Hyphenated chromatographic techniques for structural characterization and determination of masked mycotoxins

Journal of Chromatography A, 1255 (2012) 145–152

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Review

Hyphenated chromatographic techniques for structural characterization and determination of masked mycotoxins M. Cirlini, C. Dall’Asta, G. Galaverna ∗ Department of Organic and Industrial Chemistry, University of Parma, Parco Area delle Scienze, 17/a, 43124 Parma, Italy

a r t i c l e

i n f o

Article history: Available online 3 March 2012 Keywords: Deoxynivalenol-3-glucoside Zearalenone-14-glucoside Hidden fumonisins Zearalenone-14-sulphate

a b s t r a c t Mycotoxins are secondary metabolites produced by fungi that can contaminate a wide range of food and feed commodities and that are harmful to humans for their poisonous and toxic effects. An increasing amount of data have been accumulated in the last years, showing that mycotoxins may also occur in modified forms originating by plant, fungi or animal metabolism or by food processing. In particular, this modified forms may be produced via conjugation with sugars or other biological components (masked mycotoxins) or may occur as non extractable forms on account of strong interaction, association or binding with macromolecules in the food matrix (bound or hidden mycotoxins). Analytical methods have been set up in order to check for the occurrence of these forms and to evaluate their amount, in order to obtain reliable data for toxicity and exposure studies. In this paper hyphenated chromatographic methods for the determination and structural characterization of masked mycotoxins are reviewed, with a particular emphasis on liquid chromatography–(tandem) mass spectrometry as the most effective approach for their determination. © 2012 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

4.

5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analytical determination of masked mycotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extraction and clean up methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Deoxynivalenol, zearalenone and derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Extraction procedures for DON and ZEN derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Fumonisins and hidden fumonisins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Extraction procedures for fumonisins and hidden fumonisins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatographic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Chromatographic analysis of deoxynivalenol, zearalenone and derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Chromatographic analysis of fumonisins and hidden fumonisins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Strategy to detect unknown conjugated mycotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. High resolution mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Mycotoxins are secondary metabolites produced by filamentous fungi in crops or during storage, transport and processing of food and feed commodities, which may exert severe toxic effects toward humans and animals upon ingestion [1]. Among the different fungal genera producing mycotoxins, the most frequently found in food

∗ Corresponding author. Tel.: +39 0521 906196; fax: +39 0521 905472. E-mail address: [email protected] (G. Galaverna). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2012.02.057

145 146 146 146 148 149 149 149 150 151 151 151 151 152

products are Aspergillus, Fusarium, Penicillium and Alternaria. On account of the important economic and social impact of this problem, a large number of countries have nowadays regulations for maximum level of the most important mycotoxins, namely aflatoxins (AFs), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FBs), and patulin (PAT). In addition to these well known food contaminants, a growing interest has aroused in the last years toward the so called masked mycotoxins, which are mycotoxin derivatives that usually escape routine analysis on account of their different chemical behaviour in comparison to their parent compounds. In particular, these

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derivatives are mainly produced by plants via enzymatic transformations related to detoxification processes (phase II metabolites) [2,3] and have been related to a resistance mechanism exerted by plants to counteract pathogen invasion [4–6]. In addition, chemical transformations of mycotoxins may also occur during food processing and/or fermentations. This topic has been the subject of a recent review, describing the most important masked or bound mycotoxins which have been identified up to now [7]. Masked mycotoxins may occur in conjugated forms, usually formed via reaction of the parent compounds with sugars or amino acids. In addition, modification of parent compounds may take place via covalent binding or non-covalent association to macromolecules, such as starch and proteins, within the food matrix. Soluble low-molecular weight derivatives can be detected by appropriate analytical methods when their structure is known and analytical standards are available. On the contrary, masked mycotoxins bound or associated to solid matrices cannot be directly detected, as they have to be previously cleaved from the matrix by chemical or enzymatic treatment [8]. As far as the toxicological aspects are concerned, the masking phenomenon may lead to a loss of toxicity, in the case that bound forms cannot be actually released in the digestive system [9]. On the other hand, other derivatives have been proven to be partially or totally cleaved under gastrointestinal conditions, being thus able to exert the same toxic effects reported for their parent compound [10,11]. Finally, masked mycotoxins may also show an own toxicity, although very few is known so far about their potential bioactivity. For all these reasons, a classification of these conjugates under a toxicological perspective is very cumbersome and requires further investigation, leading to the necessity to include the occurrence of masked mycotoxins in risk assessment studies in order to avoid possible underestimation of the total exposure. Concerning the different derivatives, plant metabolites have been identified so far mainly for ZEN and DON. In particular, zearalenone-14-glucoside (ZEN14G) [12] and deoxynivalenol3-glucoside (DON3G) [13–19] along with the two acetylated derivatives of DON (15-acetyldeoxynivalenol, 15ADON and 3acetyldeoxynivalenol, 3ADON) [20] have been proven to frequently occur in naturally infected cereals such as wheat, barley and maize. Besides them, also zearalenone-14-sulphate (ZEN14S) was described in cereal-based products [21]. There are also strong evidences for the compartmentalization of fumonisins in plants (hidden fumonisins); occurrence of these hidden forms has been proven in raw maize as well as in derived food, although the nature of the masking mechanism has not been completely clarified [8,22,23]. The most frequently occurring masked mycotoxins in foods are reported in Fig. 1. The occurrence of masked mycotoxins generally implies a possible underestimation of the mycotoxin contamination level of a certain commodity, as the structural changes induce also changes in the physicochemical properties, such as solubility and polarity, with a consequent possible difference in the suitable experimental conditions for extraction and chromatographic analysis. In addition, eventual derivatives covalently bound to macromolecules such as starch and protein are completely lost during analysis without the application of a proper cleavage step (e.g. chemical or enzymatic hydrolysis). Similarly, associative complexes formed by mycotoxins and bioorganic macromolecules are actually partially or totally stable under the commonly applied mycotoxin extraction conditions, which mainly involve the use of methanol/water or acetonitrile/water, leading also in this case to an incomplete recovery of the target compound. All these effects lead to an underestimation of the total mycotoxin content in the sample; the subsequent failure to recognize hazardous contamination by mycotoxin monitoring implies the

potential exposure of the consumer to doses exceeding tolerable limits. A solution to this problem is to extend current analytical methods for mycotoxins by including conjugates and other relevant derivatives. For all these reasons, in the last years several methods mainly based on mass spectrometry have been developed for the determination of masked mycotoxins in food and feed [7]; although a discussion on the applicability of MS technique to this subjected has been reported, the rapid outburst of new papers in the last years and the fast growing number of studies on this subject justify the preparation of this review that will address the application of modern hyphenated techniques to masked mycotoxin determination, as well as the discussion of the main clean up approaches, although the general tendency is to avoid any clean up steps exploiting the selectivity and sensitivity of MS technique. Moreover, a paragraph will be devoted to the use of high resolution mass spectrometry techniques for the structural elucidation of masked mycotoxins. 2. Analytical determination of masked mycotoxins On account of the variety of their very different chemical structures and the heterogeneity of food and feed matrices, determination of mycotoxins often implies the use of a wide range of analytical approaches and techniques. Moreover, analytical methods for qualitative and quantitative determination of mycotoxins are in general multistep processes, involving appropriate sampling plans and sample preparation (generally including an homogenization step, due to the non uniform distribution of the toxin within the matrices), extraction of analytes from the matrix (usually with mixtures of water and polar organic solvents), clean up procedures and, sometimes, concentration of the extract before the final detection and quantitative determination [24]. Nevertheless, nowadays many analytical methods are available and many others are continuously improving for the determination of the most important mycotoxins [25,26]. Concerning masked derivatives, a further complication is due to the possible occurrence of unknown masked or conjugated forms and the general unavailability of chemical standards. Generally, two different approaches may be followed for the determination of these substances: (a) use of a chromatographic technique able to directly detect them, considering that most conjugated forms are generally more polar than their parent compounds (direct methods); and (b) conversion of the conjugated mycotoxins into their parent forms before the analysis, applying different treatments, usually during the extraction step (indirect methods) [8]. In the following paragraphs, the different approaches currently used for the extraction, the eventual clean up, the separation and the detection techniques are described. The available analytical methods currently used for the determination of masked mycotoxins and discussed below are summarized in Table 1 along with their performance parameters. 3. Extraction and clean up methods 3.1. Deoxynivalenol, zearalenone and derivatives Deoxynivalenol (DON) and zearalenone (ZEN) are produced by several species of Fusarium sp. and are associated with Fusarium head blight in wheat. Conjugation with glucose converts DON into DON3G [14] and ZEN into ZEN14G [27]. Biotransformation of ZEN may also accumulate unconjugated metabolites, such as ␣zearalenol (␣-ZOL) and ␤-zearalenol (␤-ZOL), produced by several microorganisms [28], by mammals [29], or by plants [30], which can be glycosilated too (␣-ZOLG and ␤-ZOLG, respectively) [30]. Plants and fungi can also convert ZEN into ZEN14S [11,30,31]. All these modified toxins might contribute to the overall toxicity of the

Table 1 Scheme of the available analytical methods and their performances for the determination of masked mycotoxins. Matrix

Extraction mixture

Clean up procedure

Separation technique

Detection technique

Recovery

Ref.

DON, 3ADON

Cereal (wheat and corn) based food Wheat, maize kernels

Acetonitrile/water, hexane

Florisil column; TMS derivatization

GC: DB-5 column (linear temperature ramp)

Electron impact MS: SIM modality

85–88%

[49]

Acetonitrile/water (84/16)

Mycosep 230 column

HPLC (RP-C18): methanol/water (15/85 isocratic)

59–80%

[12]

ZON, Z14G, Z14S

Arabidopsis thaliana

Acetonitrile/water (75/25)

None

100%

[29]

DON, 3ADON, 15ADON, D3G, ZON, Z14G, Z14S, HFB1

Cereals

Acetonitrile/water (acetic acid)

None

QTrap-MS/M: ESI source, MRM mode (positive and negative ionization)

Not reported

[23,48]

DON, D3G

Wheat

Acetonitrile/water (84/16)

C18 column

UV-MS

70–94%

[36]

DON, ZON, D3G, 3ADON, Z14G, Z14S

Beer, porridge, pasta, corn flour

Acetonitrile/water (neutral 80/20; 80/20 1% acetic acid, polar 40/60, 40/60 1% acetic acid)

QTrap-MS/M: ESI source (negative ionization); APCI (SRM mode)

0–105% (depending on cleanup method)

[40]

DON, ZON, D3G, 3ADON, Z14G, Z14S

Cereal based food

Acetonitrile/water (acetic acid)

C18-SPE, DON-prep, DON-test, Easi-Extract Zearalenone, Zearatest, ZearaSTAR, MycoSep 226, MycoSep 230, PSA None

HPLC (RP-C18): methanol/5 mM aqueous ammonium acetate (linear gradient) HPLC (RP-C18): methanol/water/acetic acid (in different proportions for eluent A and B) added with ammonium acetate 5 mM HPLC (RP-C18): methanol/water 70/30 isocratic HPLC (RP-C18): acetonitrile/water added with ammonium acetate 5 mM (linear gradient)

UV-Q-Trap MS/MS (APCI or ESI source, negative mode, MRM acquisition) QTrap-MS/M: ESI source, MRM mode

DON, D3G, 3ADON, ZON, FB1, FB2, FB3

Certified matrix, wheat, barley Beer and malt

Acetonitrile/water (0.1% formic acid)

None

Deionized water, acetonitrile/water (84/16)

IAC column: DONPREP

HFB1, PHFB1, PHFB2

Corn flakes

C18 and Fumoni Test column

HFB1

Corn based food

HFB1, HFB2, HFB3

Corn based food

2 N aqueous potassium hydroxide, after methanol/acetonitrile/water (25/25/50) and 1% SDS steps, for the hydrolysis; ethyl acetate for extraction. Methanol/acetonitrile/water (25/25/50), 1% SDS, 2 N KOH, then methanol/ethylendiamine tetracetic acid or methanol/acetonitrile/water (25/25/50) 2 N NaOH or KOH after water/acetonitrile/methanol step. Extraction: ethyl acetate or acetonitrile

DON, D3G, 3ADON, 15ADON

DON, D3G, 3ADON, 15ADON

HPLC (RP-C18): acetonitrile/water added with ammonium acetate 5 mM (linear gradient) UPLC (RP-C18): ammonium formate/methanol (linear gradient) UPLC (RP-C18): Ammonium formate/methanol (linear gradient) HPLC (RP-C18): aqueous formic acid (1%) and acetonitrile/methanol (1/1) (linear gradient)

QTrap-MS/M: ESI source (negative ionization)

Not specified

[20]

TOF-MS (ESI source, positive and negative mode); Orbitrap-MS TOF-MS (ESI source, negative mode), Orbitrap-MS Fluorescence detection/ESI-MS/MS: positive ionization, SIM mode

>70%

[38]

88–99%; 74–91%

[37,39]

63–86%

[45]

FumoniTest IAC column; OASIS HLB column

HPLC (RP-C18): aqueous formic acid (1%) and acetonitrile/methanol (1/1) (linear gradient)

Fluorescence detection/ESI-MS/MS: positive ionization, SIM mode

76–83%

[5]

None

HPLC (RP-C18): water and methanol acidified (0.1–0.2%) with formic acid

ESI-MS/MS: positive ionization, MRM mode

92–98%

[23,47,48]

M. Cirlini et al. / J. Chromatogr. A 1255 (2012) 145–152

Analytes of interest

147

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M. Cirlini et al. / J. Chromatogr. A 1255 (2012) 145–152 O

OH

O

HO

O

O

HO

OH

CH3 O

HO

O

CH3 O

S O

OH

O O

O

OH

Zearalenone -14-glucoside

Zearalenone -14-sulphate

HO H3C

O

H H O

O

OH

O O

OH OH

OH HO

Deoxynivalenol-3-glucoside Fig. 1. Structure of the most frequently occurring masked mycotoxins in food.

ingested food as the attached group might be cleaved by enzymes during transit into the gastrointestinal tract releasing the native toxin. As an example, ZEN14G has been found to be converted to ZEN during ingestion of contaminated feed in swine [10] and ZEN14S expresses hyperestrogenic activity to rats probably as a result of hydrolytic release of ZEN during digestion [11]. ␣-ZOL and ␤-ZOL can be produced by mammals after consumption of ZEN [32] and are able to bind to the mammal estrogen receptor, ␣-ZOL being the most effective [33]. Also, recently it was demonstrated that DON3G is at least partly cleaved to DON by various intestinal bacteria [34]. In the last few years, several investigations on the contents of masked mycotoxins in cereals or cereal-based food have been performed [4–6,9,12–14,35,36]. 3.1.1. Extraction procedures for DON and ZEN derivatives Usually, the extraction procedure of masked mycotoxins requires the use of a solvent mixture as for their native counterparts: mostly used are water, methanol and/or acetonitrile, in different percentages. Extraction mixtures for the determination of deoxynivalenol (DON) and zearalenone (ZEN) and their, respectively, natural occurring masked forms, DON3G, 3ADON, 15ADON, ZEN14G and ZEN14S, are based on acetonitrile and water in different ratios [13,30,37,38], eventually acidified with acetic acid [8,21,24] or formic acid [39]. Also pure deionized water has been described as extraction solvent [40]. Since the glycosilated forms of ZEN and DON are more polar than their parent compounds, Vendl et al. [41] studied the efficiency of different extraction mixture in neutral, acidic, neutral polar and acidic polar conditions, using maize samples spiked with multi-mycotoxin standard solutions containing DON, ZEN, DON3G, 3ADON, ZEN14G and ZEN14S. Results showed that neutral and acidic mixtures lead to comparable recoveries for most analytes, while higher water content causes lower recoveries for some analytes (ZEN and ZEN14S), in particular when acid polar mixtures were applied. An improved recovery for DON and ZEN was obtained choosing acidic extraction conditions: after optimization, the mixture acetonitrile/water/acetic acid in the ratio 79/20/1 was found to be the best for determination of different mycotoxins in wheat and maize. After solvent extraction, several methods for the determination of DON, ZEN and their masked derivatives involve some cleanup steps before the chromatographic analysis: these are generally based on the use of multifunctional column, immunoaffinity

column, solid-phase extraction, etc. [15] and are applied with the aim of enhancing signal to noise ratio for extracts obtained by complex matrices with heavy matrix effects. Berthiller et al. [13] used MycoSep 230 column for cleanup of DON3G for the investigation of its natural occurrence in Fusarium infected wheat and maize obtaining a recovery of 59 ± 11%. Sasanya et al. [37] obtained a recovery of 70% for the same analyte with a purification step on C18 cartridge for the quantification of DON and DON3G in hard red spring wheat and Kadota et al. [42] obtained good recoveries ranging from 84% to 115% using Multisep #227 cleanup for trichothecenes, derivatives and precursors including DON3G. Moreover, several authors obtained also good recovery for D3G by using immunoaffinity columns [39]. Vendl et al. [41] tested two different cleanup approaches for DON, DON3G, 3ADON, ZEN, ZEN14G and ZEN14S: the first, using C18-SPE columns or primary and secondary amine cartridges (PSA); the second, using immunoaffinity columns specifically dedicated for DON and ZEN, respectively. In particular, several immunoaffinity stationary phases were tested, in order to check for the ability of antibodies to retain the masked forms. Flow-through cartridges (MycoSep) were also checked for their applicability to multianalyte purification in a certain polarity range. Results showed that immunoaffinity columns, in the vast majority of cases, did not permit to efficiently recover masked mycotoxins on account of the inability of antibodies to retain the modified mycotoxins. Concerning SPE-based cartridges, the authors showed that push-through cleanup cartridges (MycoSep 226 and 230) allowed to recover only 3ADON in an exhaustive percentage, while C18-SPE allowed for a better recovery of 3ADON, ZEN14G but the percentage of ZEN14S was very low and DON3G was not recovered at all. Finally, PSA cartridges showed recovery percentages similar to those obtained without any cleanup procedure. In any case, the wide range of polarity of the considered analytes did not allow the adoption of a single cleanup strategy. For this reason, the final conclusion of the authors was to avoid any clean up step and to directly use raw extracts for the analysis, exploiting the sensitivity and selectivity of mass spectrometer detection. Indeed, the same authors [21] used such an approach for the analysis of DON, DON3G, 3ADON, ZEN, ZEN14G and ZEN14S in cereal-based food without any cleanup procedure. Nevertheless, very recently, an UPLC–TOFMS method for the determination of DON, DON3G and 3ADON in beer based on clean up step on DON specific immunoaffinity column was presented [40], which allow very good recovery values for the selective

M. Cirlini et al. / J. Chromatogr. A 1255 (2012) 145–152

isolation of all the three analytes simultaneously. This finding is peculiar for this type of immunoaffinity column (DONPREPTM ) and probably depends on the characteristics of these antibodies which show important cross-reactivity not generally found with other type of commercially available immunoaffinity columns. Indeed, cross reactivity of DON and its masked forms ADONs and DON3G have been previously investigated also for a number of commercially available ELISA kits [39], and, although potentially the use of kits contributed to overestimate mycotoxin quantities, other crossreacting interferences may be at the base of the phenomenon. The cross-reactivity of antibodies in commercially available test kits and immunoaffinity columns for DON and ZEN detection has been recently furtherly investigated [43,44]. In particular, DON IACs showed cross reactivity for several DON derivatives, including DON3G, while ZEN IACs showed only limited cross-reactivity for some ZEN derivatives. Although potentially very interesting as sample cleanup strategies to recover all the possible derivatives, the cross reactivity of the produced batches has to be regularly evaluated. Apart from these last examples, most emergent methods are based on simple and rapid extraction procedures coupled to very selective MS hyphenated chromatographic analysis without any cleanup steps. In this context, a modified QuEChERS (quick easy cheap effective rugged and safe) sample preparation procedure, based on the partition between water and acetonitrile induced by the addition of mixture of salts (NaCl and MgSO4 ), was recently proposed in combination with UPLC/TOF-MS instrumentation for the analysis of Fusarium mycotoxins in cereals. Although the proposed approach was very effective for the extraction and purification of several mycotoxins with good recovery values, unfortunately, DON3G showed poor recovery value, probably on account of its high polarity which does not effectively allow the transfer into the acetonitrile layer [39]. 3.2. Fumonisins and hidden fumonisins Fumonisins are relatively heat stable up to 100 ◦ C, although it is known that processing induces significant decrease of the detectable mycotoxins: chemical degradation may take place via Maillard-type reactions at high temperature or hydrolysis via loss of the two tricarballylic moieties in the presence of alkali. Indeed, hydrolysed fumonisins frequently occur in thermally treated plus alkali-treated products (tortillas and other nixtamalized products). Recent results have shown that their decrease might be due not only to chemical degradation but also to possible modifications of the mycotoxin structure by interaction with other food components, especially in thermally extruded products (corn flakes) [22]. This fact was ascribed to the existence of interactions of FB1 with food macroconstituents such as protein or starch, which lead to non detectable “hidden forms”. Thus, according to these hypotheses, the loss of fumonisins during heat processing may be ascribed to binding to proteins [45]. Indeed, several publications have demonstrated the presence of fumonisins potentially bound or strongly associated with proteins or other food components, which escape conventional analysis and can be determined only in an indirect way through the application of a hydrolysis step [8,23,46–48]. Thus, it has been observed that performing alkaline hydrolysis of contaminated corn products (especially, extruded products such as corn flakes) the amount of released hydrolysed fumonisins was often higher than that stoichiometrically derived by the conversion of the fumonisins detectable by the routine analytical methods. In this context, several authors have suggested that, besides thermal effect that could give rise to covalent bond formation, also another masking phenomenon could be operative, based probably on a physical entrapment of the mycotoxins within the structure

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of macromolecular components (such as starch) [8,46], which may have strong influence on the accuracy of fumonisin measurement. Thus, other masking mechanisms such as complexation or physical entrapment should be taken into account for the evaluation of the occurrence of hidden fumonisins in non thermally or mildthermally treated foods. In particular, Dall’Asta et al. reported for the first time the occurrence of hidden fumonisins in raw maize, suggesting that such non covalent interactions were responsible for the phenomenon [23]. 3.2.1. Extraction procedures for fumonisins and hidden fumonisins The general approach to the evaluation of fumonisin hidden forms is based on an alkali treatment of the sample which induces the loss of the tricarballylic side chains of fumonisins, releasing the hydrolysed fumonisins (HFBs), that can be easily quantified by LC–MS. Indeed, comparing the results obtained after the hydrolysis step (amount of total fumonisins) with the amount of fumonisins determined with the normal approach (free fumonisins), it is possible to evaluate the amount of bound or hidden forms. The hydrolysis approach to detect hidden fumonisins was reported for the first time by Kim et al. [46] for the determination of the supposed protein-bound fumonisin FB1 (HFB1) in corn flake samples. In particular, treatment of the sample with 1% sodium dodecylsulphate (SDS) for protein dissolution and hydrolysis with 2 N potassium hydroxide showed a high increase of the so-called “total fumonisins” in comparison to those found by applying the conventional extraction (also referred as “free fumonisins”). A main drawback of this method was due to SDS, which has to be carefully removed as it seriously affects the performance of the chromatographic separation and ESI MS detection. An improvement of this procedure was proposed by Park et al. [8]: the hydrolysis step was directly applied on the matrix with 2 N KOH, avoiding the use of SDS as extraction agent. The hydrolysed sample was then extracted with different solvents (methanol-ethylenediamine tetracetic acid 0.01 M or methanol/acetonitrile/water 25/25/50) and finally cleaned up by OASISTM HLB columns. Recently, Dall’Asta et al. [47] proposed the simultaneous determination of the main fumonisins (FB1, FB2 and FB3) and their masked derivates as hydrolysed forms (HFB1, HFB2 and HFB3) in maize and maize-based foods with an LC–MS/MS method which do not require any sample clean up. For the extraction of bound fumonisins, the residue obtained from the determination of free forms underwent to a hydrolysis step with 2 N NaOH for 60 min at 25 ◦ C. The aqueous phase was then extracted with ethyl acetate, dried under nitrogen stream and redissolved in water/acetonitrile before LC/MS/MS analysis. The estimated recovery for HFB1, HFB2 and HFB3 resulted ranged from 92% to 98% with a low quantification limit (70 ␮g/kg for HFB1, HFB2 and HFB3). Further slight modifications of the method were reported by the same authors [23,48], allowing for a more effective extraction in terms of time and recovery rate. 4. Chromatographic analysis As all the procedures requiring a cleanup step could be affected by recovery problems, on account of the very different chemical behaviour of the masked derivatives compared to their parent forms, a common approach to overpass these difficulties is the use of LC/MS or GC/MS techniques. The selectivity of these detection approaches allows to oversimplify the sample preparation and to avoid clean up steps: this approach is particularly useful for the simultaneous determination of multiple mycotoxins and multiple metabolites, a condition frequently encountered in a large part of the analysed samples. Moreover, as only three metabolites

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(DON3G, 3ADON and 15ADON) are nowadays commercially available as reference standards to be used for monitoring purposes, the identification power of the MS detection may be fully exploited also for the identification of unknown derivatives [24]. 4.1. Chromatographic analysis of deoxynivalenol, zearalenone and derivatives In the first paper demonstrating the natural occurrence of DON3G in cereals, deoxynivalenol and its masked forms DON3G, 3ADON and 15ADON were identified and quantified by QTrapLC–MS/MS analysis, using both an APCI ionization source and an UV detector. The MRM mode and the UV signal at 220 nm were used for quantification while enhanced product ion (EPI) and MS/MS/MS scans were used to gain structural information about the molecules [13]. In this case, the chromatographic separation was performed on a RP-18 Aquasil column, with methanol/water as eluent at 22 ◦ C. Comparing the results obtained for the determination of DON with a GC-ECD method with those obtained by HPLC–MS/MS, the authors found good data agreement, but with the latter more selective and sensitive technique it was actually possible to identify and directly determine DON-conjugates. Using the same instrumentation and a methanol aqueous ammonium acetate as eluent, Berthiller et al. [30] monitored in 2006 also the biotransformation of ZEN in the model plant Arabidopsis thaliana in order to identify the metabolites of this mycotoxins potentially occurring as a results of plant enzymatic activity. An array of 17 different metabolites, most prominently glucosides, malonylglucosides, dihexose- and hexose–pentose disaccharides of zearalenone, and ␣- and ␤-zearalenol, were detected in the samples. The multiple reaction monitoring (MRM) was selected for the analysis, while for identification and characterization of the two metabolites enhanced product ion (MS/MS) and multiple mass spectrometry (MS3 ) modalities were chosen. Recoveries for all the considered compounds were close to 100%. With the aim of setting up MS methods able to simultaneously detect different mycotoxins and different potentially occurring metabolites, a wide series of MS-based multimycotoxins methods were recently proposed. In particular, as pioneering work in this field, two multi-mycotoxins methods were developed, the first for the determination of 39 mycotoxins and the second that allows the simultaneous detection of 90 compounds [24,49]. Among the different mycotoxins, the methods included DON, 3ADON, 15ADON, DON3G, ZEN, ZEN14G, ZEN14S and hydrolysed FB1. The analysis was performed on a Qtrap LC–MS/MS, utilizing a Gemini RP-C18 column at 25 ◦ C and a mixture of methanol/water/acetic acid in different proportions as eluent. Detection was performed by ESI source, utilizing the MRM mode both in positive and negative polarity in two separate chromatographic runs, fully exploiting the selectivity of MS detection and avoiding any clean up steps in a oversimplified extract, dilute and shoot approach. The use of acetic acid allows to control the pH of the eluent, as this is often a critical point as some mycotoxins such as fumonisins may otherwise show peak tailing and low efficiency in chromatographic separations. Sasanya et al. [37] in 2008 described a relatively simple method for the analysis of DON and its masked metabolites by HPLC–UVMS, utilizing as mobile phase methanol/water, obtaining good results for the quantification of DON3G. In 2009 Vendl et al. [41] proposed a method for the simultaneous evaluation of DON, 3ADON, DON3G, ZEN, ZEN14G and ZEN14S in cereal based food. The analyses were performed on a QTrap triplequadrupole linear ion-trap MS/MS system, using both ESI (negative mode) and APCI (SRM mode) probes. The method involved the use of a RP-C18 column (Synergi Polar) and a water/acetonitrile mobile phase containing 5 mM ammonium acetate. The recoveries of the

compounds ranged from 72% for DON3G to 105% for ZEN14G. This method was applied to the analysis of beer, porridge, pasta and corn flour samples [41] and, subsequently, on 84 samples of cereal products, such as snacks, crackers, popcorn, biscuits, etc. [21]. DON and D3G were also successfully detected in bread and wholemeal crackers by application of a linear ion trap LC–MS/MS analysis and the results were exploited for the optimization of the processing parameters [50,51] A novel approach for the analysis of several mycotoxins in cereals, included DON3G, was proposed by Zachariasova et al. [39] using both an UPLC/TOF-MS and an U-HPLC/Orbitrap-MS systems. The chromatographic conditions were tested in order to obtain an optimal peak separation with a ternary mobile phase system composed by 5 mM ammonium formate at pH 5.6, 5 mM ammonium formate containing 0.1% of formic acid at pH 2.7 and methanol. The two different composition of eluent A were used in two separate chromatographic runs: the acid mobile phase was used for detection of fumonisins, while the other one for trichothecenes. The Orbitrap-MS technique was applied to directly analyse crude extracts from complex matrix because some difficulties were experienced using TOF-MS, even if comparable limits of detections were obtained. More recently, a rapid method for the simultaneous determination of DON, DON3G and ADONs in beer samples was validated [41]. The analyses were performed using an UPLC system coupled with a TOF mass spectrometer, using a C18 column with 5 mM ammonium formate and methanol as eluents. The detection was performed in ESI negative mode and the analysis was performed in only 4 min, although 3ADON and 15ADON coeluted as a single peak. Moreover, the same authors [38] developed another method for the determination of DON and DON3G based on the use of an UPLC–Orbitrap-MS system: the method was applied to the study of the effect of milling and baking on the mycotoxin content in contaminated wheat. In this work, the authors studied the fate of the two forms within milling and baking technologies; a distinct increase of DON3G occurred in fermented dough when bakery improvers (mainly characterized by amylolytic activity) were used, thus suggesting a possible linkage between this compound and cell polysaccharides. The study of deoxynivalenol and its acetyl derivates may be also performed by GC–MS, although glycosylated derivatives are too much polar for this approach. In particular, the use of a 5% phenyl–95% methyl-siloxane stationary phase with a temperature gradient starting from 120 ◦ C and increasing oven temperature of 5 ◦ C/min to 270 ◦ C and then of 10–290 ◦ C and held for 8 min allows the separation and detection of the three compounds [52]. Helium was used as a carrier gas at a flow-rate of 1.1 ml/min and EI ionization was used. After extraction of the samples, mycotoxins were purified with Fluorisil column and derivatized with TMS reagent containing trimethylsilylimidazole–trimethylchlorosilane–ethyl acetate (1/0.2/9) for the GC–MS analysis. Although by this approach the determination of masked forms is not feasible, their evaluation could be performed upon hydrolysis. Indeed, as an example of indirect determination, GC–MS was applied to quantify conjugated deoxynivalenol in corn and wheat upon hydrolysis via trifluoromethanesulphonic acid by Tran et al. [53]. The hydrolysis procedure was applied to the evaluation of total DON and conjugated DON in different cereal samples: upon hydrolysis, an increase of 8–70% for DON in corn and of 7–75% in wheat was observed. Really, in this case the released DON may originate not only from the most known glycoside derivative but also from other unknown forms of covalent binding with matrix components. The method is certainly suitable for determining total DON concentration in a sample, although it is not applicable for identifying the nature of the masking mechanism.

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4.2. Chromatographic analysis of fumonisins and hidden fumonisins For the separation and detection of hidden fumonisins, a very useful approach is to convert the molecules into their hydrolysed counterparts by chemical or enzymatic hydrolysis before the chromatographic analysis. Kim et al. [46] separated HFB1 and HFB2 on a C18 column, in a LC–MS system, using 0.1% aqueous formic acid and methanol/acetonitrile as mobile phase and positive ESI as detection mode. More recently, a method for the detection of hidden fumonisins after alkaline hydrolysis in the forms of HFB1, HFB2 and HFB3 was validated by Dall’Asta et al. [47]. An LC–MS/MS system was used, were LC was coupled with a triple quadrupole mass spectrometer equipped with an ESI interface (positive ion mode). The chromatographic conditions used water and methanol as eluents, both added with 0.1% of formic acid. Detection of the analytes was achieved by multiple reaction monitoring (MRM) mode. The method allowed to determine free and masked fumonisins without any clean up step. The authors then applied the method to the quantification of these interesting compounds in maize-based foods [47] and in gluten-free foods [48]. A similar approach was used by Park et al. [8] in 2004 for the determination of HFB1 in heat-processed corn-food. The method was based on the use of a HPLC separation with fluorescence detection and HPLC–MS/MS for confirmation. For this last analysis, the separation was performed on a Synergi Polar PR column, using acetonitrile/methanol and 0.02% aqueous formic acid as eluents. The detection was achieved on a tandem mass spectrometer using electrospray ionization in positive ion mode (SIM mode). The occurrence of bound fumonisin FB1 in the analysed samples was confirmed by using both HPLC-FD and HPLC–MS/MS.

5. Strategy to detect unknown conjugated mycotoxins Mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the most important methods for structure elucidation of phase II metabolites. In particular, the method of choice for the identification of unknown masked mycotoxins in processed or unprocessed food is high resolution MS, followed by NMR and/or other spectroscopic measurements (e.g. UV, infrared). Indeed, isolation of unidentified conjugated compounds by normal phase or reverse phase preparative chromatography followed by onedimensional (1D)- or two-dimensional (2D)-NMR measurements is usually required for the unambiguous structure elucidation [54]. However, when a metabolite can only be identified by means of MS due to the small amount available, the candidate compound should be synthesized using a chemical and/or a biochemical strategy; after full characterization of the natural-identical molecule, its spectral behaviour should be compared with that recorded for the natural conjugate to univocally assign the chemical structure of the novel conjugate. Very recently, MS techniques for mycotoxin conjugates identification have been reviewed by Berthiller et al. [7]. A different approach should be used when an associative binding between mycotoxins and macromolecules such as starch or proteins is investigated. In this case, the study of the interaction is usually quite troublesome, since indirect methods should be used. In particular, when proteins of known tridimensional structure are involved, the application of in silico docking methods could be very helpful for supporting the interaction hypothesis. Since this review is aimed at the application of hyphenated techniques for the determination and characterization of masked mycotoxins, only the HR-MS approach for structural identification will be addressed.

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5.1. High resolution mass spectrometry In spite of the limited structural information obtained by mass spectrometry, this technique is successfully used in the structure elucidation of metabolites, since additional information such as the structure of the parent compound and its general fragmentation pathways are usually available. The most used MS methods are those based on soft ionization modes such as electrospray (ESI) or atmospheric pressure chemical ionization (APCI). In particular, collision-induced dissociation (CID) experiments with tandem mass spectrometry (MS/MS) can be used for analysing the dissociation of the pseudo-molecular ions preferentially generated by the soft ionization methods. The MS/MS analysers mainly used for structure elucidation are triplequadrupoles and ion traps, together with hybrid instruments such as combinations of quadrupole and linear ion trap, quadrupole and time-of-flight (Q-TOF), linear ion trap and Fourier transform ion-cyclotron resonance (FT-ICR). In particular, the latter techniques provide high-resolution data of the pseudo-molecular and fragment ions, being thus particularly powerful for structure elucidation of unknown compounds. The application of multiple tandem mass spectrometric experiments (MSn ) are well suited for structure elucidation of conjugates since they permit the differentiation between the fragments of the conjugate (recording in the MS2 spectra) and of the precursor, by investigation of the collisional-induced dissociation in MSn spectra. For the screening of unknown compounds, full scans over a wide mass range are often used. Time of Flight (TOF) and Fourier transform (e.g. Orbitrap) instruments are superior to quadrupole mass spectrometers in terms of full-scan sensitivity, mass accuracy and resolving power. A recent application of high-resolution LC–Orbitrap MS has been published for the identification of two new Fusarium mycotoxin glucosides in wheat grain. In particular, fusarenon X-glucoside and nivalenol-glucoside were identified on the basis of accurate mass measurement of characteristic ions and MS/MS fragmentation patterns, although the absolute structure could not be clarified [55]. Ion traps show good full-scan sensitivity, but also a lack of resolution in mass accuracy. Besides the acquisition of accurate masses of analytes, other additional post-data acquisition mathematical tools can be applied to help the molecular identification. Fourier transform MS has been rarely used for novel masked mycotoxin detection so far. More often, TOF instruments have been used to confirm postulated chemical formulas of mycotoxin metabolites [55–59]. Further, MS/MS can be very useful for the screening of mycotoxin conjugates. At first, precursor ion scan experiments allow to find out higher molecular weight conjugates when the mass of the native mycotoxin is fixed in the third quadrupole. Moreover, neutral loss scan experiments can identify the loss of a well-known group from the conjugate, such as glucose. The main drawback of these experiments is the low sensitivity over a wide mass range. A better sensitivity can be achieved by performing product ion scans. On the other hand, this modality requires the presumption of the monoisotopic mass of the unknown conjugate ions ([M+H]+ , [M+NH4 ]+ , [M−H]− ) to be used for product ion screening. Moreover, the acquired fragmentation pattern can confirm the existence of the parent mycotoxin. A review reporting the MS approach to study in general phase II metabolites has been published [60].

6. Conclusion In the most recent years the awareness of the potential occurrence of masked derivatives of mycotoxins in our foods has increased the efforts of the scientific community for the unravelling of the different aspects, related to their identification, structural

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characterization and quantification. A particular emphasis has been put on the development of suitable analytical methodology to characterize and analyse all the possible metabolites produced by plants, microorganisms and mammals in order to clarify their significance and their fate during food processing or gastrointestinal digestion in humans and, thus, as a key point, its toxicological significance. LC–MS plays a key role both for the discovery and characterization of unknown compounds as well as for the study of their occurrence and transformation reactions, mostly on account of its selectivity, sensitivity and structural characterization ability. In particular, the possibility to avoid clean up steps and perform the analysis on the crude extract has been the key factor in metabolite discovery and quantification. Among the many different compounds identified up to now, only some were found to occur in food items. For these ones, the commercial availability of analytical standards, eventually isotopically labelled, is a key point for the development and validation of new analytical methods: at the moment, some important conjugated standards are already available and other will be probably produced in the next future. The rapid outburst of new analytical methods for these compounds may help in better defining both their significance for the metabolic pathways of plants (i.e., a method for studying and developing Fusarium resistant plant varieties) and animals, as well as their importance for human health, on the base of toxicity and bioavailability studies. References [1] [2] [3] [4]

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