Mycotoxins are secondary metabolites that are produced by microfungi which are capable of causing disease and death in humans and other animals (Bennett and Klich, 2003).

According to a risk assessment overview provided by the European Food Safety Authority (EFSA) (EFSA CONTAM Panel) related to the main contaminants in food and feed, mycotoxins represented 15% of the overall risk for human and animal health, for the period between 2003 and 2012 (EFSA, 2012).

Mycotoxins are produced by several fungal species and they can affect agricultural commodities, causing worldwide yield and economic losses (Steyn, 1995), due to their negative impact on the safety and quality of these commodities.

Five mycotoxin classes are considered to be of great economic and toxicological importance for grain in several areas throughout the world: aflatoxins (AFBs) and ochratoxin (OTA), produced by the Aspergillus and Penicillium genera, deoxynivalenol (DON), zearalenone (ZEA), and B-fumonisins (FBs), which are mainly produced by Fusarium species (Atkins and Norman, 1998).

Among the agricultural commodities affected by mycotoxins, cereals are the most contaminated (Placinta et al., 1999) and in particular maize and wheat, which, in temperate areas, could mainly be affected, in field conditions, by fungal disease caused by several Fusarium species (Logrieco et al., 2002). These Fusarium species are the main cause of FB and type-B trichothecene DON production and accumulation, in maize and in wheat, in temperate areas.

The incidence and the concentration of different mycotoxins is clearly variable over the years and from area to area through the world. In fact, mycotoxin contamination depends on the co-existence of host susceptibility and environmental conditions that are favorable to fungal infection, growth and toxinogenesis (Munkvold, 2003). Unfortunately, since the same plant tissue may be colonized by various mycotoxigenic species, it is possible that several mycotoxins could co-occur in the same food or feed matrix, with consequent possible additive or synergic toxicological effects due to their co-presence (Sanhueza and Degrossi, 2004).

The toxicological effects of the mycotoxins that are regulated by the European Commission [EC] (2007, 2010) are well known, but only few data are available in literature about toxicological studies of their combined toxicity. Lee and Ryu (2017) summarized the most relevant early studies that reported additive or synergistic effects due to the co-occurrence of mycotoxins and their interactive toxicity.

Any other mycotoxins and fungal metabolites which till now have not received detailed scientific attention, are commonly referred to as “novel” or “emerging” mycotoxins such as moniliformin (MON), enniatins (ENNs), nivalenol (NIV), etc. (Streit et al., 2013). Furthermore, in addition to the emerging mycotoxins it is also important to remember the “masked” mycotoxins which are the fraction of biologically modified mycotoxins that were conjugated by plants resulting from plant defense reactions after fungal infection which can have higher toxicity (i.e., 15-acetyldeoxynivalenol (15-ADON)) or be released, hydrolyzed, biotransformed and absorbed (i.e., deoxynivalenol-3-glucoside (DON-3-G)) in the gastrointestinal tract, primarily as the parent compound and should be considered an additional contributing factor of the total dietary exposure to the native form (i.e., DON) (Berthiller et al., 2013). The EFSA is currently working on gathering data for establishing a scientific opinion on the risks to public health related to the presence of emerging and masked mycotoxins in feed and food (EFSA, 2014, 2017, 2018).

Little is known about the toxicological effects of these compounds, some of which could be potentially toxic to humans and livestock. The main risks are in particular related to their additive or synergistic effects, if present together with regulated mycotoxins, for which their toxicological effects have mainly been defined regarding their individual presence.

In order to avoid an underestimated increased level of risk for the end-consumer, there is an urgent need to develop accurate, precise and sensitive multi-residue analytical methods but which are also fast and easily applicable in routine analysis of both regulated and emerging mycotoxins in order to acquire data on their co-occurrence.

So far, due to the different chemical properties of mycotoxins, their routine analysis has usually been performed through the determination of single compound one by one or, of certain classes of mycotoxins, through multi-determination by means of HPLC coupled with non-confirmatory UV or fluorescence detectors (Krska and Molinelli, 2007). For these reasons, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become a prominent tool for multiresidue analysis over the last few years, as pointed out in the updates published by Berthiller et al. (2015, 2016, 2017). Since LC-MS/MS methods suffer from matrix effects, the most common difficulties in developing multiresidue methods concern finding a compromise between reducing the matrix effects as much as possible, using different clean-up procedures, and increasing the number of the mycotoxins surveyed over a wide range of polarities. Most of the multiresidue LC-MS/MS methods reported in literature have adopted an intensive clean-up with sequential solid-phase extraction (SPE) (Biselli and Hummert, 2005; Klötzel et al., 2006), which limits the number of surveyed mycotoxins and is highly time-consuming, or other clean-up procedures such as accelerated solvent extraction (ASE) (Royer et al., 2004), the QuEChERS-like method (Desmarchelier et al., 2010), or the more versatile dilute-and-shoot type methods (Sulyok et al., 2006; Spanjer et al., 2008; Beltrán et al., 2009; Malachova et al., 2014), that offer the opportunity of extending the number of surveyed mycotoxins by reducing or even avoiding the sample clean-up, but could compromise the quantification (LOQ value). In addition, the applicability of all the previously described clean-up procedures, except for the dilute-and-shoot approaches, to the simultaneous analysis of ENNs and MON has yet to be proven, and is probably not feasible (Sulyok et al., 2006).

In this scenario, the current research is aimed at investigating the applicability of the developed multiresidue analytical methods to a high-throughput routine analysis for the screening and confirmation of the main EU-regulated and most relevant emerging mycotoxins to which EFSA has paid attention through the use of a TQ. For this purpose, two fast, reliable and repeatable multiresidue HPLC-ESI-MS/MS methods were developed and compared for the quantitative determination of: AFB₁, AFB₂, AFG₁, AFG₂, 3-acetyldeoxynivalenol (3-ADON), 15-ADON, DON, DON-3-G, enniatins A, A₁, B, B₁ (ENNA, ENNA₁, ENNB, ENNB₁), FB₁, FB₂, MON, NIV, and ZEA in maize and wheat.

Multiresidue LC-MS/MS methods have become a fundamental tool to investigate the overall contamination as a result of the co-occurrence of mycotoxins in the same sample, and to provide important data to design new, combined toxicological and exposure assessments.

Over the last few years, several multi-mycotoxin LC-MS/MS methods have been proposed in literature (Sulyok et al., 2006; Ren et al., 2007; Frenich et al., 2009; Desmarchelier et al., 2010; Malachova et al., 2014; De Santis et al., 2017) for different targeted mycotoxins and uses.

In the present experiment, it was decided to compare two modified sample preparation procedures that were considered to limit the preparation steps of the sample as much as possible with the aim of being faster, and therefore applicable for routine analysis, and of guaranteeing a powerful and reliable tool to simultaneously analyze multiple mycotoxins in the same feed and food matrix. The mycotoxins that were chosen for inclusion in the developed methods were both the regulated mycotoxins that are commonly found in cereals and the main emerging and masked mycotoxins to which EFSA has paid attention and requested occurrence data, that is, MON, DON-3-G, 3-ADON, 15-ADON, ENNs (EFSA, 2014, 2017, 2018). To date, the inclusion of the previous mycotoxins in a multi-mycotoxins method together with the regulated mycotoxins was only be proposed in few researches in which the analysis was carried out by more advanced and performing mass spectrometers (Sulyok et al., 2006; Malachova et al., 2014).

To the best of the authors’ knowledge, this is the first time that Oasis® PRiME HLB columns have been applied for the clean-up in a multiresidue analysis of the mycotoxins considered in the present survey. Moreover, their use was always associated with other steps, such as QuEChERS extraction and dSPE steps, which allow a greater purification of the matrix from proteins, starch and polar sugars, but which also lengthen the analytical time and increase the propagation of errors. As a result of the elimination of these two steps, the procedure applied in the present survey has become more comparable with the dilute-and-shoot method, while ensuring shorter analytical times and satisfactory results in terms of precision, accuracy and reliability. Although Desmarchelier et al. (2010) reported that a defatting step with n-exane, followed by a two-step sequential reconstitution in CH₃OH/H₂O, could be a valid alternative to Oasis® HLB columns, Oasis® PRiME HLB columns remain one of the best choices to move further toward green analytical chemistry, or at least to limit the use of chemical reagents during the analytical steps, if a fast clean up step in the sample purification method is desired.

As far as chromatographic separation is concerned, the HPLC mobile phases commonly used for mycotoxin analysis are composed of CH₃OH and H₂O acidified with acetic or formic acid and/or buffered with acetate or a formate buffer. CH₃OH is usually favored over CH₃CN, for sensitivity reasons (Berthiller et al., 2005). These acids are generally added in order to increase the ionization efficiency in the ESI source and to obtain a better peak shape for acidic compounds, whereas a buffer could aid the chromatographic separation and the sensitivity of some compounds that show more attitude to form adduct ions (Sulyok et al., 2006). No buffer additions have been considered in the developed methods because they were shown to cause ESI ion suppression of some of the detected mycotoxins. Furthermore, although other studies (Delmulle et al., 2006; Sulyok et al., 2006) reported the use of 5 mM ammonium acetate in the mobile phases, their results showed high noises in chromatograms, similarly to those obtained in the present survey. It was instead considered essential to acidify both of the mobile phases in order to increase the overall sensitivity and to obtain a better peak shape of FB₁ and FB_2. All mycotoxins were detected, through a fast determination, in 28 min applying two separate chromatographic runs, one in negative and one in positive ionization mode acquisition, took 15 min and 13 min, respectively. However, it was not possible to avoid the co-elution of some mycotoxins and, as previously reported (Delmulle et al., 2006; Sulyok et al., 2006), the co-elution was accepted because the related compounds show different transitions.

The recovery percentage range established by Regulation (European Commission [EC], 2006). No. 401/2006 was respected for all the legislated mycotoxins determined through the use of both of the sample preparation methods and for both cereal matrices, except for ZEA when the Oasis® PRiME HLB method was used. The low R_E values of FB₁ in the wheat are of limited relevance for the evaluation of the applicability of the methods, because fumonisins are the main maize mycotoxins and they were never found on the wheat, if not in traces. The recovery values were in agreement with previous report (Delmulle et al., 2006; Sulyok et al., 2006; Spanjer et al., 2008). Moreover, the application of both of the developed methods to the analysis of the maize and wheat reference materials, certified for their mass concentrations of DON in wheat and of AFBs, DON, ZEA, and FBs in maize, showed that the experimentally determined concentrations were in agreement with the certified values for both methods, and even revealed and quantified the presence of several other mycotoxins within these matrices, thus confirming a real and concrete occurrence of some of these mycotoxins.

The quantification was conducted by means of matrix-matched calibration curves, because they were considered the best alternative to the too expensive isotopically labeled internal standard (which was not available for all the analyzed mycotoxins) to compensate for both losses during extraction and matrix effects generated during the ionization of the analytes. This quantification procedure, although tested successfully, still requires the availability of matrices that are free of any of the surveyed mycotoxins, or with a contamination as low as possible.

The LOQ values assessed for AFB₁, AFB₂, AFG₁, and AFG₂ were high compared to other methods (Sulyok et al., 2006; Malachova et al., 2014; De Santis et al., 2017), and this was above all due to the high dilution factor of the two methods (eight for the dilute-and-shoot method and four for the Oasis® PRiME HLB method), that is not compatible with not-last-generation TQ, due to its lower instrumental sensitivity than other mass spectrometers.

The two developed analytical methods offer different advantages and suffer from different disadvantages. The main advantages of the dilute-and-shoot method are the low costs and the extremely reduced sample preparation times, while the main disadvantage is related to the instrumental detection limit that can be achieved with a not-last-generation TQ. On the other hand, the main advantage of the clean-up method that uses the Oasis® PRiME HLB columns is a higher purification level of the sample, which protects the instrument more from becoming dirty, while the main disadvantage is that, in order to obtain the same results and analytical performance for the different determined mycotoxins, an additional cost is foreseen for the clean-up columns.

Nevertheless, in conclusion the results suggest that the two analytical methods and both of the sample preparation techniques considerably reduce the analytical time and costs and therefore result to be both promising and applicable for high-throughput routine analysis through the use of a TQ, in order to collect data on the co-occurrence of both regulated and the most relevant among emerging and masked mycotoxins in cereals.

All datasets generated for this study are included in the manuscript and/or the supplementary files.

VS and MB conceived and designed the experiments. VS performed the experiments and run laboratory analysis. VS analyzed the data. VS contributed reagents, materials, and analysis tools. VS wrote the main manuscript text. All authors reviewed the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.