Method validation for the determination of ochratoxin A in green and soluble coffee by immunoaffinity column cleanup and liquid chromatography

A method was validated for the determination of ochratoxin A (OTA) in soluble and green coffee. Performance parameters evaluated included selectivity, accuracy, intermediate precision, linearity, limit of detection, limit of quantitation, and ruggedness. The method was found to be selective for OTA in both matrices tested. Recovery rates from soluble coffee samples ranged from 73.5 to 91.2%, and from green coffee samples from 68.7 to 84.5%. The intermediate precision (RSDr) was between 9.1 and 9.4% for soluble coffee and between 14.3 and 15.5% for green coffee analysis. The linearity of the standard calibration curve (r²) was <0.999 for OTA levels of 1.0–20.0 μg/kg in coffee samples. The limit of detection was determined to be 0.01 ng of OTA on column, while the limit of quantitation was found to be 0.03 ng on column. The limit of quantitation is equivalent to 0.6 μg/kg in soluble coffee samples and 0.3 μg/kg in green coffee samples. The results of the ruggedness trial showed two factors are critical for soluble coffee analysis: the extraction method, and the flow rate of the mobile phase. For green coffee analysis two critical factors detected were the extraction method and the storage temperature of the immunoaffinity column. Five samples of soluble coffee and 42 of green coffee were analysed using the validated method. All soluble coffee samples contained OTA at levels that ranged from 8.4 to 13.9 μg/kg. Six of the 42 green coffee samples analysed (14.3%) contained OTA at levels ranging from 0.9 to 19.4 μg/kg. The validated method can be used to monitor OTA levels in Colombian coffee for export or for local consumption.     

Einfluss der lagerdauer und des feuchtegehaltes auf die bildung von ochratoxin A und citrinin bei körnerleguminosen aus ökologischem und konventionellem anbau

Influence of storage time and moisture content on the development of ochratoxin A and citrinin in legumes kernel of ecological and conventional provenance Mould growth can cause the occurrence of mycotoxins in grain and legumes. Less information is known for legumes of ecological provenance. For this reason a storage trial was carried out with peas and horse beans, to examine the production of ochratoxin A (OTA) and citrinin (CT) in legumes kernel from ecological provenance. For that purpose kernels from legumes were remoistened to different moisture contents (MC, 14%/19%) and stored 24 weeks in a research granary (tower silo). This experiment should simulate the storage situation in farm scale from winter to summer. Every four weeks, the CO₂-content was determined and samples taken for the analysis of moisture, OTA and CT. At week 24 and a MC of <18% 1.9 μg OTA/kg of beans and 0.7 μg OTA/kg of peas (conventionally produced) were found.

Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Ochratoxin A (OTA) in Kaffee — Vergleichende Auswertung bundesweiter Daten der Jahre 1995–1999 und 2000–2002

Ochratoxin A concentrations from a total of 1223 samples from green, roasted and ground, decaffeinated and low acid and instant coffee analysed during 1995–1999 and 2000–2002 by German Official Food Control Laboratories were compared. Median concentrations, determined by statistical process analysis decreased within each group from the early group up to 2002. The rates of rejection were calculated on the basis of the recent data for green and roasted and ground coffee which may occur when particular maximum residue limits were set. Contamination rates from green coffee showed significant differences in their OTA concentrations depending on their geographical origin.

Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Mycotoxins in several Polish food products in 2004–2005

In 2004–2005, samples of several selected Polish foods such as cereal products, nuts, dried fruits, coffee and culinary spices collected from Warsaw market and taken from food producers were analyzed on presence of aflatoxin B₁, B₂, G₁, G₂ (AF), ochratoxin A (OTA), zearalenone (ZEA) and deoxynivalenol (DON). After extraction and clean-up of extracts on immunoaffinity columns (IAC), mycotoxin analyses were carried out by HPLC using fluorescence and UV detectors. The concentrations of aflatoxins and ochratoxin A depending on the kind of sample ranged from 0.02 to 7.8 (one sample, of peanuts) and 0.02–11.9 μg/kg (one coffee sample), respectively. The levels of ZEA and DON were found to be below 50 °g/kg.     

Ochratoxin A in airborne dust and fungal conidia

Farm workers are often exposed to high concentrations of airborne organic dust and fungal conidia, especially when working with plant materials. The purpose of this investigation was to study the possibility of exposure to the mycotoxin ochratoxin A (OTA) through inhalation of organic dust and conidia. Dust and aerosol samples were collected from three local cowsheds. Aerosol samples for determination of total conidia and dust concentrations were collected by stationary sampling on polycarbonate filters. Total dust was analysed by gravimetry, and conidia were counted using scanning electron microscopy. A method was developed for extraction and determination of OTA in small samples of settled dust. OTA was extracted with a mixture of methanol, chloroform, HCI, and water, purified on immunoaffinity column, and analysed by ion-pair HPLC with fluorescence detection. Recovery of OTA from spiked dust samples (0.9–1.0 μg/kg) was 74% (quantitation limit 0.150 μg/kg). OTA was found in 6 out of 14 settled dust samples (0.2–70 μg/kg). The total concentration of airborne conidia ranged from < 1.1 × 10⁴ to 3.9 × 15⁵ per m³, and the airborne dust concentration ranged from 0.08 to 0.21 mg/m³. Conidia collected from cultures of Penicillium verrucosum and Aspergillus ochraceus contained 0.4–0.7 and 0.02–0.06 pg OTA per conidium, respectively. Testing of conidial extracts from these fungi in a Bacillus subtilis bioassay indicated the presence of toxic compounds in addition to OTA. The results show that airborne dust and fungal conidia can be sources of OTA. Peak exposures to airborne OTA may be significant, e.g., in agricultural environments. This revised version was published online in August 2006 with corrections to the Cover Date.     

A new validated HPLC-FLD method for detecting ochratoxin A in dry-cured meat and in blue cheese

In the present study, a fast and sensitive method for the quantification of ochratoxin A in two lipidicproteic food matrices has been developed. In particular, the sample preparation procedure has been optimized for dry-cured meat products and blue cheeses and tested for several validation parameters (LOD, LOQ, recovery, repeatability and within-laboratory precision). The procedure has been then applied to several dry-cured meat products and blue cheeses from the market. Ochratoxin A has been occasionally found in dry-cured and smoked ham from the market and the contamination occurred both in the outer and in the inner part of the products. Concerning the blue cheese, the occurrence of ochratoxin A is reported for the first time: OTA was occasionally found at low levels (0.1–3 μg/kg) in commercial samples of Roquefort from France and Gorgonzola from Italy, opening a new issue for risk assessment and quality control. Presented at the 29^th Mykotoxin-Workshop, Fellbach, Germany, May 14–16, 2007 Financial support: This work was partially granted by Emilia-Romagna region (projects: SIQUAL and “Safety and quality of typical pork meat production chain”)     

Co-occurrence of mycotoxins in swine feed produced in Portugal

A total of 404 samples of commercial swine feed from Portugal feed mills were analysed by HPLC methods for the presence of mycotoxins: 277 samples of feed for fattening pigs were analysed for ochratoxin A (OTA), zearalenone (ZEA), and deoxynivalenol (DON), and 127 samples of feed for sows were analysed for ZEA and fumonisins (FB₁ + FB₂). Concerning feed for fattening pigs, 21 (7.6%) samples were positive for OTA, (2–6.8 μg/kg), 69 (24.9%) were positive for ZEA (5–73 μg/kg), and 47 (16.9%) were positive for DON (100–864 μg/kg). In feed for sows, the results showed 29.9% of positive samples for ZEA (5–57.7 μg/kg) and 8.7% positive samples for FB₁ and FB₂ (50–391.4 μg/kg). Co-occurrence of DON/ZEA was found most frequently, but simultaneous contamination with OTA/ZEA and OTA/DON was also found.     

Occurrence of deoxynivalenol (DON) and ochratoxin A (OTA) in dog foods

The commercially available dog food samples (29 dry foods and 11 wet foods) were analysed for deoxynivalenol (DON) and ochratoxin A (OTA) using ELISA. All (100%) dry foods were contaminated with DON with various amount of the toxin (22-1837 μg/kg). In wet food 3 samples were found to be positive for DON in the range of 95-170 μg/kg. There were a few samples contaminated with OTA: 3 samples in dry foods (7-40 μg/kg) and 2 samples in wet foods (45 and 115 μg/kg).     

Mycotoxin production by different ochratoxigenic <Emphasis Type="Italic">Aspergillus</Emphasis> and <Emphasis Type="Italic">Penicillium</Emphasis> species on coffee- and wheat-based media

Ochratoxin A (OTA) is one of the most widespread mycotoxins, and is produced by several Aspergillus or Penicillium species. Human exposure to OTA is mainly by intake of contaminated food, with cereal products, followed by coffee and red wine as the main sources of OTA. In this study, the OTA production of four ochratoxigenic fungi (two Aspergillus and two Penicillium species) was investigated in four different media, i.e. wheat and coffee model media as food-based media and two standard laboratory media (malt extract glucose agar, MEA and yeast extract sucrose agar, YES). Colony growth was documented and OTA concentrations in cultures were determined at day 2, 4 and 8 of incubation at 25°C by high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). OTA production clearly depended upon time of incubation, fungal species, and medium composition. On coffee based medium, moderate OTA levels were produced by A. ochraceus BFE635 (9.8 μg/g) and by A. niger BFE632 (10.6 μg/g) on day 8 of incubation. In wheat-based medium, these strains produced much more OTA than in coffee. The highest OTA concentration (83.8 μg/g on day 8) was formed by A. ochraceus BFE635 followed by the other Aspergillus niger BFE632 (49 μg/g). Lower OTA levels were produced by P. verrucosum BFE550 and P. nordicum BFE487, in both wheat and in YES medium, whilst OTA was hardly detectable in coffee and in MEA in case of P. nordicum. Colony growth of the tested strains on different media was not indicative of OTA production. Guttation droplets developed on wheat-based medium with the Aspergillus strains within a week, and this phenomenon coincided with the high OTA amounts formed by these species. Results from this study add to our knowledge on the behaviour of ochratoxigenic fungal species when cultured on food based media.     

Natural Occurrence of Mycotoxins in Staple Cereals from Ethiopia

The occurrence of mycotoxins in barley, sorghum, teff (Eragrostis tef) and wheat from Ethiopia has been studied. Samples were analyzed for aflatoxin B₁ (AFB1), ochratoxin A (OTA), deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) using high performance liquid chromatography (HPLC) and for fumonisins (FUM) using enzyme linked immunosorbent assay (ELISA). AFB1 and OTA were detected in samples of all the four crops. AFB1 was detected in 8.8% of the 352 samples analyzed at concentrations ranging from trace to 26 μg kg⁻¹. OTA occurred in 24.3% of 321 samples at a mean concentration of 54.1 μg kg⁻¹ and a maximum of 2106 μg kg⁻¹. DON occurred in barley, sorghum and wheat at 40–2340 μg kg⁻¹ with an overall incidence of 48.8% among the 84 mainly ‘suspect’ samples analyzed; NIV was co-analyzed with DON and was detected at 40 μg kg⁻¹ in a wheat sample and at 50, 380, and 490 μg kg⁻¹ in three sorghum samples. FUM and ZEN occurred only in sorghum samples with low frequencies at concentrations reaching 2117 and 32 μg kg⁻¹, respectively. The analytical results indicate higher mycotoxin contamination in sorghum, which could be related to the widespread storage of sorghum grain in underground pits leading to elevated seed moisture contents. This is the first report on the occurrence of OTA in teff.     

Determinatıon of ochratoxin A and total aflatoxin levels in corn samples from Turkey by enzyme-linked immunosorbent assay

Forty-seven samples of corn were collected from various street bazaars and market outlets in different regions of Turkey and total aflatoxin (AF) and ochratoxin A (OTA) levels were determined by enzyme-linked immunosorbent assay (ELISA) following sample preparation. Levels of AF and OTA in corn samples ranged between 1.75–120.3 μg/kg and 1.08–8.57 μg/kg, respectively. Although 53% of the samples analysed had no detectable levels of AF, 4% of similar samples were found to contain AFs above the acceptable limit of 10 μg/kg in Turkey. For OTA, 4% of the corn samples had levels above the acceptable limit (3 μg/kg) in Turkey, with over 43% samples not found to contain this mycotoxin. Although the levels of mycotoxins analysed in this study were not found to be high and the percentage of samples contaminated above permitted limits were low, the importance of overall daily dietary intake should not be underestimated and control of these fungal metabolites in corn must be explored to minimise the hazards they may cause in humans.     

Bestimmung von Citrinin in Getreide

A simultaneous reversed-phase HPLC determination of citrinin and ochratoxin A in cereals is proposed. Both mycotoxins are eluted on a RP-amid C16 column using a gradient eluent acidified with phosphoric acid. The limits of detection, for a signal-to-background ratio of 3, are 1 μg/kg for citrinin and 0,4 μg/kg for ochratoxin A.

Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Natural multi-occurrence of mycotoxins in rice from Niger State,Nigeria

Twenty-one rice samples from field (ten), store (six) and market (five) from the traditional rice-growing areas of Niger State, Nigeria were analysed for aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisin B₁ (FB₁) and B₂ (FB₂), and patulin (PAT) by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) respectively. T-2 toxin was determined using TLC only. AFs were detected in all samples, at total AF concentrations of 28–372 μg/kg. OTA was found in 66.7% of the samples, also at high concentrations (134–341 μg/kg) that have to be considered as critical levels in aspects of nephrotoxicity. ZEA (53.4%), DON (23.8), FB₁ (14.3%) and FB₂ (4.8%) were also found in rice, although at relatively low levels. T-2 toxin was qualitatively detected by TLC in only one sample. Co-contamination with AFs, OTA, and ZEA was very common, and up to five mycotoxins were detected in a single sample. The high AF and OTA levels as found in rice in this study are regarded as unsafe, and multi-occurrences of mycotoxins in the rice samples with possible additive or synergistic toxic effects in consumers raise concern with respect to public health.     

Combinatory effects of citrinin and ochratoxin A in immortalized human proximal tubule cells

Ochratoxin A (OTA) and citrinin (CIT) are two mycotoxins often occurring together in grains and cereals. Although both are nephrotoxic and can induce apoptosis, combination effects have not been examined up to now. Therefore, the aim of this study was to take a close look at the interactions of citrinin and OTA in cultured human proximal tubule-derived cells (IHKE cells). The cytotoxicity of both mycotoxins was studied, measuring the metabolic activity and the cell number. Furthermore, caspase 3-activation as a marker for apoptosis was examined for both mycotoxin alone and in combination. The results show that citrinin had an antagonistic effect on ochratoxin A induced caspase 3-activation in concentrations of 2.5 and 5 μmol/l. Higher concentrations (7.5 and 15 μmol/l) lead to additive effects, lower citrinin concentrations (0.25 and 1 μmol/l) did not show any effect at all. The observed decrease in caspase 3-activity was specific for the combination with OTA, since the combination of citrinin with cisplatin did not show any effect. Citrinin did not influence of the OTA-induced apoptosis when added two hours after applying ochratoxin A. Also the combination of both toxins decreased the uptake of OTA into the cells which might be an explanation for the antagonistic effect of citrinin in certain concentrations. However, the transport into cells can not be the only explanation. so further examinations are necessary. Presented at the 27^th Mykotoxin-Workshop. Dortmund, Germany, June 13–15, 2005.     

Silibinin pretreatment protects against Ochratoxin A-mediated apoptosis in primary rat hepatocytes

The inhibitory effect of silibinin on ochratoxin A (OTA)-mediated apoptosis on primary rat hepatocytes was investigated. Rat hepatocytes were prepared by two different methods: the classical enzymatic digestion method by collagenase perfusion and a new EDTA-perfusion method. The EDTA-perfusion method yielded hepatocytes, which were stably cultivated without DNA fragmentation for up to 96 h, whereas the collagenase-prepared hepatocytes showed apoptosis events as early as from the start of preparation even in the absence of OTA. Treatment with 12.5 μmol/l OTA of cultured hepatocytes prepared under ETDA perfusion developed DNA-laddering after 24–36 h. Lipopolysaccharide (LPS) of 0.1 up to 12.5 μg/ml showed no apoptotic DNA-effects under these conditions. A low concentration of 26 μmol/l silibinin given prior to OTA slightly prevented OTA-mediated DNA-laddering, whereas a five times higher concentration of silibinin (130 μmol/l) completely inhibited OTA-mediated apoptosis. Under the same conditions, caspase-3 activity in hepatocytes increased in a time-dependent manner under OTA exposure within 12–24 h but was blocked by 130 μmol/l silibinin. In contrast, LPS incubation for 12 and 24 h did not alter caspase-3 activity. To measure viability of OTA-/LPS-treated hepatocytes, the MTT-test and Live/Dead kit were applied. The results demonstrated that the used OTA concentration of 12.5 μmol/l only moderately decreased viability for up to 24 h but showed cytotoxic effects depending on longer incubation times (≥36 h). In contrast, LPS up to 12.5 μg/ml exhibited no cytotoxic effects up to 48 h. In summary, our results showed contrasting effects on apoptosis in primary rat hepatocytes by OTA (produces apoptosis) versus LPS (produces no apoptosis), also depending on the method of hepatocyte preparation. Silibinin at 130 μmol/l showed significant hepatoprotective and antiapoptotic effects against OTA-mediated cell damage on cultured rat hepatocytes.     

Ochratoxin A production in aniseed-based media by selected fungal strains and in anise fruits (<Emphasis Type="Italic">Pimpinella anisum</Emphasis> L.)

The growth conditions and ochratoxin A (OTA) production of Aspergillus strains were studied in aniseed (Pimpinella anisum L.)-based media. The results showed that methanol/NaHCO₃ (50:50, v/v) mixture for extraction and competitive direct ELISA analytical method are capable of detecting low OTA concentrations in this raw material, which were confirmed by HPLC with fluorescence detection (R ² = 0.994). In aniseed meal extract agar artificially contaminated with selected fungi, the higher OTA values obtained were 283.8 ± 28.1 μg L^-1 for A. carbonarius and between 1.7 ± 0.1 μg L^-1 and 16.5 ± 12.8 μg L^-1 for A. steynii strains. While the optimal conditions of growth for A. carbonarius and A. steynii are 28°C and 0.98 a _w, the optimal production of OTA was observed at 0.99 a _w for both A. carbonarius and A. steynii but at 22°C and 28°C, respectively. Except in one sample, all the aniseed samples analysed were negative for OTA natural contamination. This study demonstrates that aniseed can be a matrix capable to contamination with OTA, at least produced by A. carbonarius and A. steynii strains, regardless of the antimicrobial properties of aniseed essential oil.     

Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells

In order to better characterize the ochratoxin A (OTA)-induced DNA damage and to further investigate factors which may modulate dose-effect relationships in cells, the induction of micronuclei was studied in V79 Chinese hamster fibroblast cells and in primary cultures of porcine urothelial bladder epithelial cells (PUBEC). OTA was able to induce micronuclei in PUBEC and V79 cells at concentrations below those which were overtly cytotoxic. OTA concentrations between 0.03 and 1 μM caused a dose-dependent increase of micronuclei in V79 cells (up to 3-fold compared to controls); but the lowest tested concentration of 0.01 μM OTA did not induce a higher frequency of micronuclei than in the solvent control, indicative of an apparent threshold. Clear evidence for genotoxic effects was also found in PUBEC cultures treated with OTA concentrations of 1 μM and more, although the dose-effect relationship in PUBEC was more variable for several freshly isolated cell batches, pointing to differences in susceptibility to OTA between bladder cells from different donor animals. The chromosomal genotoxicity of OTA demonstrated in this study is in general accord with previous findings on the induction of clastogenic effects and oxidative DNA damage by OTA. In both cases, the shape of the dose-response curve at very low OTA concentrations supports the existence of a threshold for its genotoxicity. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Occurrence of aflatoxin B1 and ochratoxin A in Lebanese cultivated wheat

An extensive survey of filamentous fungi isolated from wheat grown and consumed in Lebanon and their capacity to produce aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) was conducted to assess fungi potential for producing these toxins in wheat. From the 468 samples of wheat kernel, collected at preharvest stage from different locations during 2008 and 2009 cultivation seasons, 3,260 fungi strains were isolated with 49.4% belonging to Penicillium spp. and 31.2% belonging to Aspergillus spp. Penicillium spp. was detected on wheat samples with a high amount of P. verrucosum (37.0%). Among the different Aspergillus spp. isolated, A. niger aggregate was predominant and constituted 37.3%. whereas the isolation rate of A. flavus and A. ochraceus was 32.2 and 25.6%, respectively. The ability to produce OTA and AFB₁ by isolates belonging to Aspergillus spp. and Penicillium spp. was analyzed by high performance liquid chromatography with fluorescence detector (HPLC-FLD). It was found that 57.0% of Penicillium spp. and 80% of A. ochraceus isolates tested produced OTA, respectively, at maximum concentrations of 53 and 65 μg/g CYA. As for the aflatoxinogenic ability, 45.3% of A. flavus produced AFB₁, with maximum concentration of 40 μg/g CYA. A total of 156 wheat samples were analyzed for the levels of OTA and AFB₁ by HPLC-FLD. The results showed that 23.7% were contaminated with OTA, at a concentration higher than 3 μg/kg and 35.2% of these samples were contaminated with AFB₁ at concentration higher than 2 μg/kg. The risks originating from toxin levels in wheat produced in Lebanon should be monitored to prevent their harmful effects on public health.     

Monitoring of mycotoxin biomarkers in the Czech Republic

AFM₁ was determined in 72 (72%) samples of human urine, range 19-6064 pg/g creatinine, mean 367 pg/g creatinine, median 158 pg/g creatinine and 90% percentile 755 pg/g creatinine in 1997. AFM₁ was determined in 46 (43.8%) samples of human urine, range 21-19219 pg/g creatinine, mean 414 pg/g creatinine, median 96 pg/g creatinine and 90% percentile 415 pg/g creatinine in 1998. OTA was determined in 2077 (94.2%) samples of human serum, range 0.1–13.7 μg/L, mean 0.28 μg/L, median 0.2 μg/L and 90% percentile 0.5 μg/L in 1994–2002. OTA was determined in 12 (40%) samples of human kidneys, range 0.1–0.2 μg/kg, mean 0.07 μg/kg, and median 0.05 μg/kg in 2001. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004.