Investigation of ochratoxin a, B and citrinin contamination in various commercial foods

Ochratoxin A (OTA), ochratoxin B (OTB) and citrinin (CIT) in commercial foods were simultaneously determined and confirmed with high-performance liquid chromatography (HPLC) and liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The samples examined were made up of cereal, fruit, coffee, and cacao products. The limits of quantification (S/N> or =10) of OTA, OTB and CIT were 0.1 microg/kg or less. Aflatoxins (AF), deoxynivalenol (DON) and fumonisins were also surveyed. Of 157 samples examined, 44 were contaminated with OTA at levels of 0.11 to 4.0 microg/kg. At least 2 positive samples were labeled as domestics. In most positive samples, the OTA level was low, less than 1 microg/kg. The highest incidence of OTA was observed in cacao powder (10/12), followed by instant coffee (5/7), cocoa (5/8) and raisin (6/13). OTB was found in fruit and cacao products containing relatively high levels of OTA. Co-occurrence of OTA, CIT and DON was found in cereal products, and co-occurrence of OTA and AF was found in cacao products. Approximately 30% of naturally contaminated OTA in roasted coffee bean moved into the extract solution when brewed with paper filter.     

Determination of ochratoxin A in red wine and vinegar by immunoaffinity high-pressure liquid chromatography

A method is described for the determination of ochratoxin A (OTA) in red wine and vinegar using an acidic chloroform extraction, an immmunoaffinity clean-up step, and a high-performance liquid chromatographic determination with fluorescence detection. The detection limit was estimated at 0.002 microg/liter. The mean recovery factors were found at 91.3 and 96.6% for wine and vinegar, respectively. Thirty-one samples of red wine originating from Mediterranean sea countries and 15 samples of vinegar were examined for the presence of OTA. All red wine samples contained OTA. Seventy-two percent of these samples were found to be contaminated over 0.1 microg/liter. Among them, nine samples contained ochratoxin A in the range of 0.5 to 3.4 microg/liter, 12 samples in the range of 0.10 to 0.50 microg/liter (median: 0.176 microg/liter), and 9 samples in the range of 0.010 to 0.100 microg/liter (median: 0.041 microg/liter). All 15 vinegar samples showed the presence of OTA. The most contaminated ones were three balsamic vinegar samples containing 0.156 microg/liter, 0.102 microg/liter, and 0.252 microg/liter of OTA. In the remaining 12 samples, ochratoxin A levels ranged from 0.008 microg/liter to 0.046 microg/liter (median: 0.012 microg/liter). These data are in good agreement with the hypothesis that wine originating from Southern countries might contain significant OTA concentration and showed the possible occurrence of traces of OTA in vinegar.     

Ochratoxin A concentrations in Greek domestic wines and dried vine fruits.

A survey for the presence of ochratoxin A (OTA) was conducted from 1995 to 1999 on 268 locally produced commercial wines, and on 81 samples of domestic dried vine fruits (currants and sultanas) collected between 1998 and 2000 from sites of primary storage and processing. The OTA concentration in red dry wines (n = 104, median = 0.09 microgram l(-1)) was not significantly different from that for white (n = 118, median = 0.06 microgram l(-1)) and rosé (n = 20, median = 0.08 microgram l(-1)) wines. Eighteen samples of dessert wines (sweet, semi-sweet, semi-dry) and eight samples of retsina wine showed larger OTA concentrations with medians of 0.33 and 0.27 microgram l(-1), respectively. Our data indicate that the geographic region of origin influences OTA contamination for the red dry wines. In fact, a trend of increasing OTA contamination was observed for red wines from northern to southern Greece. Regarding the OTA levels in dried vine fruits, sultanas (n = 27, median = 0.6 microgram kg(-1)) were less contaminated than currants (n = 54, median = 1.3 microgram kg(-1)). Also, sultanas produced in 2000 and currants produced in 1999 showed the lowest incidence of OTA contamination, with medians of 0.3 and 0.9 microgram kg(-1), respectively.     

Incidence of microflora and of ochratoxin A in green coffee beans (Coffea arabica).

Coffee is produced in tropical countries around the Equator where climatic conditions are favourable for fungal development and mycotoxin production; however, mycotoxins do not only occur in the tropical countries. The aim was to evaluate the mycoflora and possible incidence of ochratoxin A (OTA) in 60 samples of green coffee beans from Brazil. The mycological evaluation was carried out using a conventional method and the OTA was determined using sequential phenyl silane and immunoaffinity column cleanup followed by HPLC. The detection limit was 0.2 microg kg(-1). Practically all samples (91.7%) were contaminated with moulds. The dominant fungal genus was Aspergillus, including A. niger (83.3%), A. ochraceus (53.3%) and A. flavus (25.0%). The occurrence and the levels of the genus Cladosporium (16.6%) and Penicillium (10.0%) were substantially lower than Aspergilli. Twenty samples (33.3%) of 60 were contaminated with the toxin at levels ranging from 0.2 to 7.3microg kg(-1). The average concentration was 2.38 microg kg(-1). All positive samples showed OTA levels below the limit suggested by the European Union (8 microg kg(-1)).     

Simultaneous determination of ochratoxin A, B and citrinin in foods by HPLC-FL and LC/MS/MS

Methods using high-performance liquid chromatography with fluorescence detection (HPLC-FL) and using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for simultaneous determination of ochratoxin A (OTA), ochratoxin B (OTB) and citrinin (CIT) in cereal, fruit, and coffee products. The samples were extracted with ethyl acetate under an acidic condition, and then cleaned up with liquid-liquid separation. The test solutions were analyzed by reverse-phase HPLC-FL and LC/MS/MS. Mass spectral acquisition was performed in positive ion mode by applying multiple reaction monitoring. The performances of both detectors were almost equivalent. The recoveries of OTA and OTB were 87-111%, and that of CIT were 70-88%. The limits of quantification (S/N> or =10) of OTA, OTB and CIT was 0.1 mug/kg or less. These methods were considered to be useful for the determination of the three mycotoxins at low levels (0.1 microg/kg).     

Development and application of analytical methods for the determination of mycotoxins in organic and conventional wheat.

The aim of this study was to develop a multicomponent analytical method for the determination of deoxynivalenol (DON), ochratoxin A (OTA) and zearalenone (ZEN), nivalenol (NIV), 3-acetyl-DON (3-acDON), 15-acetyl-DON (15-acDON), zearalenol (ZOL) and citrinin (CIT) in wheat. It also aimed to survey the presence and amounts of DON, OTA and ZEN in Belgian conventionally and organically produced wheat grain and in wholemeal wheat flours. After solvent extraction, an anion-exchange column (SAX) was used to fix the acidic mycotoxins (OTA, CIT), whilst the neutral mycotoxins flowing through the SAX column were further purified by filtration on a MycoSep cartridge. OTA and CIT were then analysed by high-performance liquid chromatography (HPLC) using an isocratic flow and fluorescence detection, while the neutral mycotoxins were separated by a linear gradient and detected by double-mode (ultraviolet light fluorescence) detection. The average DON, ZEN and OTA recovery rates from spiked blank wheat flour were 92, 83 and 73% (RSDR = 12, 10 and 9%), respectively. Moreover, this method offered the respective detection limits of 50, 1.5 and 0.05 microg kg-1 and good agreement with reference methods and inter-laboratory comparison exercises. Organic and conventional wheat samples harvested in 2002 and 2003 in Belgium were analysed for DON, OTA and ZEN, while wholemeal wheat flour samples were taken from Belgian retail shops and analysed for OTA and DON. Conventional wheat tended to be more frequently contaminated with DON and ZEN than organic samples, the difference being more significant for ZEN in samples harvested in 2002. The mean OTA, DON and ZEA concentrations were 0.067, 675 and 75 microg kg-1 in conventional samples against 0.063, 285 and 19 microg kg-1 in organically produced wheat in 2002, respectively. Wheat samples collected in 2003 were less affected by DON and ZEN than the 2002 harvest. Organic wholemeal wheat flours were more frequently contaminated by OTA than conventional samples (p < 0.10). The opposite pattern was shown for DON, organic samples being more frequently contaminated than conventional flours (p < 0.10).     

Isolation, identification and toxigenic potential of ochratoxin A-producing Aspergillus species from coffee beans grown in two regions of Thailand

In 2006 and 2007, 32 Thai dried coffee bean samples (Coffea arabica) from two growing sites of Chiang Mai Province, and 32 Thai dried coffee bean samples (Coffea canephora var. robusta) from two growing sites of Chumphon Province, Thailand, were collected and assessed for the distribution of fungi with the potential to produce ochratoxin A (OTA). The overall percentage of fungal contamination in coffee was 98% and reduced to 60% after surface disinfection. There were remarkable ecological differences in the composition of ochratoxigenic species present in these two regions. Arabica coffee bean samples from the North had an average of 78% incidence of colonization with Aspergillus of section Circumdati with Aspergillus westerdijkiae and A. melleus as the predominant species. Aspergillus spp. of section Nigri were found in 75% of the samples whereas A. ochraceus was not detected. Robusta coffee beans from the South were 98-100% contaminated with predominantly A. carbonarius and A. niger. A. westerdijkiae was only found in one sample. The diversity of the fungal population was probably correlated with the geographical origin of the coffee, coffee cultivar, and processing method. Representative isolates of section Circumdati (52) and Nigri (82) were examined for their OTA production using HPLC with fluorescence detection. Aspergillus westerdijkiae (42 isolates out of 42), A. steynii (13/13), and A. carbonarius (35/35) in general produced large amounts of OTA, while one isolate of A. sclerotiorum produced intermediate amounts of OTA. 13% of the A. niger isolates produced OTA in intermediate amounts. OTA levels in coffee bean samples were analyzed using the Ridascreen OTA ELISA kits. Of the 64 coffee bean samples analyzed, 98% were contaminated with OTA in levels of <0.6-5.5 microg/kg (Arabica) and 1-27 microg/kg (Robusta). Presence of OTA in representative coffee samples was also confirmed by LC-MS/MS after ion-exchange purification.     

Ochratoxin A in sultanas from Turkey I: Survey of unprocessed sultanas from vineyards and packing-houses.

A method for the determination of ochratoxin A (OTA) in sultanas from Turkey using extraction with a sodium bicarbonate solution (2% NaHCO3) followed by immunoaffinity clean-up and liquid chromatography with fluorescence detection was used to assess the frequency of occurrence and level of OTA. In-house validation was carried out with spiked samples at levels of 0.15, 1.5, 5.0 and 10 microg kg-1 and average recoveries were 91, 93, 87 and 89%, respectively. The limits of detection and limit of quantification in Turkish sultanas were 0.026 and 0.09 microg kg-1, respectively. A survey for the presence of OTA was carried out on 264 unprocessed sultana samples during the production seasons between 1998 and 2000 collected annually from vineyards and from packing-houses. The analyses of unprocessed sultanas showed that 32.2% of the total number of samples contained no detectable OTA, whereas 9.8% of sultana samples had OTA concentrations above 10 microg kg-1, and the remaining 58% had levels within the range 0.026-10 microg kg-1. There were big differences in median concentrations between years. Considering the year of production, it appears that sultanas produced in 1998 and 2000 showed the lowest incidence of OTA contamination (median<0.02 microg kg-1), whereas 2002 showed the highest incidence (median=4.3 microg kg-1). The overall mean OTA concentration was calculated as 3.4 microg kg-1, and the overall median as 0.9 microg kg-1. Among the samples analysed, the highest detected level of OTA was 54 microg kg-1.     

Occurrence of ochratoxin A in Italian wines.

A total of 96 red wines and 15 white dessert wines produced mostly in the years 1995-97 in 19 Italian regions were analysed for ochratoxin A (OTA). The amount of OTA ranged from < 1 to 3856 ng/l the median (mean) was found to be 90 (419) ng/l for the red wines and 8 (736) ng/l for the white dessert wines. Our survey shows that the geographic region of origin has a strong influence on OTA contamination, both for red and for dessert wines: in fact, wines produced in southern Italy were markedly more contaminated. The overall median (mean) OTA concentration in the red wines produced in the four Italian areas (northwest, northeast, centre and south) was 2 (11), 90 (81), 134 (295) and 1264 (1233) ng/l. The same trend was observed for the white dessert wines: OTA concentrations of over 1000 ng/l were found in four out of five samples from southern Italy (1185, 2454, 3477, 3856 ng/l), while central and northern samples showed very low contamination. The contribution of wine to mean daily OTA intake can be considered negligible in the case of people drinking wine manufactured in northern and central Italy; this is not true if a medium drinker constantly consumes red wine produced in southern Italy in this case wine alone could supply the diet with an amount of OTA equal to or even above the tolerable daily intake of 5 ng/kg body weight recommended by the Scientific Committee on Food of the European Commission.     

Ochratoxin A in raisins and currants: basic extraction procedure used in two small marketing surveys of the occurrence and control of the heterogeneity of the toxins in samples.

A basic extraction procedure for analysis of ochratoxin A (OTA) in currants and raisins is described, as well as the occurrence of OTA and a control of heterogeneity of the toxin in samples bought for two small marketing surveys 1999/2000 and 2001/02. Most samples in the surveys were divided into two subsamples that were individually prepared as slurries and analysed separately. The limit of quantification for the method was estimated as 0.1 microg kg(-1) and recoveries of 85, 90 and 115% were achieved in recovery experiments at 10, 5 and 0.1 microg kg(-1), respectively. Of all 118 subsamples analysed in the surveys, 96 (84%) contained ochratoxin A at levels above the quantification level and five samples (4%) contained more than the European Community legislation of 10 microg kg(-1). The OTA concentrations found in the first survey were in the range < 0.1-19.0 microg kg(-1) with a median concentration of 0.9 microg kg(-1). In the 2001/02 study, the range was < 0.1-34.6 microg kg(-1) with a median of 0.2 microg kg(-1). Big differences were often achieved between individual subsamples of the original sample, which indicate a wide heterogeneous distribution of the toxin. Data from the repeatability test as well as recovery experiments from the same slurries showed that preparation of slurries as described here seemed to give a homogeneous and representative sample. The extraction with the basic sodium bicarbonate-methanol mixture used in the surveys gave similar or somewhat higher OTA values on some samples tested in a comparison with a weak phosphoric acid water-methanol extraction mixture.     

Immunoaffinity column clean-up and thin layer chromatography for determination of ochratoxin A in green coffee.

An immunoaffinity clean-up-based method for determining ochratoxin A (OTA) in green coffee aiming at one-dimensional thin layer chromatography (TLC) analysis was established. OTA was extracted with a mixture of methanol and aqueous sodium hydrogen carbonate solution, purified through an immunoaffinity column, separated on normal or reversed-phase (RP) TLC plates and detected and quantified by visual and densitometric analysis. The linear equation of the standard calibration curve by densitometric analysis gave R(2) > 0.999 (0.04-84 ng). The mean recovery (R) of OTA from spiked samples (1.8-109 microg kg(-1)) by densitometric and visual analyses were 98.4 and 103.8%, respectively. The relative standard deviations (RSD) for densitometric and visual analysis varied from 1.1 to 24.9% and from 0.0 to 18.8%, respectively. The RSD for naturally contaminated samples by densitometry (three levels of contamination, n = 3) varied from 11.1 to 18.1%. The correlation (R(2)) between high-performance liquid chromatography (HPLC) and densitometry, and between visual and densitometric analysis for spiked samples were > 0.99. The limit of detection (LOD) of the method was 0.5 microg kg(-1) for normal TLC. Toluene-ethyl acetate-88% formic acid (6:3:1 v/v/v) and acetonitrile-methanol-water-glacial acetic acid (35:35:29:10 v/v/v/v) were regarded as the suitable TLC solvents for eluting both standards and samples on normal and RP TLC plates, respectively. Toluene-acetic acid (99:1 v/v) was chosen as the spotting solvent among several others for giving the best sensitivity and resolution of OTA on TLC plates as well as the best recovery of OTA from standard and sample extract residues. Preliminary studies were carried out to investigate the reuse of the immunoaffinity column and the interference of caffeine in the OTA recovery.     

FTIR-ATR infrared spectroscopy for the detection of ochratoxin A in dried vine fruit

A method of screening sultanas for ochratoxin A (OTA) contamination, using mid-infrared spectroscopy/Golden Gate single-reflection ATR (attenuated total reflection), is described. The main spectral characteristics of sultanas from different sources were identified in a preliminary acquisition and spectral analysis study. Principal component analysis (PCA) showed that samples of various origins had different spectral characteristics, especially in water content and the fingerprint region. A lack of reproducibility was observed in the spectra acquired on different days. However, spectral repeatability was greatly improved when water activity of the sample was set at 0.62. A calibration curve of OTA was constructed in the range 10-40 µg OTA kg^-1. Samples with OTA levels higher than 20 µg kg^-1 were separated from samples contaminated with a lower concentration (10 µg OTA kg^-1) and from uncontaminated samples. The reported methodology is a reliable and simple technique for screening dried vine fruit for OTA.     

Contribution to the study of ochratoxin A in Spanish wines.

With the aim of assessing ochratoxin A (OTA) contamination in wines from a Spanish northern region and the influence of harvest conditions, the following samples were analysed: 40 wines (28 red and 12 white) obtained from grapes cultivated in three different places of the northern Spanish region of Navarra, but in different years, 20 samples in 1997 and 20 in 1998. Wine samples were provided by a viticultural experimental station with very consistent and controlled cultural and enological practices. A high-performance liquid chromatography (HPLC) method with fluorescence detection and immunoaffinity columns was employed (LOD = 0.05 ng ml(-1); method recovery = 101%). Eighty five per cent of the samples from 1997 showed OTA levels >0.05 ng ml(-1) (range 0.056-0.316 ng ml(-1)) and 15% of the samples from 1998 showed OTA levels above the LOD (range 0.074-0.193 ng ml(-1)). Averages detected in 1997 positive samples were 0.185 ng OTA ml(-1) wine (SD = 0.023) in white wine samples (n = 6) and 0.160 ng ml(-1) (SD = 0.119) in the red wine samples (n = 11). These differences between contamination by OTA in the samples from the two different years were attributed to the different quality of the grapes, due to the bad climate in 1997. The possibility of the loss of the mycotoxin was excluded by the analysis of OTA in contaminated wine during 12 months. This study showed that OTA is stable in wine for at least 1 year.     

Determination of ochratoxin A in pig liver-derived patés by high-performance liquid chromatography.

A solid phase extraction procedure followed by HPLC analysis with fluorescence detection has been developed for ochratoxin A (OTA) in patés derived from pig liver. After a previous extraction of OTA with 60% acetonitrile, all the samples were purified through C8 columns. The percentage recovery was 85.7% and the lower limits calculated for accurate detection and quantitation were 0.56 ng/g and 0.84 ng/g respectively. The HPLC procedure showed a good linearity in the interval of equivalent OTA concentrations of 0.84 to 3 ng/g. Values <10% were obtained for precision in HPLC determinations performed (n = 3) and (n = 9). Stability of calibration standards and samples during the analytical procedure was also demonstrated. This method was successfully applied to 38 pig-derived patés and three samples were found to be positive with OTA levels above the detection limit. The highest concentration (1.77 ng/g) has been found in a home-made paté.     

Survey of Romanian slaughtered pigs for the occurrence of mycotoxins ochratoxins A and B, and zearalenone

Blood serum, kidney, liver and muscle sample per animal were collected from slaughtered pigs (n = 52). The samples were analysed for ochratoxin A (OTA) and B (OTB) by HPLC methods. Zearalenone (ZEA) in serum was analysed by enzyme immunoassay. A total of 98% serum samples were OTA positive in the range of 0.05-13.4 ng/ml and 85% contained under 5 ng OTA/ml. The incidences of OTA in kidney and liver were very similar (79%, 75%) with mean levels of 0.54 ng/g and 0.16 ng/g, respectively. The lowest incidence (17%) and the lowest mean level contamination (0.15 ng/g) were in muscle samples. The mean distribution in tissues followed the pattern serum > kidney > liver > muscle (100%: 26%: 8.5%: 2.57%). No kidney, liver or muscle sample was found OTA positive above the maximum admitted limit in Romania (5 ng/g). No sample was found to be positive for OTB. A very similar OTA contamination (mean = 4.19 ng/ml, coefficient of variation = 34.4%) was observed in the serum samples (n = 10) collected from the same farm. A possible difference in regional distribution of OTA in Romania is suggested. Zearalenone was detected only in 17.3% of the serum samples with a maximum concentration of 0.96 ng/ml. This study shows the presence of OTA and ZEA in Romanian slaughtered pigs at levels comparable to those reported in other countries.     

Ochratoxin A in wine and grape juice sold in Canada.

Ochratoxin A (OTA) was determined in 251 samples of wines and grape juice collected over 3 years in Canada. In total, 25/84 samples of red wine, 22/96 samples of white wine, 3/46 red grape juices and 1/25 white grape juices contained OTA levels above the limit of quantitation (LOQ). Canadian wines, when compared with imported products, showed both a lower OTA occurrence, noted as positive (19 versus 48% above the limit of detection (LOD) for wines), and a lower level of OTA contamination (upper bound mean of 17.5 versus 163pg ml(-1) for wines). Wines from the USA contained no quantifiable levels of ochratoxin A. OTA was found in Canadian and US grape juice samples, with 12.9% above the LOD and an upper bound mean of 13.3pg ml(-1). It was extracted from a wine or grape juice sample by passing it through an immunoaffinity column. The sample matrix was washed off the column with water. OTA was eluted from the column with methanol and quantitatively determined by liquid chromatography using a fluorescence detector. The presence of OTA was confirmed by esterification with boron trifluoride-methanol. The LOQ of OTA was estimated as 20 pg ml(-1) in white wine (S/N 10:1) and 40 pg ml(-1) in red wine, white grape juice and red grape juice (S/N 20.1). The LOD was estimated as 4pgml(-1) for white wine and 8pgml(-1) for red wine and white and red grape juices (S/N 3:1).     

Comparative analysis of zinc status, food products' frequency intake and food habits of 11-year-old healthy children.

Children are particularly vulnerable to zinc (Zn) deficiency during periods of rapid growth and development such as infancy and adolescence. The aim was to find the relationship between food frequency, intake, food habits and zinc status in 11-year-old healthy children from southern Poland. The study group comprised children (n = 157) in the age range 11.0 +/- 0.4 years. The level of Zn in serum, erythrocytes and hair samples was measured using atomic absorption spectrophotometry. The parents of children examined completed a special food frequency questionnaire. The Zn concentration in hair (boys 182.98 +/- 65.63 microg x g(-1), n = 78; girls 203.82 +/- 39.80 microg x g(-1), n = 79; p = 0.0171), erythrocytes (8.60 +/- 2.76 mg x l(-1), n = 50) and blood serum (0.79 +/- 0.15 mg x l(-1)) correlated significantly (p < 0.05) with frequency intake of different products (hair: meat, rolls, fruit juices without additives, brawn, pate, barley, black pudding, fish canned, chips, margarine used for cooking, bacon; erythrocytes: fruits, matured cheese, dishes of meal, white cottage cheese, fruit juices without additives, cakes and cakes with cream, margarine used for bread spread; blood serum: bread, fruits, milk, kefir, yoghurt). The relationship of the Zn amount in food products, food frequency intake and the concentration in different healthy children tissues is influenced by many internal and external factors.     

Occurrence of ochratoxigenic fungi and ochratoxin A in grapes from a Tunisian vineyard

The occurrence of ochratoxin A (OTA) and the identification of the ochratoxigenic microbiota in Tunisian grapes were studied for the first time. Black aspergilli were the dominant genus among the filamentous fungi isolated from grapes and were the only potential OTA-producing fungi found. The most abundant species were member of Aspergillus niger aggregate (63%) and Aspergillus carbonarius (36%). Uniseriate aspergilli were rarely present (1%). Of the A. carbonarius isolates, 97% were OTA positive but only 3% of the A. niger aggregate isolates were OTA positive. During grape maturation, the frequency of black aspergilli increased due to increase of the numbers of A. carbonarius. Musts (n=24) obtained from grapes collected at the different sampling times were analyzed for their OTA content. Up to 37% of the musts contained OTA at levels varying between 0.59 and 2.57 microg/l. The amounts of OTA in musts increased as grapes matured. These results indicate that A. carbonarius is the main cause of OTA contamination of Tunisian grapes.     

Deoxynivalenol and ochratoxin A in German wheat and changes of level in relation to storage parameters

The occurrence of the mycotoxins deoxynivalenol (DON) and ochratoxin A (OTA) in the winter wheat of 1997 and 1998 grown under organic farming conditions was investigated using ELISAs (R-Biopharm®) for quantification. The influence of delayed drying of the grain after harvest on the development of DON and OTA was determined in storage trials (moisture: 17% and 20%; temperature: 20°C; duration: four and six weeks). The Tox5 PCR assay was used both to detect Fusarium species with the potential to produce trichothecenes and as a measure of their relative DNA content during the storage trials. The intensity of the PCR signals was correlated with the DON concentration. Fusarium species were identified microscopically by standard methods. All the freshly harvested grain samples were contaminated with DON and showed further increases in the DON concentration during storage. OTA contamination was found in 14.3% of the 1997 samples and in 24.1% of the 1998 samples. OTA increased during storage trials of the 1997 samples but not in the 1998 samples.     

Evaluation of aluminium concentrations in samples of chocolate and beverages by electrothermal atomic absorption spectrometry

Samples of chocolate, cocoa, tea infusions, soft drinks and fruit juice have been examined by, electrothermal atomic absorption spectrometry (ETA-AAS) for the presence of aluminium (Al). Fruit juices and chocolate were analysed after an adequate sample preparation; the other products were evaluated directly. Sampling was performed in duplicate for 248 independent samples. The mean Al concentration in chocolate was 9.2 +/- 7.5 mg kg(-1), and individual values were correlated with the per cent of cocoa in samples (Y = 0.63 + 0.27X, r = 0.78, p < 0.0001). Al concentration in commercial tea infusions ranged from 0.9 to 3.3 mg l(-1) (mean = 1.80 +/- 65 mg l(-1), whereas in laboratory-prepared samples it was 2.7 +/- 0.93 mg l(-1). In soft drinks, the concentrations of Al were lower, ranging from 9.1 to 179 microg l(-1); the highest values were observed in samples of orange squash (mean = 114 +/- 56 microg l(-1)). Apricot juice showed the highest Al level (mean = 602 +/- 190 microg l(-1)), being statistically, different from that of pear (mean = 259 +/- 102 microg l(-1)), but not different from that of peach juice (mean = 486 +/- 269 microg kg(-1)). Toxicologically, the amount of Al deriving from the consumption of these products is far below the acceptable daily intake of 1 mg kg(-1) body weight indicated by the FAO/WHO, and it is a verv low percentage of the normal Al dietary intake.