Carla Soler

Analysis of mycotoxins in barley using ultra high liquid chromatography high resolution mass spectrometry: comparison of efficiency and efficacy of different extraction procedures.

The effectiveness of four extraction methods (modified QuEChERS, matrix solid-phase dispersion (MSPD), solid-liquid extraction (SLE) and solid-phase extraction (SPE) clean-up) were evaluated for simultaneous determination of 32 mycotoxins produced by the genus Fusarium, Claviceps, Aspergillus, Penicillium and Alternaria in barley by ultra high pressure liquid chromatography coupled to ultra-high resolution mass spectrometry (UHPLC-Orbitrap(®) MS). The efficiency and efficacy of extraction methods were evaluated and compared in number of extracted mycotoxins and obtained recoveries. From the one point of view, QuEChERS procedure was fast and easy, as well as it was able to successfully extract all selected mycotoxins. On the other hand, SLE method, MSPD and SPE clean-up method did not extract adequately all selected mycotoxins and recoveries were not suitable enough. Thereby, method employing QuEChERS extraction connected with UHPLC-Orbitrap(®) MS was developed to quantify 32 mycotoxins in barley within this study. Analytical method was validated and recoveries ranged from 72% to 101% for selected mycotoxins with only one exception nivalenol (NIV) and deoxynivalenol-3-glucoside (D3G), which were lower than 67%. Relative standard deviations (RSD) were lower than 17.4% for all target mycotoxins. The lowest calibration levels (LCLs) ranged from 1 to 100 μg/kg. Validated method was finally used for monitoring mycotoxins in a total of 15 Czech barley samples, when only Fusarium toxins representatives were detected in 53% of samples and the mycotoxins with the highest incidence were enniatins.

Evaluation of matrix solid-phase dispersion (MSPD) extraction for multi-mycotoxin determination in different flours using LC–MS/MS

An existing matrix solid-phase dispersion (MSPD) method for aflatoxins (AFs) and ochratoxin A (OTA) extraction was extended by further 14 mycotoxins. After it careful optimization, this method was applied to determine the occurrence of these mycotoxins on commercial flour samples (with different cereals composition) collected from local markets. In a total of 49 samples investigated, 9 mycotoxins were identified. Nivalenol (NIV) and Beauvericin (BEA) were the mycotoxins found most frequently. The samples that presented major contamination were wheat flours and bakery preparations. Despite of the great number of positives finding, only one wheat flour sample exceeded the maximum limits (ML) for OTA established by the European Union (EU). However, it would be interesting to calculate the total ingest of these mycotoxins along the years.

Rapid mycotoxin analysis in human urine: A pilot study

A simple and rapid method effective for quantitative determination of deoxynivalenol (DON), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEN), ochratoxin A (OTA), aflatoxins (AFs) B(1), B(2), G(1) and G(2) and fumonisins FB(1) and FB(2) in urine was developed. The urine was diluted with phosphate buffer solution (PBS) and thoroughly mixed. For clean-up and extraction, the mixture was loaded on a MYCO 6in1 IAC. Hybrid triple quadrupole-linear ion trap mass spectrometer (QTrap) was used for the detection. Extra tools for confirmation of selected mycotoxins in positive samples, Information Dependent Acquisition (IDA) experiments, were also developed. The use of immunoaffinity columns followed by the LC-MS/MS analysis showed acceptable average recoveries between 83% and 116% and reached acceptable precision values (relative standard deviation (RSD) ≤ 14%). In a pilot study with 27 volunteers, OTA, DON and AFG(2) were detected. However, this study needs to be extended in order to understand the relation between the mycotoxins intake and mycotoxin levels in human urine.

Optimization of Matrix Solid-Phase Dispersion method for simultaneous extraction of aflatoxins and OTA in cereals and its application to commercial samples

A method based on Matrix Solid-Phase Dispersion (MSPD) has been developed for the determination of 5 mycotoxins (ochratoxin A and aflatoxins B and G) in different cereals. Several dispersants, eluents and ratios were tested during the optimization of the process in order to obtain the best results. Finally, samples were blended with C(18) and the mycotoxins were extracted with acetonitrile. Regarding to matrix effects, the results clearly demonstrated the necessity to use a matrix-matched calibration to validate the method. Analyses were performed by liquid chromatography-triple quadrupole-tandem mass spectrometry (LC-QqQ-MS/MS). The recoveries of the extraction process ranged from 64% to 91% with relative standard deviation lower than 19% in all cases, when samples were fortified at two different concentrations levels. Limits of detection ranged from 0.3 ng g(-1) for aflatoxins to 0.8 ng g(-1) for OTA and the limits of quantification ranged from 1 ng g(-1) for aflatoxins to 2 ng g(-1) for OTA, which were below the limits of mycotoxins set by European Union in the matrices evaluated. Application of the method to the analysis of several samples purchased in local supermarkets revealed aflatoxins and OTA levels.

The Role of the Liquid Chromatography-Mass Spectrometry in Pesticide Residue Determination in Food

The use of liquid chromatography (LC) in pesticide residue determination was usually limited to groups of compounds or single compounds for which no suitable gas chromatographic (GC) conditions were available. However, recent developments have significantly enlarged the LC scope in this field of analysis. One of the most important advances was the on-line coupling of efficient LC separation with mass spectrometry detectors (LC-MS and LC-MS/MS) that makes this technique an excellent method for the determination of pesticides and their transformation products in complex matrices such as food. This review considers the application of LC-MS/MS in this field. Emphasis is placed on the tandem MS applications: advantages of the technique; the sensitive and unequivocal confirmation of the presence of pesticides in food; and, important factors affecting the performance of LC-MS/MS instruments, like the type of mass analyzer or the ionization source design which would be discussed on the particular framework of pesticide and their metabolite analysis. This review also highlights a number of problems associated with the LC-MS/MS analysis of pesticides such as the matrix effects that make quantification difficult.

Natural co-occurrence of mycotoxins in wheat grains from Italy and Syria

This article describes the application of an analytical method for the detection of 25 mycotoxins in wheat grain based on simultaneous extraction using matrix solid-phase dispersion (MSPD) followed by liquid chromatography coupled to tandem mass spectrometry, a hybrid triple quadrupole-linear ion trap mass spectrometer (QTrap®). Information Dependent Acquisition (IDA), an extra confirmation tool for samples that contain the target mycotoxins, was used. The analysis of 40 Syrian and 46 Italian wheat grain samples interestingly showed that Syrian samples were mainly contaminated with ochratoxin A and aflatoxins, whereas Italian samples with deoxynivalenol and 15-acetyldeoxynivalenol. Emerging Fusarium mycotoxins were predominant in Italian samples compared to the Syrian. Among the analysed samples, only one was found containing zeralenone with level above the maximum European recommended concentration (100 ppb). These results confirm that climatic differences between Syria and Italy, both in Mediterranean basin, play a key role in the diversity of fungal genera and mycotoxins in wheat grains.

Evaluation of mycotoxins and their metabolites in human breast milk using liquid chromatography coupled to high resolution mass spectrometry.

Humans can be exposed to mycotoxins through the food chain. Mycotoxins are mainly found as contaminants in food and could be subsequently excreted via biological fluids such as urine or human breast milk in native or metabolised form. Since breast milk is usually supposed as the only food for new-borns, the occurrence of mycotoxins in thirty-five human milk samples was evaluated by a newly developed method based on QuEChERS extraction and UHPLC-HRMS detection. The method described here allows the detection of target mycotoxins in order to determine the quality of this initial feeding. The method has been fully validated, with recoveries ranging from 64% to 93% and relative standard deviations (RSD, %) being lower than 20%. Using the method described, non-metabolised mycotoxins such as ZEA, NEO, NIV, ENA, ENA1, ENB, ENB1 and metabolites, such as ZEA metabolites, HT-2, DOM and T-2 triol were detected in human milk samples. Results obtained help to estimate the exposure of mothers and infants to mycotoxins. Moreover, to the best of our knowledge, this is the first work describing the simultaneous detection, quantification and screening of mycotoxins and their metabolites in human mature milk.

Rapid whole protein quantitation of staphylococcal enterotoxins A and B by liquid chromatography/mass spectrometry

Staphylococcus aureus is an important pathogen and has been indicated as the fifth causative agent of food-borne human illness throughout the world. Staphylococcal enterotoxins (SEs) are toxic compounds excreted mainly by strains of S. aureus. Among these toxins, enterotoxins A (SEA) and B (SEB) are both of the most prevalent compounds in staphylococcal food poisoning. In this work, reverse phase liquid chromatography coupled to ESI mass spectrometry (LC-ESI/MS) has been applied for its rapid identification and quantification. Limit of detection (LOD) values were 0.5 and 0.2 ng for SEA and SEB, respectively and limit of quantification (LOQ) value was 1 ng for both enterotoxins. SEA and SEB have been analyzed as intact proteins in milk and fruit juices. Analytical methods are essential for routine monitoring purposes and safeguard public health and the proposed technique can detect and quantify successfully SEA and SEB in food samples.

One-year monitoring of aflatoxins and ochratoxin A in tiger-nuts and their beverages

A sensitive and selective liquid chromatography-triple quadrupole-tandem mass spectrometry (LC-ESI-MS-MS) method was developed for the routine analysis of aflatoxins (AFB(1), AFB(2), AFG(1) and AFG(2)) and ochratoxin A (OTA) in tiger nuts and tiger-nut beverage (horchata). A matrix solid phase dispersion was adapted to eliminate lipidic interferences. The solid support was C(18), while the elution solvent was acetonitrile. Mean recoveries obtained at two fortification levels were 72-83% and 71-81% for horchata and tiger nut respectively with relative standard deviations (RSDs) <13% and 15% respectively. The LC-MS-MS method allowed quantification and identification at low levels in two matrices. The method was applied for the routine analysis of tiger-nuts and horchata samples collected from different supermarkets of Valencia (Spain) during one year (March 2009-March 2010). A total of 238 samples were analysed and 32 samples were found positives for OTA, AFB(1), AFB(2) and AFG(2).

Applicability of hybrid linear ion trap-high resolution mass spectrometry and quadrupole-linear ion trap-mass spectrometry for mycotoxin analysis in baby food

Recent developments in mass spectrometers have created a paradoxical situation; different mass spectrometers are available, each of them with their specific strengths and drawbacks. Hybrid instruments try to unify several advantages in one instrument. In this study two of wide-used hybrid instruments were compared: hybrid quadrupole-linear ion trap-mass spectrometry (QTRAP®) and the hybrid linear ion trap-high resolution mass spectrometry (LTQ-Orbitrap®). Both instruments were applied to detect the presence of 18 selected mycotoxins in baby food. Analytical parameters were validated according to 2002/657/CE. Limits of quantification (LOQs) obtained by QTRAP® instrument ranged from 0.45 to 45 μg kg⁻¹ while lower limits of quantification (LLOQs) values were obtained by LTQ-Orbitrap®: 7-70 μg kg⁻¹. The correlation coefficients (r) in both cases were upper than 0.989. These values highlighted that both instruments were complementary for the analysis of mycotoxin in baby food; while QTRAP® reached best sensitivity and selectivity, LTQ-Orbitrap® allowed the identification of non-target and unknowns compounds.