Quantification of T-2 and HT-2 mycotoxins in cereals by liquid chromatography-multimode ionization-tandem mass spectrometry
Highlights
► A new LC-MS/MS method is developed and validated for T-2 and HT-2 mycotoxins. ► Different ion modes should be applied for the toxins in the multimode ion source. ► Strata-XL SPE cartridge results in the best recovery for both toxins in flour. ► The method was applied to cereal proficiency test samples with success.
Introduction
Mycotoxins are considered a major public health concern. In terms of chronic toxicity they are the most hazardous of all food contaminants. T-2 and HT-2 belong to trichothecenes mycotoxins, which are mostly produced by Fusarium moulds, and other fungi such as Trichoderma, Stachybotrys and Myrothecium. Different types (A, B, C and D) of trichothecenes are now known depending on their macrocyclic or non-macrocyclic structure. T-2 and HT-2 are classified into type A having non-macrocyclic form and ester group at C-8 position (Fig. 1). Trichothecenes appear mainly in cereals and cereal-based foods, and consequently, the consumption of infected foods can cause immunosuppressive effects and diseases as diarrhoea, skin irritation. Furthermore, reduction in protein, DNA and RNA synthesis were also observed [1]. The European Union (EU) therefore set maximum level (ML) concentrations for mycotoxins in food of plant origin [2], however, in the case of T-2 and HT-2 no ML has been set yet. Legislative limits for T-2 and HT-2 are currently under consideration by the European Commission [3]. The determination of T-2 and HT-2 is in focus now, since the European Union will establish maximum level concentrations for these mycotoxins in cereal products in the near future.
Recently several methods, including screening and confirmatory approaches, have been published for the analysis of T-2 and HT-2 mycotoxins in cereals reviewed by Meneely et al., Turner et al., Zöllner et al., and Lattanzio et al. [4], [5], [6], [7]. Screening methods are based on immunoassay or biosensor determinations that allow the simple, fast and relatively inexpensive analysis of target compounds. These methods are now quite rapid [4], however, they cannot be used for confirmation [8], [9]. Confirmatory approaches require the most selective and accurate determinations, which can be carried out using chromatographic separation. T-2 and HT-2 mycotoxins can be separated using both gas and liquid chromatographic (GC and LC) techniques [4], [5], [6], [7]. GC determination involves an addition derivatization step prior to analysis [10], [11], [12], which extends the analysis time. For LC separation ultraviolet (UV) detection results in high analytical limits and non-selective separation due to the low UV absorbance of these mycotoxins, and consequently, it is not recommended for analysis of complex matrices [4]. In a recent study, however, 8 μg/kg limit of detection (LOD) could be achieved in analyzing oat and wheat samples using ultra performance liquid chromatography separation and photo diode array detection (UPLC-PDA) [13]. The fluorescence detection (FLD) of T-2 and HT-2 mycotoxins enables enhanced separation and sensitivity, but this also requires a pre-column derivatization using 1-anthroylnitrile, coumarin-3-carbonyl chloride or other derivatization agents [14], [15]. Nowadays, liquid chromatographic separation and subsequent mass spectrometric detection (LC-MS) have been considered as the most suitable technique for trichothecenes analysis in complex matrices [12], [16], [17], [18], [19], [20], [21], [22]. Earlier LC-MS methods reported single quadrupole or ion trap detections, but recently, the triple quadrupole methods (LC-MS/MS) have been generally applied, which allow an improved quantification [4], [5], [6], [7]. Unless, the MS/MS detection can result in a reduction in LOD, it should be pointed out that there are big differences in the sensitivity of LC-MS/MS instruments. The reason of these deviations is mainly in the ion sources [23], [24]. Existing LC-MS/MS methods employed adequate electrospray (ESI) or atmospheric pressure chemical ionization (APCI) techniques for the ionization of T-2 and HT-2 toxins. These ion sources allowed good ion production for both mycotoxins, and therefore, low analytical limits could be achieved for them [5], [6], [7]. To the best of our knowledge there is no study in the literature describing LC-MS/MS method for T-2 and HT-2 toxins utilizing multimode ion source (MMI). This ion source contains both ESI and APCI parts, and therefore, its geometry radically differs to adequate ones [23]. Significantly, LC-MMI-MS/MS methods require thorough optimizations to reach good sensitivity for target compounds and to detect such low levels set by EU [24], [26], [27], [28], [29], [30].
In this study a liquid chromatography-tandem mass spectrometric method was developed for the rapid analysis of T-2 and HT-2 in cereals. During the method development different reversed-phase (RP) solid-phase extraction (SPE) cartridges were tested in order to reduce ion suppression or enhancement effect of matrices in MMI and to get enhanced recovery for mycotoxins. Our laboratory has analyzed a number of flour samples in every year, thus the method was developed and validated for flour matrix in accordance with Commission Regulation (EC) No 401/2006 and European Union 2002/657/EC decision [8], [9]. The studied performance characteristics were selectivity, identification, linearity, recovery, within-laboratory reproducibility, limit of detection, and limit of quantification. Finally, the optimized method was applied to naturally contaminated and spiked cereal proficiency test samples in the year of 2009. The study highlighted the applicability of the described method.
Section snippets
Reagents, equipments, samples and instruments
A 101.3 μg/mL stock solution of T-2 in acetonitrile and a 100.5 μg/mL stock solution of HT-2 in acetonitrile were obtained from Biopure (Tulln, Austria). Stock solutions were stored at − 20 °C in dark up to 1 year. A working standard solution was diluted from stock solutions with methanol and stored at − 20 °C in dark for a month. This standard solution contained both T-2 and HT-2 in 1 μg/mL concentration and was used for spiking and for calibration. HPLC gradient grade methanol and acetonitrile were
Optimization of mass spectrometric conditions
The MS/MS detection was optimized using flow injection analysis (FIA). The injector outlet of HPLC system was directly connected to the ion source of MS/MS analyzer. A mobile phase composition of methanol–water containing 5 mM ammonium acetate and 0.05% acetic acid (70/30, v/v) was applied with a flow rate of 0.3 mL/min. T-2 and HT-2 standard solutions (1 μg/mL) diluted individually with methanol were injected (1 μL) into the FIA system. First, the precursor ions were optimized using MS2 scan mode
Proficiency test study
The method was applied to naturally contaminated and spiked cereal products in proficiency test organized by IRMM European Union Reference Laboratory (EU-RL) for mycotoxins (Geel, Belgium) in the year 2009. The test materials were two naturally contaminated and one spiked samples (sample 1, 2, and 3). All samples were prepared and analyzed in duplicate. Submitted results were the average of the two independent measurements. All samples contained both toxins at different concentration levels.
Conclusions
A new LC-MS/MS method was developed for the quantitative analysis of T-2 and HT-2 mycotoxins in cereals. The multimode ion source required thorough optimization to get reliable results for both toxins at low concentration levels. T-2 was ionized with ESI ion mode, while APCI mode resulted in the highest response for HT-2 in the MMI. Sample clean-up was tested with different SPE columns, and Strata-XL (3 mL, 200 mg) cartridge enabled the best SPE condition for the simultaneous analysis of selected
Acknowledgement
We would like to thank Dr. Szabolcs Fekete for useful comments for improving the paper. We wish to thank Péter Imrik and Tas Szász Vadász (Gen-lab Ltd) who continuously supported the equipments for the measurements. We acknowledge Gábor Domány (Head of laboratory, Food Toxicological NRL) for his support.
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