Validation of an UHPLC-MS/MS method for simultaneous analysis of 11 mycotoxins in wheat flour Using Immunoaffinity column

Document Type : Research article


1 Department of Pathobiology, Science and Research Branch, Islamic Azad University, Tehran , Iran.

2 Department of Medical Parasitology and Mycology, School of Public Health, Food Microbiology Research Center, Tehran University of Medical Sciences, Tehran, Iran.

3 Department of Toxicology and Pharmacology, School of Pharmacy, Shahid Beheshti University of Medical Sciences,Tehran, Iran.


This study focuses on optimization and validation of an Ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous analysis of 11mycotoxins: Aflatoxin B1, T-2 Toxin, Ochratoxin A, Deoxynivalenol and Zearalenone in wheat matrix. Sample extraction and cleanup procedure is based on a single extraction step using acetonitrile/water/acetic acid mixture (79.5/20/0.5 v/v/v) and rapid clean-up of samples were performed with the Myco6in1+ Immunoaffinity column. Electrospray ionization at positive mode was operated to the simultaneously analysis of selected mycotoxins in a single run time of 15 min. Multiple Reaction Monitoring (MRM) mode was selected for quantification and detection of the mycotoxins. Analysis was accepted and validated for the simultaneous quantification of 11 mycotoxins at different levels (2-150 ngg-1 for AFs, T-2 TOXIN, OTA; 20-1500 ngg-1 for ZER, HT-2 TOXIN; and 100-1500 ngg-1 for DON and FB1+B2) in wheat. Calibration curves were plotted with spiked wheat flour blank samples without internal standard. Limits of detection (LOD) ranged from 0.7 to 33.3ngg-1 and limits of quantification (LOQ) ranged from 2 to 100 ngg-1. The recoveries of selected mycotoxins were in the range of 72.2-122 %.The recoveries and repeatabilities were in accordance with the criteria determined by European Union (EU) Recommendation 519/2014.


Main Subjects

(1) Varga E, Glauner T, Köppen R, Mayer K, Sulyok M,
Schuhmacher R, Krska R and Berthiller F. Stable
isotope dilution assay for the accurate determination
of mycotoxins in maize by UHPLC-MS/MS. Anal.
Bioanal. Chem. (2012) 402: 2675–86.
(2) Malachová A, Stránská M, Václavíková M, Elliott,
C T, Black C, Meneely J, Jana Hajšlová J, Ezekiel
CN, Schuhmacher R and Krska R. Advanced LC–
MS-based methods to study the co-occurrence
andmetabolization of multiple mycotoxins in cereals
and cereal-based food. Anal. Bioanal. Chem. (2018)
410: 801–25.
(3) Commission Regulation (EC) No 1881/2006,
Commission Directive 2006/1881/EC of 19
December 2006. setting maximum levels for certain
contaminants in food stuffs. Official Journal of the
European Communities (2006) L364: 5–24.
(4) ISIRI. Maximum tolerated limits of mycotoxins in
foods and feeds. National Standard No.5925 Institute
of Standard and Industrial Research of Iran (2002).
(5) Amirahmadi M, Shoeibi SH, Rastegar H, Elmi M and
Mousavi A KH. Simultaneous analysis of mycotoxins
in corn flour using LC/MS-MS combined with a
modified QuEChERS procedure. Toxin Rev. (2017)
37: 187–95.
(6) Yazdanpanah H, Miraglia M, Calfapietra FR and
Brera C. Natural occurrence of aflatoxins and
ochratoxin A in corn and barley from Mazandaran
and Golestan in north provinces of Iran. Mycotoxin
Res. (2001) 17: 21-30.
(7) Köppen R, Koch M, Siegel D, Merkel S, Maul R
and Nehls I. Determination of mycotoxins in foods:
current state of analytical methods and limitations.
Appl. Microbiol. Biotechnol. (2010) 86:1595-612.
(8) Langseth W and Rundberget T. Mycotoxin production
and cytotoxicity of Fusarium strains isolated from
Norwegian cereals. J. Chromatogr. A (1998) 815:
(9) Schollenberger, M, Müller H H, Rüfle M and
Drochner W. Natural occurrence of 16 Fusarium
toxins in edible oil marketed in Germany. Food
Control (2008) 19: 475-82
(10) Zollner P and Mayer-Helm B. Trace mycotoxin
analysis in complex biological and food matrices
by liquid chromatography-atmospheric pressure
ionization mass spectrometry. J. Chromatogr. A.
(2006) 1136 (2): 123–69.
(11) Razzazi-Fazeli E, Rabus B, Cecon B and Bohm J.
Simultaneous quantification of A-trichothecene 
Heidari G et al. / IJPR (2019), 18 (Special Issue): 182-189
mycotoxins in grains using liquid chromatography–
atmospheric pressure chemical ionisation mass
spectrometry. J. Chromatogr. A. (2002) 968: 129–
 (12) Frenich AG, Vidal JLM, Romero-Gonzalez R and
Aguilera-Luiz MM. Simple and high-throughput
method for the multi mycotoxin analysis in cereals
and related foods by ultra-high performance liquid
chromatography/tandem mass spectrometry. Food
Chem. (2009) 117: 705-12.
(13) Sulyok M, Berthiller F, Krska R and Schuhmacher
R. Development and validation of a liquid
chromatography/ tandem mass spectrometric
method for the determination of 39 mycotoxins in
wheat and maize. Rapid Comm. Mass Spectrom.
(2006) 20: 2649–59.
(14) Cavaliere C, Foglia P, Pastorini E, Samperi R and
Laganà A. Development of a multiresidue method
for analysis of major Fusarium mycotoxins in
corn meal using liquid chromatography/tandem
mass spectrometry. Rapid Comm. Mass Spectrom.
(2005) 19: 2085–93.
(15) Spanjer MC, Rensen PM and Scholten JM. LCMS/MS multi-method for mycotoxins after
single extraction, with validation data for peanut,
pistachio, wheat, maize, cornflakes, raisins and
figs. Food Addit. Contam. (2008) 25: 472 –89.
(16) Zhang J M, Wu YL and Lu Y B. Simultaneous
determination of carbamate insecticides and
mycotoxins in cereals by reversed phase liquid
chromatography tandem mass spectrometry using
a quick, easy, cheap, effective, rugged and safe
extraction procedure. J. Chromatogr. B (2013)
915–16: 13-20
(17) Shimshoni JA, Cuneah O, Sulyok M, Krska R,
Galon N, Sharir B and Shlosberg A. Mycotoxins
in corn and wheat silage in Israel. Food Addit.
Contam.: Part A (2013) 30:1614–25.
(18) Tanaka H, Takino M, Sugita-Konishi Y and Tanaka
T. Development of a liquid chromatography/
time-of-flight mass spectrometric method for the
simultaneous determination of trichothecenes,
zearalanone and aflatoxins in foodstuffs. Rapid
Commun. Mass Spectrom. (2006) 20:1422–28.
(19) Warth B, Parich A, Atehnkeng J, Bandyopadhyay
R, Schuhmacher R, Sulyok M and Krska R.
Quantitation of Mycotoxins in Food and Feed from
Burkina Faso and Mozambique Using a Modern
LC-MS/MS Multitoxin Method. J. Agric. Food
Chem. (2012) 60: 9352−63.
(20) Blandino M, Scarpinoa V, Vanaraa F, Sulyok M,
Krska R and Reyneri A. Role of the European
corn borer (Ostrinia nubilalis) on contamination of
maize with 13 Fusarium mycotoxins. Food Addit.
Contam.: Part A (2015) 32: 533–43.
(21) Åberg A T, Solyakov A and Bondesson U.
Development and in-house validation of an
LCMS/MS method for the quantification of the
mycotoxins deoxynivalenol, zearalenone, T-2 and
HT-2 toxin, ochratoxin A and fumonisin B1 and
B2 in vegetable animal feed. Food Addit. Contam.:
Part A (2013) 30: 541–49.
(22) Vicam. Myco6in1+
 LC-MS/MS manual instruction
71500 1718EN Rev D. (2013).
(23) Lattanzio VMT, Ciasca B, Powers S and Visconti
A. Improved method for the simultaneous
determination of aflatoxins, ochratoxin A and
Fusarium toxins in cereals and derived products by
liquid chromatography–tandem mass spectrometry
after multi-toxin immunoaffinity clean up. J.
Chromatogr. A (2014) 1354: 139–43.
(24) Commission Regulation (EU) No 519/2014.
2014/519 EC of 16 May 2014, amending Regulation
(EC) No 401/2006 as regards methods of sampling
of large lots, spices and food supplements,
performance criteria for T-2, HT-2 toxin and
citrinin and screening methods of analysis. Official
Journal of the European Union (2014) L147, 29-43
(25) Ren YP, Zhang Y, Shao SL, Cai ZX, Feng L, Pan
HF and Wang ZG. Simultaneous determination
of multi-component mycotoxin contaminants
in foods and feeds by ultra-performance liquid
chromatography tandem mass spectrometry. J.
Chromatogr. A. (2007) 1143: 48-64.
(26) Mavungu JDD, Monbaliu S, Scippo ML, MaghuinRogister G, Schneider YJ, Larondelle Y, Callebaut
A, Robbens J, Van Peteghem C and de Saeger S.
LC-MS/MS multi-analyte method for mycotoxin
determination in food supplements. Food Addit.
Contam.: Part A (2009) 26: 885-95.
(27) Boyd RK, Basic C and Bethem RA. Trace
Quantitative Analysis by Mass Spectrometry. John
Wiley and Sons Ltd., Manchichester (2008).
(28) Kokkonen M and Jestoi M. A Multi-compound LCMS/MS Method for the Screening of Mycotoxins
in Grains. Food Anal. Method. (2009) 2: 239. DOI:
(29) Monbaliu S, Van Poucke C, Detavernier C,
Dumoulin F, Van de Velde M, Schoeters E, Van
Dyck S, Averkieva O, van Peteghem C and de
Saeger S. A Occurrence of mycotoxins in feed
as analyzed by a multimycotoxin LC-MS /MS
method. J. Agric. Food Chem. (2010) 58: 66–71.
(30) Berthiller F, Brera C, Iha M H, Krska R, Lattanzio
V M T, MacDonald M, Malone R J , Maragos
M, Solfrizzo M, Stranska-Zachariasova M, Stroka
J and Tittlemier S A. Developments in mycotoxin
analysis: an update for 2015-2016. World
Mycotoxin J. (2017). 10: 5-29.