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Analytical and Bioanalytical Chemistry

, Volume 410, Issue 22, pp 5567–5581 | Cite as

In-house validation of a rapid and efficient procedure for simultaneous determination of ergot alkaloids and other mycotoxins in wheat and maize

  • Natalia Arroyo-Manzanares
  • Karl De Ruyck
  • Valdet Uka
  • Laura Gámiz-Gracia
  • Ana M. García-Campaña
  • Sarah De Saeger
  • José Diana Di Mavungu
Research Paper
Part of the following topical collections:
  1. Food Safety Analysis

Abstract

A fundamental step in addressing the global problem of mycotoxins is the development of highly sensitive, multi-class extraction and detection methods. This constitutes a field of research that has in recent years enjoyed a steady advance. Such methods, generally based on liquid chromatography coupled to mass spectrometry, are widely reported successfully detecting various mycotoxins in different food and feed samples. In this work, an innovative approach to multi-class mycotoxin control is proposed, offering specific advantages: a broader inclusion of more mycotoxin classes, robust and thorough extraction for all target compounds despite their varied chemical properties, and determination of all analytes from a single injection. The method involved the extraction and quantification of the main mycotoxins produced by Aspergillus, Fusarium, and Penicillium fungi, as well as their reported derivatives, together with 12 other compounds most commonly produced by Claviceps purpurea. The popularly reported QuEChERS technique has been reduced to a simple “salting-out liquid-liquid extraction” (SO-LLE) to obtain the most efficient extraction of the aforementioned mycotoxin classes in a very short time. This is in particular extremely important in ensuring correct determination of individual ergot alkaloids, for which short and robust sample preparation as well as short analytical sequences were key for minimizing the epimerization during analysis. The analyses of wheat and maize samples were performed using ultra-high performance liquid chromatography coupled with tandem mass spectrometry. Matrix-matched calibration curves were established and limits of quantification were below the maximum levels established by the EU regulation. The precision (repeatability and intermediate precision) was lower than 13% in all cases and recoveries ranged between 60 and 98% in maize and between 62 and 103% in wheat, fulfilling the current legislation. The method was applied to study the co-occurrence of these mycotoxins in wheat (n = 13) and maize (n = 15) samples from six European countries. A successful quantification of 23 different mycotoxins, from all major classes, in 85% of wheat and 93% of maize samples was achieved.

Keywords

Mycotoxins Co-occurrence Cereals Ultra-high performance liquid chromatography Mass spectrometry QuEChERS SO-LLE 

Introduction

According to the annual report of the Rapid Alert System for Food and Feed (RASFF) [1], notifications reporting on risks identified in food on the market due to mycotoxins rose dramatically in 2015 (116 notifications more than in 2014). Cereals continue to be an important source of mycotoxin contamination and the notifications concerning these matrices also increased. In fact, there were 11 notifications (six in 2014) related to the presence of deoxynivalenol (DON) in cereals and cereal products, mainly maize and maize products, and five notifications (three in 2014) related to the presence of fumonisins in maize and maize products.

Moreover, the complex ecology of fungus growth and mycotoxin production can produce mixtures of mycotoxins in foods and feeds, especially in cereals [2], and in many of these notifications, the co-occurrence of several mycotoxins has been reported. Specifically, contamination by DON was combined with high levels of zearalenone (ZEN), and the presence of fumonisins was combined with high levels of aflatoxins. Supporting this, several surveys have been carried out, all over the world, also reporting the natural co-occurrence of mycotoxins, with 116 mycotoxin combinations found in cereals and derived cereal product samples; aflatoxins + fumonisins, DON + ZEN, and fumonisins + ZEN being the most common combinations [3]. This co-occurrence of mycotoxins can therefore increase the total mycotoxin contamination level in a certain foodstuff, and in some cases its toxicity [4]. In this sense, there is an increasing concern about exposure to mycotoxin mixtures [5].

The adverse effects (acute and chronic effects) of individual mycotoxins are well known and they have been considered as toxic, teratogenic, carcinogenic, and immunomodulating agents for humans and animals [6]. Particularly, aflatoxin B1 (AFB1) has been included in group 1 of compounds explicitly carcinogenic to humans by the International Agency for Research on Cancer (IARC). Unfortunately, the toxicity of mycotoxin combinations cannot always be predicted based upon their individual toxicities since multi-exposure may lead to additive, synergistic, or antagonistic toxic effects [7]. A recent study showed that development of one or another effect is dose dependent [4].

On the other hand, the European Union (EU), based on adverse effects of some individual mycotoxins, has set maximum levels for aflatoxins, ochratoxin A (OTA), DON, ZEN, and fumonisins in cereals and several foodstuffs derived from cereals, included in the Commission Regulation No. 1881/2006 [8] and subsequent amendments. However, these regulations do not consider the combined effects of mycotoxins, even at individual concentrations lower than maximum permitted levels.

In this sense, multi-class methods that allow a simple and fast monitoring of the combined presence of mycotoxins are in demand. Over the last decade, several multi-class mycotoxin methods have been developed [9, 10, 11, 12], mainly using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), due to the high potential of this technique for simultaneous determination and identification of compounds with a wide range of chemical properties, such as mycotoxins. However, most of these methods do not include the six major ergot alkaloids (EAs) (namely, ergometrine (Em), ergotamine (Et), ergocristine (Ecr), ergokryptine (Ekr), ergosine (Es) and ergocornine (Eco)) and their epimers; a class of indole metabolites produced by grain and grass pathogens such as Claviceps spp. and whose monitoring is recommended by the EU since 2012 [13]. Only a few multi-mycotoxin LC–MS/MS methods that also include the six major EAs and their epimers have been reported [14, 15, 16]. However, these methods required two separate chromatographic runs per sample, using both positive and negative polarities, and in some cases using different mobile phases for each run [14].

Therefore, the aim of the presented study was to propose an ultra-high performance liquid chromatography (UPLC) method coupled with MS/MS detection for the determination of 35 mycotoxins, including the main ones produced by Aspergillus, Fusarium, and Penicillium fungi, in one chromatographic run. The selected mycotoxins include AFB1, aflatoxins B2 (AFB2), G1 (AFG1), and G2 (AFG2), sterigmatocystin (STE), citrinin (CIT), fumonisin B1 (FB1), fumonisin B2 (FB2), fumonisin B3 (FB3), HT-2 toxin (HT-2), T-2 toxin (T-2), ochratoxin A (OTA), nivalenol (NIV), DON, neosolaniol (NEO), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), roquefortine C (ROQ-C), deoxynivalenol-3-glucoside (DON-3-Glu), fusarenon-X (F-X), diacetoxyscirpenol (DAS), alpha zearalenol (α-ZOL), and ZEN, as well as the six major EAs (Em, Et, Ecr, Ekr, Eco, Es) and their epimers (ergotaminine (Etn), egometrinine (Emn), egocristinine (Ecrn), ergokryptinine (Ekrn), ergocroninine (Econ), and ergosinine (Esn). In addition, a QuEChERS-based extraction procedure was also optimized, in order to avoid an additional clean-up step.

The proposed method was fully validated on spiked cereal samples (wheat and maize) to assess linear dynamic range, limits of detection (LOD) and quantification (LOQ), matrix effect (ME), and trueness. Finally, the method was applied in naturally contaminated samples, and the natural co-occurrence of mycotoxins could be explored.

Materials and methods

Standards

Standards of AFB1, AFB2, AFG1, AFG2, OTA, STE, FB1, FB2, T-2, DON, deepoxy-deoxynivalenol (DOM, internal standard), and ZEN were purchased from Oskar Tropitzsch (Marktredwitz, Germany). FB3 was obtained from Promec Unit (Tygerberg, South Africa) and NIV, HT-2, and F-X from Fermentek (Jerusalem, Israel). NEO, 3-ADON, ROQ-C, and DON-3-Glu were supplied by Biopure (Tulln, Austria) and DAS, CIT, zearalanone (ZAN, internal standard), and α-ZOL by Sigma Aldrich (Bornem, Belgium). NIV and NEO were obtained as solutions at 100 μg/mL, and DON-3-glu at 50 μg/mL in acetonitrile (MeCN). The rest of the stock solutions were prepared in methanol (MeOH) at a concentration of 1 mg/mL, except FB2 and FB3 stock solutions were prepared in MeCN/ddH2O (50/50, v/v). All stock solutions were stored at − 18 °C except FB2 and FB3, which were stored at 4 °C.

Fine film-dried ergot alkaloid standards Em, Et, Es, Eco, Ekr, Ecr, Emn, Esn, Etn, Econ, Ekrn, Ecrn were purchased from Coring System Diagnostix GmbH (Gernsheim, Germany). As indicated by the manufacturer, the film-dried standards were reconstituted in 5 mL of solvent (MeCN), to give concentrations of 100 μg/mL and 25 μg/mL, for the main EAs and for the -inine isomers, respectively. Methylergometrine (MeEm, as methylergometrine maleate) was obtained from VWR International (Zaventem, Belgium) and dihydroergotamine (DhEt, as dihydroergotamine tartrate) from Sigma Aldrich. Stock solutions at 1 mg/mL were prepared in MeOH/MeCN (50/50, v/v) and MeCN for MeEm and DhEt, respectively. Because of the rapid epimerization of EAs in solution, dried standard residues were made from the freshly prepared standard solutions as follows: individual or mixed standard solutions were aliquoted into dark brown or aluminum-covered glass tubes, evaporated to dryness at 40 °C under a stream of nitrogen, and stored at − 20 °C. These residues were reconstituted in the required amount of solvent immediately before use. Handling the ergot alkaloid standards in this way, they were stable for at least 1 year [17].

Chemicals and reagents

Solvents were LC–MS grade. Formic acid and ammonium formate eluent additives for LC–MS, sodium chloride (NaCl), and magnesium sulfate (MgSO4) were obtained from Sigma Aldrich. MeCN and MeOH were obtained from Biosolve Chimie (Dieuze, France). Ultrapure water (18.2 MΩ/cm3, Milli-Q Plus system, Millipore, Bedford, MA, USA) was used throughout the work. Ultrafree centrifugal filters with 0.22 μm Durapore® membrane (Merck KGaA, Darmstadt, Germany) were used for filtration of samples prior to their injection into the chromatographic system.

Instruments and equipment

Separations were performed on an UPLC Waters Acquity system (binary solvent manager, auto sampler, and column heater units) from Waters Corporation (Milford, MA, USA). The mass spectrometer measurements were performed on a triple quadrupole mass spectrometer with electrospray ionization (ESI), a Xevo TQ-S, also from Waters Corp. An ACQUITY HSS UPLC T3 (150 mm × 2.1 mm, 1.8 μm) column from Waters Corp. was used. The instrumental data were collected using the MassLynx® Software version 4.1 SCN876 from Waters Corp.

A SIGMA 3-16PK refrigerated centrifuge from Sartorius AG (Göttingen, Germany), an L46 variable-speed vortex mixer from Labinco (Breda, The Netherlands) and a TurboVap LV evaporator system from Biotage (Uppsala, Sweden) were also used.

LC-MS conditions

UPLC separations were performed using a mobile phase consisting of 0.3% formic acid aqueous solution with 5 mM ammonium formate (solvent A), and MeOH with 0.3% formic acid and 5 mM ammonium formate (solvent B) at a flow rate of 0.4 mL/min. The eluent gradient profile was as follows: 0 min: 5% B; 0.5 min: 5% B; 20 min: 94% B; 21 min: 94% B, 24 min: 5% B, and 28 min: 5% B. The temperature of the column was 30 °C and the injection volume was 10 μL.

The mass spectrometer was operated in positive ESI mode under the multiple reaction monitoring (MRM) conditions shown in Table 1. The ionization source parameters were source temperature 150 °C; nebulizer gas (nitrogen) 7 bar; source offset voltage +50 V; cone gas flow of 150 L/h and desolvation gas set to 400 °C, with flow at 1000 L/h. Four identification points that included RT (1 point), one precursor and two product ions (1.5 points per transition) were obtained for each mycotoxin, therefore fulfilling the requirements established by the EU for confirmation of food contaminants [18]. The most abundant transition was used for quantification and the other one for confirmation purpose. To minimize epimerization of EAs, autosampler temperature was maintained at 4 °C and sample sequence was limited to a maximum of 24 h.
Table 1

Mass spectrometric parameters for the different target analytes

Mycotoxin

Retention time (min)

Precursor ion (m/z)

Molecular ion

Cone voltage (V)

Product ions (m/z)b

Collision energy (eV)

NIV

3.44

313.1

[M + H]+

35

174.9 (Q)

10

176.9 (C)

10

DON-3-Glu

4.22

476.0

[M + H]+

35

248.0 (Q)

20

296.0 (C)

15

DON

4.83

297.1

[M + H]+

40

249.1 (Q)

12

203.0 (C)

16

Em

5.55

326.2

[M + H]+

30

223.0 (Q)

23

208.2 (C)

30

DOMa

6.50

281.2

[M + H]+

40

233.2 (Q)

10

109.1 (C)

17

F-X

6.53

355.1

[M + H]+

16

247.1 (Q)

10

137.1 (C)

23

MeEma

6.58

340.3

[M + H]+

28

223.3 (Q)

25

208.2 (C)

35

NEO

7.10

400.1

[M + NH4]+

25

305.3 (Q)

13

365.1 (C)

10

Emn

7.20

326.2

[M + H]+

33

208.0 (Q)

25

223.0 (C)

25

3-ADON

8.44

339.1

[M + H]+

14

231.2 (Q)

10

137.3 (C)

12

15-ADON

8.44

339.1

[M + H]+

14

231.2 (Q)

10

137.3 (C)

12

AFG2

9.90

331.1

[M + H]+

25

313.1 (Q)

19

245.1 (C)

18

AFG1

10.41

329.1

[M + H]+

35

243.1 (Q)

25

311.1 (C)

20

AFB2

11.04

315.1

[M + H]+

25

259.1 (Q)

35

286.9 (C)

40

Es

11.06

548.3

[M + H]+

20

223.3 (Q)

33

208.1 (C)

40

Esn

11.30

548.3

[M + H]+

20

223.3 (Q)

33

208.1 (C)

40

DAS

11.45

384.3

[M + NH4]+

38

307.3(Q)

9

349.3 (C)

9

Et

11.45

582.5

[M + H]+

22

208.2 (Q)

25

268.4 (C)

45

AFB1

11.60

313.0

[M + H]+

70

285.1 (Q)

22

241.1 (C)

35

Eco

11.64

562.5

[M + H]+

20

223.3 (Q)

25

268.3 (C)

40

Etn

11.65

582.5

[M + H]+

22

208.2 (Q)

25

268.4 (C)

45

DHEta

11.78

584.5

[M + H]+

20

270.2 (Q)

30

225.3 (C)

35

CIT

12.05

251.0

[M + H]+

25

205.0(Q)

25

191.0 (C)

30

Ecr

12.35

610.4

[M + H]+

23

208.1 (Q)

25

268.4 (C)

45

Ekr

12.51

576.3

[M + H]+

21

208.1 (Q)

25

268.4 (C)

45

Econ

12.59

544.4

[M-H2O + H]+

39

277.5 (Q)

26

223.3 (C)

37

ROQ-C

13.01

390.2

[M + H]+

25

322.2 (Q)

30

193.0 (C)

42

HT-2

13.11

442.3

[M + NH4]+

40

263.2 (Q)

12

215.2 (C)

12

Ekrn

13.22

558.5

[M-H2O + H]+

38

223.3 (Q)

35

305.1 (C)

27

Ecrn

13.40

592.4

[M-H2O + H]+

35

223.3 (Q)

35

305.4 (C)

29

FB1

14.10

722.4

[M + H]+

40

334.3 (Q)

40

353.4 (C)

36

T-2

14.38

484.3

[M + NH4]+

40

305.2 (Q)

12

185.2 (C)

20

α-ZOL

15.14

321.4

[M + H]+

40

303.0 (Q)

8

175.0 (C)

24

FB2

15.17

706.4

[M + H]+

70

336.3 (Q)

32

318.3 (C)

32

ZANa

15.36

319.0

[M + H]+

18

205.0 (Q)

13

275.0 (C)

13

ZEN

15.36

319.0

[M + H]+

40

283.1 (Q)

12

301.1 (C)

10

OTA

15.42

404.1

[M + H]+

40

239.0 (Q)

24

358.2 (C)

14

STE

15.78

325.1

[M + H]+

40

281.1 (Q)

34

310.1 (C)

24

FB3

15.91

706.4

[M + H]+

70

336.3 (Q)

32

318.3 (C)

32

NIV: nivalenol, DON-3-Glu: deoxynivalenol-3-glucoside, DON: deoxynivalenol, Em: ergometrine, DOM: deepoxy-deoxynivalenol, F-X: fusarenon-X, MeEm: methylergometrine, NEO: neosolaniol, Emn: egometrinine, 3-ADON: 3-acetyldeoxynivalenol, 15-ADON: 15-acetyldeoxynivalenol, AFG2: aflatoxin G2, AFG1: aflatoxin G1, AFB2: aflatoxin B2, Es: ergosine, Esn: ergosinine, DAS: diacetoxyscirpenol, Et: ergotamine, AFB1: aflatoxin B1, Eco: ergocronine, Etn: ergotaminine, DHEt: dihydroergotamine, CIT: citrinin, Ecr: ergocronine, Ekr: ergokryptine, Econ: ergocroninine, ROQ-C: roquefortine C, HT-2: HT-2 toxin, Ekrn: ergokryptinine, Ecrn: egocristinine, FB1: fumonisin B1,T-2: T-2 toxin, α-ZOL: alpha zearalenol, FB2: fumonisin B2, ZAN: zearalanone, ZEN: zearalenone, OTA: ochratoxin A, STE: sterigmatocystin, FB3: fumonisin B3

aInternal standard

bProduct ions: (Q) transition used for quantification, (I) transition used for confirmation

Sample preparation

Sample preparation was based on the first step (extraction/partition process) of the QuEChERS procedure [19]. A 2-g portion of cereal sample and 8 mL of water were placed into a 50-mL screw cap test tube with conical bottom, which was shaken by vortex for 10 s. Subsequently, 10 mL of 5% formic acid in MeCN was added to the tube and shaken by vortex for 2 min. A mixture of salts (4 g MgSO4 and 1 g NaCl) was added and the tube was vigorously shaken by hand for 1 min and by vortex for 2 min. Then, a centrifugation step was performed at 4500 rpm for 5 min at 4 °C, and 5 mL of the supernatant was transferred to a glass tube. It was evaporated to near dryness under a gentle stream of N2 at 40 °C, reconstituted with 0.2 mL of MeOH:H2O (50:50, v/v), centrifuged in a Ultrafree®-MC centrifugal device for 5 min at 14,000g at 4 °C and transferred into amber vials for injection into the LC-MS system.

Method validation

The developed method was validated for all mycotoxins targeted in this study in wheat and maize. The validation was performed according to Commission Decision 2002/657/EC [18].

ME was evaluated at two concentration levels for each mycotoxin in both matrices. It was calculated as 100 × [(signal of analyte in extract − signal of analyte in neat solvent)/signal of analyte in neat solvent]. The studied concentrations were as follows: 1 to 20 μg/kg for AFB1, AFB2, AFG1, AFG2; 0.5 to 20 μg/kg for STE; 2 to 200 μg/kg for OTA; 1 to 100 μg/kg for T-2, FB2, FB3, NEO; 10 to 100 μg/kg for DAS; 10 to 200 μg/kg for HT-2, ROQ-C, ZEN, α-ZOL; 5 to 100 μg/kg for FB1; 10 to 150 μg/kg for CIT; 20 to 400 μg/kg for DON, 3-ADON, F-X; 100 to 500 μg/kg for NIV; 10 to 200 μg/kg for the six major EAs and their epimers. The ratio of areas of the analyte and the internal standard was considered as analytical response. ZAN at 100 μg/kg was used as internal standard for AFB1, AFB2, AFG1, AFG2, STE, OTA, CIT, T-2, HT-2, FB1, FB2, FB3, ROQ-C, ZEN, and α-ZOL; DOM at 200 μg/kg for DON, 3-ADON, F-X, DAS, NEO, and NIV; MeEm at 50 μg/kg for Em and Emn; and DhEt at 200 μg/kg for the rest of the EAs. The usefulness of the chosen internal standards in correcting for volume differences that are inherent to the analytical procedure was already demonstrated in previous validated methods [11, 17, 20] and by reliable results in routine use and good z-scores in diverse proficiency testing programs. As described later, the matrix-matched calibration approach was implemented to compensate for the matrix effects.

Linearity was calculated by least-square regression. The LODs and LOQs were determined according to “Guidance Document on the Estimation of LOD and LOQ for Measurements in the Field of Contaminants in Feed and Food” (European Union, EUR 28099 EN) [21].

The precision of the proposed method was evaluated in terms of repeatability (intraday precision) and intermediate precision (interday precision). Repeatability was assessed by application of the whole procedure to wheat and maize samples spiked at three different concentration levels of each mycotoxin. Each sample was prepared in triplicate (technical replicates), giving a total of nine samples for each matrix. All the samples were processed on the same day, and each extract was injected in triplicate (instrumental replicates). Intermediate precision was evaluated with a similar procedure, spiking and analyzing three different samples (technical replicates) on three different days.

Finally, in order to assess the trueness of the proposed method, recovery experiments were carried out on wheat and maize samples, previously analyzed to establish the absence of detectable mycotoxins. None of them gave a positive result above the LODs of the method. These samples were spiked at three different concentration levels, processed as described previously. In total, nine samples for each matrix were prepared. These samples were injected in triplicate into the UPLC-MS/MS system.

Results and discussion

Optimization of sample treatment

As described in Section 2.5., sample treatment was based on the extraction/partition process of the QuEChERS procedure, a suitable tool for multi-class, multi-residue methods, which tries to extract a wide variety of different analytes from the same sample [19]. In addition, the QuEChERS procedure is fast and easy to perform, requires a minimal amount of chemicals (especially solvents), and provides a certain degree of selectivity while still covering this wide array of analyte–matrix pairs. Because of the large range of chemical properties different mycotoxins may have, and the wide variety of cereal matrices that they may contaminate, sample treatment based on the QuEChERS procedure has already been proposed. This methodology has been applied for multi-class analysis of mycotoxins in cereals or derived cereal products [9, 22, 23].

In this work, a previous analytical method for determination of 15 mycotoxins in rice and pseudocereals [9] was adapted for simultaneous determination of the six major EAs and their epimers along with 23 other mycotoxins. In the published method, Arroyo-Manzanares et al. extracted mycotoxins under acidic conditions using MeCN with 5% of formic acid as extraction solvent. However, EAs are neutral at high pH and positively charged in acidic solutions (pKa 4.8–6.2), and can therefore be extracted either with polar solvents under acidic conditions or with non-polar organic solvents under alkaline conditions [17]. In light of this, the percentage of formic acid in MeCN as extraction solvent was evaluated between 0 and 7.5% in order to obtain the highest extraction efficiency for all studied mycotoxins. Wheat samples were used as the representative matrix during this optimization step. The results of this assay are shown in Fig. 1. Extraction efficiencies below 60% were obtained for CIT with percentages of formic acid below 5%, and for OTA when formic acid was not added to MeCN. In all cases, the extraction recoveries of EAs were above 60%, although a light decrease was observed when the percentage of formic acid was higher than 1%, changing from 70–85% to 60–75%. Consequently, in order to obtain recoveries above 60% for all the analytes, 5% of formic acid in MeCN was selected for further experiments. Determination of EAs is often hampered by the epimerization that often occurs during the analytical process. This problem has tremendously limited the interest of different researchers both in the development and in the application of analytical methodologies for this class of compounds. In several methods for multi-class analysis of mycotoxins, EAs have been simply omitted. When EAs have to be analyzed, it is of utmost importance to avoid epimerization to achieve reliable and reproducible results. Therefore, unlike in some other QuEChERS-based methods [23], the sample preparation in the presently proposed method was reduced to a simple “salting-out liquid-liquid extraction” (SO-LLE). This was extremely important to minimize the epimerization of EAs during analysis and ensure their correct determination. In addition, as described under section “Materials and Methods,” other measures such as a careful handling of the standards or limiting and shortening the analytical sequence were concurrently applied for reliable and robust determination of EAs.
Fig. 1

Influence of percentage of formic acid added to extraction solvent on the extraction efficiency of the analytes from wheat

Optimization of LC-MS method

Initially, the influence of mass spectrometer parameters (cone voltage and collision energy) on the intensity of each analyte was studied. With this purpose, individual standard solutions of 1 mg/L in 0.1% aqueous formic acid/MeCN (50/50, v/v) were infused into the mass spectrometer and all the compounds were tested using ESI positive and negative modes. ESI positive showed the best results in terms of sensitivity for most of the mycotoxins and protonated precursor ions [M + H]+ were selected for most of them. However, ammonium adducts [M + NH4]+ were selected for T-2, HT-2, NEO and DAS, and [M-H2O + H]+ for Econ, Ecrn and Ekr, as previously described [17]. Each compound was characterized by its retention time and by two product ions. The most intense product ion was used for quantification, while the second one was used for confirmation purposes. The results of this experiment are shown in Table 1.

Subsequently, the stationary phase and the mobile phase were studied. Acidic mobile phases and reverse-phase LC have usually been used for multi-class determination of mycotoxins [9, 10, 11, 12]. However, these methods do not include the six major EAs and their epimers. In the case of ergot alkaloid determination, acidic [24] as well basic [25] mobile phases have been used. Since FB’s and OTA are better analyzed under low pH conditions, acidic mobile phase was therefore selected for further experiments.

Two reverse-phase columns, namely ACQUITY HSS T3 column (150 mm × 2.1 mm, 1.8 μm) and ZORBAX RRHD Eclipse Plus C18 (100 mm × 2.1 mm, 1.8 μm), with different combinations of mobile phases, consisting of water (solvent A) and MeOH (solvent B) with 5 mM ammonium formate, and different percentages of formic acid (between 0.1 and 0.3%) were tested. The HSS T3 column and mobile phase with 0.3% formic acid provided better peak shapes and more intense MS signals, and hence were selected for subsequent studies. In addition, a gradient elution was needed to elute all analytes within reasonable time, achieving a good separation and good peak shape. However, under applied LC conditions (see section 2.4), the pairs of mycotoxins 3-ADON/15-ADON showed the same retention time and product ions. Therefore, their determination was approximated as the sum of 3-ADON and 15-ADON, using 3-ADON as the external calibration standards.

Finally, under the selected chromatographic conditions, the ionization source parameters were investigated to obtain the following optimal values: source temperature 150 °C; nebulizer gas (nitrogen) 7 bar; source offset voltage + 50 V; cone gas flow of 150 L/h and desolvation gas set to 400 °C, with flow at 1000 L/h.

Validation of the method

An exhaustive validation was carried out in order to evaluate the suitability of the proposed method for the determination of mycotoxins in wheat and maize. Thus, linear dynamic ranges, LODs and LOQs, ME, precision, and trueness were evaluated for each matrix.

Calibration curves and performance characteristics

Firstly, ME was evaluated at two concentration levels for each mycotoxin in both matrices. It was calculated as 100 × [(signal of analyte in extract − signal of analyte in neat solvent)/signal of analyte in neat solvent]. Table 2 shows the values of the ME and, as can be seen, signal suppression was significant for all the compounds and therefore matrix-matched calibration curves were necessary for quantification purposes.
Table 2

Matrix effect (ME) (%) from wheat and maize, calculated as 100 × [(signal of analyte in extract − signal of analyte in neat solvent) / signal of analyte in neat solvent]

 

Wheat

Maize

Level 1

Level 2

Level 1

Level 2

AFB1

− 70.4

− 76.6

− 81.8

− 79.9

AFB2

− 83.1

− 81.7

− 86.6

−  85.9

AFG1

− 73.5

− 70.5

− 79.9

− 79.7

AFG2

− 83.3

− 81.8

− 83.3

− 74.7

STE

− 61.4

− 66.6

− 79.9

− 79.6

OTA

− 70.5

− 69.0

− 81.8

− 76.2

T-2

− 70.7

− 77.5

− 77.9

− 74.2

HT-2

− 71.6

− 71.5

− 79.3

− 74.2

FB1

− 74.6

− 75.9

− 87.8

− 74.9

FB2

− 61.0

− 70.9

− 87.9

− 72.5

FB3

− 45.2

− 50.7

− 61.8

− 48.0

CIT

− 72.2

− 78.2

− 79.0

− 78.6

ROQ-C

− 82.2

− 73.5

− 81.0

− 79.9

ZEN

− 89.8

− 75.5

− 79.5

− 73.6

α-ZOL

− 80.9

− 81.8

− 89.4

− 88.0

DON

− 87.0

− 73.6

− 79.2

− 86.1

DAS

− 69.1

− 62.0

− 69.7

− 77.4

3-ADON

− 67.1

− 57.6

− 78.9

− 73.2

F-X

− 73.6

− 62.0

− 79.4

− 77.4

NEO

− 55.0

− 54.3

− 69.9

− 68.8

NIV

− 81.7

− 70.4

− 79.4

− 76.0

Em

− 64.4

− 77.5

− 83.1

− 87.4

Emn

− 72.4

− 76.6

− 88.9

− 86.3

Et

− 76.5

− 84.0

− 86.4

− 80.6

Etn

− 80.0

− 84.1

− 86.3

− 81.5

Ecr

− 81.4

− 76.6

− 80.0

− 73.3

Ecrn

− 77.9

− 71.2

− 84.9

− 80.3

Ekr

− 82.0

− 77.4

− 81.1

− 84.3

Ekrn

− 75.5

− 78.7

− 77.2

− 80.9

Eco

− 73.8

− 77.5

− 83.8

− 84.9

Econ

− 74.4

− 75.5

− 87.1

− 80.9

Es

− 73.4

− 77.2

− 81.7

− 87.5

Esn

− 71.4

− 77.2

− 83.1

− 87.4

Level 1: AFB1, AFB2, AFG1, AFG2: 1 μg/kg; OTA: 2 μg/kg; STE: 0.5 μg/kg; T-2, FB2, FB3, DAS: 10 μg/kg, NEO: 1 μg/kg; HT-2, CIT, ROQ-C, ZEN, α-ZOL: 10 μg/kg; FB1: 5 μg/kg; DON, 3-ADON, F-X: 20 μg/kg; NIV: 100 μg/kg; Ergot alkaloids: 10 μg/kg

Level 2: AFB1, AFB2, AFG1, AFG2: 10 μg/kg; OTA: 100 μg/kg; STE: 10 μg/kg; T-2, FB2, FB3, DAS, NEO: 50 μg/kg;HT-2, CIT, ROQ-C, ZEN, α-ZOL: 150 μg/kg; FB1: 100 μg/kg; DON, 3-ADON, F-X: 300 μg/kg; NIV: 400 μg/kg; Ergot alkaloids: 150 μg/kg

AFB1: aflatoxin B1, AFB2: aflatoxin B2, AFG1: aflatoxin G1, AFG2: aflatoxin G2, OTA: ochratoxin A, T-2: T-2 toxin, HT-2: HT-2 toxin, FB1: fumonisin B1, FB2: fumonisin B2, FB3: fumonisin B3, CIT: citrinin, ROQ-C: roquefortine C, ZEN: zearalenone, α-ZOL: alpha zearalenol, DON: deoxynivalenol, DAS: diacetoxyscirpenol, 3-ADON: 3-acetyldeoxynivalenol, F-X: fusarenon-X, NEO: neosolaniol, NIV:nivalenol, Em: ergometrine, Emn: egometrinine, Et: ergotamine, Etn: ergotaminine, Ecr: ergocronine, Ecrn: egocristinine, Ekr: ergokryptine, Ekrn: ergokryptinine, Eco: ergocronine, Econ: ergocroninine, Es: ergosine, Esn: ergosinine

Matrix-matched calibration curves were obtained using wheat and maize blank samples spiked at five or six different concentration levels of mycotoxins, processed in duplicate, and injected in triplicate. Since the isomers 3-ADON/ and 15-ADON show a similar behavior in LC-MS [20], calibration curves for their respective sums were carried out using only 3-ADON standards, respectively. Moreover, an approximate quantification of DON-3-glu was carried out in this study using DON as standard, assuming that they present similar analytical behaviors, as it was previously reported for the determination of other secondary metabolites, such as several analogs of OTA [26] and destruxins [27]. The studied concentrations were as follows: 1 to 20 μg/kg for AFB1, AFB2, AFG1, AFG2; 0.5 to 20 μg/kg for STE; 2 to 200 μg/kg for OTA; 1 to 100 μg/kg for T-2, FB2, FB3, NEO; 10 to 100 μg/kg for DAS; 10 to 200 μg/kg for HT-2, ROQ-C, ZEN, α-ZOL; 5 to 100 μg/kg for FB1; 10 to 150 μg/kg for CIT; 20 to 400 μg/kg for DON, 3-ADON, F-X; 100 to 500 μg/kg for NIV; 10 to 200 μg/kg for the six major ergot alkaloids and their epimers.

Tables 3 and 4 summarize the results of linearity, LODs and LOQs for wheat and maize, respectively, and as can be seen satisfactory determination coefficients (R2 > 0.98) were obtained confirming that all analytical responses were linear over the studied ranges. Low LOQs were obtained for most mycotoxins, being below the legislated maximum levels for those mycotoxins in maize and wheat intended for direct human consumption (AFB1 < 2 μg/kg, sum of four aflatoxins < 4 μg/kg, OTA < 3 μg/kg, DON < 750 μg/kg, ZEN < 75 μg/kg, and sum of FB1 and FB2 < 400 μg/kg) [8], or below the recommended maximum levels (sum of T-2 and HT-2 < 100 μg/kg in maize and < 200 μg/kg in wheat) [28]. The highest LODs were obtained for NIV, which was attributed to its lower ionization compared to the other target mycotoxins and possibly to signal suppression effects induced by matrix. LOQs of EAs (ranged between 3.1 and 9.8 μg/kg) were somewhat higher than those obtained using LC-MS/MS methods focused only on EAs determination [17], but lower [29] or similar [14] to those obtained with multi-class LC-MS/MS methods.
Table 3

Performance characteristics of the proposed method in wheat

Mycotoxin

Equation

Linear range (μg/kg)

Linearity R2

LOD (μg/kg)

LOQ (μg/kg)

AFB1

y = 0.0184× + 0.0104

0.88–20

0.993

0.27

0.88

AFB2

y = 0.0855× + 0.02

0.44–20

0.993

0.13

0.44

AFG1

y = 0.0136× + 0.003

0.89–20

0.992

0.27

0.89

AFG2

y = 0.2135× + 0.003

0.76–20

0.993

0.23

0.76

STE

y = 0.0132× + 0.0001

0.46–20

0.996

0.14

0.46

OTA

y = 0.023× + 0.8281

1.90–200

0.991

0.58

1.90

T-2

y = 0.0153× + 0.292

0.90–100

0.992

0.27

0.90

HT-2

y = 0.0017× + 0.0177

8.61–200

0.991

2.61

8.61

FB1

y = 0.0031× + 0.0226

1.28–100

0.991

1.28

4.24

FB2

y = 0.0053× + 0.0969

0.82–100

0.993

0.25

0.82

FB3

y = 0.0102× + 0.1689

0.89–100

0.990

0.27

0.89

CIT

y = 0.0079× − 0.0038

8.69–150

0.992

2.63

8.69

ROQ-C

y = 0.0001× + 0.0015

9.41–200

0.990

2.85

9.41

ZEN

y = 0.0043× − 0.0212

8.92–200

0.991

2.70

8.92

α-ZOL

y = 0.0005× + 0.0038

8.12–200

0.986

2.46

8.12

DON

y = 0.0054× + 0.1556

18.05–400

0.993

5.47

18.05

DAS

y = 0.2812× + 4.3334

8.15–100

0.991

2.47

8.15

3-ADON

y = 0.0071× − 0.0275

15.87–400

0.994

4.81

15.87

F-X

y = 0.0062× − 0.0377

18.37–400

0.991

5.57

18.37

NEO

y = 0.1163× + 0.9727

3.06–100

0.992

0.93

3.06

NIV

y = 0.0005× + 0.0036

79.17–500

0.990

23.99

79.17

Em

y = 0.0067× + 0.0321

8.39–200

0.991

2.54

8.39

Emn

y = 0.0245× − 0.042

9.79–200

0.987

2.97

9.79

Et

y = 0.0001× + 0.0001

8.91–200

0.990

2.70

8.91

Etn

y = 0.0017× + 0.0092

9.77–200

0.996

2.96

9.77

Ecr

y = 0.001× + 0.0023

8.60–200

0.991

2.61

8.60

Ecrn

y = 0.0007× + 0.0039

6.53–200

0.987

1.98

6.53

Ekr

y = 0.0021× − 0.0051

5.19–200

0.984

1.57

5.19

Ekrn

y = 0.0003× + 0.0066

9.02–200

0.993

2.73

9.02

Eco

y = 0.0032× − 0.0083

9.17–200

0.991

2.78

9.17

Econ

y = 0.0004× + 0.0025

8.86–200

0.992

2.69

8.86

Es

y = 0.0052× + 0.0523

8.85–200

0.996

2.68

8.85

Esn

y = 0.0049× − 0.0006

9.22–200

0.990

2.88

9.52

AFB1: aflatoxin B1, AFB2: aflatoxin B2, AFG1: aflatoxin G1, AFG2: aflatoxin G2, OTA: ochratoxin A,T-2: T-2 toxin, HT-2: HT-2 toxin, FB1: fumonisin B1, FB2: fumonisin B2, FB3: fumonisin B3, CIT: citrinin, ROQ-C: roquefortine C, ZEN: zearalenone, α-ZOL: alpha zearalenol, DON: deoxynivalenol, DAS: diacetoxyscirpenol, 3-ADON: 3-acetyldeoxynivalenol, F-X: fusarenon-X, NEO: neosolaniol, NIV:nivalenol, Em: ergometrine, Emn: egometrinine, Et: ergotamine, Etn: ergotaminine, Ecr: ergocronine, Ecrn: egocristinine, Ekr: ergokryptine, Ekrn: ergokryptinine, Eco: ergocronine, Econ: ergocroninine, Es: ergosine, Esn: ergosinine, y: ratio of the peak area of the analyte to that of IS, x: analyte concentration

Table 4

Performance characteristics of the proposed method in maize

Mycotoxin

Equation

Linear range (μg/kg)

Linearity R2

LOD (μg/kg)

LOQ (μg/kg)

AFB1

y = 0.0014× + 0.0044

0.66–20

0.995

0.20

0.66

AFB2

y = 0.0301× − 0.0152

0.84–20

0.995

0.26

0.84

AFG1

y = 0.0019× + 0.0019

0.58–20

0.996

0.18

0.58

AFG2

y = 0.1403× − 0.07

0.79–20

0.992

0.24

0.79

STE

y = 0.0015× − 0.0002

0.46–10

0.995

0.14

0.46

OTA

y = 0.0311× + 0.7277

1.72–200

0.992

0.52

1.72

T-2

y = 0.0054× + 0.0356

3.95–100

0.991

1.20

3.95

HT-2

y = 0.0006× + 0.0062

7.40–200

0.991

2.24

7.40

FB1

y = 0.0016× − 0.007

3.82–100

0.989

1.16

3.82

FB2

y = 0.0049× + 0.0647

4.16–100

0.993

1.26

4.16

FB3

y = 0.0116× + 0.0847

0.90–100

0.991

0.27

0.90

CIT

y = 0.0052× − 0.0145

7.78–150

0.991

2.36

7.78

ROQ-C

y = 5E-05× − 0.0002

9.29–200

0.989

2.81

9.29

ZEN

y = 0.0039× + 0.0334

9.74–200

0.992

2.95

9.74

α-ZOL

y = 0.0009× − 0.0042

8.19–200

0.991

2.48

8.19

DON

y = 0.0066× + 0.3252

17.38–400

0.989

5.27

17.38

DAS

y = 0.1729× + 1.8256

8.44–100

0.991

2.56

8.44

3-ADON

y = 0.0111× + 0.136

18.90–400

0.992

5.73

18.90

F-X

y = 0.0092× − 0.0706

19.16–400

0.991

5.81

19.16

NEO

y = 0.0415× + 0.1655

5.31–100

0.995

1.61

5.31

NIV

y = 0.001× − 0.0092

90.66–500

0.992

27.47

90.66

Em

y = 0.0101× + 0.0679

9.12–200

0.9888

2.77

9.12

Emn

y = 0.0162× + 0.0476

7.64–200

0.9839

2.32

7.64

Et

y = 0.0262× + 0.3157

9.84–200

0.9859

2.98

9.84

Etn

y = 0.023× + 0.1148

7.00–200

0.9828

2.12

7.00

Ecr

y = 0.0192× + 1.3763

3.14–200

0.9840

0.95

3.14

Ecrn

y = 0.0052× − 0.0302

9.34–200

0.9786

2.83

9.34

Ekr

y = 0.0133× + 0.0859

9.52–200

0.9791

2.89

9.52

Ekrn

y = 0.003× + 0.0184

9.87–200

0.9723

2.69

8.87

Eco

y = 0.0152× − 0.0628

6.73–200

0.9812

2.04

6.73

Econ

y = 0.0032× − 0.011

6.46–200

0.9804

1.96

6.46

Es

y = 0.0367× − 0.3513

9.10–200

0.9779

2.76

9.10

Esn

y = 0.0356× − 0.2979

7.95–200

0.9802

2.41

7.95

AFB1: aflatoxin B1, AFB2: aflatoxin B2, AFG1: aflatoxin G1, AFG2: aflatoxin G2, OTA: ochratoxin A, T-2: T-2 toxin, HT-2: HT-2 toxin, FB1: fumonisin B1, FB2: fumonisin B2, FB3: fumonisin B3, CIT: citrinin, ROQ-C: roquefortine C, ZEN: zearalenone, α-ZOL: alpha zearalenol, DON: deoxynivalenol, DAS: diacetoxyscirpenol, 3-ADON: 3-acetyldeoxynivalenol, F-X: fusarenon-X, NEO: neosolaniol, NIV: nivalenol, Em: ergometrine, Emn: egometrinine, Et: ergotamine, Etn: ergotaminine, Ecr: ergocronine, Ecrn: egocristinine, Ekr: ergokryptine, Ekrn: ergokryptinine, Eco: ergocronine, Econ: ergocroninine, Es: ergosine, Esn: ergosinine

y: ratio of the peak area of the analyte to that of IS, x: analyte concentration

Precision

The precision of the proposed method was evaluated in terms of repeatability (intraday precision) and intermediate precision (interday precision). Repeatability was assessed for wheat and maize samples by application of the whole procedure to three samples (technical replicates) spiked at two different concentration levels of each mycotoxin. All the samples were processed on the same day, and each extract was injected in triplicate (instrumental replicates). Intermediate precision was evaluated with a similar procedure, spiking and analyzing three different samples on three different days. The results of precision study, expressed as relative standard deviation (RSD, %) of the ratio of analyte peak area/internal standard peak area, are shown in Tables 5 and 6 for wheat and maize, respectively. In all cases, RSD values lower than 13% were obtained, conforming with current legislation [30].
Table 5

Precision study (% RSD of the ratio between analyte peak area/internal standard peak area) in wheat samples

 

Repeatability (n = 9)

Intermediate precision (n = 9)

Level 1

Level 2

Level 1

Level 2

AFB1

9.2

5.2

10.9

5.8

AFB2

6.6

5.5

10.5

8.9

AFG1

8.9

6.4

11.4

8.6

AFG2

7.6

6.6

8.0

7.1

STE

5.5

6.9

8.0

7.8

OTA

3.6

5.8

6.2

10.7

T-2

4.0

3.3

10.7

12.6

HT-2

6.7

5.3

9.7

8.8

FB1

9.1

9.1

9.2

10.7

FB2

8.9

7.0

9.8

11.1

FB3

6.1

7.8

11.2

7.8

CIT

4.3

5.4

8.8

9.1

ROQ-C

9.5

8.1

11.5

8.1

ZEN

5.7

8.8

9.1

10.8

α-ZOL

7.4

8.8

8.7

10.1

DON

9.9

2.9

9.3

8.9

DAS

9.0

4.4

9.2

5.2

3-ADON

5.1

8.3

11.8

11.6

F-X

4.6

8.1

8.9

8.1

NEO

7.0

8.0

7.0

10.5

NIV

5.9

4.3

7.9

6.6

Em

4.0

2.1

8.8

3.7

Emn

1.8

1.2

6.3

7.0

Et

8.2

5.7

8.5

6.2

Etn

6.1

7.8

9.7

11.9

Ecr

6.6

7.9

9.3

11.1

Ecrn

7.9

5.9

11.7

7.7

Ekr

7.5

3.9

10.5

9.5

Ekrn

7.1

5.3

8.9

11.8

Eco

3.9

4.0

5.6

10.3

Econ

9.0

8.8

12.1

8.8

Es

4.9

4.1

10.9

5.0

Esn

4.4

4.4

7.7

7.2

Level 1: AFB1, AFB2, AFG1, AFG2: 1 μg/kg; OTA: 2 μg/kg; STE: 0.5 μg/kg; T-2, FB2, FB3, DAS: 10 μg/kg; NEO: 1 μg/kg; HT-2, CIT, ROQ-C, ZEN, α-ZOL: 10 μg/kg; FB1: 5 μg/kg; DON, 3-ADON, F-X: 20 μg/kg; NIV: 100 μg/kg; Ergot alkaloids: 10 μg/kg

Level 2: AFB1, AFB2, AFG1, AFG2, OTA: 10 μg/kg; OTA: 100 μg/kg; STE: 10 μg/kg; T-2, FB2, FB3, DAS, NEO: 50 μg/kg;HT-2, CIT, ROQ-C, ZEN, α-ZOL: 150 μg/kg; FB1: 100 μg/kg; DON, 3-ADON, F-X: 300 μg/kg; NIV: 400 μg/kg; Ergot alkaloids: 150 μg/kg

AFB1: aflatoxin B1, AFB2: aflatoxin B2, AFG1: aflatoxin G1, AFG2: aflatoxin G2, OTA: ochratoxin A, T-2: T-2 toxin, HT-2: HT-2 toxin, FB1: fumonisin B1, FB2: fumonisin B2, FB3: fumonisin B3, CIT: citrinin, ROQ-C: roquefortine C, ZEN: zearalenone, α-ZOL: alpha zearalenol, DON: deoxynivalenol, DAS: diacetoxyscirpenol, 3-ADON: 3-acetyldeoxynivalenol, F-X: fusarenon-X, NEO: neosolaniol, NIV: nivalenol, Em: ergometrine, Emn: egometrinine, Et: ergotamine, Etn: ergotaminine, Ecr: ergocronine, Ecrn: egocristinine, Ekr: ergokryptine, Ekrn: ergokryptinine, Eco: ergocronine, Econ: ergocroninine, Es: ergosine, Esn: ergosinine

Table 6

Precision study (% RSD of the ratio between analyte peak area/internal standard peak area) in maize samples

 

Repeatability (n = 9)

Intermediate precision (n = 9)

Level 1

Level 2

Level 1

Level 2

AFB1

6.6

9.8

9.9

10.7

AFB2

8.5

4.2

10.4

8.9

AFG1

7.2

6.7

8.3

7.5

AFG2

8.3

2.3

9.5

8.1

STE

9.2

9.6

10.7

10.5

OTA

7.0

4.6

7.3

5.6

T-2

9.1

9.9

9.6

10.7

HT-2

7.9

9.4

9.9

10.7

FB1

6.7

9.6

8.2

10.8

FB2

6.0

7.7

8.5

8.9

FB3

5.2

5.2

6.8

8.0

CIT

5.5

8.3

8.1

9.1

ROQ-C

8.7

9.5

9.4

10.2

ZEN

4.6

4.0

7.2

9.2

α-ZOL

9.9

7.6

10.0

9.9

DON

8.0

7.4

8.8

9.5

DAS

9.9

8.7

10.0

9.1

3-ADON

8.3

3.6

9.4

9.6

F-X

6.7

8.8

8.2

9.9

NEO

8.6

6.8

9.9

9.6

NIV

6.5

4.6

8.7

9.7

Em

10.4

8.0

11.8

10.2

Emn

9.5

8.5

10.7

11.3

Et

9.0

8.5

10.1

9.8

Etn

9.9

8.8

10.9

11.6

Ecr

10.0

9.3

12.1

11.1

Ecrn

4.5

8.1

8.9

10.8

Ekr

9.7

9.8

11.2

11.8

Ekrn

6.9

7.0

9.9

10.4

Eco

7.8

9.0

8.2

10.8

Econ

10.1

9.5

11.5

10.9

Es

10.7

10.2

10.8

11.0

Esn

8.5

6.8

9.1

10.1

Level 1: AFB1, AFB2, AFG1, AFG2: 1 μg/kg; OTA: 2 μg/kg; STE: 0.5 μg/kg; T-2, FB2, FB3, DAS: 10 μg/kg; NEO: 1 μg/kg; HT-2, CIT, ROQ-C, ZEN, α-ZOL: 10 μg/kg; FB1: 5 μg/kg; DON, 3-ADON, F-X: 20 μg/kg; NIV: 100 μg/kg; Ergot alkaloids: 10 μg/kg

Level 2: AFB1, AFB2, AFG1, AFG2, OTA: 10 μg/kg; OTA: 100 μg/kg; STE: 10 μg/kg; T-2, FB2, FB3, DAS, NEO: 50 μg/kg; HT-2, CIT, ROQ-C, ZEN, α-ZOL: 150 μg/kg; FB1: 100 μg/kg; DON, 3-ADON, F-X: 300 μg/kg; NIV: 400 μg/kg; Ergot alkaloids: 150 μg/kg

AFB1: aflatoxin B1, AFB2: aflatoxin B2, AFG1: aflatoxin G1, AFG2: aflatoxin G2, OTA: ochratoxin A, T-2: T-2 toxin, HT-2: HT-2 toxin, FB1: fumonisin B1, FB2: fumonisin B2, FB3: fumonisin B3, CIT: citrinin, ROQ-C: roquefortine C, ZEN: zearalenone, α-ZOL: alpha zearalenol, DON: deoxynivalenol, DAS: diacetoxyscirpenol, 3-ADON: 3-acetyldeoxynivalenol, F-X: fusarenon-X, NEO: neosolaniol, NIV: nivalenol, Em: ergometrine, Emn: egometrinine, Et: ergotamine, Etn: ergotaminine, Ecr: ergocronine, Ecrn: egocristinine, Ekr: ergokryptine, Ekrn: ergokryptinine, Eco: ergocronine, Econ: ergocroninine, Es: ergosine, Esn: ergosinine

Recovery studies

In order to assess the trueness of the proposed method, recovery experiments were carried out on wheat and maize samples, previously analyzed to establish the absence of detectable mycotoxins. None of them gave a positive result above the LODs of the method. These samples were spiked at three different concentration levels, processed as described previously and injected in triplicate into the UPLC-MS/MS system. Table 7 summarizes the results of recovery studies, which illustrates recoveries above 60% were obtained in all cases, ranging between 60 and 98% in maize and between 62 and 103% in wheat, fulfilling requirements of the current applicable legislation [30]. Slightly lower recoveries were obtained in maize for most mycotoxins except for CIT. However, similar values were obtained in both matrices for DAS, 3-ADON, F-X, NEO, and NIV. The most important difference was observed for ZEN, ranging between 82 and 88% in wheat and between 67 and 74% in maize. In the case of EAs, the recoveries were between 60 and 89%, not observing any significant difference between the main EAs and their epimers.
Table 7

Recovery (%) study in wheat and maize (% RSD)

 

Wheat (n = 9)

Maize (n = 9)

Level 1

Level 2

Level 1

Level 2

AFB1

100.2 (9.2)

96.8 (5.2)

85.3 (6.9)

89.9 (7.4)

AFB2

99.2 (6.6)

87.7 (5.5)

86.4 (9.2)

88.4 (4.2)

AFG1

98.5 (8.9)

84.5 (6.4)

74.7 (7.1)

76.5 (6.7)

AFG2

97.5 (7.6)

87.8 (6.6)

82.8 (5.7)

80.4 (1.9)

STE

101.9 (5.5)

90.7 (6.9)

74.6 (9.0)

81.6 (9.5)

OTA

92.7 (3.6)

87.0 (5.8)

88.1 (7.0)

88.2 (3.7)

T-2

94.2 (4.0)

80.4 (3.3)

91.0 (9.1)

79.9 (9.2)

HT-2

90.6 (6.7)

85.1 (5.3)

88.6 (7.9)

80.1 (6.9)

FB1

99.3 (9.1)

87.3 (9.1)

79.5 (6.7)

66.3 (9.4)

FB2

80.1 (8.9)

77.1 (7.0)

71.2 (6.0)

70.9 (7.1)

FB3

100.6 (6.1)

93.2 (7.8)

81.8 (5.2)

78.6 (5.0)

CIT

70.8 (4.3)

68.8 (5.4)

95.3 (5.5)

84.5 (7.5)

ROQ-C

99.5 (9.5)

89.7 (8.1)

84.6 (8.7)

82.6 (8.6)

ZEN

87.8 (5.7)

82.7 (8.8)

66.8 (4.6)

71.2 (4.0)

α-ZOL

81.0 (7.4)

77.2 (8.8)

71.3 (9.9)

66.5 (7.2)

DON

95.6 (9.9)

91.4 (2.9)

76.7 (8.0)

74.7 (6.6)

DAS

93.6 (9.0)

92.7 (4.4)

91.1 (9.6)

92.1 (5.8)

3-ADON

87.7 (5.1)

89.7 (8.3)

96.0 (8.3)

87.5 (3.2)

F-X

86.0 (4.6)

88.0 (8.1)

92.3 (6.7)

85.9 (7.9)

NEO

91.9 (7.0)

97.3 (8.0)

89.3 (8.6)

94.5 (6.8)

NIV

78.0 (5.9)

82.8 (4.3)

76.5 (6.5)

82.9 (4.3)

Em

61.5 (4.0)

66.6 (2.1)

60.4 (10.4)

67.1 (8.0)

Emn

65.5 (1.8)

70.2 (1.2)

67.7 (9.5)

63.3 (8.5)

Et

79.8 (8.2)

81.8 (5.7)

64.4 (9.0)

62.7 (8.5)

Etn

73.2 (6.1)

70.0 (7.8)

70.5 (9.9)

63.4 (8.8)

Ecr

72.0 (6.6)

75.5 (7.9)

64.8 (10.0)

63.1 (9.3)

Ecrn

74.5 (7.9)

77.4 (5.9)

60.5 (4.5)

60.0 (8.1)

Ekr

67.0 (7.5)

68.6 (3.9)

59.9 (9.7)

61.8 (9.8)

Ekrn

78.4 (7.1)

78.9 (5.3)

62.4 (6.9)

65.9 (7.0)

Eco

67.1 (3.9)

66.1 (4.0)

69.6 (7.8)

64.1 (9.0)

Econ

61.8 (9.0)

65.4 (8.8)

60.1 (10.1)

68.0 (9.5)

Es

64.1 (4.9)

66.2 (4.1)

60.7 (10.7)

65.6 (10.2)

Esn

65.6 (4.4)

63.9 (4.4)

66.8 (8.5)

70.0 (6.8)

Level 1: AFB1, AFB2, AFG1, AFG2: 1 μg/kg; OTA: 2 μg/kg; STE: 0.5 μg/kg; T-2, FB2, FB3, DAS: 10 μg/kg; NEO: 1 μg/kg; HT-2, CIT, ROQ-C, ZEN, α-ZOL: 10 μg/kg; FB1: 5 μg/kg; DON, 3-ADON, F-X: 20 μg/kg; NIV: 100 μg/kg; Ergot alkaloids: 10 μg/kg

Level 2: AFB1, AFB2, AFG1, AFG2, OTA: 10 μg/kg; OTA: 100 μg/kg; STE: 10 μg/kg; T-2, FB2, FB3, DAS, NEO: 50 μg/kg;HT-2, CIT, ROQ-C, ZEN, α-ZOL: 150 μg/kg; FB1: 100 μg/kg; DON, 3-ADON, F-X: 300 μg/kg; NIV: 400 μg/kg; Ergot alkaloids: 150 μg/kg

AFB1: aflatoxin B1, AFB2: aflatoxin B2, AFG1: aflatoxin G1, AFG2: aflatoxin G2, OTA: ochratoxin A, T-2: T-2 toxin, HT-2: HT-2 toxin, FB1: fumonisin B1, FB2: fumonisin B2, FB3: fumonisin B3, CIT: citrinin, ROQ-C: roquefortine C, ZEN: zearalenone, α-ZOL: alpha zearalenol, DON: deoxynivalenol, DAS: diacetoxyscirpenol, 3-ADON: 3-acetyldeoxynivalenol, F-X: fusarenon-X, NEO: neosolaniol, NIV: nivalenol, Em: ergometrine, Emn: egometrinine, Et: ergotamine, Etn: ergotaminine, Ecr: ergocronine, Ecrn: egocristinine, Ekr: ergokryptine, Ekrn: ergokryptinine, Eco: ergocronine, Econ: ergocroninine, Es: ergosine, Esn: ergosinine

Analysis of naturally contaminated samples

In order to further demonstrate the fit for purpose of the method, it was applied to the analysis of 13 wheat samples and 15 maize samples sourced from different countries of Europe (Belgium, Spain, Italy, Switzerland, Lithuania, and Hungary). The overall mycotoxin contamination is summarized in the Electronic Supplementary Material (ESM) in Tables S1 and S2 for wheat and maize, respectively. It has to be noted that the reported concentrations were not rounded up and measurement uncertainty was not taken into account. 85% (11/13) of wheat samples and 93% (14/15) of maize samples gave results above the LOQs for one or more mycotoxins. The most common mycotoxins were fumonisins and EAs in maize and wheat samples, respectively. The following mycotoxins were not detected in any of the wheat or maize samples: AFG1, AFG2, STE, OTA, CIT, ROQ-C, α-/β-ZOL, DON-Glu, F-X and NEO. Additionally, T-2, HT-2 and EAs were not detected in maize, while AFB1, AFB2, FB3 and DAS were not present in wheat samples. Concerning the regulated mycotoxins, maximum limits were exceeded by a wheat sample contaminated with ZEN at 78.7 (maximum limit, 75 μg/kg), and 11 maize samples contaminated with ZEN, DON (maximum limit, 750 μg/kg), and fumonisins (maximum limit for sum of fumonisins, 400 μg/kg). Aflatoxins were detected in only one maize sample, exceeding the maximum limit, since AFB1 was found at 3 μg/kg (maximum limit, 2 μg/kg) and AFB2 at 2 μg/kg (maximum limit for the sum of four aflatoxins, 4 μg/kg).

EAs were detected in 10 of 13 analyzed wheat samples. Et and its epimer Etn were found to be the most frequently occurring EAs, reaching also the highest levels of contamination. Co-occurrence of EAs with NIV (90% of samples) was the most frequent, followed by co-occurrence with DON (7 of the 10 contaminated samples with EAs) and co-occurrence with HT-2 (2 of the 10 contaminated samples with EAs).

In addition, one sample showed co-occurrence of EAs, fumonisins (FB1, FB2), T-2, ZEN, and DON and its derivates (3-ADON/15ADON).

In maize, co-occurrence of fumonisins and ZEN was detected in 57% of samples, while fumonisins with DON and its derivates (3ADON/15ADON or DAS) were found in 67% of contaminated maize samples.

Interestingly, while DON co-occurred with its derivatives 3ADON/15ADON in 75% of maize samples positive for DON, this co-occurrence in wheat samples amounted only to 13%.

Conclusions

As previously mentioned, the European Union’s food and feed safety alert system is recording an annual increase in positive detections of mycotoxins. The degree to which this is representative of increased fungal proliferation or simply a consequence of improved detection methods shall be disambiguated in time. However, the fact remains that staple crops from all over the world are contaminated by an increasingly well-characterized array of mycotoxins, and this reality is thoroughly corroborated by the international research community.

Mycotoxin targets produced by Claviceps spp. with those from Aspergillus, Fusarium and Penicillium fungi were included in the presently described method. This constitutes a more comprehensive single-analysis approach than previously published methods. Furthermore, optimized extraction conditions for all target compounds at the sample preparation stage were shown to support the very high sensitivity of UPLC-MS/MS proposed method.

Analysis of naturally contaminated samples confirms the prolific co-occurrence of mycotoxins, and reflects the reality of cereal co-contamination by multiple mycotoxins that may themselves be produced by multiple, even non-related fungal species. This significant result underlines the importance and applicability of the method to ongoing food and feed safety screening programs. To this end, it is of particular note that, despite its broad efficacy, the described method requires relatively inexpensive consumables, and little specialized training in utilizing sample preparation protocol to be effectively deployed. The simplicity and speed of the SO-LLE procedure in combination with short analytical sequences were key for a robust and correct determination of the individual ergot alkaloids and their corresponding epimers.

Results produced by such a rigorous method constitute integral support for other lines of investigation in all three temporal dimensions. In the present, samples determined to be contaminated above maximum levels may be immediately diverted from the food and feed supply. Secondly, characterizing the heterogeneity of mycotoxin contamination between sample sets informs epidemiological studies of fungal proliferation. Analysis of mycotoxin primary production, and assessments of intervention strategies at the farm level are vital to prevent mycotoxins from reaching agricultural products. Finally, identification of common contamination profiles provides a fundamental basis for elucidating the real-world health effects produced by these contaminants. Though the pathogenic mechanisms underlying mycotoxicity are often studied in idealized dose-response systems, both in vivo and in vitro, assessment of real-world health effects attributable to mycotoxin co-exposure relies on accurate data characterizing the extant contamination.

Notes

Acknowledgements

The authors thank the Spanish Ministry of Economy and Competitiveness (Project Ref: AGL2015-70708-R). Natalia Arroyo-Manzanares received a post-doctoral grant from the University of Granada.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_1018_MOESM1_ESM.pdf (134 kb)
ESM 1 (PDF 133 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Natalia Arroyo-Manzanares
    • 1
    • 2
  • Karl De Ruyck
    • 1
  • Valdet Uka
    • 1
  • Laura Gámiz-Gracia
    • 2
  • Ana M. García-Campaña
    • 2
  • Sarah De Saeger
    • 1
  • José Diana Di Mavungu
    • 1
  1. 1.Laboratory of Food Analysis, Faculty of Pharmaceutical SciencesGhent UniversityGhentBelgium
  2. 2.Department of Analytical ChemistryUniversity of GranadaGranadaSpain

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