analyte elute at different preparation of matrix reference material containing retention times and the co-eluting matrix components could incurred OTA in pork-based products was published vary between these different retention times. Therefore, by the Italian National Reference Laboratory for Feed the applicability of structurally related internal standards and Food, in connection with the European Commission must be fully assessed. mandate regarding the standardisation for methods of analysis for mycotoxins in food. Here, the pork meat In the studies mentioned above, comparison of the stable products were bought at the local market (in sufficient isotope dilution method with classical external matrix- amount of approximately 2 kg of each product), chopped matched calibration was also performed, and despite the and ground repeatedly (at least 8 times) in the meat slightly better performance characteristics of stable isotope grinder, and analysed for OTA. The rest of the material dilution, the matrix-matched quantification approach was portioned (10-11 g) and distributed in plastic tubes, showed satisfactory results for all of the mycotoxins tested labelled, sealed and stored at -20 °C. The samples with (Debevere et al., 2019; Li et al., 2019a). Therefore, when appropriate OTA contamination levels were chosen as the a laboratory cannot perform the isotope dilution-based matrix reference materials (ham with low, medium, and quantification, the matrix-matched calibration still can be high OTA at concentrations of 0.77, 2.22 and 12.3 μg/kg, considered as a fully satisfactory approach, at least for less respectively; liver with a medium OTA concentration of complex cereal-based matrices. 2.80 μg/kg; and chopped pork meat with a low OTA level of 0.66 μg/kg). The materials were tested for homogeneity For adequate quality assurance of methods for mycotoxins and stability, and will be further used within an inter- analysis, certified matrix reference materials (CRMs) with laboratory method validation study (De Santis et al., 2020). properly defined and metrologically traceable assigned values and uncertainties are essential, especially for An indispensable aspect in quality control of analytical evaluating extraction efficiency. Earlier research showed methods is the participation in proficiency tests (PTs) and that matrix reference materials with incurred (i.e. naturally interlaboratory studies. For these, the design of the study occurring) mycotoxins are preferred over fortified material, (or PT) itself plays a crucial role in interpreting results and especially when analysing plant-based matrices with low evaluating laboratory proficiency. This topic was nicely moisture content, because the incurred mycotoxins are reviewed by Guo et al. (2019a). The authors referred to incorporated deeper and stronger within the matrix. Use differences in trueness and uncertainty of results from of fortified matrix for recovery determination could be two different interlaboratory comparisons, where either over-optimistic and misleading (Dzuman et al., 2014). the provided, or a laboratory’s own analytical standards Using this knowledge, the aim of educated suppliers were used. When the common standards prepared by of CRMs is to replace the traditional frequently used organisers were obligatorily utilised for quantification, single-analyte CRMs prepared by fortifying matrix there was significantly better agreement of results (Guo et with standards) by the matrix reference material where al., 2019a). The second important message highlighted in mycotoxins are present as naturally incurred analytes. this review is the apparent advantage of using isotopically Along with the benefit of monitoring method trueness, labelled mycotoxins as internal standards for quantification. another advantage of CRMs with incurred analytes is Use of isotope labelled standards resulted in significantly the natural co-occurrence of multiple mycotoxins, which lower reported relative standard deviations (RSDs) and addresses the needs of laboratories aligning with the trend higher recoveries in comparison with results generated towards multi-mycotoxin methods. In response to this by the classical external calibration approach. This fact current trend and its associated demands, the US National is being gradually reflected in the change of algorithms Institute of Standards and Technology and US Food and setting the assigned value for test materials used in PTs Drug Administration developed a CRM containing all and interlaboratory comparisons. Recently, the increasing mycotoxins that are regulated and significantly relevant number of PT organisers used the assigned value generated to health (i.e. AFs, DON, FBs, OTA, ZEN, and HT- by utilising the isotopically labelled mycotoxins as internal 2/T-2) in maize, by mixing different batches of naturally standards as opposed to the consensus value determined contaminated maize to yield the concentrations of interest. from the results submitted by participants (Guo et al., 2019a).

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4. Chromatography with MS/MS A similar approach to that described above compared a ‘dilute and shoot’ procedure to using hydrophilic-lipophilic There were several multi-mycotoxin methods applied balanced copolymer SPE clean up (Oasis PRiME HLB, to various matrices published over the past year. The Waters) for maize and wheat matrices (Scarpino et al., multi-mycotoxin methods vary in the compounds that 2019). Both methods used the same extraction [acetonitrile/ are analysed in each of the methods. The methods are water/acetic acid (79:20:1, v/v/v)], however the ‘dilute and tailored sometimes for the matrices that are being tested, shoot’ method diluted the extract with an equal volume such as pairing mycotoxins with pesticides for the cannabis of acetonitrile/water/acetic acid (20:79:1, v/v/v) in order industry or testing multiple Fusarium mycotoxins and to reduce signal suppression and/or enhancement. It is ionophores for grains. Multi-mycotoxin analysis is being also important to note that acetic acid was added to the performed more routinely with advances in the sensitivity extraction to obtain better recovery for the fumonisins. of high-performance liquid chromatography (HPLC) The diluted extract was then filtered and analysed by LC- coupled with triple quadrupole mass spectrometry, as MS/MS using a calibration curve prepared in solvent. well as new techniques in matrix purification. Several The other approach of this trial used the copolymeric SPE methods discuss the purification process that is necessary cartridge to remove fats and phospholipids that can cause or recommended to receive better recovery, sensitivity, signal suppression and enhancement. The SPE was used and selectivity. Current mycotoxin regulations or advisory in cartridge format, without QuEChERS extraction and levels for food and feed apply to a number of different dispersive SPE steps. Quantitation was performed using individual mycotoxins, emphasising the importance of matrix-matched calibration standards. Mycotoxins analysed multi-mycotoxin methods. for this study were DON, ENN A, ENN A1, ENN B, ENN B1, FB1, FB2, ZEN, 3-ADON, 15-acetyl-deoxynivalenol The simultaneous determination of multiple mycotoxins in (15-ADON), deoxynivalenol-3-glucoside (DON-3G), Korean soybean paste (doenjang) by HPLC-MS/MS with NIV, AFB1, AFB2, AFG1, and AFG2. Both methods used immunoaffinity clean-up addresses the need for purification CRMs, and values obtained were within the specified in complex matrices and screening for more mycotoxins concentrations of the CRMs. Limits of quantitation (Woo et al., 2019). The authors capitalised on the cross (LOQs) for the ‘dilute and shoot’ procedure tended to be reactivity of a commercially-available immunoaffinity higher than the SPE procedure. The signal suppression column (‘Myco 6in1’; Vicam, Milford, MA, USA) to a and enhancement for both matrices were different. Maize number of mycotoxins structurally related to those for had high signal suppression for the aflatoxins, 15-ADON, which the column is marketed towards. The method was and NIV in both methods. However, 3-ADON, DON, and used to analyse for twenty mycotoxins including AFB1, DON-3G only showed suppression when using the SPE aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 method. Both maize and wheat CRMs were also found to (AFG2), aflatoxin M1 (AFM1), OTA, ZEN, ZON, α-ZOL, contain 3-ADON, 15-ADON, and DON-3G. The wheat α-ZAL, β-ZOL, β-ZAL, T-2, HT-2, DON, 3-acetyl- CRM also contained ENN B, ENN B1, and ZEN. Matrix- deoxynivalenol (3-ADON), NIV, fumonisin B1 (FB1), matched calibrations were also used due to the expense of fumonisin B2 (FB2) and fumonisin B3 (FB3). Doenjang is the isotopically labelled standards (which are not available a fermented soybean paste produced in Korea and has a for all the mycotoxins that were analysed in this trial). salt content of 7-23%, high protein content, and contains There are advantages to both methods, however the main brown pigments, which all make it a difficult matrix to advantages to using the added SPE step is reduction of signal analyse. During fermentation, the doenjang is susceptible suppression and enhancement in addition to increased to contamination by mycotoxigenic fungi. Most methods instrument protection from matrix residue. used for extraction, purification, and analysis of doenjang utilise solid phase extraction (SPE), immunoaffinity column Many challenges arise with the separation and purification purification, and C18 or ion-pairing materials, which of analytes from complex foods and feeds in multi- result in poor removal of the salts and other polar matrix mycotoxin methods. Swine, poultry, and dairy feeds were components. Woo et al. (2019) utilised a two-step extraction analysed utilising QuEChERS extraction with an additional using water followed by methanol with the addition of NaCl, 1% formic acid soak, clean-up with dispersive C18, and dilution with phosphate buffer, and further clean up with then addition of 13C-labelled isotopic standards prior to an immunoaffinity column prior to LC-MS/MS analysis. analysis by LC-MS/MS (Nualkaw et al., 2020). The method Commercial and homemade doenjang samples were tested analysed 17 different mycotoxins and had limits of detection with this method and both categories were found to contain (LODs) ranging from 0.25 to 40 μg/kg and LOQs from 0.5 multiple mycotoxins at various concentrations. This method to 100 μg/kg. The method was used on 300 feed samples of purification may be helpful when applied to other difficult in which the Fusarium mycotoxins were among the most matrices with similar compositions, or when needing to prevalent. This method demonstrated a potential for improve the recovery, sensitivity, and selectivity when analysing these difficult matrices, however larger sample analysing problematic matrices. sizes may need to be tested for better representation of the

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contamination found in the feed as only 1 g of ground feed ENN B1, and ENN A1, as well as BEA, AME, HT-2, ZEN, was extracted for analysis. Feed is a complex mixture of and β-ZAL. This method could facilitate more research various components, therefore evidence supporting the use on the environmental fate and impact of mycotoxins in of such a small test portion would complement this study. aquatic ecosystems.

A new multi-mycotoxin method that incorporated a much 5. Chromatography with targeted high wider variety of Fusarium-produced mycotoxins than resolution mass spectrometry typically analysed was used by Ekwomadu et al. (2020). This method included free, conjugated, and emerging In the last decade HRMS turned from a research-only mycotoxins and was used to analyse 123 maize samples technique into a valuable tool in laboratories dedicated collected from agriculture regions of South Africa. to routine testing and high throughput food analysis. Many of the samples contained FB1, FB2, FB3, and the Among the approximately 1,800 Orbitraps and LC- very infrequently studied fumonisins B4 and A1. DON, (Q)-ToF instruments sold worldwide in 2018, probably ZEN, HT-2, T-2, and NIV were also present. Conjugated 10-20% were installed in laboratories of the food sector Fusarium mycotoxins that were found in the samples and official control labs (G. van ‘t Slot, Bruker Daltonik included DON-3G, ZEN-sulphate, hydrolysed FB1, and GmbH, personal communications). While at the beginning, HT-2-glucoside. Emerging mycotoxins that were analysed pesticides were the focus of HRMS methods, nowadays the were monoacetoxyscirpenol, diacetoxyscirpenol (DAS), regulated mycotoxins are shifting into the focus of method neosolaniol (NEO), culmorin, 15-hydroxyculmorin, development. Similar to what has been observed for MS/ 5-hydroxyculmorin, MON, beauvericin (BEA), ENN B, ENN MS analysis in the first decade of this century, the initially B1, ENN B3, aurofusarin, bikaverin, butenolid, epiequisetin, published methods had to set their own performance equisetin, apicidin, deoxyfusapyron, fusapyron, fusaric acid, and quality criteria with only few official guidelines and fusarinolic acid, 7-hydroxypestalotin, acuminatum B and C, published protocols. Based on these experiences, new chlamydospordiol, chlamydosporol, chrysogin, and siccanol. criteria found their way into official regulations and Sample preparation was performed by extraction and recommendations for HRMS. For instance, for pesticide dilution utilising acidified aqueous acetonitrile solutions, analysis such guidelines were prepared by the European centrifugation, and then analysed by LC-MS/MS. and EU national reference laboratories and published as documents of the EU Directorate-General for Health and Freshwater ponds are also being evaluated for mycotoxins; Food Safety (Pihlström et al., 2019). According to these it has been shown that mycotoxins can be transferred into recommendations for HRMS, two ions with accurate ponds through excess water that has been in contact with mass within 5 ppm of the calculated mass are required for contaminated plants. These mycotoxins can then potentially identification. One of these ions is required to be a fragment affect the organisms found in the ponds (Goessens et al., in ion while the second ion should be the (de-)protonated press). A method using SPE clean-up and UPLC-MS/MS molecular ion or an adduct ion thereof. Additional was validated for 20 mycotoxins in freshwater from ponds requirements published in the document are for instance in Belgium by Goessens et al. Six stable isotope-labelled that two extracted ion chromatograms must fully overlap internal standards were used to improve precision; for all with a signal to noise ratio of 3 for each trace. In case of analytes the relative standard deviation from analysis of absence of noise, the ion has to be present in at least five three replicates decreased when quantitation used internal consecutive scans. Interestingly, no specific requirements standards. The improvement varied amongst analytes, for an ion ratio, as common for single reaction monitoring with a minimal to a 3× decrease in RSD. Matrix-matched transitions in triple quad mass spectrometry, are set. This calibration standards prepared using natural pond water is explained by the different modes of recording for mass analysed previously and found to be free of mycotoxin spectra and fragmentation as well as the impact of matrix analytes were used to mitigate the apparent low extraction ions on the mass spectrometer (Pihlström et al., 2019). efficiency of alternariol monomethyl ether (AME; mean Relevant aspects of these guidelines could be incorporated 7% as compared to 75-109% for the other 19 mycotoxins). into guidance for mycotoxin analysis using HRMS. Besides The method was deemed to be sensitive, as LODs ranged minimum requirements, recommendations for optimal from 0.04 to 28 ng/l and concentrations less than 100 ng/l resolution, which is probably depending on the instrument, were regarded as of little environmental importance with have not been published, but strongly vary among the respect to toxicity towards aquatic organisms. The authors papers dealing with Orbitrap analyser, as reported below. managed the low extraction recovery of AME by using matrix-matched calibration curves and internal standard An interesting study dealing with the impact of mass during quantitation. The apparent mean recovery of AME resolution for the analysis of feed samples such as forage ranged from 89-117% with precision ranging from 11-38% maize has been published by Jensen et al. (2019). The for the recovery experiments performed at the LOQ of 10 authors follow European recommendations regarding both ng/l. Mycotoxins found in water samples included ENN B, selectivity of the ion extraction as well as the requirements

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for the presence of confirmatory ions (Jensen et al., 2019). (8+2) +0.1% formic acid and a 2 ml aliquot was treated with The resolution of Orbitrap mass spectrometers can be MgSO4 and C18 material. Following centrifugation, the defined by the duration of the scanning time, more precisely obtained extract was evaporated to dryness and used for by the duration of the recording of the axial frequency. analysis. The authors compared treatment with C18 material Accordingly, the Orbitrap mass spectrometer used in this for lipid removal with a freezing out procedure and found publication offered resolution (full width half maximum, the first one more efficient and better applicable to their for m/z 200) settings of 17,500, 35,000, 70,000, and 140,000. workflow. Determined LOQ values as low as 1.25 µg/kg for For the forage maize matrix, a resolution of at least 35,000 DON and its acetylated derivatives as well as 0.013 µg/kg was required to obtain signals for all analytes with a mass for ZEN were obtained, which are factors of 20 to 5,000 accuracy of 5 mg/kg while for silage maize a resolution lower than the detection capability reported by Jensen et al. of 70,000 was required. Fragment spectra were obtained (2019). However, besides differences in the sample matrices, in the data-dependent acquisition (DDA) mode with the a direct comparison is also not possible as Castaldo et al. same resolution and a mass extraction window of 1 Da. The (2019) did not exactly specify their signal requirements for reported scan rate was ≥3 Hz. The authors propose that the LOD and LOQ. While Jensen clearly states that European complexity of the feed samples and especially the separation criteria were applied, Castaldo et al. (2019) only state that of analyte signals with low intensity from background the LOQ was ‘the lowest concentration of the analyte noise is critical and thus resolution should be evaluated that produced a chromatographic peak with precision for every matrix. Interestingly, the authors observed a and accuracy <20%’. Whether or not the confirmatory ion strong increase of signal intensity when they shifted the was observed at that level is not clearly stated. Further ionisation of the source from ‘standard’ heated electrospray comparison by means of SSE are not possible as these values (HESI) to atmospheric pressure chemical ionisation (APCI). are missing from Jensen et al. (2019). Castaldo et al. (2019) This way, a factor of 7 increase of signal intensity for DON stated that SSE was between 75 and 98%, and therefore and derivatives (except DON-3G) was obtained. Signals of little effect. In comparison to Jensen et al. (2019), the for ZEN, α-/β-ZOL, and DON-3G were at least doubled Orbitrap was equipped with a HESI ion source and operated when APCI was applied. APCI is usually favoured by less in positive as well as negative mode. The Orbitrap was polar analytes. While APCI is also known to be less prone adjusted to a resolution of 35,000 for full scan and 17,500 for to matrix effects, matrix was not the decisive factor for the additional all ion fragmentation (AIF) mode, resulting APCI selection as APCI resulted in better signal intensities in a scan rate of 2 Hz. with neat solvents as well (T. Jensen, Christian-Albrechts- Universität Kiel, personal communication). With APCI for Another variation of a QuEChERS approach was published ionisation and a resolution of 70,000 for both matrices, by Narváez et al. (2020). Based on a previously published detailed method validation was performed for the Fusarium approach for food supplements, the authors studied the toxins DON, DON-3G, DOM, 3-/15-ADON, ZEN, and optimal purification of the QuEChERS extract by comparing α-/β-ZOL. With this method, RSD for inter- and intra-day primary secondary amine, graphitised carbon black, C18, precision were in the range between 2 and 16% and apparent and Z-sep+ materials being added to the acetonitrile phase recoveries between 95 and 108% were obtained. The authors together with MgSO4. The Z-sep+ material consists of defined a decision limit and detection capability with the zirconium and C18 bi-functionalised silica particles and is latter ranging between 20 µg/kg for 3- and 15-ADON well known as dispersive SPE material for pesticide analysis. in maize silage and 141 µg/kg for DON in forage maize. This material is claimed to be optimal for removal of large Unfortunately, a detailed description of the matrix effects quantities of fat (samples containing >15% fat) as C18 is in form of signal suppression/enhancement (SSE) values is known to adsorb lipophilic impurities such as tri- and missing in this publication. Such a table would clarify the diglycerides and zirconium acts as strong Lewis acid and challenges posed by this matrix and help readers who want can bind the vicinal hydroxy groups of mono-glycerides. to apply the methods to understand what signal intensities This clean-up procedure produced quite good SSE values are to be expected on their mass spectrometer. In summary, between 80 and 120% for the analysed mycotoxins (Narváez this publication highlights the remaining critical aspects et al., 2020). However, no rationale was provided for the that have to be considered for modern LC-HRMS analysis. authors’ selection of investigated mycotoxins. Aflatoxins, It shows the current limits of HRMS with regard to the ZEN, ZOLs, and ZAN, the enniatins (ENN) as well as T-2, screening of a broad analyte spectrum, since in this case HT-2, and neosolaniol were examined, but much more a certain selectivity was unavoidable due to the required frequently occurring DON and the ADONs were missing. sample preparation. Fumonisins, although known to be difficult to analyse with QuEChERS, were also not considered. It would have been Castaldo et al. (2019) published an ultra-high performance beneficial for the publication if the authors had investigated liquid chromatography (UHPLC)-Q-Orbitrap method for a broader range of analytes, including those more commonly feed material, in this case dry pet feed. In their approach, regulated by food standards. Even if sample clean-up 2 g of sample were extracted with 10 ml acetonitrile/water for DON does not work out (perhaps due to affinity of

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zirconium for the adjacent hydroxyl groups of DON) it Concerning human exposure, aflatoxin biomonitoring has would not have been detrimental to the publication, but an outstanding relevance due to the strong carcinogenic would have provided useful information for other groups properties and the high acute toxicity. Common target working in this area. analytes and validated biomarkers in urine are the AFB1 metabolite AFM1 and the AFB1-N7-guanine adduct which is Biomonitoring of mycotoxins with a known relevance to an excretion product of AFB1 bound to DNA. Debegnach et humans and animals has been successfully performed by al. (2020) studied the applicability of a UHPLC-Q-Orbitrap LC-MS for many years. For horses, alkaloids produced by system for the analysis of these compounds as well as AFB1 endophytic fungi of pasture grasses such as ergovaline, in human urine samples using both, dilute and shoot and ergotamine, peramine, lolitrem B, N-acetylloline, immunoaffinity clean-up methods. While AFM1 and AFB1 N-formylloline or paxilline are of especially high relevance were commercially available, AFB1-N7-guanine adduct as they are regularly associated with diseases, such as was synthesised by the authors as a qualitative reference. ergotism, fescue toxicosis, and rye grass staggers. In To support accurate quantification, stable isotope labelled case of acute toxicosis, urine and blood samples are the analogues of AFM1 and AFB1 were added before clean-up. physiological matrix of choice, but long term (chronic) Ionisation of the analytes was done using HESI and full exposure cannot be monitored in these materials due to MS as well as data dependent MS2 spectra were recorded. the short biological half-lives of the alkaloids. To that end Identification criteria included the detection of the parent Rudolph et al. (2019) developed a method for the detection ion within 0.1 min of the expected retention time with a of these compounds in equine hair. For analysis, 4 cm mass window of 5 ppm. Additionally, one characteristic sections of horsehair were taken, ground, and extracted fragment ion was required for successful identification. with a mixture of acetonitrile and methanol (1+1). Polar LOD and LOQ were defined by measurement uncertainty analytes of this extract were directly analysed by hydrophilic according to JRC technical report EUR 28099 (Wenzl et al., interaction liquid chromatography, while an additional 2016). For maximum signal intensity, the authors decided clean-up step was required for the less polar substances. to sum up the signals for protonated and sodiated AFM1 During method development, the authors found out that as both ions were observed in comparable quantities. purification using Waters HLB columns was successful However, this can be critical for accurate quantification except for paxilline and lolitrem B. However, to avoid costly as ionisation efficiencies for both ions can be different. Also, SPE, the authors preferred a duplicate extraction of the an impact of the sodium load of the individual samples and initial extract by liquid-liquid extraction. In a first step, this calibration solutions cannot be excluded. An additional was done with 1-chloropropane at pH 9, while the second source of error might be that sodiated and protonated liquid-liquid extraction was done with dichloromethane at ions can produce different fragment spectra as only the pH 14 to maximise the extraction efficiency of peramine. latter one can perform charge migration fragmentations The extracts of the halogenated solvents were combined, (Demarque et al., 2016). However, due to the application evaporated to dryness and subsequently analysed by of stable isotope labelled standards, any effects by varying UHPLC-HRMS. The LOD was defined as the lowest ratios between protonated and sodiated AFM1 should have concentration where a ‘reliable identification by MS2 been compensated. By applying an analyte enrichment and spectra’ was possible, which seems to be comparable to the purification using immunoaffinity columns; a LOD of 1.5 current European requirements for pesticides (Pihlström pg/ml for AFM1 could be obtained, which is considerably et al., 2019). Extraction efficiencies of this method were lower compared to 40 pg/ml determined with the dilute between 59 and 91% for the alkaloids. For the mycotoxins, and shoot approach and in this case are essential for the LODs between 1 and 25 µg/kg were obtained and RSDs detection of any analyte in this study. All determined for intra- as well as inter-day precision were in the range AFM1 concentrations except one were between LOD and 3%-11%. Analysis of hair samples from eight horses with the LOQ of 5 pg/ml. Debegnach et al. (2020) were then suspected fescue toxicosis or ryegrass staggers, as well as facing a challenging situation: keeping the strict validation non-affected control samples, revealed only the presence of criteria and accepting that no quantitative comparison of a N-acetylloline and N-formylloline in varying concentrations recorded dataset is possible or lowering the performance in all tested samples. The authors suggest that this could be requirements for the method to lower the LOD. The authors explained by the slow process of alkaloid transfer from skin did both; they accepted a higher degree of uncertainty for into the growing hair, but also point out that the symptoms assigned concentrations to the samples in the range between do not necessarily have to be caused by the alkaloids. In LOD and LOQ but additionally they clearly stated that the summary, the methodology except for paxilline and lolitrem values were below the defined LOQ and have thus elevated B has successfully been established, but confirmation that uncertainty. To readers, it would have been interesting to the method is suitable for retrospective detection of alkaloid get information on where the LOD of the method would exposure has not yet been provided. have been, if it was estimated based on the signal-to-noise ratio according to Pihlström et al. (2019). Finally, compared to the other publications dealing with Orbitrap analysers

10 World Mycotoxin Journal 14 (1) Developments in mycotoxin analysis 2019-2020

that were reviewed in this chapter, Debegnach et al. (2020) Combining targeted HRMS analysis with NTA was unfortunately did not mention the applied mass resolution presented for the analysis of mixed feed rations for nor the extraction window, which, as stated above, can have mycotoxins (Facorro et al., 2020). This demonstrates the an impact on sensitivity and selectivity. real power of DIA HRMS analysis, where you are able to quantify a number of compounds and then use the same 6. Chromatography with non-targeted high datasets for NTA screening. This workflow allowed the resolution mass spectrometry authors to quantify 26 mycotoxins in their feed samples and they were also able to detect 12 additional compounds, Low resolution LC-MS/MS has been the gold standard which could then be added to future quantification analysis. for mycotoxin analysis for more than two decades. In the Similarly, Castaldo et al. (2019) used AIF HRMS analysis last decade with the development of Q-ToF and Orbitrap for the targeted quantitation of 28 mycotoxins in dry pet instruments, there has been a shift toward the use of food. They then performed post-target screening for an HRMS targeted analysis for compound identifications. As additional 245 fungal and bacterial metabolites. Their a result of these instruments becoming more mainstream, analysis showed that nearly all pet food had some mycotoxin application of complementary non-targeted methods, such contamination, but it was mostly below EU regulatory as data-independent acquisition (DIA), DDA, and AIF limits. The NTA work demonstrated that a number of modes have also increased. The benefits of these techniques fungal metabolites were common contaminants and should are that no prior knowledge of the compounds in the sample be included in future targeted work. Additionally, Izzo et al. are needed, additional compounds are easily added without (2020) applied DDA HRMS for the analysis of mycotoxins the need for new method development, and the spectral and pharmacologically active substances in milk with data can be archived and retrospectively mined for new retrospective screening. The method was used to target or emerging mycotoxins as they become of concern. This 30 mycotoxins in 56 milk samples with the results indicating has led to advancements in open source software, such as that none of these compounds were present. Interestingly, XCMS, MZmine and MS-DIAL for data processing. It has retrospective screening identified 7 mycotoxins [AFM2, also inspired the development of spectral databases for penicillic acid, tentoxin (TTX), DOM-1, hydrolysed FB2, rapid compound searching, such as MASSBANK, METLIN T-2-triol, FB3] in 10 of the milk samples. These three and mzCloud. Since the previous review in mid-2019, a studies, in feed, pet food and milk all demonstrate the number of papers describing advances in the use of non- value in combining targeted analysis using HRMS with targeted (NTA) HRMS for mycotoxin analysis have been non-targeted screening regardless of the NTA technique published. These papers describe analysis of feed, pet food, used (DIA, DDA or AIF). milk, during thermal food processing, quantification in cereals without standards, and metabolite determination NTA also played an important role in the determination of for biomonitoring studies. phase I and glucuronide phase II metabolites produced from microsomal incubations with 17 mycotoxins (Slobodchikova One of most notable studies from the last year involved the et al., 2019). In vitro metabolism of the 17 mycotoxin development of an approach for addressing the challenge of standards [T-2, HT-2, 3-ADON, 15-ADON, fusarenone-X quantification for non-target or suspect screening without (FUS-X), DON, NIV, AFB1, AFB2, AFG1, AFG2, ZEN, the need for authentic standards (Liigand et al., 2020). The α-ZOL, β-ZOL, ZAN, α-ZAL, β-ZAL] resulted in 188 authors note that HRMS analysis is gaining importance metabolites, more than half of which had not previously due to the fact that 100s or even 1000s of compounds can been reported. DDA HRMS analysis was essential for be identified in a single sample. Their approach involved this work to first characterise all possible metabolites, quantifying compounds based on their predicted ionisation and second so that the data could be used to build a efficiency values as determined from a training set of comprehensive library for future human biomonitoring compounds. This would allow transfer of this technique analysis. The combination of targeted and NTA screening between eluents, LC setups, and instruments via a regression in this study is another good example of the power of using model. The work was validated by predicting pesticide and HRMS for both quantification and biomonitoring screening mycotoxin concentrations in cereal samples. Using this for exposure. approach, they were able to obtain an average prediction error (defined as the difference between measured and The studies presented all support the idea of combining predicted concentrations) of 5.4 times as compared to 526 targeted analysis for quantification with non-target analysis times if equal responses to all analytes is assumed, which for screening and to inform future targeted studies. It is they indicate is within the range necessary for toxicological not feasible for all labs to purchase 1000s of standards determinations. There is still more work to be done in this and run them routinely. Screening of samples using NTA area but the ability to obtain approximate concentrations will allow analysts to set priorities for future analysis. As from analysis of samples in complex matrices is a game indicated in the first study, one of the big challenges of NTA changer for NTA-HRMS. is quantification of compounds without standards. There is

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a lot of work ongoing in this area and it will only continue 15 μg/kg), LOQs (4-45 μg/kg), and recoveries (61-116%) to improve in the future. The use of HRMS analysis for of the ergot alkaloids were comparable to other published screening will continue to gain ground in routine analysis as methodologies. All analytes were analysed by HPLC with databases improve and software packages for data process fluorescence detection with a total run time of less than 40 are better equipped to handle these datasets. min. Application of lysergic acid diethylamide during the validation of this method contributes greatly to its success 7. Chromatography with non-mass as some more polar ergot alkaloids have recovery issues. spectrometry detection Two methods were published for the simultaneous detection Chromatographic analysis of mycotoxins by HPLC, UHPLC, of AFs and OTA by HPLC with fluorescence detection. and gas chromatography (GC) coupled with non-mass One method focused on cereals with the utilisation of a spectroscopy detectors are recognised as reference methods multi-toxin immunoaffinity clean-up column (Aflaochra in the food and feed industry. HPLC and GC with non-MS Prep) with no aflatoxin derivatisation (Dhanshetty and detectors are commonly requested methods because of the Banerjee, 2019). The cereals were extracted with an reference status, ease of use compared to MS, and their 80% methanol to water solution followed by an extract lower cost to purchase and operate. The current trend for dilution with phosphate buffered saline and multi-antibody the chromatographic analysis of mycotoxins with non-MS immunoaffinity clean-up. Low LOQs for AFs and OTA were detection is the development of multi-toxin analysis with 0.25 and 1 ng/g, respectively, were facilitated by using a large fluorescence and ultraviolet (UV) detection, automation, volume flow cell in the fluorescence detector. In rice, the dispersive liquid to liquid micro-extraction techniques recoveries for AFs ranged from 84-105% and >72% for OTA (DLLME), and the utilisation of detection technologies that with a RSD for all toxins of <12%. The other AFs and OTA have become increasing sensitive, such as near-infrared method focused on cannabis and similarly utilised multi- (NIR) spectroscopy. antibody immunoaffinity clean-up and additionally the validation of an automated in-line reusable immunoaffinity Multiple methods were published for the application of cartridge (Wilcox et al., 2020). Cannabis was extracted with multi-mycotoxin analysis with non-MS detection in 2019 a 75% acetonitrile to water solution followed by an extract to date. One method was published for the simultaneous dilution with a 10% Tween 20 in phosphate buffered saline determination of AFs, OTA, ochratoxin B (OTB), and ZEN solution. The diluted extract was passed through a multi- in animal feed (Muñoz-Solano and González-Peñas, 2020). toxin immunoaffinity clean-up column or directly loaded This method was validated for use with swine, cow, sheep, onto an automated system that passed the diluted extracts and poultry feeds with global recoveries of 74 to 88% for through an in-line reusable immunoaffinity cartridge for all toxins with a RSD of <7%. The samples were extracted AFs and separately OTA. Both the manual and automated with an acetonitrile, water, and orthophosphoric acid methods were independently validated and utilised post solution, purified with a SPE cartridge, and subjected to column Kobra cell derivatisation for AFB1 and AFG1. The a DLLME procedure. The purified samples were injected manual method recoveries of AFs and OTA ranged from 96- on an HPLC with fluorescence detection with post column 98% and 88% with RSDs of 5.6-6.6% and 4.8%, respectively. photochemical derivatisation for AFB1 and AFG1 with a The automated method recoveries of AFs and OTA ranged total runtime of approximately 40 min. This method is from 102-107% and 99% with RSDs of 1.8-2.9% and 2.0%, quite advantageous for laboratories optimising equipment respectively. Automation of mycotoxin analysis reduces risk time and consumables. The method was validated for of human error and is optimal for industry laboratories that complex matrices which implies that for simple matrices, analyse large volumes of similar matrices on a routine basis. samples could be further concentrated allowing for lower LOQs. Practical methods should be developed to achieve a A multi-toxin method for the simultaneous determination LOQ for mycotoxin determination which are significantly of DON, DON-3G, NIV, and nivalenol-3-glucoside (NIV- less than the regulatory limit of interest so that decision 3G) in baby formula and rice utilising HPLC with UV makers can properly apply a decision rule with a high level detection was also published (Lee et al., 2019). This method of confidence. involves a simple sample extraction with purification by immunoaffinity clean-up. Recovery ranged from 79 A separate multi-toxin method was recently published to 107% with a RSD of <12%, for 4 analytes. The LODs for the quantification of 12 ergot alkaloids (ergometrine, achieved ranged from 1-4 μg/kg with LOQs of 4-13 μg/kg. ergotamine, ergosine, ergocornine, ergocryptine, The capability to quantify the 3-β-D-glucosides of DON ergocristine and their corresponding inine-epimers) in rye and NIV with a simple extraction adds great value to the (Holderied et al., 2019). This method utilised ethyl acetate, method and will be increasingly beneficial as more research methanol, and aqueous ammonia for extraction, followed is performed on the toxicity of modified mycotoxins. by aluminium oxide purification and the addition of lysergic acid diethylamide for an internal standard. All LODs (1-

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Two methods were published for the determination of to distinguish between barley that is highly contaminated AFs by HPLC with fluorescence detection with sample (>1,250 μg/kg) and a low level of contamination. The partial preparation by DLLME. One method focused on AFs least squares-discriminant analysis classification model in oats, rice, coconut, almond, and birdseed, as well as permitted observation of 90 to 82% in the cross-validation plant-based milk and milk-based products enriched with set, while the partial least squares-regression quantification oats, almonds, and walnuts (Hamed et al., 2018). Analytes presented low error values. This method may be suitable for were extracted from the samples with DLLME techniques an initial screening of barley for DON utilised in tandem and were analysed with post column photochemical with other screening technologies. This publication is an derivatisation. Recovery ranged from 82 to 104% with interesting development as many organisations have tried a RSD of <10% and LOQ of <0.5 μg/l for all AFs. The unsuccessfully to develop NIR methods for mycotoxin other method focused on grains with a modified DLLME screening, however caution is needed with such methods technique (Asadi, 2020). DLLME variables were optimised, as their specificity is low. It is not apparent if the signal is including sample volume, sample pH, salt concentration, being produced by the fungi, changes to sample matrix, type of extract, and volume extracted. After optimisation, or something else. a RSD of 5-8% was obtained for AFB1 and AFB2. The LOD achieved was 15 ng/kg for AFB1 and 10 ng/kg for AFB2 8. Multiplex biosensors with a linear range of 50-500 ng/kg and 35-400 ng/kg, respectively. Biosensors and assays based upon biological binding elements, such as antibodies (immunosensors and A method for the extraction of AFs and detection by HPLC immunoassays) and aptamers (aptasensors) continue to with fluorescence detection coupled with post column be very active areas of investigation. Much, if not most, of photochemical derivatisation utilising commercial bare the research in these areas is being conducted in Asia and magnetic Fe3O4 nanoparticles as sorbents for sample we are fortunate that many of the authors have elected to preparation was published (Zhang et al., 2020a). Vegetable publish their results in English. The breadth and depth of oil was diluted with hexane followed by the direct addition of research into rapid bio-based detection methods has led bare magnetic Fe3O4 nanoparticles performing a magnetic to publication of reviews into the various aspects of this solid phase extraction. Significant variables that can affect area. While biosensors are often classified by the type of magnetic SPE were optimised and post optimisation the toxin-binding element (e.g. antibody, aptamer, imprinted method was validated. Recovery ranged from 83% to 106% polymers, etc.), they are also frequently classified by the with a RSD of <10%, for all 4 AFs. The LODs achieved were technology used for signal transduction and detection (e.g. 0.01-0.16 μg/kg. The application of commercially available optical, electrochemical, piezoelectric, etc.). A wide-ranging bare magnetic Fe3O4 nanoparticles and validations of its review of mycotoxins, analytical techniques for detecting extraction efficiency allows for the continued development microorganisms, and mycotoxin biosensors, was provided of magnetic SPE development, as synthesis of magnetic by Oliveira et al. (2019). This paper reviews the various Fe3O4 nanoparticles is complex. transduction and detection platforms, with a focus on the most recent literature. Several reviews were also provided A multicommutated optosensor fluorometric method for certain segments of the field. A review of biosensors was developed for the quantification of ZEN in maize and based upon electrochemical devices was provided by cereal stuffs (Llorent-Martínez et al., 2019). A QuEChERS Evtugyn and Hianik (2019). This is a general review with extraction method was applied to maize and other maize- basic information on antibody or aptamer/toxin interactions based feed products. Once the test portions were extracted, and the basis of electrochemical biosensors, in particular the they were injected on a modified luminescence spectrometer various measurement modes available. The use of materials utilising 50% methanol/water (v/v) as a mobile phase with that mimic enzymes in functionality (nanozymes) and a flow cell filled with a C18 silica gel. The ZEN under these their use in detection of a variety of analytes, including conditions was retained in the flow cell where the detector mycotoxins, was reviewed by Zhang et al. (2019). Among records its signal. Recovery ranged from 93 to 107% with the biosensors that use optical devices for detection are LOD and LOQ sufficient for levels established by the EU a subgroup that use photoactive materials. A review of for the regulation of ZEN. This method could be utilised photoelectrochemical biosensors was provided by Zhou as a screening technique, however it is recommended that and Tang (2020). The photoelectrochemical biosensors all positive results be verified by alternative technologies use light as an excitation source and photocurrent as the with greater specificity. recognition signal. With regard to optical biosensors, Nabok et al. (2019) reviewed a subset of optical sensors that use Even though it did not include a chromatographic process, a total internal reflection ellipsometry with localised surface method using NIR spectroscopy with chemometric tools to plasmon resonance for detection. The latter also includes screen DON in barley showed some promise (Caramês et al., an introduction to a newer type of optical planar waveguide 2020). The purpose of this study was to verify if it is possible polarisation interferometer.

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Measurement of multiple analytes can be accomplished Most multiplexed LFIA are either duplex or triplex systems. in several ways: by having signals separated temporally, by Recently a 5-plex system was reported for AFB1, ZEN, having signals separated spatially, by measuring specific DON, T-2, and FB1 in cereals (Liu et al., 2020). A 3-D attributes of each analyte, or by measuring labels specific printed device attached to a smartphone allowed for two for each analyte. Each of these approaches has been applied modes for data collection: fluorescence or colour. The to immunoassays for mycotoxins. Because speed is a major former used a UV-LED excitation source and antibodies reason for using immunoassays, temporal separation is labelled with fluorescent Europium microspheres. The less commonly used. Distinguishing between multiple latter used a visible LED excitation source and antibodies mycotoxins is most often accomplished using spatial labelled with AuNPs. Software allowed the investigators separation. The best examples are the separation of lines on to select between the two modes. The LFIA were of the lateral flow immunoassays (LFIAs, or ‘test strips’), enzyme antigen-immobilised type. The test strips were held for linked immunosorbent assay wells (e.g. microwell ELISAs), 5 min in a mixture of sample extract that had been pre- and spots (e.g. arrays). incubated for 3 min with the labelled antibody probes. Switching the probes used in the pre-incubation permitted While colloidal gold nanoparticles (AuNP) remain the switching between the two detection endpoints. The labels/ most popular label for LFIAs, a wide variety of other endpoints were used to establish LODs based upon a visual labels have also been reported. Among these are labels threshold (vLOD), and a quantitative threshold (qLOD: composed of lanthanide chelates. Lanthanides have long defined as 80% B/Bo). As might be expected, the qLODs fluorescent lifetimes, which make them excellent labels were lower than the vLODs. For AFB1, ZEN, DON, T-2, for time-resolved fluorescence (TRF). The use of TRF can and FB1 the qLODs were: 0.59, 0.24, 0.32, 0.9, and 0.27 µg/ reduce background noise and thereby improve sensitivity. kg when the endpoint was colour. When the endpoint was Nanospheres of europium were used to label primary fluorescence the assays were more sensitive: 0.42, 0.1, 0.05, (anti-toxin) antibodies in a triplex assay for AFB1, ZEN, 0.75, and 0.04 µg/kg, respectively. Recoveries from maize, and chlorothalonil in maize and peanuts (Wang et al., for both formats, ranged from 84 to 110%. Coefficients 2019). The LFIA used an antigen-immobilised format. of variation ranged from 8 to 19%. An examination of 30 Samples were extracted with 70% methanol, centrifuged, cereal samples revealed good agreement between the two and the supernatant diluted for testing. The entire protocol, methods and an LC-MS/MS method. Given the results, including extraction, took 15 min. The analytical ranges for the greater sensitivity of the time resolved fluorescence AFB1 and ZEN in maize were 0.48-20 µg/kg and 1.56-200 microspheres mode would argue for its preference. µg/kg, respectively. For peanuts the analytical ranges were 0.54-20 µg/kg and 1.71-200 µg/kg, respectively. Recoveries While most LFIAs use spatial separation to distinguish ranged from 86 to 111%. With eight naturally contaminated between toxins, an alternative approach was recently samples of maize and peanuts, good agreement was found described (Di Nardo et al., 2019). The LFIA was a duplex between LFIA and LC-MS/MS results. A similar approach assay for AFB1 and B-type fumonisins in raw and processed was used in a triplex assay for AFB1, ZEN, and OTA in foods. Rather than having a separate test line for each maize, soybean, sorghum, wheat, rice, and oats (Chang et analyte, there was a single, combined, test line that changed al., 2020). Two incubations, one with the sample before hue depending upon the presence of the toxin(s). Primary application to the test strip and a second, on the test strip, antibodies were labelled with either blue AuNP (for FBs) totalled 14 min. A portable multi-channel reader was used or red AuNP (for AFB1). With no toxin present the test to measure the TRF. The LODs for AFB1, ZEN, and OTA line was purple (red +blue). In the presence of FB1 alone in the 6 grains were 0.04, 0.2, and 0.1 µg/kg, respectively. the test line was red, while in the presence of AFB1 alone Samples of each of the 6 commodities were spiked at three the test line was blue. In the presence of both toxins the levels: the LOD, twice the LOD, and 4 times the LOD. test line was colourless. Visual cut-off levels were 1 ng/ml Each sample was tested 6 times in duplicate over three and 50 ng/ml for AFB1 and FB1, respectively. Images from consecutive days. The average intra-assay and inter-assay the LFIA were also captured via smartphone and the recoveries ranged from 72 to 120%, with an RSD of less colour and intensity used as a semi-quantitative measure than 15%. The triplex assay was applied to 60 grain samples, of toxin content. Samples of wheat, fresh pasta, pasta, and 10 for each commodity. Of these, 9 tested positive for one French pastry were spiked with each toxin at two levels or more of the toxins. No false positives or false negatives corresponding to 2.5 and 5 µg/kg for AFB1 or 125 and were found. In all cases, the levels found were in good 250 µg/kg for FB1. All samples were correctly classified as agreement with those found using an LC-MS/MS method. contaminated/non-contaminated. No false positives were In each of these reports, the use of TRF seems to have been reported from 30 blank samples, and no false negatives effective at reducing background signals. However, this were found amongst 60 fortified samples. By combining test benefit may be offset somewhat by the need for specialised lines, the test strips could be made shorter, which might be instrumentation to measure TRF, an aspect that will likely an advantage of the technique, as shorter test strips could improve with time. potentially be more rapid than longer strips. The time

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between sample application and measurement was 10 min, according to toxin. To quantify the amount of antibody, which is good. Unfortunately, there was no description as a 532 nm laser was used to induce fluorescence from the to whether such LFIA were indeed faster than those having phycoerythrin. Milk samples were treated with acetonitrile, two detection lines, which was the expected benefit from centrifuged, and a portion of the supernatant was diluted the approach. for testing. The assays were sensitive, with LODs of 0.045, 0.94, 7.5, and 2.5 ng/ml for AFM1, OTA, DON, and FB1, Combining aspects of both spatial and optical discrimination respectively. Recoveries from spiked milk ranged from 76 was recently accomplished by using LFIA with surface- to 95%. The LOD for AFM1 was close to the EU regulatory enhanced Raman spectroscopy (SERS) for detection (Zhang limit of 0.05 ng/ml. The inclusion of OTA, DON, and FB1 as et al., 2020b). The LFIA-SERS technique was used to detect targets was a bit unusual, as milk is not typically considered AFB1, ZEN, DON, FB1, T-2, and OTA in maize. The LFIA to be a significant source of potential exposure for these was constructed in the antigen-immobilised format, with toxins. One limitation for this format was the extensive three test lines and one control line. Each test line was used analysis time: approximately 2 h, which is considerably for the detection of two toxins. To distinguish between longer than most commercial tests for these toxins. the two toxins at each line two Raman reporter molecules were used: 5,5-dithiobis-2-nitrobenzoic acid (DTNB) and Surface plasmon resonance is one of the oldest biosensor 4-mercaptobenzoic acid (MBA). The reporter molecules technologies that has been applied to mycotoxins. In most were incorporated into gold-silver core shell nanoparticles reports the technology has been used to indirectly measure which were, in turn, used to label the primary (anti-toxin) the change in mass that occurs on the surface of the sensor antibodies. In this fashion, at each test line DTNB served as when biomolecules bind to it. Several mechanisms have the label for one analyte and MBA served as the label for the been explored to amplify the binding event. One such other. Each of the toxins was identified by a combination of mechanism involves immobilisation of silver nanoparticles location (test line) and reporter molecule (DTNB or MBA). (AgNP) on the sensor surface (Jiang et al., 2020). Following Spiked maize was extracted with 70% methanol/water, AgNP immobilisation, bovine serum albumin conjugates mixed, and centrifuged. The test solution was prepared by of citrinin (CIT), AFB1, and OTA were also immobilised. diluting the supernatant 10-fold and adding it to wells of Toxins competed with the immobilised antigens for a microplate containing the labelled primary antibodies. anti-toxin antibody. The amount of antibody bound was After 2 min the test strip was added, held for 10 min, and determined after the addition of a secondary antibody- the SERS spectra were collected with a portable Raman horseradish peroxidase conjugate, washing, and addition of system. The solution LODs were: 0.001 ng/ml (AFB1), a substrate that yielded a chemiluminescent product. The 0.006 ng/ml (ZEN), 0.11 ng/ml (DON), 0.26 ng/ml (FB1), luminescence of the product was enhanced approximately 0.009 ng/ml (T-2), and 0.02 ng/ml (OTA). Because the two-fold through proximity to the immobilised AgNP. In ratio of solvent:maize in the extraction was 10:1, and an buffer solution, the assay was very sensitive, with LODs additional 10-fold dilution was made, the LODs in maize of 0.39, 0.44, and 0.83 pg/ml for CIT, AFB1, and OTA, could be expected to be roughly 100-fold greater than the respectively. Assays times were relatively long: 72 min. solution values. This suggests the assay would be sensitive When three samples of red yeast rice were spiked at 0.1 enough to detect all six toxins at relevant levels in maize. ng/ml recoveries ranged from 83 to 106%. Unfortunately, Recoveries ranged from 79 to 106% with coefficients of there was no description of how the spiking was performed, variation below 16%. which made it difficult to determine the sensitivity for the assay in the red yeast rice. If the spiking level of 0.1 ng/ml Multiple labels are also commonly employed in biosensor was based on the level in the purified and diluted extract, formats to distinguish among analytes. One type of then this corresponded to a spiking level of 160 µg/kg in biosensor that uses this approach uses microspheres that the red yeast rice. However, because the spiking level was can be distinguished from one another based upon their much greater than the LODs (0.39 to 0.83 pg/ml), it may be fluorescence characteristics, with each type of sphere possible that the assay is sensitive enough to detect AFB1 associated with a particular analyte. The fluorescence and OTA at relevant levels. of antibodies labelled with a fluorophore such as phycoerythrin is used to indirectly quantify the analyte Applications continue to be explored for the multiplex using a flow cytometer. Recently, a similar approach was detection of mycotoxins with novel instrument platforms. used to measure AFM1, OTA, DON, and FB1 in milk (Qu One such platform based upon white light reflectance et al., 2019). Paramagnetic microspheres coated with toxin- spectroscopy was used to measure AFB1 and FB1 in wheat ovalbumin conjugates competed with free toxin for binding and maize (Anastasiadis et al., 2020). The technique was to anti-toxin antibodies. The anti-toxin antibodies were based upon the shift in the interference spectrum occurring then labelled with a secondary antibody-phycoerythrin as biomolecules bound to the surface, analogous to biolayer conjugate. A 635 nm laser was used to induce emission interferometry. Regions of interest corresponding to each from the microbeads, allowing the beads to be classified toxin had different thicknesses of SiO2 which allowed them

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to be separated by deconvoluting the interference spectrum Lastly, a multiplexed assay was reported that did not fit collected from a single probe. The authors termed this conveniently in any of the other sections of this review variation of white light reflectance spectroscopy ‘multi because it was neither a biosensor, nor was it based upon area reflectance spectroscopy’. From an immunoassay mass spectrometry. The sensor, which was developed for standpoint, the format was that of an immobilised antigen AFB1, ZEN, and DON in maize, was based upon SERS. assay, with AFB1-bovine serum albumin and FB1-ovalbumin The SERS spectra of the toxins were recorded following attached to the probe. Diluted extracts of samples were their interaction with a 3-dimensional ‘cauliflower-shaped’ mixed with antibody and passed through the device for surface (Li et al., 2019b). The sensor surface was formed 7 min. Amplification was achieved by adding a secondary by coating an anodic aluminium oxide substrate with antibody (5 min). The chips were then regenerated using polydimethylsiloxane and then with AuNPs. To extract HCl (3 min). Testing time, excluding extraction, was 15 min maize samples, 20% methanol was added (2:1 solution:solid). which is comparable to other multiplexed biosensors such Samples were sonicated and then centrifuged. The purified as imaging surface plasmon resonance. The multi area extracts were spiked with toxins, spotted onto the sensor reflectance spectroscopy instrument lacked moving parts, surface, then dried before collection of the spectrum. which may facilitate the development of a portable device Solution LODs were 1.8 ng/ml (AFB1), 48 ng/ml (ZEN), and may be an advantage in terms of ruggedness. LODs and 25 ng/ml (DON). Recoveries of AFB1 spiked at 10 ng/ml were 0.05 and 1.0 ng/ml for AFB1 and FB1, respectively. to 1,000 ng/ml ranged from 94 to 110%. Recoveries of ZEN The quantification ranges were 0.15 to 5.0 ng/ml (AFB1) spiked at 500 to 50,000 ng/ml ranged from 94 to 104%. and 3.3 to 50 ng/ml (FB1). Maize and wheat were extracted Recoveries of DON spiked at 100 to 10,000 ng/ml ranged with a 5-fold ratio of solvent:solid and then diluted 1:20 from 93 to 120%. A limitation of the method was the for analysis. Based upon this, the expected LODs in maize extraction and sample clean up, with the sonication and or wheat may be extrapolated as 5 µg/kg (AFB1) and centrifugation steps taking 30 min and 20 min respectively. 100 µg/kg (FB1). Recoveries ranged from 85 to 115%. Because the purified extracts, rather than the samples, were While the sensitivities here were not as good as for some spiked it is unclear how well the recoveries would equate of the other biosensors, the potential to combine the to real samples. It is also unclear whether the extraction technique with a portable device suggests further research solution (20% methanol) would have sufficient solvent is warranted. strength to fully extract the AFB1 and ZEN from naturally contaminated maize. Despite these drawbacks, the potential Recently a 5-plex electrochemical immunosensor was of directly detecting the toxins is intriguing and warrants developed for potential bioterrorism agents that included further investigation of the SERS technique. three mycotoxins (Schulz et al., 2019). The mycotoxins, T-2, roridin-A (ROR-A), and AFB1, were detected in human 9. Single mycotoxin, or single mycotoxin family, serum. The format was a competitive immunoassay using biosensors pairs of anti-toxin (mAb1) and anti-idiotype antibodies (mAb2). For T-2 toxin and AFB1 the mAb1 were immobilised More than 45 papers focusing on single mycotoxin/ onto electrodes, while for ROR-A mAb2 was immobilised family biosensors have been published over the period instead. This determined the nature of the reagents that from mid-2019 to mid-2020. Rather than covering all were mixed with the toxins in the competition reaction. available studies, this section aims to select and discuss For T-2 and AFB1 the reagents combined with the toxin the more mature methods suitable to be applied by other standards were conjugates of mAb2 with β-galactosidase researchers, which have been subjected to extensive (β-gal). For ROR-A, because mAb2 was immobilised, validation, evaluated against reference methods and/or the toxin standards were combined with a mAb1-β-gal matrix reference materials, or in PTs. In addition, a selection conjugate. Following washing steps to remove unbound of new approaches, even if not yet fully validated, but which β-gal, the proportion of bound enzyme was measured hold promise for further improvements in immunoassays through the generation of an electrochemically active analytical performances and fitness-for-purpose will be product. Assays took 13.4 min. The LODs in assay buffer reviewed. were 0.4, 0.7, 0.5, and 0.6 ng/ml for T-2, HT-2, ROR-A, and AFB1, respectively. In human serum spiked at 4, 10, With respect to methods based on well-established techno­ and 100 ng/ml, the recoveries of HT-2 ranged from 52 to logies, such as LFIAs, ELISAs, or fluorescence polarisation 79%. The recoveries of ROR-A ranged from 88 to 101%, immunoassays (FPIAs), some examples of single mycotoxin and the recoveries of AFB1 ranged from 101 to 115%. An assays validated according to EU official guidelines for advantage of using anti-idiotype antibodies instead of toxin- mycotoxin screening methods (EC, 2014) can be found in protein conjugates was that because they did not contain the literature from mid-2019 to mid-2020. The experimental actual toxin, the chance of being exposed to toxin during design outlined in the EU regulation is definitely easy to production of the conjugates was eliminated. apply and is highly informative about method performance.

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The process of evaluating and comparing the performance sample extracts before they were introduced into the sensor. of LFIA and ELISA methods for screening AFM1 in milk The immobilised conjugate competed with CPA for the was reported by Pecorelli et al. (2020). Blank samples primary antibody. Detection of the primary antibody was (<0.5 ng/kg) and samples containing AFM1 at 25, 50, and enhanced by the addition of a secondary antibody labelled 75 ng/kg were used for the validation. The target screening with gold nanoparticles. The response, using matrix- concentration was 50 ng/kg and the cut-off values obtained matched calibration, was inversely proportional to the toxin were 38 and 48 ng/kg for the LFIA and ELISA formats, content. After evaluation of some analytical parameters for respectively. With regard to blank samples, a false suspect quantitative CPA detection, such as recoveries (89 to 126%), rate of 0.1% was found for both formats. For positive samples repeatability (RSDr ≤16%) and LODs (17 and 6 µg/kg in containing AFM1 at levels higher than 50 ng/kg, the false maize and cheese, respectively), validation as a screening negative rate was 0.4% for both formats. The intermediate method was performed to determine if the iSPR method was precision was <32% for the LFIA and <15% for the ELISA. able to differentiate samples spiked at the target screening Results from the validation study were confirmed through concentration, or higher, from blank samples. Because there long term quality control measurements of intra-laboratory is no official regulatory limit for CPA, a target screening variability. For cow’s milk naturally contaminated with concentration of 40 µg/kg was used. The validation included AFM1 at levels between ‘non-detected’ (<0.5 ng/kg) and 50 a comparison of the iSPR method to an ELISA method for ng/kg, the results from both formats were also correlated measurement of CPA in naturally contaminated maize with those obtained using the AOAC Official Method samples. The results indicated that the iSPR method was 2000.08. The article also contains a brief discussion of able to correctly discriminate between samples containing the fitness for purpose of validated immunoassays that CPA above or below 40 µg/kg with a false negative rate of encompasses factors other than analytical performance, ≤5%. Furthermore, a qualitative agreement was observed including the analysis time, skill and training of the operator, between the ELISA and the iSPR methods with a Pearson and the location where the assays are performed. correlation of 0.92. One of the main advantages of this biosensor technology is claimed to be sensor reusability. A further example of the use of EU official guidelines In this report, the durability of the sensor chips showed for immunoassay validation was reported by Lippolis et no significant changes in response over 25 analysis cycles. al. (2019). The assay was an FPIA for the simultaneous determination of T-2, HT-2, and their relevant glucosides, The overall performance of immunoassays strongly with results expressed as the sum of the analytes. The FPIA depends on the quality of the selected immunoreagents, was based upon an HT-2-specific antibody and was highly e.g. antibody. For this reason, development and testing of sensitive to HT-2 toxin (IC50 = 2.0 ng/ml). The cross- new antibodies continues to deserve considerable efforts reactivity to T-2 and the T-2 and HT-2 glucosides were in current research. This year, Maragos et al. (2019) 100%, and 80%, respectively. For application to wheat, two undertook the challenge of developing new monoclonal extraction protocols were proposed, one aqueous and the antibodies for the detection of citreoviridin (CTV) and its other a mixture of methanol and water (90:10, v/v). Both geometric isomer, iso-citreoviridin (iso-CTV) to improve methods demonstrated good analytical performance in the sensitivity of immunoassays targeting these toxins. terms of sensitivity (LOD of 10 µg/kg), recovery (92-97%) Performances of the newly developed antibodies were tested and precision (RSDr ≤13%). These capabilities fulfil the EU by setting up a competitive ELISA. Analytical performance criteria for acceptability for the quantitative determination of the immunoassay were evaluated in fortified white rice of T-2 and HT-2. The fitness for purpose of the FPIA was using matrix-matched calibration for mixtures of CTV/ also evaluated using a target screening concentration iso-CTV. Satisfactory recoveries were obtained, averaging of 100 µg/kg, which corresponds to the indicative level 97±10% over the range of 0.4 to 7.2 mg/kg, indicating the established by the EU for sum of T-2 and HT-2 in wheat. developed monoclonal antibodies and ELISAs as fit for The applicability of the screening method for assessing the purpose to screen white rice for CTV and iso-CTV at the content of these toxins in wheat at regulatory levels levels that are toxicologically relevant. was demonstrated by satisfactory intermediate precision (RSDip ≤25%), cut-off level (80 and 96 µg/kg for the two Compared to new monoclonal antibodies, polyclonal ones methods) and rate of false positives (<0.1%). are less expensive and easier to obtain, but they can still exhibit excellent performances in the determination of To detect cyclopiazonic acid (CPA) in maize and cheese, an small molecules. A new polyclonal antibody for CIT has imaging surface plasmon resonance immunoassay (iSPR) been made available in the period from mid-2019 to mid- was developed (Hossain et al., 2019). iSPR is a form of SPR 2020 (Singh et al., 2019) and applied to develop an ELISA where multiple regions of interest on the surface of the for analysis of grain-based products. When subjected to sensor are probed simultaneously. The CPA sensor used a in house validation in fortified wheat and maize flours, CPA-protein conjugate immobilised onto the sensor surface. the method exhibited recoveries ranging from 87 to 116% An anti-CPA antibody (primary antibody) was added to with RSDr of less than 20%. Individual matrix-matched

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standards of CIT were prepared in post-extraction fortified providing a detailed optimisation and characterisation of the wheat and maize matrices to avoid the possibility of matrix developed sensing device, the work produced a more mature effects confounding assay results. Furthermore, the method method targeting a sound mycotoxin/matrix combination. was evaluated by participating in a PT from the Food Sun et al.’s work also provided validation data with real Analysis Performance Assessment Scheme and obtained samples and data on sensor stability. In this work, an an acceptable z-score (-0.6) for a maize flour sample. A electrochemical immunosensor using pulse voltammetry small survey of grain-based foods was conducted using was developed for the detection of OTA in malt. The applied this method and CIT was detected in 43% of the samples strategy for sensor manufacturing utilised a self-assembling up to a concentration of 18 µg/kg. compact 2-mercaptoacetic (TGA) monolayer on the surface of the working Au electrode to form the Au/TGA/bovine In the area of new antibody development and testing, serum albumin-OTA/anti-OTA monoclonal antibody nanobodies, particularly VHH nanobodies which are composite probes. Sensor characterisation included stability derived from the Alpaca heavy chain immunoglobulin G testing performed through measuring the test responses to antibody, are being explored more and more within the OTA after storing the electrode in buffer at 4 °C for 10 days. new generation of mycotoxin assays due to the possibility The results indicated that the current signals were stable in to produce them in vitro in prokaryotic or eukaryotic acceptable range with RSDr <5% (n=3). Method accuracy, expression systems. An interesting example has been reliability, and potential application to real samples was reported by Tang et al. (2020) who proposed a nanobody- evaluated by fortifying OTA-free malt samples with three mediated Förster resonance energy transfer (FRET)-based OTA levels (5, 10, 25 µg/kg). Recoveries ranged from 72 competitive immunoassay using quantum dots for the to 97% with RSDr less than 28%. Toxin quantification was detection of OTA. Besides the application of nanobodies, performed by standard calibration since available data this example covers two relevant advanced topics in the showed matrix interferences to be negligible. Overall field of mycotoxins immunoassays: the use of the FRET method performance looked promising, however analysis technique, a recently explored strategy for homogeneous of matrix reference materials and/or comparison with immunoassays as well as labelling with quantum dots, reference methods would be needed to confirm method which are often proposed as alternative labels to AuNP reliability. to get a lower LOD. FRET refers to the transmission of photoexcitation energy from a donor molecule to a nearby Within new technologies and new devices proposed for rapid acceptor molecule. Quantum dots have been demonstrated immunosensing of mycotoxins, it is worth mentioning the to be simultaneously effective as the donor and acceptor paper-based microfluidic device for DON quantification in FRET. Since the energy transfer efficiency in FRET set up by Jiang et al. (2019). A colorimetric competitive inversely correlates to the distance between the donor immunoassay was obtained by applying the traditional and acceptor, the authors hypothesised that the use of a detection technique based on AuNP labelling on a new paper miniaturised antibody, such as a nanobody, could contribute based microfluidic device. The sensor was manufactured by to shorten the effective FRET distance and improve the printing the channel pattern (conjugate pad, test line and energy transfer efficiency for a lower LOD. The optimised control line, adsorbent pad) onto a nitrocellulose membrane assay used standard calibration, since matrix effects were through a wax ink printer. This automated procedure is overcome by applying different dilution factors (from 10 expected to ensure high reproducibility of manufactured to 100-fold) depending on the cereal matrix. Analytical chips, however no data were reported to confirm this performances (LOD, recoveries, and precision) were assumption. A portable imaging system was developed to evaluated in several cereal samples fortified with OTA over enable on-site signal reading. Calibration curves were built the range 0.02-2 μg/kg. The average recovery rate and the using standard DON solutions, whereas different dilution RSDr of intra-assay measurements ranged from 81 to 111% factors were proposed for the different sample matrices and from 3.0 to 9.4%, respectively. With respect to inter- to manage matrix effects and ensure operation within the assay measurements, the average recovery rate and RSDr calibration range (a dilution factor of 1000 was applied for were in the range of 80-109% and 4.5-9.0%, respectively. samples of distiller’s dried grains with solubles; 500 for maize, feed, and rice samples; and 100 for wheat samples). Recovery In the area of electrochemical immunosensors, most experiments were performed with uncontaminated maize articles published over the period from mid-2019 to mid- samples fortified with DON in the range 0.05-1.8 mg/kg. The 2020 provide proof of concept of selected electrochemical recoveries of DON were in the range of 90-105% with RSDr techniques for the detection of mycotoxins, going deeply values lower than 10%. In addition, the DON concentrations into the characterisation of the developed modified were measured with the paper-based microfluidic device in electrode. However, in most cases no optimisation of the 21 food, feed, and feed ingredient samples in comparison sample preparation protocol is reported, relying instead with a commercial DON ELISA (Elabscience Biotechnology, on previously published methods. The work by Sun et al. Houston, TX, USA). Results obtained with the two methods (2020) was selected for this review because in addition to showed linear correlation.

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A new competitive immunoassay format exploiting A further trending topic which is worth mentioning is the pressure changes generated by O2 production from H2O2 application of TRF for sensitive mycotoxin detection in was developed by Zhang et al. (2020c). The assay strategy, immunochromatography assays (TRFICA). The technique, similar to classical ELISA, employed platinum nanoparticles exploiting the fluorescence signal arising from antibodies (PtNPs) which were proposed as an alternative to or antigens labelled with lanthanide-chelate-embedded horseradish peroxidase to catalyse O2 production from nanoparticles or microbeads, provides increased detection H2O2. Gold labelled PtNPs (Au@PtNPs) and Au@PtNPs sensitivity compared with that of the traditional AuNP- conjugated with a secondary antibody (Au@PtNPs-IgG) based strip assay. TRF requires the use of an expensive were synthesised for the competitive immunoassay for detector which limits is application in field and in AFB1 detection. Au@PtNPs-IgG was used to catalyse the routine analysis. However, the development of a portable production of O2 from H2O2. The assay was conducted TRFICA reader, made commercially available in the last in a sealed microwell and the pressure of developed five years, is enhancing the TRFICA-based strip test O2 (inversely proportional to AFB1 concentration) was potential application for rapid and cost-effective analysis. recorded by a handheld barometer. AFB1 quantification A review on TRFICA applications for AFB1, AFM1, and was performed by standard calibration curve. Analytical total AF detection in different agricultural products parameters were evaluated in peanuts at three spiking has been published this year (Li et al., 2020) discussing levels (from 5 to 20 µg/kg), resulting in recoveries from potential advantages of this new technique with respect to 83 to 112%, whereas intra-assay and inter-assay variability conventional immunochromatographic assays. ranged from 2.7 to 5.8% and from 2.3 to 6.4%, respectively. A preliminary evaluation of the trueness of results generated 10. Assays with antibody analogues by the new assay was carried out by comparison with an immunoaffinity column – HPLC-based method on a set While antibodies and their analogues remain the gold of 6 maize and peanuts samples contaminated with AFB1 standard in affinity reagents for mycotoxin analysis, other in the range 4.8-21.6 µg/kg. The results generated by the alternative technologies for detection have emerged in two methods showed a satisfactory agreement. recent years, motivated by the need to address the practical limitations of antibodies (e.g. batch to batch variation, Among the newest alternative signal amplification shelf life, cost) that could be overcome with a synthetic strategies, oligonucleotides amplification via PCR-like substitute. Among the most common alternatives are reactions is a trending topic in the recent review period. aptamers and molecularly imprinted polymers (MIPs). An example is represented by the biobarcode based Aptamers are synthetic biomolecules, typically nucleic acids immunoassay developed by Chen et al. (2020). Bio-barcode but sometimes peptides, selected through a combinatorial detection involves the use of a large number of identical screening process for their affinity and specificity for a oligonucleotides as probes, which are fixed on the surface molecular target. MIPs, often known as ‘plastic antibodies’, of nanoparticles via chemical bonds. In the assay by Chen are synthetic polymers that have a pre-determined et al. (2020) this strategy was applied to the detection selectivity for a particular analyte due to the incorporation of OTA. Gold labelled nanoparticles were coated with of a molecular template during their synthesis. oligonucleotides (barcode DNA) and anti-OTA antibodies, whereas the antigen (OTA) functionalised with magnetic To rival conventional antibody-based detection systems, nanoparticles was used as competitor in the homogeneous aptamer and MIP-based detection strategies have to immunoassay. After magnetic separation from unbound exploit the inherent practical benefits of these synthetic AuNPs and target OTA, the barcode DNA eluted from equivalents: primarily, their robust nature and the low cost magnetic nanoparticle-bound AuNP was amplified via of their production. Aside from use as affinity reagents for catalytic hairpin assembly. The amplified product gave chromatographic separation, synthetic antibody analogues, a fluorescent signal which was inversely proportional to such as MIPs and aptamers, have found applications in OTA concentration in the sample. A preliminary evaluation a variety of assays and biosensor platforms. Mycotoxin of assay quantitative performances was obtained with assays that are inexpensive, have long shelf lives, and do fortified sample extracts (maize, wheat, and peanuts) not require refrigeration of components are highly desired showing high recoveries (93-109%) and good precision and could fill a niche in quick screening technology where (3-7% RSD). A deeper discussion about fitness for purpose extensive sampling is needed; here, aptamer and MIP-based and practicability of this innovative strategy compared to platforms could have a distinct advantage. traditional and commercially available assays, considering for instance the costs of the PCR amplification reaction Oftentimes, mycotoxin detection methods are devised (oligonucleotides probes, primers), complexity of laboratory where the MIP or aptamer affinity agent could simply equipment, ease of operation and requested technician skills replace the antibody in an established assay. In recent would help the reader to better understand the positioning work by Munawar et al. (2019), molecularly imprinted of this new approach and its relevant field of application. polymer nanoparticles (nanoMIPs) were explored as a

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replacement for the primary antibody in a competitive system capable of performing rapid OTA analysis with ELISA for FB1 detection. The nanoMIPs were produced only microliters of sample (Costantini et al., 2019). Their by radical polymerisation on FB1-immobilised glass beads. portable system, about the size of a small tablet computer, They were then coated in the wells of a microplate and consisted of a microfluidic chip on an optoelectronic ‘on- incubated with a horseradish peroxidase-FB1 conjugate. glass’ substrate for fluorescence detection. OTA aptamers Maize extract solution from 53 naturally contaminated immobilised within the microfluidic channels were loaded maize samples were analysed using their approach and with an intercalating fluorophore that is displaced upon the results were compared with those obtained with a OTA binding, leading to a loss of fluorescence and a commercially available ELISA (AgraQuant®, Romer Labs, decrease in the photocurrent detected by the photosensors Getzersdorf, Austria) and a reference HPLC method positioned below. Their lab-on-a-chip had an LOD of (AOAC No. 2001.04). Their analysis demonstrated that 1.3 ng/ml and was shown to be effective for the detection the nanoMIP-based ELISA showed satisfactory correlation of OTA (5-200 ng/ml) in beer and wheat samples with only in comparison to both the standard competitive ELISA minimal matrix effect noted. method as well as to HPLC. Another novel miniature sensor has been proposed based Similar to nanoMIPs, surface molecularly imprinted on a combination of MIP membranes and a smartphone, polymers are finding increased usage in mycotoxin analysis. which could be used for the field detection of ZEN in With this sub-category of MIPS, the recognition sites are cereals (Sergeyeva et al., 2020). A ZEN-selective urethane- distributed only on the surface of a substrate, avoiding the acrylate MIP membrane was used to capture the target preparation of excessive nonspecific recognition sites and and ZEN’s natural fluorescence was analysed by a Spotxel® yielding higher mass transfer rates than their bulk MIP Reader smartphone application (Sycasys Software GmbH, counterparts. An added advantage is that the substrate upon Germersheim, Germany). In its direct sensing mode the which the MIP is prepared can also facilitate target sensing. approach reached a detection limit of 126 µg/kg. The For example, fluorescent quantum dots recently served as sensitivity was improved to a detection limit of 1.26 µg/ the support for a surface MIP for the ‘turn-off’ fluorescence kg by using a competitive sensing mode with a highly- detection of AFB1 (Guo et al., 2019b). The fluorescence fluorescent structural analogue (2-[(pyrene-l-carbonyl) assay was found to have an LOD of 4 ng/g, estimated as amino]ethyl 2,4-dihydroxybenzoate). The method was also the concentration that quenched 3× the standard deviation tested against naturally contaminated maize flour, wheat of the blank signal divided by the slope of the calibration flour, rye flour, and Romer Labs-Check-Sample-Survey curve prepared in solvent. Notably and not unexpectedly, with good correlation to the established levels. Aside from there was substantial cross-reactivity to AFG1 and to cross-reactivity that was noted for α-ZOL and to a lesser 5,7-dimethoxycoumarin, the dummy template used in extent to β-estradiol, the portability, low-cost and simplicity the MIP preparation. When spiked coix seed and wheat of this MIP membrane-based smartphone sensor system seed samples were tested using the method, the recoveries made it very promising for use in the field. ranged from 99 to 102% with relative standard deviations below 1.5%. In the present format, the assay made use of Electrochemical sensing of mycotoxins is attractive given a standard spectrofluorometer, limiting its applicability the low cost and the potential for miniaturisation of the for in-field use. Nevertheless, given the combination of necessary instrumentation. MIP technology is particularly binding and detection in one particle, this approach could compatible with electrochemical detection given that be interesting to apply to a lateral flow assay format with electropolymerisation can be used to prepare the MIP a portable fluorescence reader. Furthermore, multiplexing directly on the sensor surface. Radi and colleagues recently could be achieved by combining selective MIPs on quantum reported on the impedimetric detection of ZEN (Radi et al., dots of different sizes (and consequently different emission 2020) and DON (Radi et al., 2019) using MIP-coated screen- wavelengths). printed gold electrodes, small, self-contained strips with working, reference and counter electrodes printed on a single One can also envision how the inherent stability and substrate. The prepared electrochemical sensors showed cost-effectiveness of synthetic receptors could enable the acceptable LODs (2 ng/g for ZEN and 1 ng/g for DON) preparation of assays that are less suited to antibodies given in phosphate buffer solution. In both cases, the authors their limited shelf life. Lab-on-a-chip technology aims to demonstrated accurate and selective detection of the target combine all necessary steps for chemical analysis onto a mycotoxin in spiked commercial corn flakes with mean single miniaturised device. These portable systems are recoveries ranging from 96 to 104% (RSDs from 2.1 to 3.8 %) appealing platforms for quick and sensitive mycotoxin for ZEN and 91 to 99% (RSDs between 1.8 and 3.5%) for analysis directly in the field, however the integrated DON. The ease of fabrication and low cost of screen-printed nature of these devices leads to more stringent stability gold electrodes and MIPs would facilitate the preparation of requirements for the affinity agent. Costantini and co- bulk quantities of these disposable electrochemical devices workers have prepared an aptamer-based lab-on-a-chip for rapid, on-site, real-time mycotoxin analyses.

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Lateral flow assays (LFA) and microfluidic paper-based 0.7 pg/l. The method worked reasonably well with real analytical devices (µPAD) are among the one-step, in- sample matrices, with OTA, AFB1, and FB1 recoveries in situ testing approaches that are well-suited for synthetic spiked cereals of 81±4% to 114±2%, 80±5% to 113±1%, mycotoxin receptors such as aptamers. µPADs are simple and 85±4% to 120±2%, respectively. The main challenge paper-based devices containing a detection zone and a with the method is the need for specialised equipment; sample zone interconnected by microchannels. Recently, a fibre optic spectrometer equipped with stereoscopic a µPAD colorimetric assay was developed using AuNPs microscope was needed to decode the barcodes while a coated with a 21-mer AFM1 aptamer (Kasoju et al., 2020). fluorescent microscope was used for the fluorescence The aptamer, in the absence of the target, protects the intensity measurements. Miniaturisation of components AuNPs from salt-induced aggregation, maintaining the red and use of on-glass platform described above (Costantini colour of the colloidal gold. In the presence of AFM1, the et al., 2019) for photocurrent detection could improve the aptamer dissociates from the AuNPs to bind to AFM1; this practicality of this approach. In addition, relevant measures leaves the particles susceptible to salt-induced aggregation of sensitivity, such as reporting LODs on a sample mass leading to a colour change to blue. When the µPAD basis, are needed to provide potential users with a clear was challenged with spiked milk samples, a blue colour picture of the potential of new methods. developed immediately, while in the case of the control, no colour change was observed. Detection limits of 3 pM A homogeneous fluorescence assay using fluorescent in buffer and 10 nM in spiked milk samples were reported upconversion nanoparticles and quenching gold nanorods and results could be read within 2 minutes. Lateral flow labelled with ZEN and FB1 aptamers has been developed assays are a similar platform where a sample is placed on a for multiplexed mycotoxin detection in solution (He et al., paper strip test device and the results are displayed within 2020). The aptamer-labelled gold nanorods are linked to minutes. A fluorophore-tagged aptamer with affinity for the upconversion nanoparticles through complementary AFB1 and AFB2 was employed by Zhao and colleagues as the DNA sequences to form quenched nanoassemblies. In affinity agent in a fluorescence-turn off LFA for screening of the presence of ZEN or FB1, the nanoassemblies are Type-B aflatoxins in nuts and dried figs (Zhao et al., 2020). dispersed as the aptamer strands release the complementary The test strip had an LOQ of 0.5 µg/kg when combined with sequences to interact with their cognate target, leading to a commercial strip test reader. This aptamer-based strip the recovery of fluorescence signal. The LODs of the assay was applied to spiked and commercial peanuts, almonds, for ZEN and FB1 are 0.01 ng/g and 0.003 pg/g, respectively, and dried figs, with recoveries from 93-112%. These rapid with average recoveries from spiked maize samples of 90 and instrument-free methods for first-level screening of to 107%. As described, the assay is not practical for field mycotoxin contamination are promising approaches for use given the need for a spectrofluorometer with 980 nm economical, high-throughput, on-site monitoring. laser excitation. However, recent reports of a portable upconversion nanoparticle LFA reader (Gong et al., 2019) The co-occurrence of multiple mycotoxins can result from suggests that this approach could be plausibly translated a single fungal species producing several toxic metabolites, to a convenient strip format. or several species can be present simultaneously, producing different toxins. Furthermore, a variety of mycotoxins Examples presented in this section were chosen due to could be present in mixtures of raw material samples, their potential for in-field testing. Unfortunately, while such as breakfast cereals and flours. Platforms capable of innovative and interesting from an academic point of view, low-cost multiplexed screening of multiple mycotoxins many aptamer and MIP biosensors and analytical methods is another area where synthetic antibody analogues are impractical for field use. Indeed, while many recently could find applications. Unfortunately, many reports on published approaches using these antibody alternatives multiplexed sensors using mycotoxin-binding aptamers can detect mycotoxins at low levels and with appreciable are too intricate and convoluted to be useful. Recently, specificity, the cost and practicality of many of these more an innovative approach to the simultaneous detection of exotic platforms is limiting. Continued progress towards OTA, FB1, and AFB1 was devised by labelling photonic portable and robust but inexpensive approaches, such as crystals with DNA molecular beacons that were comprised dipstick and other similar simple technology, in particular of a specific mycotoxin aptamer and a fluorophore in multi-mycotoxin format, should be the goal. binding G-quadruplex sequence (Zhang et al., 2020d). In the presence of the specific mycotoxin, the hairpin Acknowledgements structure of molecular beacons would open, exposing the G-quadruplex, and leading to fluorophore binding Mention of trade names or commercial products in this and an increase in fluorescence intensity. Examining the publication is solely for the purpose of providing specific corresponding reflection peaks of the blue, green, and red information and does not imply recommendation or barcodes allowed for identification of the specific mycotoxin endorsement by the U.S. Department of Agriculture. while the fluorescence was quantified to give LODs of USDA is an equal opportunity provider and employer.

World Mycotoxin Journal 14 (1) 21 S.A. Tittlemier et al.

The contribution of M. Stranska was supported by the Cowger, C., Ward, T.J., Nilsson, K., Arellano, C., McCormick, S.P. Ministry of Education, Youth and Sports (project No and Busman, M., 2020. Regional and field-specific differences in 8E18B045), METROFOOD-CZ research infrastructure Fusarium species and mycotoxins associated with blighted North project (MEYS Grant No: LM2018100), and projects OPPC Carolina wheat. International Journal of Food Microbiology 323: CZ.2.16/3.1.00/21537, CZ.2.16/3.1.00/24503 and NPU I 108594. https://doi.org/10.1016/j.ijfoodmicro.2020.108594 LO1601. De Santis, B., Gregori, E., Debegnach, F., Moracci, G., Saitta, C. and Brera, C., 2020. Determination of ochratoxin A in pork meat Conflict of interest products: single laboratory validation method and preparation of homogeneous batch materials. Mycotoxin Research 36: 235-241. 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24 World Mycotoxin Journal 14 (1) Developments in mycotoxin analysis 2019-2020

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based competitive indirect ELISA for determination of citrinin in oils using bare Fe3O4 as magnetic sorbents coupled with high- grain-based foods. Food Additives and Contaminants Part A 36: performance liquid chromatography with fluorescence detection. 1567-1573. https://doi.org/10.1080/19440049.2019.1640895 Journal of Chromatographic Science 58: 678-685. https://doi. Slobodchikova, I., Sivakumar, R., Rahman, M.S. and Vuckovic, D., 2019. org/10.1093/chromsci/bmaa026 Characterization of phase I and glucuronide phase II metabolites Zhang, K. and Xu, D., 2019. Application of stable isotope dilution of 17 mycotoxins using liquid chromatography-high-resolution and liquid chromatography tandem mass spectrometry for multi- mass spectrometry. Toxins 11: 433. https://doi.org/10.3390/ mycotoxin analysis in edible oils. Journal of AOAC International toxins11080433 102: 1651-1656. https://doi.org/10.5740/jaoacint.18-0252 Sun, C., Liao, X., Huang, P., Shan, G., Ma, X., Fu, L., Zhou, L. and Zhang, W., Tang, S., Jin, Y., Yang, C., He, L., Wang, J. and Chen, Y., Kong, W., 2020. A self-assembled electrochemical immunosensor 2020b. Multiplex SERS-based lateral flow immunosensor for the for ultra-sensitive detection of ochratoxin A in medicinal and detection of major mycotoxins in maize utilizing dual Raman labels edible malt. Food Chemistry 315: 126289. https://doi.org/10.1016/j. and triple test lines. Journal of Hazardous Materials 393: 122348. foodchem.2020.126289 https://doi.org/10.1016/j.jhazmat.2020.122348 Tang, Z., Liua, X., Sua, B., Chena, Q., Caoa, H., Yuna, Y., Xub, Y. Zhang, W., Wu, W., Cai, C., Hu, X., Li, H., Bai, Y., Zhang. Z. and Li, and Hammockc, B.D., 2020. Ultrasensitive and rapid detection of P., 2020c. A sensitive, point-of-care detection of small molecules ochratoxin A in agro-products by a nanobody-mediated FRET- based on a portable barometer: aflatoxins in agricultural products. based immunosensor. Journal of Hazardous Materials 387: 121678. Toxins 12: 158. https://doi.org/10.3390/toxins12030158 https://doi.org/10.1016/j.jhazmat.2019.121678 Zhang, X., Wu, D., Zhou, X., Yu, Y., Liu, J., Hu, N., Wang, H., Li, G. Tittlemier, S.A., Blagden, R., Chan, J., McMillan, T.L., Pleskach, K. and and Wu, Y., 2019. Recent progress in the construction of nanozyme- Izydorczyk, M.S., 2020b. Effects of processing whole oats on the based biosensors and their applications to food safety assay. Trends analysis and fate of mycotoxins and ergosterol. World Mycotoxin in Analytical Chemistry 121: 115668. https://doi.org/10.1016/j. Journal 13: 45-56. https://doi.org/10.3920/WMJ2019.2530 trac.2019.115668 Tittlemier, S.A., Cramer, B., Dall’Asta, C., Iha, M.H., Lattanzio, V.M.T., Zhao, Z., Wang, H., Zhai, W., Feng, X., Fan, X., Chen, A. and Wang, Maragos, C., Solfrizzo, M., Stranska, M., Stroka, J. and Sumarah, M., M., 2020. A lateral flow strip based on a truncated aptamer- 2020a. Developments in mycotoxin analysis: an update for 2019- complementary strand for detection of type-B aflatoxins in nuts and 19. World Mycotoxin Journal 13: 3-41. https://doi.org/10.3920/ dried figs. Toxins 12: 136. https://doi.org/10.3390/toxins12020136 WMJ2019.2535 Zhou, Q. and Tang, D., 2020. Recent advances in photoelectrochemical Wang, D., Zhu, J., Zhang, Z., Zhang, Q., Zhang, W., Yu, L., Jiang, biosensors for analysis of mycotoxins in food. Trends in Analytical J., Chen, X., Wang, X. and Li, P., 2019. Simultaneous lateral flow Chemistry 124: 115814. https://doi.org/10.1016/j.trac.2020.115814 immunoassay for multi-class chemical contaminants in maize and peanut with one-stop sample preparation. Toxins 11: 56. https:// doi.org/10.3390/toxins11010056 Wenzl, T., Haedrich, J., Schaechtele, A., Robouch, P. and Stroka, J., 2016. Guidance document on the estimation of LOD and LOQ for measurements in the field of contaminants in feed and food. EUR 28099. Publications Office of the European Union, Luxembourg. https://doi.org/10.2787/8931 Wilcox, J., Pazdanska, M., Milligan, C., Chan, D., MacDonald, S. and Donnelly, C. 2020. Analysis of aflatoxins and ochratoxin A in cannabis and cannabis products by LC-fluorescence detection using cleanup with either multiantibody immunoaffinity columns or an automated system with in-line reusable immunoaffinity cartridges. Journal of AOAC International 103: 494-503. https:// doi.org/10.5740/jaoacint.19-0176 Woo, S.Y., Ryu, S.Y., Tian, F., Lee, S.Y., Park, S.B. and Chun, H.S., 2019. Simultaneous determination of twenty mycotoxins in the Korean soybean paste doenjang by LC-MS/MS with immunoaffinity cleanup. Toxins 11: 594. https://doi.org/10.3390/toxins11100594 Zhang, D., Cai, L., Bian, F., Kong, T. and Zhao, Y. 2020d. Label-free quantifications of multiplexed mycotoxins by G-quadruplex based on photonic barcodes. Analytical Chemistry 92: 2891-2895. https:// doi.org/10.1021/acs.analchem.9b05213

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Appendix. List of abbreviations

3-ADON 3-acetyl-deoxynivalenol LC-MS liquid chromatography mass spectrometry 15-ADON 15-acetyl-deoxynivalenol LFA lateral flow assay AIF all ion fragmentation LFIA lateral flow immunoassay

AFB1 aflatoxin B1 LOD limit of detection AFB2 aflatoxin B2 LOQ limit of quantitation AFG1 aflatoxin G1 MIPs molecularly imprinted polymers AFG2 aflatoxin G2 MS mass spectrometry AFM1 aflatoxin M1 MON moniliformin AFM2 aflatoxin M2 MPA mycophenolic acid AFs aflatoxins MS/MS tandem mass spectrometry AgNP silver nanoparticles nanoMIPs molecularly imprinted polymer nanoparticles AME alternariol monomethyl ether NIR near-infrared APCI atmospheric pressure chemical ionization NIV nivalenol AuNP colloidal gold nanoparticles NIV-3G nivalenol-3-glucoside BEA beauvericin NTA non-targeted analysis CIT citrinin OTA ochratoxin A CTV citreoviridin OTB ochratoxin B CRM certified reference material µPAD microfluidic paper-based analytical device CPA cyclopiazonic acid PCR polymerase chain reaction DDA data-dependent acquisition PT proficiency test DIA data-independent acquisition PtNP platinum nanoparticle DLLME dispersive liquid to liquid microextraction Q-ToF quadrupole-time of flight DOM-1 de-epoxydeoxynivalenol QuEChERS quick, easy, cheap, effective, rugged, and safe DON deoxynivalenol ROC roquefortine C DON-3G deoxynivalenol-3-glucoside RSD relative standard deviation ELISA enzyme-linked immunosorbent assay SERS surface-enhanced Raman spectroscopy ENN A enniatin A SPE solid-phase extraction ENN A1 enniatin A1 SPR surface plasmon resonance ENN B enniatin B SSE signal suppression/enhancement ENN B1 enniatin B1 T-2 T-2 toxin EU European Union TRF time-resolved fluorescence

FB1 fumonisin B1 TRFICA time-resolved fluorescence immunochromatography FB2 fumonisin B2 assays FB3 fumonisin B3 TTX tentoxin FPIA fluorescence polarization immunoassay UHPLC ultra-high pressure liquid chromatography FUS-X fusarenone-X UV ultraviolet GC gas chromatography ZAN zearalanone HCl hydrochloric acid α-ZAL α-zearalanol HPLC high-performance liquid chromatography β-ZAL β-zearalanol HRMS high resolution mass spectrometry ZEN zearalenone HT-2 HT-2 toxin α-ZOL α-zearalenol iSPR imaging surface plasmon resonance β-ZOL β-zearalenol

IC50 half maximal inhibitory concentration

26 World Mycotoxin Journal 14 (1)