Improved sample preparation ensures accurate quantification of multiple mycotoxins in maize by Liquid Chromatography-stable Isotope Dilution Assay-Tandem Mass Spectrometry (LC-SIDA-MS/MS)

Abstract

The objective of this study was to develop and validate an improved sample preparation technique for accurate quantification of aflatoxin B1 (AFB1), AFB2, AFG1, AFG2, deoxynivalenol (DON), fumonisin B1 (FB1), FB2, Ochratoxin A (OTA), zearalenone (ZEN), HT-2 toxin and T-2 toxin in maize using liquid chromatography-isotope dilution mass spectrometry. Mycotoxin contamination in agricultural commodities poses a threat to human health. Contamination of food is recognised as a source of food borne illness by the World Health Organisation (WHO). The toxicity of mycotoxins has been evaluated by the Joint Food and Agricultural Organisation (FAO)/WHO Expert Committee on Food Additives (JECFA) and the maximum levels (MLs) for the agricultural important mycotoxins have been established. Agricultural commodities need to be tested to ensure food safety prior to human consumption; this requires accurate analytical methods for identification and quantification of these mycotoxins at the regulatory levels. Analytical methods based on liquid chromatography coupled to mass spectrometry have been developed for identification and quantification of mycotoxins. However, MS based analysis is affected by matrix effects that results from ionisation inefficiency of the target analyte due to co-eluting matrix components. Therefore, there is a need for improved sample preparation methods which can minimise, or possibly eliminate, matrix components prior to mass spectrometric analysis. Dilute-and-shoot , Quick, Easy, Cheap, Efficient, Rugged and Safe (QuEChERS) and solid phase extraction (SPE) techniques were evaluated for matrix removal efficiency in multi mycotoxin determination in maize. Isotopically labelled mycotoxin standards were used to compensate for variations during the analysis. Spiked blank maize samples and matrix reference materials were used to evaluate the performance of each sample preparation technique. Dilute-and-shoot technique was used as a first approach to estimate expected matrix effects and to verify whether isotopically labelled internal standards can compensate for matrix effects during the analysis. All the analytes were affected by the presence of matrix effects, signal suppression/enhancement (SSE) ranged between 88% - 194%. When %REC > 130% it was deemed enhanced. The QuEChERS method was ineffective in isolating mycotoxins from the matrix. Results from dilute-and-shoot and QuEChERS highlighted the need of a selective clean-up step to reduce matrix effects. Different SPE columns with different sorbents were evaluated for matrix removal efficiency and analyte retention performances. Columns with analyte(s) selective sorbents were effective in improving recoveries for those specific analytes. Also, minimum matrix effects were observed from these columns. However, for multi mycotoxin determination, an ideal clean-up step should yield good recoveries for all the mycotoxins with varying physicochemical properties. Hydrophilic-lipophilic balanced (HLB) SPE column gave good recoveries for most analytes despite relatively high matrix effects with respect to selective sorbents. A clean-up method based on HLB clean-up was optimised to improve matrix removal efficiency. An accurate, precise and robust method for the determination of multiple mycotoxins in maize was developed and validated. This method is based on ultrasonic extraction, economical HLB SPE clean-up and ultra-high performance liquid chromatography-stable isotope dilution assay-tandem mass spectrometry (UHPLC-SIDA-MS/MS). Sample extraction based on two extraction steps using acidified methanol/water mixture and HLB SPE clean-up resulted in good analyte recoveries 57% ? %REC ? 142% for most analytes. Fast polarity switching mode was used to determine all the analytes in one chromatographic run without compromising chromatographic resolution. Method performance results indicate that the method can be used to detect and quantify mycotoxins at the regulated levels. Keywords: Mycotoxins, maize, stable isotope dilution assay, ultra-high performance liquid chromatography, tandem mass spectrometry.