Two-dimensional chromatographic method for determination of aflatoxins in liver; occurrence of aflatoxins in animal liver.

A thin-layer chromatographic method is described for the analysis of aflatoxins in animal liver. Liver samples are extracted with chloroform and phosphoric acid. After filtration, an aliquot is evaporated and defatted by liquid-liquid partitioning. The extract is submitted to silica gel minicolumn cleanup and the final extract is concentrated and submitted to two-dimensional thin-layer chromatography (TLC). The identity of aflatoxins B1 and M1 is confirmed with trifluoroacetic acid (TFA) carried out on the thin-layer plate used for quantitation of these aflatoxins. The method permits the detection and confirmation of aflatoxins in liver in concentrations as low as 0.05 micrograms/kg. Average recoveries for aflatoxin M1 and aflatoxin B1 at spiking levels of 0.2 micrograms/kg were found to be 65% and 85%, respectively. With this method, 73 samples of bovine liver, 70 samples of porcine liver, and 56 samples of chicken liver taken from different slaughterhouses were investigated. In one sample of bovine liver, aflatoxins B1, B2, and M1 could be detected in concentrations of 0.10, 0.03, and 0.08 micrograms/kg, respectively.

Comparison of six methods of analysis for the determination of aflatoxin B1 in feeding stuffs containing citrus pulp.

Six published methods of analysis for the determination of aflatoxin B1 have been compared for their suitability to determine aflatoxin B1 in feeding stuffs containing citrus pulp. These methods are the official European Community (EC) procedure, four procedures proposed in the European Community to replace this method and a new procedure developed by the authors of this article. In all procedures chloroform is used for initial extraction. Various clean-up systems are then applied. For the ultimate separation and detection, use is made of high performance liquid chromatography (HPLC) in three procedures and two-dimensional thin layer chromatography (TLC) in two procedures. One method allows either HPLC or TLC. All experiments were carried out with samples of a batch of feeding stuff containing citrus pulp, artificially contaminated with aflatoxins B1, B2, G1 and G2 at levels of ca 13, 5, 10 and 4 micrograms/kg respectively. Three methods were found to be suitable: a procedure in which gel permeation clean-up and two-dimensional TLC are used; a procedure in which TLC clean-up and reverse phase HPLC with postcolumn derivatization are used: a procedure in which cartridge clean-up and either HPLC or TLC are used. The latter method is preferred because its efficient clean-up yields a very clean extract, allowing the application of various systems of HPLC or TLC. Published recovery data of these three methods for aflatoxin B1 vary from 85-90% at a level of ca 10 micrograms/kg feeding stuff.

Liquid chromatographic determination of aflatoxins in feedstuffs containing citrus pulp.

A liquid chromatographic (LC) method was developed for the determination of aflatoxins in feedstuffs containing citrus pulp. The feed-stuff sample is extracted with chloroform, followed by Sep-Pak Florisil cartridge cleanup and Sep-Pak C18 cartridge cleanup. The final eluate (water-acetone, 85 + 15, v/v) is submitted to reverse-phase liquid chromatography with water-methanol-acetonitrile (130 + 70 + 40, v/v/v) as mobile phase and postcolumn derivatization with iodine. Citrus components are removed from the extract efficiently. The limit of detection for aflatoxin B1 is less than 1 microgram/kg. Other aflatoxins can also be detected and measured. Recoveries of aflatoxins B1, B2, G1, and G2 for dairy rations spiked at 13, 5, 10, and 4 micrograms/kg were 87, 86, 81, and 82%, respectively. Corresponding coefficients of variation were 3.1, 3.6, 5.2, and 3.8%, respectively.

Thin layer chromatographic method for analysis and chemical confirmation of sterigmatocystin in cheese.

A semi-quantitative method is described for the analysis of sterigmatocystin in cheese. The method is based on extraction of cheese with MeOH--4% KCl (9 + 1), followed by Florisil and polyamide column cleanup and 2-dimensional thin layer chromatography (TLC). Visualization of sterigmatocystin on the TLC plates is enhanced by an AlCl3 spray reagent. The identity of sterigmatocystin is confirmed by a 2-dimensional TLC test based on reaction with trifluoroacetic acid (TFA) on the plate after first development. The reaction product formed is visualized by spraying with AlCl3. The method allows detection and confirmation of sterigmatocystin in cheese at concentrations as low as 5 micrograms/kg. The method has been applied to cheese samples ripening in warehouses and naturally molded with Aspergillus versicolor.

Confirmatory test for aflatoxin M1 on a thin layer plate.

The identity of aflatoxin M1 can easily be confirmed directly on a thin layer plate by reacting aflatoxin M1 with trifluoroacetic acid (TFA). This confirmation reaction is carried out on the thin layer plate which has been developed in 2 dimensions and used for the quantitation of aflatoxin M1 in the sample. TFA is superimposed on the separated M1 spot. The plate is kept in the dark 3 min, heated to 75 degrees C for 5 min, and developed with chloroform-methanol-acetic acid-water (92 + 8 + 2 + 0.8). The Rf value of the blue-fluorescent derivative is compared with that for the M1 standard. The method was used successfully on extracts of milk, cheese, and liver. M1 quantities on the plate as low as 0.5 ng can be confirmed by this method. The method is also suitable for simultaneous confirmation of aflatoxin B1.

Differences Between Aspergillus flavus Strains in Growth and Aflatoxin B1 Production in Relation to Water Activity and Temperature.

The optimum and limiting conditions of water activity (aw) and temperature for growth of and aflatoxin B1 production by various Aspergillus flavus strains were determined. Agar media were used in which the aw was adjusted by addition of sucrose or glycerine. Optimum temperatures for aflatoxin B1 production at high aw varied with the strain tested being 13-16, 16-24, or 31 C. Strains with a low temperature optimum for aflatoxin B1 production showed fast growth at 37 C without aflatoxin B1 production. A reduced aw (0.95 and less) together with a moderate or low temperature inhibited toxin production more than growth. However, at a high temperature one strain showed stimulation of aflatoxin B1 production on the glycerine medium at reduced aw No differences were noted between aflatoxinpositive and aflatoxin-negative strains with respect to growth under various conditions.

Patulin Production by Some Fungal Species in Relation to Water Activity and Temperature.

The combined effects of water activity (aw) and temperature on growth and patulin production by strains of Penicillium expansum , Penicillium patulum , and Aspergillus clavatus were determined. Malt agar media were used, in which the aw was adjusted by addition of sucrose or glycerine. The minimum aw values for patulin production by P. expansum , P. patulum , and A. clavatus were 0.99, 0.95, 0.99, respectively. The temperature ranges for patulin production by P. expansum , P. patulum , and A. clavatus were 0-24, 4-31, and 12-24 C, respectively. The optimum temperatures for patulin production by P. expansum and A. clavatus were low compared with those for growth. Optimum temperatures for patulin production at high aw by P. patulum varied with the strain tested and were 8 or 31 C. The temperature range for patulin production in apples by P. expansum was determined. The minimum temperatures for rotting and patulin production were 1 C in Cox Orange cv. and 4 C in Golden Delicious cv. The amount of patulin accumulating in rotten tissue of six apple varieties differed greatly. The invasiveness of and patulin production by various strains of four patulin-producing fungal species were tested. All P. expansum strains tested caused rot containing patulin. The increase of rot and patulin production by P. crustosum and A. clavatus depended on the strains tested. None of the P. patulum strains was able to invade Golden Delicious apples.

Limitations in protection afforded by gloves in laboratory handling of aflatoxins.

The permeability of latex and vinyl laboratory gloves by aflatoxins in chloroform or dimethyl sulfoxide (DMSO) has been investigated. Latex gloves provide good protection against permeation by aflatoxins in DMSO, but aflatoxins in chloroform permeate all types of laboratory gloves. If gloves are contaminated when aflatoxins in chloroform are handled, an immediate change of gloves is recommended.

Ochratoxin A Production by Some Fungal Species in Relation to Water Activity and Temperature.

The effects of water activity (aw) and temperature on growth of and ochratoxin A (OA) production by strains of Aspergillus ochraceus , Penicillium cyclopium , and Penicillium viridicatum were investigated. On agar media in which the aw had been adjusted by addition of sucrose or glycerol, the minimum aw values for OA production by A. ochraceus , P. cyclopium and P. viridicatum lay between 0.83-0.87, 0.87-0.90, and 0.83-0.86, respectively. At 24 C, optimum aw values for OA production by A. ochraceus and P. cyclopium were 0.99 and 0.95-0.99, respectively, whereas that of P. viridicatum varied and was 0.95 and 0.99 for the two strains tested. At optimum aw, the temperature range for OA production by A. ochraceus was 12-37 C, whereas that of P. cyclopium and P. viridicatum was 4-31 C. Optimum temperature for OA production by A. ochraceus was 31 C, whereas that of P. cyclopium and P. viridicatum was 24 C. On Edam cheese of 0.95 aw the minimum temperature for OA production by P. cyclopium was 20 C. On barley meal, P. viridicatum produced maximal quantities of OA at 0.97 aw and could produce OA at temperatures as low as 12 C.

Penicillic Acid Production by Some Fungal Species in Relation to Water Activity and Temperature.

The combined effects of water activity (aw) and temperature on growth of and penicillic acid (PA) production by strains of Penicillium cyclopium , Penicillium martensii , and Aspergillus ochraceus were determined. On malt agar media in which the aw had been adjusted by addition of sucrose or glycerol, the minimum aw for PA production by P. cyclopium and A. ochraceus was 0.97 and that of P. martensii was 0.99. The temperature range for PA production by P. cyclopium and P. martensii was 4-31 C, whereas that of A. ochraceus was 8-31 C. Optimum temperature for PA production by P. cyclopium and A. ochraceus varied with the strain tested and was 24-31 C. The only strain of P. martensii tested showed an optimum temperature of 16-24 C. On Gouda and Tilsiter cheese of 0.96-0.98 aw, temperature ranges for growth of P. cyclopium , a common mold on cheese, were 0-24 and 4-16 C, respectively. When a strain of P. cyclopium known to be able to produce PA in culture media, was grown on Gouda cheese incubated at different temperatures, no PA was detectable in the moldy cheese at the time the average colony diameter was 30 mm. However, in a culture on Gouda cheese incubated for a prolonged time (42 days) at 16 C, PA was detectable. On poultry feed, A. ochraceus produced PA aw as low as 0.88, whereas the minimum aw for PA production by P. cyclopium was 0.97.

EC-collaborative study on the determination of aflatoxin B1 in animal feeding stuffs.

A collaborative study was conducted to test a method, proposed in the European Community (EC) as a candidate-official method for the determination of aflatoxin B1 in compounded feeding stuffs. The study was undertaken on behalf of the European Commission's Community Bureau of Reference (BCR). It involved 25 laboratories from 11 EC countries. The method, based on chloroform extraction and Sep-Pak Florisil and C18 cartridge clean-up, offered either reverse-phase high performance liquid chromatography (HPLC) with iodine post-column derivatization, or two-dimensional thin-layer chromatography (TLC) as determinative steps. In the study, 22 laboratories applied HPLC, three laboratories applied TLC. The study involved six unknown samples. These consisted of blind duplicate samples of compounded feeding stuff, with target concentrations of aflatoxin B1 at less than 2, 8 and and 14 micrograms/kg. Statistical analysis of the HPLC data was carried out according to ISO 5725. For the less than 2 micrograms/kg sample, all reported aflatoxin concentrations were less than 2 micrograms/kg. At the 8 and 14 micrograms/kg level (pooled), repeatability (r) and reproducibility (R), expressed as ratios, were 1.4 and 1.7 respectively, and within-laboratory and between-laboratory coefficients of variation were 11% and 18% respectively. The study revealed that admittance of daylight in the laboratory caused losses of aflatoxin B1 and must be avoided. New glassware coming into contact with aqueous solutions containing aflatoxin B1 was found to be a potential cause of loss of aflatoxin B1. The method has been recommended to the European Commission to be considered for adoption in EC regulations.

Detection of Mold in Food by Enzyme-Linked Immunosorbent Assay.

Evaluation of the enzyme-linked immunosorbent assay (ELISA) for detecting a mold-specific, heat-stable and water-soluble antigen demonstrated the potential of the method for detecting molds in food products. The mold antigen, as produced by Penicillium spp. and Aspergillus spp., was present in all food samples containing aflatoxin B1. The amount of mold antigen present in the test samples was related in each case to the aflatoxin B1 content. Experiments done with samples artificially inoculated with mycotoxin-producing molds revealed that mold contamination could be detected by ELISA at a very early stage. The minimum detectable amount of mold mycelium for three different species of Penicillium was 38 ng/g of sample.

Paralytic shellfish poison reference materials: an intercomparison of methods for the determination of saxitoxin.

Within the framework of the European Commission's Measurements and Testing Programme (BCR) a project has been undertaken to develop shellfish reference materials for Paralytic Shellfish Poisons (PSP). In a preliminary phase of the project, an intercomparison study of methods was undertaken. In this exercise 18 laboratories were asked to analyse solutions of saxitoxin and PSP-containing shellfish extracts with a method of their choice. The study revealed that: all the methods considered (four HPLC methods, one ELISA method) were in principle adequate for the quantification of saxitoxin in solution in the absence of interfering substances (Coefficient of variation (CV) 33% at a concentration of 0.5 microgram/ml); three of the HPLC methods used were able to quantify saxitoxin in PSP-positive mussel extract, the fourth method gave significant overestimation; the CV of all HPLC results was 53% at a mean saxitoxin mass fraction of 2.06 mg/kg mussel meat, the recoveries varied from 59-173%; and the ELISA method grossly overestimated the saxitoxin content in mussel extract, probably due to cross reactions of the antibodies with other PSP. The feasibility of preparing a homogeneous batch of ampouled mussel extracts (CV 3.5% at a saxitoxin concentration of approximately 1.5 mg/kg shellfish), sufficiently stable for at least 4 months storage both at 4 degrees C and approximately 20 degrees C, was demonstrated. The performance of the different methods for the analysis of PSP other than saxitoxin has not yet been evaluated, due to the current lack of PSP standards. Some of the problems observed in the intercomparison study were partly due to the nature of the chromatographic columns used, the composition of the HPLC mobile phase (pH, ion strength), non-optimal conditions for derivatization and matrix interference. Following the outcome of this study, a three year multistage project involving 15-20 European laboratories has been initiated, aimed at improving the accuracy and comparability of PSP measurements as well as preparing reference materials for PSP.

The development of five animal feed reference materials, certified for their aflatoxin B1 content.

The development of three peanut meal and two compound feed reference materials and the certification of their aflatoxin B1 content is described. The materials were prepared and certified within the Measurements and Testing Programme of the Commission of the European Communities as part of a broad activity to improve accuracy and agreement of results of measurements on food and agriculture. RM 262 (peanut meal) was prepared from uncontaminated peanut products. RM 263 and RM 264 (peanut meals) were prepared from naturally contaminated peanuts which were blended with uncontaminated ones, to achieve the desired aflatoxin B1 mass fractions. RM 375 and RM 376 (compound feeds) were made by blending decontaminated dairy feed together with commercial feed ration and contaminated dairy feed with several feed compounds, respectively. Details are given of the preparation and the investigations to verify homogeneity and stability of the materials. The certification exercise was carried out by 17 laboratories using a variety of extraction and clean-up procedures. Most laboratories used liquid chromatography as the determinative step, although operating under a variety of chromatographic conditions. A few laboratories applied thin layer chromatography with densitometric quantification. Peanut meal RM 262 was certified as containing aflatoxin B1 at a mass fraction of < 3 micrograms/kg, RM 263 at 43.3 +/- 2.1 micrograms/kg and RM 264 at 204 +/- 10 micrograms/kg. Compound feed RM 375 was certified as containing aflatoxin B1 at a mass fraction of < 1 micrograms/kg and RM 376 at 9.2 +/- 0.5 micrograms/kg. The materials can be employed either to establish or confirm a calibration curve, or to check the performance of a method.

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. I: Interlaboratory studies of methods of analysis.

This paper describes the first part of a project undertaken to develop mussel reference materials for Paralytic Shellfish Poisoning (PSP) toxins. Two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX) in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the second part of the project: the certification exercise. In the first study, 18 laboratories were asked to measure STX and dc-STX in rehydrated lyophilized mussel material and to identify as many other PSP toxins as possible with a method of their choice. In the second interlaboratory study, 15 laboratories were additionally asked to determine quantitatively STX and dc-STX in rehydrated lyophilized mussel and in a saxitoxin-enriched mussel material. The first study revealed that three out of four post-column derivatization methods and one pre-column derivatization method sufficed in principle to determine STX and dc-STX. Most participants (13 of 18) obtained acceptable calibration curves and recoveries. Saxitoxin was hardly detected in the rehydrated lyophilized mussels and results obtained for dc-STX yielded a CV of 58% at a mass fraction of 1.86 mg/kg. Most participants (14 out of 18) identified gonyautoxin-5 (GTX-5) in a hydrolysed extract provided. The first study led to provisional criteria for linearity, recovery and separation. The second study revealed that 6 out of 15 laboratories were able to meet these criteria. Results obtained for dc-STX yielded a CV of 19% at a mass fraction of 3.49 mg/kg. Results obtained for STX in the saxitoxin-enriched material yielded a CV of 19% at a mass fraction of 0.34 mg/kg. Saxitoxin could not be detected in the PSP-positive material. Hydrolysis was useful to confirm the identity of GTX-5 and provided indicative information about C1 and C2 toxins in the PSP-positive material. The methods used in the second interlaboratory study showed sufficiently consistent analysis results to undertake a certification exercise to assign certified values for STX and dc-STX in lyophilized mussel.

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. II: Certification study.

This paper describes the second part of a project undertaken to develop certified mussel reference materials for paralytic shellfish poisoning toxins. In the first part two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin and decarbamoyl-saxitoxin in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the certification exercise. Fifteen laboratories participated in this certification study and were asked to measure saxitoxin and decarbamoyl-saxitoxin in rehydrated lyophilized mussel material and in a saxitoxin-enriched mussel material. The participants were allowed to use a method of their choice but with an extraction procedure to be strictly followed. The study included extra experiments to verify the detection limits for both saxitoxin and decarbamoyl-saxitoxin. Most participants (13 of 15) were able to meet all the criteria set for the certification study. Results for saxitoxin.2HCl yielded a certified mass fraction of <0.07 mg/kg in the rehydrated lyophilized mussels. Results obtained for decarbamoyl-saxitoxin.2HCl yielded a certified mass fraction of 1.59+/-0.20 mg/kg. The results for saxitoxin.2HCl in enriched blank mussel yielded a certified mass fraction of 0.48 +/- 0.06 mg/kg. These certified reference materials for paralytic shellfish poisoning toxins in lyophilized mussel material are the first available for laboratories to test their method for accuracy and performance.