A strong trend toward using highly selective high-resolution mass spectrometry technologies for rapid screening of multiple mycotoxins has been observed in recent years. Consequently, within the European Union, specific performance requirements for screening methods for mycotoxins have been issued in the Regulation 519/2014/EU that apply not only to bioanalytical methods based on immuno-recognition, but also to mass spectrometry approaches. This regulation establishes that the "aim of the validation is to demonstrate the fitness for purpose of the screening method", and focuses the entire validation procedure on the determination of cut-off value, false negative and false suspect rates. However, performance characteristics such as sensitivity, selectivity, and precision are embedded in these parameters. In the present study, a liquid chromatography high-resolution mass spectrometry method was developed for the simultaneous determination of the major Fusarium toxins in wheat, namely deoxynivalenol, 3- and 15-acetyl deoxynivalenol, T-2 and HT-2 toxins, zearalenone, enniatins A, A1, B, B1, and beauvericin. A sample preparation protocol was optimized, based on a double extraction (methanol followed by acetonitrile/water mixture), and purification through solid phase extraction C18 columns, achieving satisfactory recoveries for the target mycotoxins covering a wide range of polarities. Analytical performances of the developed method were evaluated through both in house validation and small scale interlaboratory study. The validation study, designed according to the Commission Regulation 519/2014/EU, was performed at screening target concentrations (STC) close to EU maximum permitted or indicative levels. The in house validation procedure provided the precision of the response under repeatability conditions and the intermediate precision (the latter resulting lower than 42% for all mycotoxins), the matrix effect from different wheat varieties on the precision, the cut off value, and the rate of false positive results for samples containing the target mycotoxins at 20% of the STC, that resulted lower than 1% in all cases. A small scale collaborative study was therefore carried out to determine reproducibility and robust and laboratory independent cut off values. Finally, analysis of naturally contaminated samples and reference materials confirmed method suitability for screening of Fusarium mycotoxins in wheat, to check compliance with EU maximum permitted or recommended levels
LIQUID CHROMATOGRAPHY - HIGH RESOLUTION MASS SPECTROMETRY SCREENING OF FUSARIUM TOXINS IN WHEAT: EVALUATION OF ANALYTCAL PERFORMANCES THROUGH IN HOUSE VALIDATION AND SMALL SCALE COLLABORATIVE STUDY
Biancamaria Ciasca;Michelangelo Pascale;
2017
Abstract
A strong trend toward using highly selective high-resolution mass spectrometry technologies for rapid screening of multiple mycotoxins has been observed in recent years. Consequently, within the European Union, specific performance requirements for screening methods for mycotoxins have been issued in the Regulation 519/2014/EU that apply not only to bioanalytical methods based on immuno-recognition, but also to mass spectrometry approaches. This regulation establishes that the "aim of the validation is to demonstrate the fitness for purpose of the screening method", and focuses the entire validation procedure on the determination of cut-off value, false negative and false suspect rates. However, performance characteristics such as sensitivity, selectivity, and precision are embedded in these parameters. In the present study, a liquid chromatography high-resolution mass spectrometry method was developed for the simultaneous determination of the major Fusarium toxins in wheat, namely deoxynivalenol, 3- and 15-acetyl deoxynivalenol, T-2 and HT-2 toxins, zearalenone, enniatins A, A1, B, B1, and beauvericin. A sample preparation protocol was optimized, based on a double extraction (methanol followed by acetonitrile/water mixture), and purification through solid phase extraction C18 columns, achieving satisfactory recoveries for the target mycotoxins covering a wide range of polarities. Analytical performances of the developed method were evaluated through both in house validation and small scale interlaboratory study. The validation study, designed according to the Commission Regulation 519/2014/EU, was performed at screening target concentrations (STC) close to EU maximum permitted or indicative levels. The in house validation procedure provided the precision of the response under repeatability conditions and the intermediate precision (the latter resulting lower than 42% for all mycotoxins), the matrix effect from different wheat varieties on the precision, the cut off value, and the rate of false positive results for samples containing the target mycotoxins at 20% of the STC, that resulted lower than 1% in all cases. A small scale collaborative study was therefore carried out to determine reproducibility and robust and laboratory independent cut off values. Finally, analysis of naturally contaminated samples and reference materials confirmed method suitability for screening of Fusarium mycotoxins in wheat, to check compliance with EU maximum permitted or recommended levelsI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
