Development of reference materials for paralytic shellfish poisoning toxins.

A project was undertaken to develop mussel reference materials that were certified for their mass fractions of saxitoxin and decarbamoyl-saxitoxin. Fifteen laboratories from various European countries participated. Three of these had major responsibility for substantial parts of the work and overall coordination of the project. The project involved 4 main activities: (1) procurement and characterization of calibrants; (2) improvement of analytical methodology; (3) preparation of reference materials, including homogeneity and stability studies; (4) 2 interlaboratory studies and a certification exercise. The joint activities resulted in 3 homogeneous and stable reference materials: 2 lyophilized mussel materials with and without naturally incurred paralytic shellfish poisoning (PSP) toxins, and a saxitoxin enrichment solution. The reference materials were certified with respect to their saxitoxin and decarbamoyl-saxitoxin content. The lyophilized mussel material with PSP toxins (CRM 542) contained <0.07 mg saxitoxin x 2HCl/kg and 1.59 +/- 0.20 mg decarbamoyl-saxitoxin x 2HCl/kg. The lyophilized mussel material without PSP toxins (CRM 543) contained <0.07 mg saxitoxin x 2HCl/kg and <0.04 mg decarbamoyl-saxitoxin x 2HCl/kg. The certified value of the saxitoxin mass fraction in the saxitoxin enrichment solution (CRM 663) was 9.8 +/- 1.2 microg/g.

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.

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 effect of commercial processing on the paralytic shellfish poison (PSP) content of naturally-contaminated Acanthocardia tuberculatum L.

A study was undertaken to determine if any reduction in contamination of Acanthocardia tuberculatum L. (Mediterranean cockle) by paralytic shellfish poisons (PSP) could be enhanced by operations carried out during the industrial canning process, allowing contaminated raw material to be commercially marketed in safe conditions for edible purposes. A general decrease in PSP levels was consistently observed when comparing raw materials and their corresponding final products, these dropping to acceptable levels. PSP levels were determined by mouse bioassay and a fluorometric method, and saxitoxin was determined by HPLC. The detoxifying effects averaged over 71.7% and 81.8% (mouse bioassay), 70.6% and 90.9% (fluorometric method), 77.9% and 83.5% (HPLC), for boiling and sterilizing operations respectively. The highest level detected in raw material was 800 micrograms/100 g by mouse bioassay.

Occurrence of Aflatoxin and Aflatoxigenic Molds in Foods and Feed in Spain.

A survey was carried out to obtain data on the occurrence of aflatoxin and aflatoxigenic mold contamination of foods in Spain. A variety of commodities amounting to 338 samples were analyzed, comprising cereal grains, mixed feeds, edible nuts, wheat flour for bread-making, biscuits, sliced bread, soya beans and breakfast cereals. The results reveal a rather low incidence of aflatoxin contamination in samples tested. Aflatoxins were detected in 4 of 27 samples of mixed feeds at levels below 5 µg/kg; one sample of peanuts was contaminated with 120 µg aflatoxin B1/kg and 22 µg aflatoxin B2/kg. Aflatoxins B1 and B2 were also detected in a lot of whole maize flour, averaging 8 µg/kg and 3 µg/kg, respectively. Of a total of 288 samples tested, 100% showed variable incidences of fungal contamination. Maize samples were the ones most frequently contaminated with Aspergillus flavus (54.5%). Strains of A. flavus isolated from maize samples also showed the highest proportion of aflatoxigenic molds (17.2%) compared with those isolated from other sources.