Ochratoxin A in artisan salami produced in Veneto (Italy)

Fifty samples of artisan salami purchased in Veneto (Italy) were analysed for the determination of ochratoxin A (OTA). The analytical method, based on a sample preparation procedure with immunoaffinity columns (IACs), together with analysis by high-performance liquid chromatography with fluorescence detection (HPLC-FD), has guaranteed a high rate of recovery (about 97%), limit of detection (LOD) and limit of quantification (LOQ), respectively, of 0.06 µg kg^?1 and 0.20 µg kg^?1. OTA was detected in five samples, but only one exceeded the guideline value (1 µg kg^?1) established by the Italian Ministry of Health for pork meat and derived products. The results would seem to suggest that salami made with the traditional, non-industrial production method can be considered safe as regards contamination by OTA. However, the very high concentration observed in one sample proves that a high OTA contamination is also possible in this type of product. Thus, the controls of mycotoxin contamination must consider also salami.     

Improved sensitivity of ochratoxin A analysis in coffee using high-performance liquid chromatography with hybrid triple quadrupole-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS)

A novel and sensitive method utilising high-performance liquid chromatography coupled to triple quadrupole-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS) was developed in order to analyse the content of ochratoxin A (OTA) in coffee samples. The introduction of the triple-stage MS scanning mode (MS³) has been shown to increase greatly sensitivity and selectivity by eliminating the high chromatographic baseline caused by interference of complex coffee matrices. The analysis included the sample preparation procedure involving extraction of OTA using a methanol–water mixture and clean-up by immunoaffinity columns and detection using the MS³ scanning mode of LC-QqQLIT-MS/MS. The proposed method offered a good linear correlation (r² > 0.998), excellent precision (RSD < 2.9%) and recovery (94%). The limit of quantification (LOQ) for coffee beans and espresso beverages was 0.010 and 0.003 µg kg^?1, respectively. The developed procedure was compared with traditional methods employing liquid chromatography coupled to fluorescent and tandem quadrupole detectors in conjunction with QuEChERS and solid-phase extraction. The proposed method was successfully applied to the determination of OTA in 15 samples of coffee beans and in 15 samples of espresso coffee beverages obtained from the Latvian market. OTA was found in 10 samples of coffee beans and in two samples of espresso in the ranges of 0.018–1.80 µg kg^?1 and 0.020–0.440 µg l^?1, respectively. No samples exceeded the maximum permitted level of OTA in the European Union (5.0 µg kg^?1).     

Analysis of Ochratoxin A in Wine by High-Resolution UHPLC-MS

A method for determination of ochratoxin A (OTA) in wines using a new-solid phase extraction clean-up procedure followed with ultra performance liquid chromatography (UHPLC)-Orbitrap MS based on two scan events (full-scan Fourier transform mass spectrometer [FTMS] and higher energy-induced collision dissociation[HCD] data-dependent MS/MS) in positive ionization mode has been developed. The limit of detection (LOD) was estimated at 0.46 μg l^?1 for white wine, 0.53 and 0.54 μg l^?1 for rosé and red wines, respectively. The limit of quantification (LOQ) was estimated at 1.57 μg l^?1 in white wine, 1.77 and 1.81 μg l^?1 in rosé and red wines. Recovery experiments were carried out with spiked samples at three concentration levels (2, 5 and 10 μg l^?1). The OTA recoveries in spiked white wine samples varied from 69.6 % to 99.8 %, while the recoveries for rosé and red wine samples were in the range of 63.0–110.2 % and 63.6–103.2 %, respectively. Finally, based on the results, it is concluded that the combination of C18 cartridge with conventional particle packed columns and UHPLC LTQ-Orbitrap XL is an appropriate procedure for OTA analysis in wines.     

Determination of Ochratoxin A in Pig Muscle Using Dispersive Liquid-liquid Microextraction Combined with High-Performance Liquid Chromatography

A new method was developed for the rapid analysis of ochratoxin A (OTA) in pig muscle using dispersive liquid-liquid microextraction (DLLME) combined with high-performance liquid chromatography (HPLC). OTA was extracted with methanol and then cleaned up with DLLME procedure. After being blown dry and redissolved, the sample was determined using high-performance liquid chromatography with fluorescence detector (HPLC-FLD). Under the experimental conditions, the LOD was 0.21 μg kg^?1, LOQ was 0.70 μg kg^?1, and the recovery was higher than 80 %. Intra- and inter-day repeatability expressed as RSD were less than 5 %. The established method is more efficient, easier, and cheaper than conventional cleaning up procedure.     

Evaluation of extraction methods for ochratoxin A detection in cocoa beans employing HPLC

Cocoa is an important ingredient for the chocolate industry and for many food products. However, it is prone to contamination by ochratoxin A (OTA), which is highly toxic and potentially carcinogenic to humans. In this work, four different extraction methods were tested and compared based on their recoveries. The best protocol was established which involves an organic solvent-free extraction method for the detection of OTA in cocoa beans using 1% sodium hydrogen carbonate (NaHCO₃) in water within 30 min. The extraction method is rapid (as compared with existing methods), simple, reliable and practical to perform without complex experimental set-ups. The cocoa samples were freshly extracted and cleaned-up using immunoaffinity column (IAC) for HPLC analysis using a fluorescence detector. Under the optimised condition, the limit of detection (LOD) and limit of quantification (LOQ) for OTA were 0.62 and 1.25 ng ml^–1 respectively in standard solutions. The method could successfully quantify OTA in naturally contaminated samples. Moreover, good recoveries of OTA were obtained up to 86.5% in artificially spiked cocoa samples, with a maximum relative standard deviation (RSD) of 2.7%. The proposed extraction method could determine OTA at the level 1.5 µg kg^–1, which surpassed the standards set by the European Union for cocoa (2 µg kg^–1). In addition, an efficiency comparison of IAC and molecular imprinted polymer (MIP) column was also performed and evaluated.     

Co-occurrence of aflatoxins,ochratoxin A and citrinin in “egusi” melon (Colocynthis citrullus L.) seeds consumed in Ireland and the United Kingdom

The natural co-occurrence of aflatoxins (AFs), ochratoxin A (OTA) and citrinin (CIT) in melon seed samples obtained from retailers and households in Ireland and the United Kingdom (UK) was evaluated. AFs and OTA were determined by HPLC with fluorescence detection while CIT was analysed by HPLC-MS/MS. AFB₁ was detected in all (100%) samples (mean = 9.7 μg kg^?1; range = 0.2–66.5 μg kg^?1). Mean total AFs was 12.0 μg kg^?1 (range = 0.3–82 μg kg^?1). Commercially retailed samples showed a significantly higher AFB₁ contamination (p < 0.05) than the household samples. OTA occurred in 3 (13.6%) samples, while 4 (18.2%) were contaminated with CIT at very low levels. In this study, 68% of the melon seed samples were contaminated above the 2 μg kg^?1 EU limit for AFB₁ in oilseeds. These results highlight the need for the development of strategies to reduce AF contamination in “egusi” for human consumption.     

Polycyclic aromatic hydrocarbons in cereal products on the Turkish market

The contamination level of four EU marker polycyclic aromatic hydrocarbons (PAHs) in some cereal-derived products was surveyed in this study. Thirty-eight samples, 20 bread and 18 breakfast cereals, were purchased from retail shops and local markets of East Black sea region in Turkey. The samples were analysed for four EU marker PAHs, using ultrasonic extraction, solid-phase extraction (SPE) clean up and stable-isotope dilution gas chromatography with mass-spectrometric (GC/MS) detection. The method was validated with the parameters linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ) and uncertainty. Total content of the four PAHs in bread varied from 0.19 to 0.46 µg kg^?1 and in breakfast cereals from 0.10 to 0.87 µg kg^?1.     

Occurrence of ochratoxin A and citrinin in Czech cereals and comparison of two HPLC methods for ochratoxin A detection

The aims of the study were to obtain information about the occurrence of ochratoxin A (OTA) and citrinin (CIT) in cereals harvested in the Czech Republic and to compare two analytical procedures for detecting OTA. A total of 34 cereal samples, including two matrix reference materials (R-Biopharm, Germany), were analysed. The results were compared with the limit for raw cereal grains used as a foodstuff according to Commission Regulation No. 1881/2006, which allows a maximum OTA level of 5 µg kg^?1. Compared were two methods based on the high-performance liquid chromatography principle, one using the immunoaffinity columns OchraTest? (VICAM) and the second based on solvent partition (PART), both followed by fluorescence detection. The highest OTA contents were found in two barley samples. According to the method employed, the results for the first sample (malting barley) were VICAM = 31.43 µg kg^?1 and PART = 44.74 µg kg^?1. For the second sample (feeding barley) they were VICAM = 48.63 µg kg^?1 and PART = 34.40 µg kg^?1. Two samples of bread wheat had an OTA content approaching the legal limit (VICAM = 4.71 µg kg^?1 and PART = 6.03 µg kg^?1; VICAM = 4.12 µg kg^?1 and PART = 3.95 µg kg^?1). CIT was analysed using the PART method only, and its highest content (93.64 µg kg^?1) was found for the malting barley sample with high OTA content (44.74 µg kg^?1 as analysed using PART).     

Moniliformin analysis in maize samples from North-West Italy using multifunctional clean-up columns and the LC-MS/MS detection method

A fast clean-up method has been developed to purify maize extracts and to detect moniliformin (MON) in maize samples from North-West Italy over a four-year period (2008–2011). The method is based on the use of MycoSep® 240 Mon clean-up columns (Romer Labs^®). Samples were extracted using acetonitrile/water (84:16, v/v), and the extracts were purified with previously described clean-up columns. The liquid chromatography–tandem mass spectrography (LC-MS/MS) analysis has been carried out by means of hydrophilic interaction chromatography (HILIC), combined with negative electrospray mass spectrometry. The method has a recovery of 76–91% (relative standard deviation, RSD%: 6–14%), a limit of detection (LOD) of 1 µg kg^–1 and a limit of quantification (LOQ) of 4 µg kg^?1. Naturally contaminated maize (108 samples) was analysed for MON content. The average percentages of positive samples was 93% with the following ranges (µg kg^?1): 33–2606 (2008); <LOD–527 (2009); <LOD–920 (2010); <LOD–409 (2011).     

Determination of ochratoxin A in traditional Chinese medicinal plants by HPLC–FLD

Traditional Chinese medicinal plants (TCMPs), commonly used as spices, additives or foods, are also widely used in China to prevent and cure human disease. Due to their provenance, TCMPs may be contaminated by various fungal species, including ochratoxigenic fungi, during growth, collection, transportation and, especially, storage. A reliable method for the determination of ochratoxin A (OTA) in TCMPs of different origins was developed to monitor OTA levels in China. Analyses involved the extraction of OTA by methanol/water, immunoaffinity column (IAC) clean-up and HPLC quantification with fluorescence detection (FLD). Positive results were further confirmed by LC–ESI–MS/MS. The limit of detection (LOD) was 0.3 µg kg^?1, based on a signal-to-noise ratio of 3 : 1. Among the total of 57 TCMPs samples collected from six different areas, 31 were visibly moldy due to inappropriate storage; 26 sample, purchased from local TCMPs markets, were not visibly moldy. The results showed that 23 of the visibly moldy samples and two of the not visibly moldy were contaminated with OTA at levels ranging 1.2–158.7 and 2.5–5.6 µg kg^?1, respectively. This is the first report of the natural occurrence of OTA in TCMPs.     

Ochratoxin A in red pepper flakes commercialised in Turkey

The aim of this study was to investigate the presence of Ochratoxin A (OTA) in red pepper flakes commercialised in Turkey. A total of 75 samples were collected from different supermarkets and traditional bazaars in Istanbul during 2012–2013. OTA analysis was performed by high-performance liquid chromatography equipped with fluorescence detection after immunoaffinity column clean-up. The method was linear in the range 0.05–40 μg kg^?1 (r² = 0.9997). Twenty-seven out of 31 (87.1%) packed red pepper flake samples contained OTA at concentrations ranging from 0.6 to 1.0 μg kg^?1, whereas 100% of the unpacked red pepper flake samples contained OTA, in the range 1.1–31.7 μg kg^?1. Overall, only 4 unpacked samples (5.3%) contained OTA, with a mean value of 23.4 μg kg^?1, which is higher than the European Union limit. It is suggested that OTA content should be carefully considered in red pepper flake samples especially marketed in unpacked form.     

Occurrence of ochratoxin A in Canadian wheat shipments, 2010–12

Randomly selected domestic and export shipments (n = 1907) of Canadian durum and other wheat that occurred between 1 January 2010 and 31 December 2012 were analysed for ochratoxin A (OTA). The majority of samples did not contain OTA above the LOQ of 1 μg kg^–1. Only 37% of samples analysed contained quantifiable OTA; the median OTA of the positive results was 2.10 μg kg^–1. Canada Western Amber Durum shipments contained OTA more frequently, and at slightly higher concentrations, than Canada Western Red Spring wheat. For both wheat classes the frequency of OTA occurrence and mean concentrations appeared to increase in the lower grades, but these increases were not statistically significant. A periodic trend of a late summer increase of mean monthly OTA concentrations in shipments appears tied to the cycle of producer deliveries of wheat to primary grain elevators.     

Validation data for HPLC/FLD determinations of ochratoxin A in red paprika and black pepper adopting a one-step clean-up procedure

Validation data for the determination of ochratoxin A (OTA) in two spice matrices, red paprika and black pepper, were obtained for samples prepared with a simplified single-step clean-up column. Extracts of finely ground samples of red paprika and black pepper were prepared and applied to a Mycosep® 229 Ochra clean-up column. The purified extract was then subjected to HPLC/FLD analysis. The relative standard deviation for repeatability (RSDr) of the method was 11.8% for red paprika and 9.9% for black pepper. The limit of detection (LOD) value (three times the noise) was estimated as corresponding to the response of an extract derived from a blank matrix (previously washed) and spiked at 1.0 µg kg^?1. The limit of quantitation (LOQ) (three times LOD) was 3.0 µg kg^?1. The performance of the one-step column clean-up procedure appears to be a suitable alternative to commonly used clean-up techniques and allows the precise determination of OTA in two complex matrices.     

A Sensitive Immunoaffinity Column-Linked Indirect Competitive ELISA for Ochratoxin A in Cereal and Oil Products Based on a New Monoclonal Antibody

A new sensitive monoclonal antibody (mAb) 1H2 against ochratoxin A (OTA) was reported herein. This mAb belonged to the immunoglobulin G1 (k chain) isotype. In the optimized indirect competitive enzyme-linked immunosorbent assay (icELISA), 1H2 showed a 50 % inhibition concentration (IC₅₀) value of 0.058 ng/mL and a detection limit (IC₁₀) of 0.001 ng/mL. The cross-reactivity of 1H2 with ochratoxin B, aflatoxins, deoxynivalenol, zearalenone, T-2 toxin, or fumonisins was below 0.3 %. Based on this mAb, an immunoaffinity column (IAC)-linked icELISA was developed for OTA detection in the cereal and oil products. The working range of the assay for solid sample was 0.36–16 μg/kg. The recoveries from spiked samples of IAC-linked icELISA ranged from 83 to 101 %. These recoveries were much higher than those of icELISA (21–78 %) and in good agreement with those obtained by using the standard high-performance liquid chromatography method (87–110 %). The results indicated that the mAb 1H2 had the values for studies of OTA in the crude agricultural products.     

Aflatoxins and ochratoxin A in export quality raisins collected from different areas of Pakistan

During 2012–2014, 170 samples of export quality raisins were collected from different vendors in Pakistan. The collected samples were analysed for the presence of aflatoxins (AFs) and Ochratoxin A (OTA) contamination using high-performance liquid chromatography technique. The limit of detection and limit of quantification of AFs/OTA were 0.12/0.10 and 0.36/0.30 µg kg^?1, respectively. Only 5% of the samples were contaminated with AFs, ranging 0.15–2.58 µg kg^?1 with a mean of 0.05 ± 0.26 µg kg^?1. None of the raisin samples exhibited AFs contamination above the maximum limit (ML = 4 µg kg^?1) as set by the European Union (EU). About 72% of the samples were contaminated with OTA, ranging 0.14–12.75 µg kg^?1 with a mean of 2.10 ± 1.9 µg kg^?1. However, in 95.3% of the tested samples, OTA level was lower than the ML of 10 µg kg^?1 as regulated by the EU. Apparently, a strict and continuous monitoring plan, including regulatory limits, improves food safety and quality for all types of commodities.     

Quantification of mycotoxins in vegetable oil by UPLC-MS/MS after magnetic solid-phase extraction

ABSTRACT

The detection of mycotoxin contamination in foodstuffs is highly significant for public health. Herein we report an analytical method based on magnetic solid-phase extraction (MSPE) and UPLC-MS/MS for the simultaneous determination of mycotoxins, including fumonisins B₁ (FB₁), zearalenone (ZON) and ochratoxin A (OTA), in vegetable oil. Magnetic nanoparticles coated with double layers of silicon dioxide were synthesised and found to be an effective MSPE adsorbent for mycotoxins. The proposed MSPE procedure serves not only for sample clean-up but also for mycotoxin enrichment that enhances greatly the assay’s sensitivity. Under the selected MSPE conditions, linear matrix-matched calibration curves were obtained for mycotoxins in a concentration range from 0.178 to 625 μg kg^–1. The limits of detection were 0.210 μg kg^–1 for FB₁, 0.0800 μg kg^–1 for OTA and 1.03 μg kg^–1 for ZON. The proposed MSPE UPLC-MS/MS method was applied for the determination of mycotoxins in vegetable oil samples, including maize oil, rapeseed oil and soybean oil. ZON was detected in a maize oil at 101 μg kg^–1, which is below the European Union limit of 200 μg kg^–1 in foodstuffs.     

Effect of post-harvest treatments on the occurrence of ochratoxin A in raw cocoa beans

Cocoa beans are the principal raw material for chocolate manufacture. Moulds have an important place in the change in the quality of cocoa beans due to their role in the production of free fatty acids and mycotoxins, namely ochratoxin A (OTA). This study investigated the impact of the key post-harvest treatments, namely the fermentation and drying methods on OTA contamination of raw cocoa beans. Analytical methods for OTA detection were based on solid–liquid extraction, clean-up using an immunoaffinity column, and identification by reversed-phase HPLC with fluorescence detection. Of a total of 104 randomly selected cocoa samples analysed, 32% had OTA contents above 2 µg kg^–1. Cocoa sourced from pods in a bad state of health had a maximum OTA content of 39.2 µg kg^–1, while that obtained from healthy pods recorded 11.2 µg kg^–1. The production of OTA in cocoa beans increased according to the pod-opening delay and reached 39.2 µg kg^–1 after an opening delay of 7 days after harvest, while 6.1 and 11.2 µg kg^–1 were observed when pods were opened after 0 and 4 days. OTA production also seemed to depend considerably to the cocoa fermentation materials. When using plastic boxes for bean fermentation, the OTA production was enhanced and reached an average OTA content of about 4.9 µg kg^–1, while the raw cocoa treated in banana leaves and wooden boxes recorded 1.6 and 2.2 µg kg^–1 on average respectively. In parallel, the OTA production was not really influenced by either the mixing or the duration of the fermentation or the drying materials.     

Occurrence of ochratoxin A,fumonisin B2 and black aspergilli in raisins from Western Greece regions in relation to environmental and geographical factors

The presence of ochratoxin A (OTA), fumonisin B₂ (FB₂) and black aspergilli in raisins from Western Greece regions (Messinia, Corinthia, Achaia, Ilia and Zante Island) was investigated in relation to the different geographic and climatic conditions in the 2011 growing season. The biseriate species Aspergillus niger “aggregate” and A. carbonarius were mainly identified. The population of A. niger “aggregate” species occurred in all raisin samples at colony-forming units (CFU) concentrations significantly higher (mean 2.2 × 10⁵ CFU g^?1 homogenate) than those of A. carbonarius population (mean 4.9 × 10³ CFU g^?1 homogenate), which occurred in 80% of the raisin samples. OTA was found in 73% of the samples at levels ranging from 0.1 µg kg^?1 to 98.2 µg kg^?1, with the highest level occurring in a raisin sample from Ilia that also contained the highest level of A. carbonarius. The European Union legal limit for OTA was exceeded in 15% of the raisin samples. FB₂ was found in 29% of the raisin samples at levels ranging from 7.1 µg kg^?1 to 25.5 µg kg^?1, with 20% of the samples co-occurring with OTA. Principal-component analysis was applied to levels of mycotoxins, fungal contamination, geographical data and environmental conditions recorded in the harvesting (August) or drying (September) period. Principal-component analysis clearly indicated a good direct correlation of rainfall and relative humidity with OTA and A. carbonarius contamination. A lack of clustering was observed when A. niger and FB₂ contamination were considered. This is the first report on the co-occurrence of the mycotoxins OTA and FB₂ in dried vine fruits from Greece.     

Determination of mycotoxins in biscuits,dried fruits and fruit jams: an assessment of human exposure

ABSTRACT

A reliable, fast and simple method using UHPLC-MS/MS was developed for the determination of aflatoxins B₁ (AFB1), G₁ (AFG1), B₂ (AFB2) and G₂ (AFG2), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEA), HT-2 toxin and T-2 toxin in crude extracts of biscuits with fruit filling, cookies, dried fruits and fruit jams. The method was successfully demonstrated on 39 samples of biscuits with fruit filling, 34 cookies, 14 dried fruits and 10 fruit jams. The mycotoxins detected in biscuits samples were ZEA, OTA, T-2 and AFB1 with an average concentrations of positive samples of 2.64, 4.10, 8.13 and 1.32 µg kg^?1, respectively; while the mycotoxins detected in jam samples were AFB1, OTA, T-2 and AFB2 with an average concentrations of positive samples of 2.00, 17.7, 4.37 and 1.15 µg kg^?1, respectively. The results showed that the majority of samples were in compliance with relevant regulations. However in eight samples of biscuits and three samples of fig jam the contents of OTA were higher than the existing OTA limits. The combined dietary exposure of selected mycotoxins was estimated for the first time for children, adolescents and adults. The estimated combined dietary exposures were all lower than the proposed value assumed to predict a possible risk scenario.     

Occurrence of chloramphenicol in cereal straw in north-western Europe

Two surveys are presented of straw analysed for naturally occurring chloramphenicol (CAP), a drug banned for use in food-producing animals. In the first study, CAP was analysed by LC-MS/MS and detected in 37 out of 105 straw samples originating from the Netherlands, France, the UK, Germany and Denmark. The highest level found was 6.3 µg kg^?1, the average 0.6 µg kg^?1 and the median 0.2 µg kg^?1. The second study included a method comparison between ELISA and LC-MS/MS and a survey of CAP in cereal straw sampled at farms in all areas of Sweden. A total of 215 samples were screened by ELISA and a subset of 26 samples was also analysed by LC-MS/MS. Fifty-four of the samples contained more than 1 µg kg^?1 CAP and the highest level found was 32 µg kg^?1 (confirmed by LC-MS/MS). The highest contents of CAP in this study were allocated to the Baltic sea coast in the south-eastern part of Sweden (the county of Skåne and the Baltic Sea isle of Gotland). These results indicate a high incidence of CAP in straw in north-west Europe and have a severe impact on the enforcement of European Union legislation.