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Journal of Chromatography A, 1354 (2014) 139–143

Contents lists available at ScienceDirect

Journal of Chromatography A

j o ur na l ho me page: www.elsev ier .com/ locate /chroma

hort communication

mproved method for the simultaneous determination of aflatoxins,chratoxin A and Fusarium toxins in cereals and derived products byiquid chromatography–tandem mass spectrometry after multi-toxinmmunoaffinity clean up

eronica Maria Teresa Lattanzioa,∗, Biancamaria Ciascaa, Stephen Powersb,ngelo Visconti a

National Research Council of Italy (CNR), Institute of Sciences of Food Production (ISPA), Via Amendola 122/o, 70126 Bari-ItalyVicam, A Waters Business, 34 Maple Street, Milford, MA 01757, USA

r t i c l e i n f o

rticle history:eceived 28 March 2014eceived in revised form 26 May 2014ccepted 27 May 2014vailable online 2 June 2014

eywords:C–MS/MSflatoxinschratoxin Ausarium toxinserealsulti-mycotoxin immunoaffinity

a b s t r a c t

An improved method for the quantitative determination of aflatoxins (B1, B2, G1, G2), ochratoxin A, fumon-isins (B1, B2), zearalenone, deoxynivalenol, nivalenol, T-2 and HT-2 toxins in cereals and derived products,at levels comparable with EU maximum permitted levels, was developed. The effective co-extraction ofthe mycotoxins under investigation was achieved in 4 min by a double extraction approach, using waterfollowed by methanol. Clean up of the extract was performed by a new multi-toxin immunoaffinity col-umn. Analytical performance characteristics were evaluated through single laboratory validation. Rawwheat and maize, corn flakes and maize snacks were chosen as representative matrices for method vali-dation. The validation assay was carried out at 50, 100 and 150% of EU maximum permitted levels foreach mycotoxin. Statistical analysis of the results (ANOVA) provided the within laboratory reproducibil-ity and the error contributions from repeatability, between day effects, and influences from differentmatrix composition. Recoveries generally higher than 70% were obtained for all tested mycotoxins withrelative standard deviation (within laboratory reproducibility) lesser than 37%. Limits of quantification

(calculated as the lowest amount of each analyte which could be determined with a precision of 10%)ranged from 1 �g/kg to 30 �g/kg. The trueness of generated data was assessed by analysis of referencematerials. The proposed method was proven to be suitable to assess, with a single analysis, compliance ofthe selected cereal based foods with the EU maximum permitted or recommended levels for all regulatedmycotoxins.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Mycotoxins are naturally occurring toxic metabolites which cane produced by fungi infecting agricultural crops during the growth,rying, and subsequent storage. The natural fungal flora associatedith foods is dominated by the genera Aspergillus, Fusarium, Peni-

illium, and Alternaria [1]. The range of foods susceptible to fungal

rowth and subsequent mycotoxin contamination is large and rep-esents many of the staple food crops worldwide. Mycotoxins aremall molecules with various chemical structures and, therefore,

∗ Corresponding author at: Institute of Sciences of Food Production (ISPA),ational Research Council of Italy (CNR), Via Amendola 122/O, 70126 Bari, Italy.el.: +39 080 5929364; fax: +39 080 5929374.

E-mail address: veronica.lattanzio@ispa.cnr.it (V.M.T. Lattanzio).

ttp://dx.doi.org/10.1016/j.chroma.2014.05.069021-9673/© 2014 Elsevier B.V. All rights reserved.

various biological effects that range from acute to chronic symp-toms [2].

In Europe, harmonized maximum permitted levels for the majormycotoxins, namely aflatoxins (AFB1, AFB2, AFG1, AFG2), ochra-toxin A (OTA), fumonisins (FB1, FB2), deoxynivalenol (DON), andzearalenone (ZEA) are currently included in the European legisla-tion [3,4] and indicative maximum levels for the sum of T-2 (T-2)and HT-2 (HT-2) toxins have been recently issued [5]. Although notregulated, attention is paid to the occurrence of nivalenol (NIV),another Fusarium toxin that frequently contaminates cereals alsoin combination with DON [6].

Reliable methods to enable enforcement of the regulations

in daily practice are highly desired. For these purposes severalLC–MS methods have been proposed for the determination of singleand multi-mycotoxins in foods [7–10]. The reliability of myco-toxin analysis data can be improved by means of interlaboratory

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alidated methods of analysis, e.g. the official methods of AOACnternational and the methods standardized by European Standard-zation Committee (CEN). To date none of the AOAC Internationalr CEN methods, which refer mainly to single or closely relatedycotoxins in different food matrices, is based on LC–MS. When

nterlaboratory validated methods are not available, a singleaboratory validation should be conducted to evaluate method per-ormances. In house method performances must be in complianceith acceptability criteria specified in the Commission Regulation

01/2006/EC [11], and in the Technical Report CEN/TR 16059:201012].

A LC–ESI-MS/MS method intended for quantitative determi-ation of 11 major mycotoxins in maize at levels comparableith EU maximum permitted levels was proposed in a previousanuscript [13]. The method was based on a double extraction

pproach, using water followed by methanol, to achieve effec-ive co-extraction of the mycotoxins under investigation, and

ulti-antibody immunoaffinity columns (IAC) for extract cleanp. When tested in maize, at EU maximum permitted levels, theethod showed satisfactory performances in terms of recoveries

nd repeatability. Modifications of this procedure, mainly dealingith the extraction step, were described by other authors [14,15].

The present manuscript describes a significant improvementf previous methods, with particular reference to the followingspects: extension of method applicability to a wider mycotoxinnd food commodity range, shortening and simplifying of samplereparation, full in house validation.

. Methods

.1. Chemicals and reagents

Acetonitrile, methanol (HPLC grade) and glacial acetic acidere purchased from Sigma–Aldrich (Milan, Italy). Ultrapure wateras produced by a Millipore Milli-Q system (Millipore, Bedford,A, USA). Ammonium acetate (for mass spectrometry), was from

igma–Aldrich (Milan, Italy). AFB1, AFB2, AFG1, AFG2, FB1, FB2, OTA,IV, DON, T-2, HT-2 and ZEA were purchased from Biopure Referen-ensubstanzen GmbH (Tulln, Austria). Whatman N. 4 filter papersere obtained from Whatman International Ltd. (Maidstone, UK).yco6in1+TM immunoaffinity columns were obtained from VICAM

A Waters business, Watertown, MA, USA). Phosphate bufferedolution at pH 7.4 (PBS) was prepared by dissolving commercialhosphate buffered saline tablets (Sigma–Aldrich, Milan, Italy) inistilled water.

.2. Samples

Maize, durum wheat, corn flakes, and maize crackers whereurchased from the Italian retail market, and analysed accordingo a previously validated multi-mycotoxin method [13] to confirmhe absence of mycotoxin contamination. The following reference

aterials were purchased from Trilogy Analytical Laboratory, Inc.Washington, USA): wheat contaminated with DON (batch n. D-W-67), maize contaminated with AFB1 and AFB2 (batch n. A-C-267)nd maize contaminated with FB1, FB2 (batch n. F-C-438).

.3. LC–MS/MS equipment and parameters

LC–MS/MS analyses were performed on a QTrap MS/MS sys-em, from Applied Biosystems (Foster City, CA, USA), equipped withn ESI interface and a 1100 series micro-LC system comprising a

inary pump and a microautosampler from Agilent TechnologiesWaldbronn, Germany). The analytical column was a Gemini® C18olumn (150 mm × 2 mm, 5 �m particles) (Phenomenex, Torrance,A, USA), preceded by a Gemini C18 guard column (4 mm × 2 mm,

gr. A 1354 (2014) 139–143

5 �m particles). The ESI interface was used in negative and posi-tive ion mode. All related instrumental parameters can be found inLattanzio et al. [13].

The mass spectrometer operated in MRM (multiple reactionmonitoring) mode as described in Table 1, by monitoring 2 tran-sitions (earning therefore 4 identification points according to ECguidelines [16]) for each compound, with a dwell time of 100 ms.Quantification of mycotoxins was performed by matrix matchedcalibration.

2.4. Mycotoxin solutions

A mixed standard solution was prepared in methanol with thefollowing mycotoxin concentrations: NIV and DON 37.5 �g/mL;T-2 and HT-2 2.5 �g/mL, ZEA 5 �g/mL; AFG1, AFG2, AFB2,0.05 �g/mL; AFB1, 0.1 �g/mL; FB1, 40 �g/mL; FB2, 10 �g/mL; andOTA, 0.15 �g/mL. This solution was used for spiking experimentsand to prepare calibrant solutions.

Calibrant solutions for external matrix-matched calibration(five points) were prepared in blank sample extract solutionspassed through Myco6in1+TM columns, according to the cleanup procedure described in the following. Appropriate volumes ofmixed standard solution were added to the column eluate beforedrying it down. Then the residue was redissolved with 200 �Lof methanol/water 20/80 by vortexing for 1 min. Matrix-matchedcalibrations were performed in the range 187.5–1500 �g/kg NIVand DON, 0.5–4 �g/kg AFB1, 0.25–2 �g/kg AFG1, AFG2, and AFB2,12.5–100 �g/kg T-2 and HT-2, 200–1600 �g/kg FB1, 50–400 �g/kgFB2, 25–200 �g/kg ZEA, and 0.75–6 �g/kg OTA. Matrix matched cal-ibrations were prepared for each food matrix considered in thisstudy using the relevant blank extract. To use exactly the samesample for matrix matched calibration, analysis of reference mate-rials was performed by the standard addition method, i.e. by addingknown amounts of reference standard of the target analytes to thesample before the extraction.

2.5. Sample preparation (extraction and clean-up)

Cereal and cereal food samples were finely ground by an UltraCentrifugal Mill ZM 200 (Retsch GmbH, Haan, Germany) to pass a500 �m sieve.

Ten grams of sample were weighed into a blender jar andextracted with 40 mL of water by blending at high speed for 2 min.Then 60 mL of methanol was added to the sample (without remov-ing the first extract) and extracted again by high speed blendingfor 2 min. The extract was filtered through paper filter (WhatmanN.4). Five millilitres (0.5 g sample) of filtered extract were evapo-rated under air stream at about 40–50 ◦C to reduce the volume toapproximately 2 mL. Then 5 mL PBS was added. The samples werepassed through the Myco6in1+TM column, then the column waswashed with 10 mL of water. After drying the column by a gentleair flow, toxins were eluted with 3 mL water and 2 mL methanol byapplying the following procedure: 1.5 mL of methanol were passedthrough the column by gravity and the eluate was collected in a10 mL glass tube vial. Then a second portion of 1.5 mL of methanolwas applied and let stand in the column bed for 5 min, then theeluate was collected in the same tube. Afterwards 2 mL water werepassed through the column and collected in the same tube. Theeluate was dried under air stream at 40 ◦C and reconstituted with200 �L of methanol/water 20:80. Twenty microliters (equivalent to50 mg sample) were injected into the LC–MS apparatus.

For recovery experiments, individual sub-samples (10 g) were

spiked with an appropriate volume of mixed mycotoxin solu-tion. Spiked samples were left overnight at room temperature toallow solvent evaporation and equilibration between analytes andmatrix.

V.M.T. Lattanzio et al. / J. Chromatogr. A 1354 (2014) 139–143 141

Table 1MRM parameters for mycotoxin detection.

Analyte Precursor ion Q1 (m/z) Q3 (m/z) DP (V) EP (V) CE (V) CXP (V)

NIV [NIV+CH3COO]− 371.1 281.159.1a

−23 −6 −19−38

−2.8−6.2

DON [DON+CH3COO]− 355.1 295.059.0a

−23 −4.5 −17−40

−2.5−7.0

AFG2 [AFG2+H]+ 331.1 313.2a

245.360 9 30

3543.5

AFG1 [AFG1+H]+ 329.1 311.2243.1a

54 10 2333

4.53.5

AFB2 [AFB2+H]+ 315.0 287.2259.0a

60 9 3035

3.73.5

AFB1 [AFB1+H]+ 313.2 241.0a

213.455 11 50

603.53.5

HT-2 [HT-2+NH4]+ 442.0 263.2a

215.020 5 17

154.03.3

T-2 [T-2+NH4]+ 483.9 245.2a

185.320 7 20

253.6

FB1 [FB1+H]+ 722.0 352.2334.1a

70 9 5353

4.54.5

FB2 [FB2+H]+ 706.2 354.4336.4a

70 9 4547

15.04.0

OTA [OTA+H]+ 404.0 239.0a

221.130 5 30

454.04.0

ZEA [ZEA−H]− 317.0 175.1130.9

−70 −7 −30−40

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1: first quadrupole; Q3: third quadrupole; DP: declustering potential; EP: entranca Transition used for analyte quantification.

.6. Single laboratory validation design

The validation experiments were conducted according to thexperimental design as shown in Fig. 1. Four different food com-odities (maize, wheat, corn flakes, maize crackers) were analysed

n three different days. In addition, two of the food commoditiesere analysed in duplicates on each day under repeatability condi-

ions, resulting in 6 independent and complete analyses per day and8 measurements in total (per each spiking level). These food com-odities were analysed after fortification with the 12 mycotoxins

t three different concentrations, namely 50%, 100% and 150% of

he relevant EU maximum permitted levels (reported in Table 2).his design was therefore applied three times, namely for sampleet fortified at the three established concentrations. The analyti-al results were then subjected to analysis of variance (ANOVA),

able 2esults from ANOVA for each mycotoxin at three different spiking levels, showing the avehe contribution of the various error sources to the overall variance of the analytical resultesults are presented for the sum of the two toxins [13].

NIV DON AFB1

Spiking level (�g/kg) 375 375 1

50% of target level Mean recovery (%) 80 82 73

RSDWLR (%) 11 15 35

Source of errors (% of total variance)Day 1 40 41

Matrix 71 55 24

Repeatability 28 5 35

Spiking level (�g/kg) 750 750 2

100% of target level Mean recovery (%) 82 82 76

RSDWLR (%) 12 26 35

Source of errors (% of total variance)Day 0 2 0

Matrix 0 16 44

Repeatability 100 83 76

Spiking level (�g/kg) 1125 1125 3

150% of target level Mean recovery (%) 73 81 73

RSDWLR (%) 18 19 31

Source of errors (% of total variance)Day 0 0 18

Matrix 70 55 29

Repeatability 30 45 53

ntial; CE: collision energy; CXP: collision cell exit potential.

separately for each concentration level as specified by ISO 5725[17]. The model that underlies the analysis of variance of the datacollected by this nested design is that each of the measurement ofthe response Yijk is defined as the true value (TV) plus the contri-bution of 3 components estimated as follows:

Yijk = TV + Di + Mij + Rijk,

where Di is the between-day variability, Mij is the between-matrixvariability and Rijk is the within-day variability. The within-day variability gives the precision under repeatability conditions,

whereas all components including the between-days and between-matrix variability give the intermediate precision. The statisticalassessment was done with the software package MINITABTM Sta-tistical Software for Windows (Version 14).

rage recoveries, the within laboratory reproducibility (intermediate precision) and. Since selective hydrolysis of T-2 to HT-2 occurs during water extraction, validation

AFB2 AFG1 AFG2 T2 + HT2 FB1 FB2 ZEA OTA

0.5 0.5 0.5 50 400 100 50 4.578 69 76 77 88 65 64 7033 25 31 26 30 17 27 18

57 62 8 33 0 0 46 024 18 79 55 99 97 34 8519 20 13 12 1 3 20 15

1.0 1.0 1.0 100 800 200 100 379 85 93 87 76 68 74 7336 22 28 18 30 23 27 31

0 0 0 3 0 0 7 4539 31 34 62 55 69 0 061 69 66 35 45 31 93 55

1.5 1.5 1.5 150 1200 300 150 1.578 79 73 74 74 63 72 7133 28 37 26 26 23 20 19

28 60 0 0 28 0 1 400 7 48 55 67 61 0 31

72 33 52 45 5 39 99 29

142 V.M.T. Lattanzio et al. / J. Chromatogr. A 1354 (2014) 139–143

Fig. 1. Experimental design for single laboratory method validation. On each day,an identical set of 4 different food commodities (M1, maize, M2, wheat, M3, cornflakes, M4, maize crackers) were analysed in duplicates. The experimental designwa

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Fig. 2. Total ion chromatogram (sum of MRM transitions) of (A) maize sampleextract spiked with: NIV, DON 1000 �g/kg; AFG2, AFB2 2.5 �g/kg, AFG1 7.5 �g/kg,AFB1 12.5 �g/kg; FB1 800 �g/kg, FB2 200 �g/kg, HT-2, T-2, ZEA 100 �g/kg, OTA

ability criteria established by EC [11].

as applied for each food matrix three times, namely samples containing the analytet 50%, 100%, and 150% of the target levels.

. Results and discussion

The present manuscript describes an improvement of a previ-us method [13] based on multi-mycotoxin immunoaffinity cleanp and LC–MS/MS detection. In the previous method, the samplereparation protocol was based on a double extraction with waternd methanol, requiring 120 min shaking and a laborious clean uptep, since the two extracts were applied separately to the IAC tovoid losses of DON, weakly retained by the relevant antibody in theolumn bed. The introduction, in the commercial IAC, of a new anti-ody able to bind DON and NIV, allowed to: (a) include NIV amonghe target analytes, and (b) shorten and simplify the clean up pro-edure. The latter improvement was due to the higher tolerancef organic solvent showed by the new antibody, enabling to load

unique extract onto the IAC, instead of keeping the two extractseparated. Furthermore the two extraction steps were carried outy high speed blending requiring 4 min instead of 120. Examples ofC–MS/MS chromatograms of spiked and naturally contaminatedaize samples are shown in Fig. 2.

The optimized procedure was tested in four different food matri-

es at three spiking levels, corresponding to 50%, 100% and 150% EUaximum permitted or recommended levels (Fig. 1).

20 �g/kg; (B) maize sample extract naturally contaminated with DON 30 �g/kg,traces of AFB1 (<LOQ), FB1 2567 �g/kg, FB2 404 �g/kg, ZEA 88 �g/kg, and OTA5 �g/kg.

In addition to evaluation of recovery rates at the different testedconcentrations, the ANOVA of analytical results provided the fol-lowing information:

- The repeatability standard deviation;- The between day variation;- Matrix effects (on the whole method performance);- The within laboratory reproducibility, which was the sum of the

above error contributions.

Recovery rates and results of the statistical assessment for eachmycotoxin are shown in Table 2. Evaluating the estimated precisionprofile of the method revealed that in all cases the relative standarddeviation (within laboratory reproducibility, RSDWLR) varied from11% to 37% meeting CEN requirements for each mycotoxin at therelevant tested concentrations [12]. Satisfactory recovery valueswere obtained, ranging from 63% to 88%, in compliance with accept-

The between-matrix variation represented the major source ofvariance in 45% (15/33) of cases. It is important to underline thatthis experimental design evaluated the matrix effect on the whole

V.M.T. Lattanzio et al. / J. Chromatogr. A 1354 (2014) 139–143 143

Table 3Analysis of reference materials: comparison between reference and measured concentrations. Samples were extracted and purified onto Myco6in1+TM columns accordingto the optimized method (see details in Section 2).

Reference value(RV, �g/kg)

RV uncertainty(uRV, �g/kg)

Measured value(MV, �g/kg)

MV uncertainty(uMV, �g/kg)

�m (�g/kg) U� (�g/kg)

DON in wheat flourTrilogy batch n.D-W-167

2900 200 2548 306 352 731

AFB1, AFB2 inmaize flourTrilogy batch n.A-C-267

AFs 14.3 1.1 12.2 2.3 2.1 5.0

FB1, FB2 in maizeflourTrilogy batch n.F-C-438

FBs 3600 400 3575 277 25 973

uMV = [(LOD/2)2 + ( ̨ × C)2]1/2 is the uncertainty of measured value and was calculated using the formula reported in the Commission Regulation (EC) No 401/2006, whereLOD is the limit of detection of the method (�g/kg), ̨ is a constant, numeric factor to be used depending on the value of C. C is the concentration of interest (�g/kg).�m = |MV − RV| is the absolute difference between measured and reference value.U� = 2*u� is the expanded uncertainty of the difference between measured and reference value.uI nt rh

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nalytical procedure, that means the impact of matrix compositionn the extraction efficiency and mainly on the interactions betweenntibodies and their relevant mycotoxins. Whereas matrix effectsn the LC–MS response were supposed to be properly compensatedy the use of matrix matched calibration. The contribution of matrixhanges to the overall variation of the analytical results was simi-ar to that given by repeated measurements (in the same day). Onhe basis of these results an easy extension of the whole analyticalrocedure to other matrices without any modification is expected.

Quantification limits were estimated as the lowest amount ofach analyte which could be determined with a precision of 10%between matrix relative standard deviation), and were: 30 �g/kgor NIV, 20 �g/kg for DON, 1 �g/kg for AFB1, AFB2, AFG1, AFG2,

�g/kg for HT-2, T-2, FB1, FB2, 10 �g/kg for ZEA, and 1 �g/kg forTA. According to CEN TR 16059 [12] quantification limits suit-ble for enforcement of the legal limit shall be: equal to or lesshan maximum level (ML) × 1/5, when ML is >100 �g/kg, ML × 2/5,hen ML is <100 �g/kg, and ML/2n in case of regulation for the

um of n mycotoxins. Therefore quantification limits obtained forhis method were fit-for-purpose for NIV, DON, sum of T-2 + HT-, fumonisins, ZEA and OTA, and slightly higher than desired forflatoxins. It is worth mentioning that detection limits of 0.5 �g/kgn maize and 0.2 �g/kg in wheat, corn flakes and maize crackers

ere easily obtained for aflatoxins by analysing samples preparedccording to the developed method by a more sensitive mass spec-rometer (API 5000TM from ABSciex).

Finally, to demonstrate the trueness of generated data, referenceaterials were analysed with the proposed method. Mycotoxin

oncentrations were obtained by means of matrix assisted calibra-ion, and their accuracy was controlled considering the referencealue. The experimentally determined concentrations of all targetnalytes were in good agreement with reference values (see detailsn Table 3).

cknowledgements

This work was carried out with the financial support of

he Project MIUR – PON02 00186 3417512, “New Strategies formprovement of Food Safety: Prevention, Control, Correction”S.I.Mi.S.A) and the Project “MICOPRINCEM” funded by the Ital-an Ministry of Agricultural, Food and Forestry Policies (MiPAAF).

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Miriam Haidukowski is also acknowledged for her support in chem-ical analysis.

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17] International Organization for Standardization (ISO), ISO 5725-3:1994(E), ISOStandards, Geneva, 1994, ICS 03:120:30.