Joint IDF-IUPAC-IAEA(FAO) interlaboratory validation for determining aflatoxin M1 in milk by using immunoaffinity clean-up before thin-layer chromatography.

A collaborative study was conducted under the auspices of the International Dairy Federation (IDF), the International Union of Pure and Applied Chemistry (IUPAC) and the International Atomic Energy Agency (IAEA), a collaborative Food and Agricultural Organization (FAO) body fully to validate a method combining immunoaffinity clean-up to thin-layer chromatography for the determination of aflatoxin M(1) in milk. Work was done in order to afford those laboratories not equipped with high-performance liquid chromatography, mainly from developing countries, with a simplified but fully validated method as an alternative to the European validated immunoaffinity-high performance liquid chromatography method published as an EN ISO Standard 14501, February 1999. The validation study was carried out on samples of aflatoxin M(1)-contaminated milk and milk powder at levels close to the tolerable level of 0.5 microg l(-1) as recommended by the Codex Alimentarius and to the regulatory level of 0.05 microg l(-1) as laid down by the European Commission. Fourteen laboratories representing 11 countries participated in the trial. The relative standard deviations for repeatability and reproducibility based on raw data were in the range 27-48 and 35-54%, respectively. The recovery rate varied from 32 to 120%. The mishandling of two crucial steps of the protocol such as matrix sample reconstitution and extract evaporation could explain the wide variation of the recovery rate. For this reason, data were then corrected for recovery. Consequently, the relative standard deviations for repeatability and reproducibility were recalculated after correction for recovery and were in the range 26-54 and 34-53%, respectively. The method will be published as a standard ISO/DIS 14674--IDF 190.

New data on the occurrence of ochratoxin A in human sera from patients affected or not by renal diseases in Tunisia.

Ochratoxin A is often found in the sera of people exposed to this mycotoxin in their food (cereals such as barley, coffee, wines, fruit juices, spices, products of animal origin such as pig and poultry offal). Ochratoxin A is suspected of playing a role in the Balkan Endemic Nephropathy, a nephropathology described in Balkan areas where ochratoxin A is often found in cereals and in pork-derived products. In North Africa like Tunisia where high incidence of chronic interstitial nephropathies of unknown aetiology are pointed out, the involvement of ochratoxin A was suspected but contradictory studies on the degree of human exposure did not succeed in evidencing the role of ochratoxin A. In the present work, sera from 47 volunteers hospitalised for nephropathic damages including bladder tumours (21 people), and from 62 patients hospitalised for disorders other than nephropathic ones, were analysed for ochratoxin A contents. The determination of ochratoxin A in sera was done by a validated immunoaffinity-HPLC method. Sera from unaffected population exhibited percentages of 74.2%, 22.6% and 3.2% containing ranges of ochratoxin A as <0.10-0.5 microg/l, 0.51-1.0 microg/l and above 1.0 microg/l respectively. For patients affected with renal diseases, percentages were 59.5%, 25.5% and 14.9% on the same ranges of ochratoxin A levels respectively. The average ochratoxin A concentration for patients with urinary tract disease excluding cancer patients was 0.99+/-1.28 microg/l while that for the non-nephropathic patients was 0.53+/-1.00 microg/l. However the average levels in the cancer patients was only 0.26+/-0.20 microg/l. Those results are in line with most of previously published works and did not confirm very high ochratoxin A contents found in other reports from same regions.

Proficiency testing for the evaluation of the ability of European Union-National Reference laboratories to determine aflatoxin M1 in milk at levels corresponding to the new European Union legislation.

In 1992, the European Union set up a network of National Reference Laboratories and charged the Community Reference Laboratory with the responsibility to design a proficiency testing scheme for assessing the analytical ability of laboratories involved in the official control of aflatoxin M1 in milk. Since 1996, two exercises of proficiency testing have been performed on samples of milk powder and liquid milk at various levels of aflatoxin M1 contents. The trials were conducted according to ISO Guide 43, in particular for the homogeneity testing of sample batches and for the calculation of laboratory z-scores. The National Reference Laboratories officially designated by their governments participated in this programme. Samples were naturally-contaminated milk obtained by feeding cows with aflatoxin B1-contaminated feed. The levels of aflatoxin M1 in the samples ranged from 0.2 to 0.7 microg/kg in milk powder and from 0.05 to 0.07 microg/l in liquid milk. These levels were chosen as being close to the European Union-regulated limit of 0.05 microg of aflatoxin M1 per litre. The results produced by laboratories were compiled and statistically analysed to detect any outlying results and to calculate the individual z-scores. Except for one laboratory in each exercise, all laboratories exhibited acceptable or questionable z-scores. The interlaboratory relative standard deviation for reproducibility (RSDR) obtained for both 1996 and 1998 exercises were in the range 15.7-30.3%. Compared with other published studies, this indicates a very good precision for the performance of this laboratory network in the analysis of traces of aflatoxin M1 in milk.

Use of immunoaffinity columns for clean-up of diarrhetic toxins (okadaic acid and dinophysistoxins) extracts from shellfish prior to their analysis by HPLC/fluorimetry.

Diarrhetic Shellfish Poisoning (DSP) is a severe gastro-intestinal disease caused by consumption of seafood contaminated by microalgal toxins, mainly okadaic acid (OA) and structurally related toxins, dinophysistoxins (DTXs). Regulatory monitoring is generally based on rodent bioassays which, however, present some technical and ethical disadvantages. The most promising technique of analysis of these toxins involves an HPLC separation with spectrofluorimetric detection after derivatization of the toxins with a fluorescent reagent. The lack of specificity of the extraction procedure (liquid-liquid partition), and the presence of interfering compounds in the matrix, does not allow the determination and the quantification of low amounts of toxins in seafood. In this paper, the authors report the development and the characterization of immunoaffinity columns (IAC), which were elaborated using anti-okadaic acid monoclonal antibodies, for a specific retention of the OA group of toxins. The coupling yield and the stability of these columns were investigated as well as their capacity to remove interfering compounds. Cross-reactivity was observed between the antibodies and the DTX-1 and the DTX-2, allowing the detection of the different toxins in a single analysis. Different spiked (1 microgram OA/g) or naturally-contaminated (mussel digestive gland: 2 micrograms OA/g; algae: 165 micrograms OA/g) matrices were tested. The recovery for OA varied from 55 to 95% according to the matrices. The IAC purification was then included as a step of a global [IAC/HPLC/spectrofluorimetric detection] method and the performance of the method was evaluated. Estimations of the linearity and the accuracy (percentages of the presumptive response for OA in the range +101% to +114%) were satisfactory in accordance with the method validation criteria.

A French monitoring programme for determining ochratoxin A occurrence in pig kidneys.

Ochratoxin A is a carcinogen and nephrotoxin which can enter the food chain resulting in human exposure. As pig herds are exposed to ochratoxin A through their feed, their kidneys, livers and pork meat are considered as a possible route of exposure for humans. France, an important producer of pork and pork products, set up a national monitoring programme which included the training of six routine public laboratories in the analysis of ochratoxin A using an immunoaffinity step followed by a HPLC-fluorimetric detection. The programme randomly sampled 300 healthy and 100 nephropathic pig kidneys in 1997 and 710 healthy pig kidneys in 1998. Less than 10% of samples were significantly contaminated by ochratoxin A : in the 1997 survey, 1% of samples contained 0.40-1.40 microg kg(-1) of ochratoxin A and in the 1998 survey 7.6 % exhibited ochratoxin A levels in the range 0.5-5 microg kg(-1). In the case of nephropathic kidneys, only traces of ochratoxin A (0.16 to 0.48 microg kg(-1)) were detected in six samples out of 100. Even if not a major route of exposure for humans, pigs are clearly exposed to this mycotoxin and monitoring of pork products and of feed for swine is necessary.

Diarrhoeal toxin production at low temperature by selected strains of Bacillus cereus.

The growth of four Bacillus cereus strains producing diarrhoeal toxin at 32 degrees C (F4433/73 and 29.155, isolated on the occasion of foodborne outbreaks, and F4581/76L and F4581/76R, two variants of a clinical strain), a weakly toxigenic strain isolated in routine analysis of food (3505M) and an emetic isolate (F3502/73) was investigated at low temperature. Biomass was determined by protein assay. Generation times were: for strain F3502/73, which grew at > or = 12 degrees C, 8.71 h (at 12 degrees C); for other strains, which grew at > or = 10 degrees C, 10.2 to approximately 18.9 h (at 10 degrees C). Toxin production during growth was evaluated by a commercial kit (Oxoid) and by a toxicity test on Chinese hamster ovary cells. Strains F4433/73 and F4581/76, secreting high levels of diarrhoeal toxin during the exponential phase at 32 degrees C, produced high levels of toxicity at 10 degrees C until the stationary phase. Strain 29.155 had decreased toxin production at 10 degrees C. Toxicities for cellular extracts remained low when compared with culture filtrates. A correlation was found between the toxicity values given by the two detection methods tested, and the suitability of both methods for the detection of potential poisoning isolates is discussed.

Production of diarrheal toxin by selected strains of Bacillus cereus.

The production of diarrheal toxin by six selected strains of Bacillus cereus was monitored during growth at 32 degrees C, a temperature described as near-optimal for growth and toxin production. Toxic activity was measured in culture filtrates and cellular extracts sampled at three different times during growth. Two alternative methods, a cytotoxicity test on Chinese hamster ovary (CHO) cells and a commercial immunological test (BCET-RPLA, Oxoid) were used. Toxin titres were in agreement with epidemiological characteristics and toxicity demonstrated by using other systems in other examinations. A comparison of intra- and extracellular toxicities measured at the exponential and stationary growth phases showed that the toxin was essentially secreted during the exponential phase. For several strains, secretion peaked during the period from the middle exponential phase to the beginning of the stationary phase. There was no important overall increase of the toxicity during full and late stationary phase. The level was stable or even lower, thus indicating that diarrheal toxin production during stationary phase was small, if any, and that the toxin was unstable under these conditions. Statistical analysis of toxicities showed that the cytotoxicity test was correlated with the immunological test (significant at a 1% level). For routine determinations, a toxicologic laboratory may use any of the two methods, depending on its facilities, the immunological test being relatively expensive.

Application of Immunoaffinity Column Cleanup to Aflatoxin M1 Determination and Survey in Cheese.

Milk products such as cheeses may be contaminated by aflatoxin M1 when manufactured with milk from dairy cattle that have consumed aflatoxin B1-contaminated feeds. The usefulness of immunoaffinity columns to determine aflatoxin M1 content in many kinds of cheeses with very good recoveries is demonstrated. The analysis of aflatoxin M1 in a 1990 to 1995 limited survey in France shows that the occurrence of this mycotoxin in cheeses is rather infrequent. With the exception of samples from 1989 to 1990 when aflatoxin B1-contaminated maize meals were incidentally imported to supplement dairy cattle feed, very few samples were found with above 0.200 µg of aflatoxin M1 per kg of cheese, the maximum acceptable level.

Procedures for destruction of patulin in laboratory wastes.

Patulin is immunosuppressive and there is limited evidence of its carcinogenicity in experimental animals. The International Agency for Research on Cancer (IARC) initiated a programme for the development of degradation techniques for the commonly investigated mycotoxins. As a part of this programme, the following techniques were tested for the degradation of patulin: treatment with ammonia, treatment with ascorbic acid, and treatment with potassium permanganate in acidic or in alkaline conditions. Patulin analysis was performed by using HPLC with UV detection. Mutagenic activity of degradation residues was tested by in Salmonella typhimurium strains TA 97a, TA 98, TA 100, and TA 102. Complete disappearance of patulin was not achieved after 92 h of treatment with ascorbic acid. All the other methods tested led to complete removal of the molecule. However, the technique using potassium permanganate in acidic conditions produced residues which were mutagenic without activation to Salmonella typhimurium strains TA 100 and TA 102, which was attributed later to Mn2+. The two other techniques gave satisfactory results and were selected for further validation studies.

Screening for okadaic acid by immunoassay.

Increasing incidences of phytoplankton blooms with the potential danger of toxin release into the food chain have necessitated the search for new diagnostic methods that can detect toxins quickly and reliably. A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantitate okadaic acid in shellfish and phytoplankton extracts. To determine the specificity of the assay, a number of toxins, such as calyculin A, brevetoxin-1, and dinophysistoxins-1, -2, and -3 were analyzed. Both dinophysistoxins-2 and -1 could be detected by the assay but in concentration ranges 10- and 20-fold higher than that for okadaic acid, respectively. Dinophysistoxin-3, calyculin A, or brevetoxin-1 could not be detected with this assay. To validate the accuracy of the method, 18 mussel and 7 phytoplankton extracts were analyzed in parallel for okadaic acid content by ELISA and liquid chromatography combined with either fluorescence or mass spectrometric detection. Very high correlation between the results was found.

Use of immunoaffinity chromatography as a purification step for the determination of aflatoxin M1 in cheeses.

A method using immunoaffinity as a purification step for the determination of aflatoxin M1 in cheese is described. A simple solvent extraction with dichloromethane followed by a washing step with N-hexane gives a prepurified extract. A comparison between two ways of aflatoxin M1 purification, by solid-phase extraction clean-up and by immunoaffinity, was carried out. The use of immunoaffinity columns containing monoclonal antibodies against aflatoxin M1 gives the best result, i.e. a very clean preparation containing purified aflatoxin M1. The quantification of aflatoxin M1 is then performed by high performance liquid chromatography using fluorometric detection. This method was successfully carried out on naturally-contaminated and spiked cheeses. Recoveries are about 75%. The limit of quantification is 0.020 microgram of aflatoxin M1 per kg of cheese. This method seems suitable for use in monitoring programmes for aflatoxin M1 contamination in dairy products such as cheese.

Cooked mussels contaminated by Dinophysis sp.: a source of okadaic acid.

A method was developed for fractionation and isolation of toxic components present in extracts prepared from Dinophysis-contaminated mussels. The major toxin present in French mussels was identified as okadaic acid by its chromatographic properties and spectral data. Large amounts of mussel tissue (digestive glands and remaining meat) can be treated easily if they are cooked, or cooked and dried and are useful for isolating significant amounts of okadaic acid.

Behavior of 14C aflatoxin M1 during camembert cheese making.

Camembert cheeses are made from raw milk spiked with aflatoxin M1. Three aflatoxin M1 levels (7.5 micrograms/L, 3 micrograms/L, and 0.3 micrograms/L) are used. In curds 35.6, 47.1, and 57.7% of aflatoxin M1, respectively, are recovered, and in wheys 64.4, 52.9, and 42.3%, respectively, are recovered. During the first 15 days of storage, the aflatoxin M1 content of different cheeses decreases 25, 55, and 75%, respectively. A similar experiment is made with milk contaminated with 14C labeled aflatoxin M1. The same results are obtained, except for the behavior of aflatoxin M1 in cheese; the same 14C activity is recovered during storage for 30 days.

Patterns of experimental contamination by Protogonyaulax tamarensis in some French commercial shellfish.

As a result of the proliferation of toxic marine dinoflagellates along European coasts and the recent discovery of paralytic poisons in French shellfish, experimental studies were conducted on four species of shellfish from the Brittany coasts. Contamination rates of a culture of toxic Protogonyaulax tamarensis, were determined for Mytilus edulis, Crassostrea gigas, Pecten maximus and Ruditapes philippinarum. Mussels and scallops were very rapidly contaminated showing high toxin accumulation rates, whereas rates for oysters and clams were low. During the decontamination phase, two stages were observed in mussels and scallops: a fast decrease in toxin, of the same order of magnitude as the accumulation, followed by a slow decrease, with the toxic rate remaining above the quarantine level of 80 micrograms/100 g. Toxin analysis, both in the culture and in the shellfish, was performed using high performance liquid chromatography. GTX3 and GTX8/epiGTX8 were the dominant toxins in the early stage of the decontamination phases, whereas GTX2 was the predominant compound during the slow phase of decontamination.

Effects of ammoniation on the 'carry-over' of aflatoxins into bovine milk.

Two experiments were performed using lactating cows fed various treated and non-treated commodities from AFB1 contaminated peanut cakes. Treatment with ammonia gas by an autoclaving process was used for detoxification. Two methods were used for AFM1 determination in every milk sample: a TLC procedure recognized by AOAC and IDF and an HPLC method with a detection limit of 0.100 and 0.010 microgram/l, respectively. In a first experiment, lactating cows were fed treated and untreated meals during periods separated by uncontaminated soya meals phases. The total excreted AFM1 was 2.6% of the total ingested AFB1 from untreated feed contaminated at 1100 micrograms/kg. During periods receiving treated meals in the diet, AFM1 contents in milk were below 0.1 microgram/l. However, by using AFM1 data obtained using the HPLC method, an AFM1/AFB1 ratio of 4.6% was found from treated feed contaminated at 40 micrograms AFB1/kg. In a second experiment, a herd of 50 lactating cows was used for a long term (16 months) feeding of mixed commodities containing 30% ammoniated peanut cakes. AFB1 residues in the treated diet were below 10 micrograms/kg, the EEC action level, and no AFM1 residue was found up to 0.1 microgram/l in collected milk throughout this experiment.

The 'carry-over' of aflatoxin into milk of cows fed ammoniated rations: use of an HPLC method and a genotoxicity test for determining milk safety.

Aflatoxin B1 (Af.B1) contaminated and detoxified commodities were used to feed lactating cows. The detoxification treatment was performed using an autoclaving process. Af.B1 contents in untreated groundnut meal, treated groundnut meal, and a mixture of treated groundnut meal and soyameal were 475.0, 6.0 and 3.0 micrograms/kg, respectively. Af.M1 determination in milk was performed using an HPLC method with a detection limit of 0.025 micrograms/l. Also, an attempt was made to apply a genotoxicity test, the SOS Chromotest, to check for the presence of genotoxic residues in the various milk samples. The total excreted Af.M1 was 1.08%, 14.7% and 6.35% of the total ingested Af.B1 from untreated, treated and mixed treated feed, respectively. An unusual Af.M1 excretion peak was observed during the first four days of feeding the treated diet. Approximately 30% of the unreacted ingested Af.B1 appeared in the milk as the metabolite Af.M1. On the following days, the amount of Af.M1 in the milk samples decreased quickly and became almost undetectable. However, the genotoxicity test assays did not show any difference between the various experimental milk samples.

Direct enzyme-linked immunosorbent assay for determining aflatoxin M1 at picogram levels in dairy products.

Protocols for detecting picogram quantities of aflatoxin M1 in dairy products were established. Milk samples were subjected to a reverse phase Sep-Pak C18 cartridge treatment before analysis by an enzyme-linked immunosorbent assay (ELISA) according to previously published procedures. M1 in yogurt, brick cheddar, and ripened Brie cheese was extracted by a modified Pons method, subjected to a normal phase silica cartridge treatment, and analyzed by ELISA. The detection limits for M1 in milk, yogurt, cheddar, and Brie were 10, 10, 50, and 25 ppt (ng/kg), respectively. Recovery for M1 added to these products was in the range 70-110%. Good agreement was found for M1 levels in several naturally contaminated milk samples analyzed by both ELISA and liquid chromatography.

Determination of aflatoxin M1 in milk by liquid chromatography with fluorescence detection.

A liquid chromatographic (LC) method is proposed for the determination of aflatoxin M1 in milk. The method was successfully applied to both liquid whole and skim milk and also whole and skim milk powder. The samples are initially extracted with acetonitrile-water followed by purification using a silica gel cartridge and a C18 cartridge. Final analysis by LC was achieved using a radial compression module equipped with a 5 micron C18 column and a fluorescence detector. The method was successfully applied to samples at levels of 10 to 0.08 ppb added aflatoxin M1 with recoveries in the range of 70-98%.