Cristina Juan

Incidence of ochratoxin A in rice and dried fruits from Rabat and Salé area, Morocco

One hundred samples of dried fruits (20 dried raisins, 20 walnuts, 20 peanuts, 20 dried figs and 20 pistachios) and 20 samples of rice purchased from retail shops in the Rabat and Salé area in Morocco were analysed for ochratoxin A (OTA) by immunoaffinity clean-up (IAC) and liquid chromatography (LC) with fluorescence detection. The limit of quantification (LOQ) (S/N = 10:1) of OTA was 0.02 ng g−1 in rice, 0.03 ng g−1 in pistachio, peanut and walnut, and 0.03 ng g−1 in dried raisins and dried figs. The incidences of occurrence of OTA in dried raisins, walnuts, peanuts, dried figs and rice were 30, 35, 25, 65 and 90%, respectively. Analytical results showed that pistachio samples contained no detectable OTA, but concentrations ranged from 0.02 ± 0.01 to 32.4 ± 2.10 ng g−1 in rice, from 0.10 ± 0.05 to 2.36 ± 0.75 in peanut, from 0.03 ± 0.01 to 1.42 ± 0.45 in dried figs, from 0.05 ± 0.02 to 4.95 ± 0.02 in dried raisins, and from 0.04 ± 0.01 to 0.23 ± 0.05 in walnuts. The results also showed that 15% of the total number of rice samples analysed exceeded the 2002 regulatory limit set by European Union regulations for cereals. This is the first report on the occurrence of OTA in dried fruits and rice available in Morocco.

Fusarium species, chemotype characterisation and trichothecene contamination of durum and soft wheat in an area of central Italy

BACKGROUND Fusarium head blight (FHB) of wheat is an important disease causing yield losses and mycotoxin contamination. The aim of the work was to detect and characterise trichothecene producing Fusarium species in durum and soft wheat cultivated in an area of central Italy in 2009 and 2010 and to determine trichothecene contamination by LC-MS/MS in the grain. RESULTS F. graminearum s. str. was the most frequent species. In 2009, the occurrence of F. avenaceum and F. poae was higher than in 2010. Among F. graminearum strains, the 15-acetyl deoxynivalenol (15-ADON) chemotype could be found more frequently, followed by nivalenol (NIV) and 3-ADON chemotypes, while all F. culmorum isolates belonged to the 3-ADON chemotype. All F. poae strains were NIV chemotypes. In vitro trichothecene production confirmed molecular characterisation. Durum wheat was characterised by a higher average DON contamination with respect to soft wheat, NIV was always detected at appreciable levels while type-A trichothecenes were mostly found in durum wheat samples in 2009 with 6% of samples exceeding the contamination level recently recommended by the European Union. CONCLUSION Climatic conditions were confirmed to be predominant factors influencing mycotoxigenic species composition and mycotoxin contaminations. However, NIV contamination was found to occur irrespective of climatic conditions, suggesting that it may often represent an under-estimated risk to be further investigated.

Levels of ochratoxin A in wheat and maize bread from the central zone of Portugal

Ochratoxigenic fungi are natural contaminants of cereal and the produced toxins are harmful to humans and animals. Ochratoxin A (OTA) is among the most important mycotoxins, and the International Agency for Research on Cancer (IARC) classifies it as possibly carcinogenic to humans (group 2B). A total of 61 samples of bread from the central zone of Portugal were analysed for OTA by liquid chromatography (LC) with fluorescence detection (FD). For confirmation two procedures were applied, methyl ester derivatization with boron trifluoride-methanol and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS). As far as we know, this is the first report where on-line LC/electrospray ionization (ESI) tandem mass spectrometry (MS/MS) was used for OTA analysis in bread. Limits of detection (LOD) and quantification (LOQ) were 0.015 and 0.03 ng/g, using LC-FD, and 0.03 and 0.09 ng/g by LC-MS/MS. The incidence of OTA was 12.9% and 70.0% for wheat and maize bread, respectively. The highest OTA levels were obtained for maize bread, having one sample exceeded the European maximum limit established for OTA in cereal products. The estimate daily intake (EDI) was below the tolerable daily intake.

Ochratoxin A in rice on the Moroccan retail market.

One hundred (100) samples of rice purchased from retail markets in five different cities (Rabat, Témara, Salé, Casablanca and Méknès) in Morocco from January to October 2006 were surveyed for the presence of ochratoxin A (OTA) using Accelerated Solvent Extraction (ASE) coupled to liquid chromatography with fluorescence detection. The identification of OTA in positive rice samples was confirmed by methyl ester derivatization. Analytical results showed a frequency of contamination of 26% of total analyzed rice samples. The percentage of contamination of samples was 24, 26.6, 16.6, 27.7 and 30% in Rabat, Témara, Méknès, Salé and Casablanca respectively. Levels of OTA in positive samples ranged between 0.08 and 47 ng/g. The average contamination of all analyzed samples was 3.5 ng/g. The highest frequency of positive samples (30%) and the most contaminated sample (47 ng/g) was found in a sample from Casablanca city. 14 out of 100 total samples exceeded the maximum level of 5 ng/g set by European regulations for OTA in cereals. Based in the results presented in this study, the estimated daily intake of OTA in rice was 0.32 ng/kg bw/day for Moroccan consumers.

Determination of ochratoxin A in maize bread samples by LC with fluorescence detection.

Ochratoxin A (OTA) is a secondary fungal metabolite produced by several moulds, mainly by Aspergillus ochraceus, A. carbonarius, A. niger and by Penicillium verrucosum. The present work shows the results of comparative studies using different procedures for the analysis of OTA in maize bread samples. The studied analytical methods involved extraction with different volumes of PBS/methanol, different extraction apparatus, and clean-up through immunoaffinity columns. The separation and identification were carried out by high-performance liquid chromatography with fluorescence detection. The optimized method for analysis of OTA in maize bread involved extraction with PBS:methanol (50:50), and clean-up with IAC column. The limit of quantification was 0.033ngg(-1). Recoveries ranged from 87% to 102% for fortifications at 2.000 and 0.500ngg(-1), respectively, within-day R.S.D. of 1.4% and 4.7%. The proposed method was applied to 15 samples and the presence of OTA was found in nine samples at concentrations ranging from nd to 2.650ngg(-1).

Development and Validation of a LC-ESI-MS/MS Method for the Determination of Alternaria Toxins Alternariol, Alternariol Methyl-Ether and Tentoxin in Tomato and Tomato-Based Products

Alternaria species are capable of producing several secondary toxic metabolites in infected plants and in agricultural commodities, which play important roles in food safety. Alternaria alternata turn out to be the most frequent fungal species invading tomatoes. Alternariol (AOH), alternariol monomethyl ether (AME), and tentoxin (TEN) are some of the main Alternaria mycotoxins that can be found as contaminants in food. In this work, an analytical method based on liquid chromatography (LC) tandem mass spectrometry (MS/MS) detection for the simultaneous quantification of AOH, AME, and TEN in tomato and tomato-based products was developed. Mycotoxin analysis was performed by dispersive liquid-liquid microextraction (DLLME) combined with LC-ESI-MS/MS. Careful optimization of the MS/MS parameters was performed with an LC/MS system with the ESI interface in the positive ion mode. Mycotoxins were efficiently extracted from sample extract into a droplet of chloroform (100 µL) by DLLME technique using acetonitrile as a disperser solvent. Method validation following the Commission Decision No. 2002/657/EC was carried out by using tomato juice as a blank matrix. Limits of detection and quantitation were, respectively, in the range 0.7 and 3.5 ng/g. Recovery rates were above 80%. Relative standard deviations of repeatability (RSDr) and intermediate reproducibility (RSDR) were ≤ 9% and ≤ 15%, respectively, at levels of 25 and 50 ng/g. Five out of 30 analyzed samples resulted positive to at least one Alternaria toxin investigated. AOH was the most common Alternaria toxin found, but at levels close to LOQ (average content: 3.75 ng/g).

Cytotoxic effects and degradation products of three mycotoxins: alternariol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol in liver hepatocellular carcinoma cells.

This work is focused in studying the cytotoxic effects on HepG2 cells of the mycotoxins alternariol (AOH), 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) by the MTT assay, as well as in the identification of the degradation products and/or metabolites originated after treatment by liquid chromatography tandem mass spectrometry (LC-MS/MS) equipment and extracted from culture media. HepG2 cells were treated at different concentrations over 24, 48 and 72 h. The IC50 values were from 65 to 96 μM, from 3.6 to 6.2 μM and from 5.2 to 8.1 μM for AOH, 3-ADON and 15-ADON, respectively. Among all three mycotoxins assayed, deoxynivalenol (DON) derivated presented the highest toxic potential. Mass spectrometry (MS) scan chromatograms of studied mycotoxins allowed to detect products from: (i) the glutathione conjugate: (ii) sulfuric acid conjugated and (iii) amino group of cysteine conjugate. At all assayed times, the increase of recoveries values was obtained in a concentration dependent manner to finally decrease in the following ranking: 72 h>24h>48 h. The abundance relative (%) obtained for AOHs gluthathione ion product oscillated between 48 and 80% while for 3-ADONs ranged from 50 to 80%.

Evaluation of Alternaria mycotoxins in strawberries: quantification and storage condition

ABSTRACT Alternariol (AOH), alternariol methyl ether (AME) and tentoxin (TEN) are Alternaria mycotoxins produced by the most common post-harvest pathogens of fruits. The production of these metabolites depends on several environmental factors, mainly temperature, water activity, pH and the technological treatments that have been applied to the product. In this study, the occurrence of AOH, AME and TEN was evaluated in strawberries samples stored at different temperatures ranges (at 22 ± 2 or 6 ± 2°C) and different periods (up to 1 month) simulating the current practice of consumer’s storage conditions. Sample extraction was performed using a liquid–liquid extraction method prior to LC-MS/MS analysis. AOH was the most prevalent mycotoxins with a 42% at strawberries stored at (22 ± 2)°C and 37% stored at (6 ± 2)°C. The highest AOH levels were found in samples conserved at (22 ± 2)°C ranging between 26 and 752 ng g–1. AME levels ranged between 11 and 137 ng g–1, which were found mainly in stored samples at (6 ± 2)°C for more than 28 days. None sample presented levels of TEN in either of the studied conditions.

Binary and tertiary combination of alternariol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol on HepG2 cells: Toxic effects and evaluation of degradation products.

Fungi producers of mycotoxins are able to synthesize more than one toxin. Alternariol (AOH) is one of the mycotoxins produced by several Alternaria species, the most common one being Alternaria alternata. The toxins 3-Acetyl-deoxynivalenol (3-ADON) and 15-Acetyl-deoxynivalenol (15-ADON) are acetylated forms of deoxynivalenol (DON) produced by Fusarium graminearum. In the present work it is determined and evaluated the toxic effects of binary and tertiary combination treatment of HepG2 cells with AOH, 3-ADON and 15-ADON, by using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide), to subsequently apply the isobologram method and elucidate if the mixtures of these mycotoxins produced synergism, antagonism or additive effect; and lastly, to analyze mycotoxins conversion into metabolites produced and released by HepG2 cells after applying the treatment conditions by liquid chromatography tandem mass spectrometry (LC-MS/MS) equipment and extracted from culture media. HepG2 cells were treated at different concentrations over 24, 48 and 72h. IC50 values detected at all times assayed, ranged from 0.8 to >25μM in binary combinations; while in tertiary it ranged from 7.5 to 12μM. Synergistic, antagonism or additive effect detected in the mixtures of these mycotoxins was different depending on low or high concentration. Among all four mycotoxins combinations assayed, 15-ADON+3-ADON presented the highest toxic potential. At all assayed times, recoveries values oscillated depending on the time and combination studied.

Presence of Enniatins and Beauvericin in Romanian Wheat Samples: From Raw Material to Products for Direct Human Consumption

In this study, a total of 244 wheat and wheat-based products collected from Romania were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) in order to evaluate the presence of four enniatins (ENs; i.e., ENA, ENA1, ENB, and ENB1) and beauvericin (BEA). For the wheat samples, the influence of agricultural practices was assessed, whereas the results for the wheat-based products were used to calculate the estimated daily intake of emerging mycotoxins through wheat consumption for the Romanian population. ENB presented the highest incidence (41% in wheat and 32% in wheat-based products), with its maximum levels of 815 μg kg−1 and 170 μg kg−1 in wheat and wheat-based products, respectively. The correlation between the concentrations of ENB and ENB1 in wheat grain samples and farm practices (organic or conventional) was confirmed statistically (p < 0.05). This is the first study that provides comprehensive information about the influence of agricultural practice on emerging Fusarium mycotoxin presence in Romanian wheat samples and the estimated daily intake of ENs and BEA present in wheat-based products for human consumption commercialized in Romania.