Collaborative Study Report: Determination of Alternaria toxins in cereals, tomato juice and sunflower seeds by liquid chromatography tandem mass spectrometry

The Institute for Reference Materials and Measurements of the Joint Research Centre, a Directorate-General of the European Commission, organised a method validation study to evaluate the performance of a method for the simultaneous determination of five Alternaria toxins in cereals, tomato juice and sunflower seed samples. The method validation study was conducted according to the International Union for Pure and Applied Chemistry harmonised protocol. The method was used for the determination of altenuene, alternariol, alternariol monomethyl ether, tentoxin and tenuazonic acid in both naturally contaminated and fortified samples. It was based on the extraction of the test materials with an acidified methanol – water mixture, followed by solid phase extraction clean-up. The determination was carried out by reversed phase high performance liquid chromatography coupled to a triple quadrupole mass spectrometric detector. The trial involved 16 participants representing a cross section of research, private and official control laboratories from 11 EU Member States and Canada. The selection of collaborators was based on the performance in the pre-trial that was organised prior to the collaborative trial with participation of 25 laboratories. Mean recoveries reported ranged from 53% to 107%. The sample reconstitution in a water-based injection solution is thought to be responsible for the low recovery obtained for alternariol monomethyl ether, which is the least polar compound from the toxins of interest. The relative standard deviation for repeatability (RSDr) ranged from 2.0 to 34.8%. The relative standard deviation for reproducibility (RSDR) ranged from 7.7 to 49.6%, reflecting HorRat values from 0.5 to 2.4 according to the Horwitz function modified by Thompson. A correction for recovery with the data generated by spiking experiments partially improve the reproducibility performance of the method. The results highlight that the performance characteristics strongly depend on the matrix analysed, despite that fact that matrix matched calibration was used. These matrix effects can be compensated using stable isotope labelled internal standards; however, stable isotope analogues for the analysed compounds are not commercially available so far. The outcome of this study however underpins its fitness-for-purpose, which is a requirement for its formal standardisation by the European Committee for Standardization (CEN).JRC.F.5 - Food and Feed Complianc

Report on the development of a method for the determination of Alternaria toxins and citrinin in wheat, tomato juice and sunflower seeds by liquid chromatography – tandem mass spectrometry

A new method was developed for the determination of five Alternaria toxins and citrinin in wheat, tomato juice and sunflower seed samples based on a liquid chromatography – tandem mass spectrometric determination. The optimized sample preparation involved a solid – liquid extraction with pure methanol, a derivatization step, and subsequent, solid-phase extraction purification on Strata-XL cartridges. The separation was carried out using HPLC column packed with core-shell C-18 particles. HPLC system was directly coupled to mass spectrometer via electrospray interface employed in negative mode. For the highest selectivity and sensitivity multiply reaction monitoring mode was used in the detector. The absolute recoveries (63.3% – 113.1%) and repeatability (1.2% – 13.9%) of the method, calculated from spiked samples, generated sufficient data.JRC.D.5 - Standards for Food Bioscienc

Report on the 2015 Proficiency Test of the European Union Reference Laboratory for Mycotoxins, for the Network of National Reference Laboratories. Determination of Cintrinin in Red Yeast Rice.

The IRMM operates the European Union Reference Laboratory (EURL) for Mycotoxins. One of its core tasks is to organise inter-laboratory comparisons (ILCs) among appointed National Reference Laboratories. This report presents the results of the ILC of the EURL for Mycotoxins which focused on the determination of citrinin in red yeast rice samples.JRC.D.5-Standards for Food Bioscienc

Simultaneous analysis of Alternaria toxins and citrinin in tomato: an optimized method using liquid chromatography – tandem mass spectrometry

Alternaria toxins and citrinin are mycotoxins produced by fungi growing on agricultural commodities. Maximum levels for these toxins in foods are currently under consideration by the European Commission as a risk management measure. In this study, a new quantitative method is described for the determination of five Alternaria toxins and citrinin in tomato and tomato juice samples based on liquid chromatography with tandem mass spectrometry detection. Samples were extracted with pure methanol, followed by a derivatization step with 2,4 dinitrophenylhydrazine to improve the determination of tenuazonic acid and to decrease the wide polarity difference between the compounds of interest. Samples were purified on hydrophilic modified styrene polymer solid-phase extraction cartridges. High performance liquid chromatographic columns packed with different core-shell materials have been tested for the separation of toxins, and a C-18 phase with 2.7 µm particle size was applied in the final method to achieve sufficient separation of all relevant analytes. A key element of this approach was to prove successful transferability of the method to three different triple quadrupole mass spectrometers. A full single laboratory method validation has been performed on two liquid chromatography – tandem mass spectrometric systems and performance characteristics met the predefined requirements. Moreover, the method has been used in an international proficiency test, and the satisfactory z-scores obtained (-0.1 – 0.8 in tomato juice samples) demonstrated the reliability of the described approach. The method will be validated in an inter-laboratory collaborative study and if criteria for method precision are met, the method will be proposed to as a new Work Item to the European Committee for Standardization.JRC.F.5-Food and Feed Complianc