title: Development and validation of an ultra-high-performance liquid chromatography tandem mass spectrometric method for the simultaneous determination of free and conjugated Alternaria toxins in cereal-based foodstuffs.
abstract: A UPLC-ESI+/--MS/MS method for the simultaneous determination of free (alternariol, alternariol monomethyl ether, altenuene, tenuazonic acid, tentoxin, altertoxin-I) and conjugated (sulfates and glucosides of alternariol and alternariol monomethyl ether) Alternaria toxins in cereals and cereal products (rice, oat flakes and barley) was developed. Optimization of the sample preparation and extraction methodology was achieved through experimental design, using full factorial design for extraction solvent composition optimization and fractional factorial design to identify the critical factors in the sample preparation protocol, which were in turn subjected to optimization. Final extracts were analysed using an Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer equipped with an electrospray interface operated in both positive and negative ionization mode. Chromatographic separation was achieved using an Acquity UPLC HSS T3 column, and the applied gradient elution programme allowed for the simultaneous determination of 10 Alternaria toxins in a one-step chromatographic run with a total run time of only 7min. Subsequently, the method, applying isotopically labelled internal standards ([2H4]-alternariol monomethyl ether and [13C6,15N]-tenuazonic acid), was validated for several parameters such as linearity, apparent recovery, limit of detection, limit of quantification, precision, measurement uncertainty and specificity (in agreement with the criteria mentioned in Commission Regulation No. 401/2006/EC and Commission Decision No. 2002/657/EC). During validation, quality of the bioanalytical data was improved by counteracting the observed heteroscedasticity through the application of weighted least squares linear regression (WLSLR). Finally, 24 commercially available cereal-based foodstuffs were subjected to analysis, revealing the presence of tenuazonic acid in both rice and oat flake samples (<LOQ - 68±7μgkg-1) and tentoxin in rice samples (<LOQ - 10.9±2.0μgkg-1).
authors.affiliation: Department of Bioanalysis, Laboratory of Food Analysis, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium. Electronic address: jeroen.walravens@Ugent.be.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Walravens
authors.affiliation: Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9, 1060 Vienna, Austria.
authors.forename: Hannes
authors.initials: H
authors.lastname: Mikula
authors.affiliation: BIOANALYTIK Weihenstephan, ZIEL Research Center for Nutrition and Food Sciences, Technische Universität München, Alte Akademie 10, 85354 Freising, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Rychlik
authors.affiliation: Chair of Analytical Food Chemistry, Technische Universität München, Alte Akademie 10, 85354 Freising, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Asam
authors.affiliation: Department of Bioanalysis, Laboratory of Food Analysis, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
authors.forename: Emmanuel Njumbe
authors.initials: EN
authors.lastname: Ediage
authors.affiliation: Department of Bioanalysis, Laboratory of Food Analysis, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
authors.forename: José Diana
authors.initials: JD
authors.lastname: Di Mavungu
authors.affiliation: Faculty of Bioscience Engineering, Laboratory of Biochemistry and Brewing, Ghent University, Valentin Vaerwyckweg 1, 9000 Ghent, Belgium.
authors.forename: Anita
authors.initials: A
authors.lastname: Van Landschoot
authors.affiliation: Department of Veterinary Public Health and Food Safety, Laboratory of Chemical Analysis, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: Lynn
authors.initials: L
authors.lastname: Vanhaecke
authors.affiliation: Department of Bioanalysis, Laboratory of Food Analysis, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
authors.forename: Sarah
authors.initials: S
authors.lastname: De Saeger
keywords: (Masked) mycotoxins; Alternaria; Experimental design; Homoscedasticity; UPLC-MS/MS; Validation