Committee on Natural Toxins

442 GENERAL REFEREE REPORTS: JOURNAL OF AOAC INTERNATIONAL, VOL. 83, NO. 2, 2000 GENERAL REFEREE REPORTS Committee on Natural Toxins GENERAL REFEREE REPORTS: JOURNAL OF AOAC INTERNATIONAL, VOL. 83, NO. 2, 2000 Mycotoxins MARY W. TRUCKSESS U.S. Food and Drug Administration, Division of Natural Products, 200 C St SW, Washington, DC 20204, USA In the past, total diet studies (market basket studies) have been conducted internationally for various contaminants such as heavy metals and pesticides. No such formalized studies have been made for mycotoxins. For the 1999 International Total Diet Conference held in Kansas City, MO, I was invited to speak about mycotoxins in foods. It was brought out that a total diet program for mycotoxins is needed. Many mycotoxins have been found in various foods. The effects of ingestion of combined toxins are largely unknown because most toxicological animal studies have been for individual or groups of toxins. The levels of the administered toxins were usually higher than those found in foods. The results on animals are then interpreted as applied on humans. For toxins such as the fumonisins, the organs affected vary with the test animal species. Animal data should be used as a guide for the quantitative evaluation of risk assessment in the absence of the needed epidemiological data. Total diet survey data to determine exposure levels and animal health effect can be used as an indicator for toxin related problems of epidemiological studies. The toxins and levels of toxins in human diets would be of much interest. 1999 has been an extremely active year in the area of mycotoxin collaborative studies as described below. Methods approved for Final Action: (1) 995.15 Fumonisin B 1,B 2, and B 3 in corn, liquid chromatographic method. (2) 995.10 Patulin in apple juice, liquid chromatographic method. Method approved for First Action: (1) 999.07 Aflatoxin in peanut butter, pistachios, figs, and paprika by immunoaffinity column cleanup, liquid chromatographic method (E. Anklam, Joint Research Centre, Ispra, Italy). These reports of the General Referees were presented at the 113th AOAC INTERNATIONAL Annual Meeting, September 26 30, 1999, Houston, Texas, USA. The recommendations were reviewed by the Committee on Natural Toxins. See the report of the committee, this issue. Collaborative studies completed: (1) Patulin in clear and cloudy apple juices and apple puree, liquid chromatographic method (Susan McDonald, MAFF/Food Safety Directorate, Norwich, United Kingdom). (2) Aflatoxins in almonds and peanuts, reusable immunoaffinity column, liquid chromatographic method (Chuck Bird, Neogen, Lansing, MI). (3) Aflatoxin M 1 in liquid milk by immunoaffinity column, liquid chromatographic method (Sylvaine Dragacci, AFSSA, Cedex, France). (4) Total aflatoxins in corn, whole cottonseed, poultry feed, pet food, and walnuts, ELISA method (Chuck Bird, Neogen). The Associate Referee found results of the study unacceptable. (5) Ochratoxin A in roasted coffee, liquid chromatographic method (Alison Williams, Leatherhead Foods RA, Surrey, UK). (6) Ochratoxin A in barley, liquid chromatographic method (Alison Williams). (7) Method modification of 991.44 Ochratoxin A in corn and barley, liquid chromatographic method. (8) Fumonisins (total) in corn, ELISA method (Larry Rice, U.S. Department of Agriculture, Ames, IA). The study will be repeated. Collaborative study protocols received: (1) Fumonisins (total) in corn, ELISA method (Chuck Bird). (2) Fumonisins in corn and sorghum, by immunoaffinity column, solution fluorimetric method and liquid chromatographic methods (Mary W. Trucksess). (3) Aflatoxin B 1 in animal feed by immunoaffinity column, liquid chromatographic method (Jörg Stroka, Joint Research Centre, Ispra (VA), Italy). (4) Aflatoxin B 1 in baby food by immunoaffinity column, liquid chromatographic method (Jörg Stroka). Selected Associate Referee Topics Sampling and Subsampling Associate Referee Thomas B. Whitaker (U.S. Department of Agriculture, North Carolina State University, Raleigh, NC) reports that several research projects to determine the variability and distributional characteristics associated with sampling agricultural commodities for mycotoxins are continuing. USDA/ARS measured the sampling, sample preparation, and analytical variability associated with testing shelled corn for

GENERAL REFEREE REPORTS: JOURNAL OF AOAC INTERNATIONAL, VOL. 83, NO. 2, 2000 443 fumonisin (1) and developed a method to predict the performance of aflatoxin sampling plans for farmers stock peanuts (2). Studies at Michigan State University measured the variability among probe samples taken from wheat contaminated with deoxynivalenol (3). The information from these studies will be used to design and evaluate the performance of fumonisin sampling plans for shelled corn, deoxynivalenol sampling plans for wheat, and aflatoxin sampling plans for farmers stock peanuts. The CODEX Committee on Food Additives and Contaminants (CCFAC) concerned with the establishment of international aflatoxin limits and sampling plans for raw shelled peanuts traded in the export market, has come to a consensus of a maximum limit of 15 ng/g total aflatoxins and an aflatoxin test procedure developed by an FAO Expert Consultation that uses a single 20 kg test sample. The sampling plan is described in detail in the FAO Nutrition Paper 55 (4). The CODEX Commission met in Rome, Italy, June 27 to July 3, 1998, and accepted the CCFAC recommended maximum limit of 15 total ng/g and the aflatoxin test procedure that uses a single 20 kg sample. There were discussions for the establishment of an expert group to develop implementation procedures for the aflatoxin test procedure outlined in FAO Nutrition Paper 55. The CODEX Commission will consider the recommendations of the expert group at its next meeting in June 2001. In January 1999, the EU announced regulations for aflatoxin limit for both raw (destined for further processing) and consumer-ready products. As an example, for raw shelled peanuts, the EU established a maximum limit of 8 ng/g B 1 and 15 ng/g total aflatoxin. For consumer-ready peanut products, the maximum limit is 2 ng/g B 1 and 4 ng/g total aflatoxin. The sample design is currently under discussion among member nations. Aflatoxin M 1 Associate Referee Hans P. van Egmond (National Institute of Public Health and the Environment, Bilthoven, The Netherlands) reports that the collaborative study on a method for aflatoxin M 1 in milk, involving immunoaffinity column cleanup with HPLC determination, in 1998 under the auspices of the Standards, Measurements and Testing Programme (SMT) of the European Commission, has been completed with a report (5, 6). The method was tested for raw milk at levels ranging from 0.023 0.103 µg/l, and RSD R values found were ranging from 21 to 27%. The collaborative study report is currently being reviewed by AOAC Methods Committee on Natural Toxins, and by the SMT Programme. If the method fulfills European requirements (7), it may be used for official purposes in the EU, where a new Community regulation is in force since January 1999, with a tolerance limit for aflatoxin M 1 in milk set at 0.05 µg/l (8). The EU Community Reference Laboratory for Milk and Milk Products conducted a laboratory proficiency study for aflatoxin M 1 in milk in November 1998. Sixteen laboratories took part in the study that involved test samples of milk contaminated at 0.05 and 0.07 µg aflatoxin M 1 /L. All laboratories used methods based on immunoaffinity cleanup except one, that used ELISA. RSD R values were at 20 and 16% for the 2 studied levels, respectively, indicating good analytical competency for the determination of aflatoxin M 1 at the EU regulation level (9). The results of the ELISA method were comparable to the results obtained with the HPLC methods. Aflatoxin M 1 standard solutions can now be obtained from the Institute for Reference Materials and Measurements (IRMM) in Geel, Belgium. The IRMM supplies aflatoxin M 1 standard solutions in chloroform (concentration, 10 µg/ml) as BCR RM (reference material) 423. A full report describing the preparation of RM 423 is available (10). The National Institute of Public Health and the Environment, Bilthoven, The Netherlands, no longer supplies aflatoxin M 1 standard solutions. A very sensitive method using column-switching LC technique for aflatoxin M 1 in human urine and milk was recently published (11). The biological fluid was diluted, centrifuged, and injected directly into the chromatographic system. A cation-exchange pre-column coupled on-line to a column-switching LC system was used for sample pretreatment and concentration. The entire analysis was performed in 40 min and recoveries were reported at 2.5 ng/l urine and milk. Surveys for aflatoxin M 1 in milk and milk products in several countries have been reported (12 16). In Brazil (12), Italy (15) and Poland (16) the aflatoxin M 1 levels in milk and milk products (corrected for dry matter percentage) were near or below 0.05 µg/kg. Analytical results of samples in Egypt (13) and India (14) were higher. The highest reported aflatoxin M 1 levels were 3.7 µg/l in raw milk in Egypt and 2 4 µg/l in milk and milk products in India. Aflatoxin Methods Associate Referee David M. Wilson (University of Georgia, Coastal Plain Station, Tifton, GA) reports that there has been moderate activity in methods development this past year. There has been more emphasis on chemical methods again and less reliance on immunochemical methodology. Progress in aflatoxin methodology included the following. The collaborative study on molar absorptivities of aflatoxin in acetonitrile, methanol, and toluene acetonitrile (9 + 1) was published and adopted by AOAC INTERNATIONAL as a method modification (17). A cooperative laboratory proficiency testing in corn and peanuts was carried out for laboratories in Thailand (18). The use of laboratory control samples for aflatoxin B 1 determination was found suitable as analyzed by 3 AOAC methods, the CB, BF, and minicolumn methods (19). The use of the black light test in corn was evaluated in Brazil. When compared with TLC values the black light test was not able to reliably indicate lots with possible aflatoxin contamination and gave many false positive results (20). Transmittance of near infra red (NIR) light was used to detect internally moldy peanuts, this technique may be valuable in removal of aflatoxin contaminated nuts (21). A direct competitive ELISA and an indirect competitive ELISA was used in China for screening and quantitative measurement of B 1 (22, 23). An HPLC method for aflatoxins using amperometric detection was developed (24) as well as a

444 GENERAL REFEREE REPORTS: JOURNAL OF AOAC INTERNATIONAL, VOL. 83, NO. 2, 2000 new method for aflatoxins Q 1, P 1, and B 1 using heptakis-di-o-methyl-β-cyclodextrin as a post column reagent (25). Preliminary results on the use of MS/DI (direct inlet system) for confirmation of aflatoxin were published (26). Also, methods were adapted for analysis of aflatoxins in date fruits (27), pistachio and cashew nuts (28, 29), and airborne corn dust (30). A method for the aflatoxin related metabolites, sterigmatocystin and o-methylsterigmatocystin in milk was developed (31). In the UK, an ELISA method for determination of the aflatoxin B 1 albumin adducts was developed (32). The Associate Referee recommends continued study on aflatoxin methods. Alternaria Toxins Associate Referee Michele Solfrizzo (National Research Council, Institute of Toxins and Mycotoxins, Bari, Italy) reports that information on Alternaria mycotoxins research continues to be limited, and few articles have been published during the last year. Sixteen out of 32 commercial samples of apple juice concentrates collected in Spain were found contaminated with alternariol (AOH; 1.35 5.42 ng/ml) and trace levels of alternariol methyl ether (AME; 33). Three strains of A. alternata isolated from indoor air, internal surface of an ventilation duct and cellulosic acousting ceiling tile, respectively were shown to produce high amounts of AOH (159 352 ng/g) and AME (114 377 ng/g) on ceiling tiles at relative humidities of 84 89% and 97% (34). A direct competitive enzyme-linked immunosorbent assay (ELISA) as a postcolumn monitoring system after high performance liquid chromatography (HPLC) was used to analyze fumonisins and tenuazonic acid (TA) in mycelia and spores of A. alternata (AAL) f. sp. lycopersici cultures (35). Underivatized sample extracts were injected into a C 18 reversed phase column eluted with a methanol-water gradient and fractions (0.5 ml each) were collected and then analyzed by ELISA. Fumonisin B 1 and TA were found in the 3 tested A. alternata cultures (grown 16 days on potato dextrose agar) at levels of 0.05 4.8 ng/g and 80 2470 ng/g, respectively. Fumonisin B 2 and B 3 were not detected whereas HPLC fractions eluting at retention time close to TA were positive in the ELISA test indicating that other AAL toxins analogs were present in the sample extracts. Citrinin Associate Referee David Abramson (Agriculture and Agri-Food Canada, Winnipeg, Canada) reports that the first commercial ELISA for citrinin has been introduced. This ELISA kit uses monoclonal antibodies, and can reportedly detect down to 15 ng/g in cereals and feeds in 30 60 min. The Ridascreen Fast Citrinin kit, is manufactured and distributed by R-Biopharm, Darmstadt, Germany. Monoclonal antibodies have been employed in an ELISA and in an immunoaffinity column (IAC) for the detection of citrinin in foodstuffs (36). The authors assayed citrinin in natural colorants derived from Monascus fungi, and in foods containing these substances. ELISA of vegetarian sausages purchased in Germany (8 samples) and of imported Asian sauces (2 samples) indicated 22 105 ng/g citrinin. Two samples of commercial food colorants contained 157 and 2800 ng/g. High incidence of positive results reflected the current use of Monascus cultures as natural food colorants. Monascus fungal species were identified as potent citrinin producers by researchers in France in 1996. Their recent studies with isotopically labelled acetate (37) indicate that citrinin arises from a tetraketide precursor in M. ruber, rather than from a pentaketide as seen in Penicillium and Aspergillus species. In M. ruber, both citrinin and the red pigment compounds are formed from the same tetraketide intermediate. The accumulation of citrinin in wheat has been studied using Penicillium citrinum (38). At 30 C and water activity of 0.810 or higher, citrinin reached maximum level after 40 45 days. Water activities of 0.810 and 0.825 produced peak levels of 65 and 460 ng/g, while water activity of 0.885 resulted in 22000 ng/g of citrinin. P. verrucosum chemotypes produced both ochratoxin A and citrinin. Citrinin had been localized around the outer layers of the spores as determined by chemical and microscopic techniques (39). The high levels of citrinin (8 24% of the spore weight) might function as a protectant against 300 320 nm light, with a consequent improvement in spore survival rate. Cyclopiazonic Acid Associate Referee Joe W. Dorner (U.S. Department of Agriculture, Dawson, GA) reports that there has continued to be interest in cyclopiazonic acid (CPA) with most of the analytical efforts focused on development of ELISA and immunochemical methodology. An improved ELISA for CPA in corn, peanuts, and mixed feed was reported (40). Detection limits for CPA in corn, mixed feed, and peanuts were estimated to be around 100, 300, and 600 ng/g, respectively. Mean recoveries for CPA added to corn, mixed feed, and peanuts were 98, 92, and 93%, respectively. An immunoaffinity column for CPA was prepared by coupling a CPA-specific monoclonal antibody to CNBr-activated sepharose 4B (41). Columns had a binding capacity of 4 µg CPA and could be regenerated at least 10 times. When columns were used to clean up extracts before ELISA analysis, detection limits for CPA in corn, peanuts, and mixed feed improved to 2.0, 4.4, and 4.7 ng/g, respectively. Recoveries of CPA added to corn, peanuts, and mixed feed extracts were 88 105, 86 100, and 90 110%, respectively. Immunoaffinity column cleanup was coupled with LC quantitation for determination of CPA in peanuts (42). Recoveries of CPA from spiked peanuts ranged from 83.7 to 90.8% with a limit of detection of 2.5 ng/g. The method was used to analyze components of farmers stock peanuts that were found to be naturally contaminated with CPA ranging from 3.0 to 8105 ng/g. Feed samples from Portugal were screened for natural occurrence of CPA and aflatoxins. 6.2% were contaminated with CPA (160 µg/kg) and 45.0% with aflatoxin B 1 (1 16 µg/kg; 43). Analysis of 45 strains of Aspergillus flavus isolated from the feed samples showed that 42.2% produced

GENERAL REFEREE REPORTS: JOURNAL OF AOAC INTERNATIONAL, VOL. 83, NO. 2, 2000 445 CPA, 51.1% produced aflatoxin B 1, and 22.2% produced both toxins. The Associate Referee recommends continued study of this toxin. Ergot Alkaloids Associate Referee George M. Ware (U.S. Food and Drug Administration, Atlanta, GA) reports no interaction between ergot alkaloids in endophyte-infected tall fescue seed and pyrrolizidine alkaloids in tansy ragwort when simultaneously fed to sheep (44). A review of separation of chiral compounds by capillary electrophoresis (CE) was reported (45). The review presents the different chiral selectors used CE for the separation of enantiomers. The use of charged cyclodextrins and synthetic micelles to separated ergot alkaloids is discussed in detail. A study was conducted to compare ergot alkaloid excretion via urinary or biliary systems and to determine the rate of appearance or clearance of these alkaloids in cattle that were grazing on ergot infected tall fescue (46). The total ergot alkaloid excretions in urinary and biliary system were quantitated using competitive ELISA. No data were presented on the method limits of detection and quantitation. Experiments were conducted with rabbits to determine the effect of endophyte-infected tall fescue seed on rabbit performance and to examine the effect of anti-ergot alkaloid immunization on rabbit performance and protection against fescue toxicosis (47). The study showed feeding contaminated fescue seed diets reduced rabbit weight gain and food intake. Immunization against ergot alkaloids provided temporary improvement in food intake and weight gains. The researchers believe that rabbits may serve as a model animal for fescue toxicosis research. The Associate Referee for ergot alkaloids recommends that antibodies developed in these studies (46, 47) be evaluated for potential use in a quantitative ELISA method. Fumonisins Associate Referee Larry Rice (U.S. Department of Agriculture, Ames, IA) reports that research interest in the fumonisins continues to remain high as shown by at least 95 referenced articles in the last 12 months alone. The release of the draft of the toxicology and carcinogenesis studies of fumonisin B 1 will only increase this research interest. Surveys of preharvest corn in Georgia (1996 1998) as well as previous surveys in Illinois, Iowa, Missouri, and Nebraska have clearly shown the fumonisins to be at best a low level contaminant of maize and in the southern United States (Texas, Georgia, North Carolina, etc.) to be consistently present at levels known to produce toxicoses in animals. The only reported outbreak of equine leukoencephalomalacia in the last 12 months was in Mexico (48). Most of the recent research work has centered on the role of the fumonisins in apoptosis and cell death (49 54) and modifications of the analytical methods of analysis for the fumonisins and their apparent biomarkers sphinganine and sphingosine (55 58). Fluorescence detection appears to remain the analytical method of choice with growing application of monoclonal antibodies cleanup. William Norred et al. (59) has recently presented a review of the health hazards and proposed mechanism of action of the fumonisins. In view of the continued interest in identification of the fumonisins internationally continued research on the fumonisins is recommended. Ochratoxins Associate Referee Benedicte Hald (Royal Veterinary and Agricultural University, Denmark) reports that at the International Symposium Mycotox 98 held in Toulouse, France, 2 4 July, 1998, papers were presented involving analytical methodology, natural occurrence, risk assessment, regulation, and toxicological properties of ochratoxin A (OA; 60). Other developments of the year were as follows. The reliability and robustness of an immunoaffinity column-based method for quantitative analysis of OA was evaluated in a collaborative study (61). Performance data, such as recovery, reproducibility, and repeatability were determined. Mean recoveries were > 90% at OA concentration of 5 µg/kg. The performance of the column method tested compared very favorably with results of other published collaborative studies for mycotoxins. Confirmation of identity of OA has become especially important as lower levels are being reported as well as natural occurrence in new and different commodities and in human and animal body fluids and tissues. A simplified procedure for the confirmation of OA in biological samples was developed for the esterification of OA, ochratoxin B, and OA-OH. When the sample was incubated for > 12 h with methanol and 6N HCl, the conversion of OA into its corresponding methylester was > 95%. Detection limit for OA was 1 ng/ml (62). A sensitive quantitative and selective method for OA in complex matrixes using LC/electrospray ionization/ms/ms method was reported (63). The minimum detection of OA detected was 20 pg/g. OA was determined in artificially contaminated cocoa beans using automated sample preparation and LC analysis. Recoveries of OA ranged from 87 106% (64). A flow-through membrane-based enzyme immunoassay for rapid detection of OA in wheat with a determination limit of 4 ng/g (65) as well as a rapid procedure for OA in wheat and oats but not applicable to barley, rye, or trout feed (66), was also developed. During the past year, there were many reports of occurrence of OA in agricultural commodities including roasted and soluble coffee (67), coffee beans and cereals (68), pork, poultry, coffee, beer and pulses (69), wheat, barley and coffee (70), beer (71), and wines of different types and grape products (72). Also reported was OA occurrence in human plasma or serum in Japan (73), Canada (74), Spain, (75) and Tuscany, Italy (76). Norwegian milk and infant formulas were analyzed for the occurrence of OA. The levels found were high enough to result in OA intake in small children greater than the allowable tolerable daily intake of 5 µg/kg body weight (77). Continued study of this topic is recommended.

446 GENERAL REFEREE REPORTS: JOURNAL OF AOAC INTERNATIONAL, VOL. 83, NO. 2, 2000 Patulin Associate Referee Myrna Sabino (Instituto Adolfo Lutz, Brazil) reports that a gas chromatographic/mass spectrometric (GC/MS) method was developed for the analysis of underivatized patulin (78). The use of an electronic pressure control, on-column injection and hexachlorobenzene as internal standard avoided the need for derivatization for the analysis of patulin in apple juice. The limit of detection of the method was 4 µg/l. A procedure combining diphasic dialysis extraction with in situ acylation and GC/MS was reported for patulin in apple juice (79). Patulin was derivatized with acetic anhydride and collected in the tubing after diphasic dialysis and was directly determined using GC/MS in the selective-ion monitoring mode without further concentration and cleanup. The limit of quantitation was 10 µg/l. Surveillance studies of patulin in apple juice and fruit juices were reported. In the United Kingdom, 4 out of 300 apple juice laboratory samples analyzed were found to contain patulin at levels > 50 µg/l (80). The highest patulin concentration found in the survey was 171 µg/l. In Brazil 117 processed fruit juices (apple, grape, pineapple, papaya, guava, banana, and mango) and 38 laboratory samples of sound fruits (apple, papaya, mango, pear, and peach) were analyzed for patulin (81). Only one of 30 test samples of apple juice was found to contain patulin, at 17 µg/l. Patulin was not detected in the other juices and fruits. In Turkey 215 apple juice concentrates from 3 different producers were collected, diluted to single strength, and analyzed (82). Of these laboratory samples, 43.5% were found to contain patulin at levels >50µg/L. The Associate Referee recommends continued study on patulin methods. Trichothecenes Associate Referee Robert M. Eppley (U.S. Food and Drug Administration, Washington, DC) reports that it is noteworthy that in the last 2 years, no new trichothecene derivatives have been reported. Most of the publications for the last year have been methodology related (83 93). One report (94) presents an indirect calorimetric technique using the inhibition of a yeast enzyme activity. A biosynthesis study (95) has identified an intermediate in the formation of sambucinol. Several reports of the natural occurrence of the 8-ketotrichothecenes were noted. The visibly moldy areas of sweet corn ears harvested during an unusually wet and cool season were found to contain deoxynivalenol (DON) at mean levels of 445 µg/g (83). A small survey of Korean corn using both GC/MS and HPLC found 5 8-ketotrichothecenes, zearalenone and fumonisins co-occurring in visibly moldy corn, with lower levels of these toxins detectable in the visibly healthy corn (84). The epidemic of Fusarium head blight (FHB) in barley in the upper midwest region of the USA since about 1993, has resulted in several studies. In one report, the development of FHB and the production of DON are correlated with stages of the barley growth and the weather conditions (85). Another study showed a weak correlation between the logarithm of DON levels and percentage of infected kernels (86). GC/MS analyses were used in this determination. DON, 15-acetylDON, 3-acetylDON, and 3,15-diacetylDON were detected in the approximate ratio of 47:4:1:1. In addition, the authors demonstrated good correlation of a commercial ELISA with the GC/MS method. In yet another study, a comparison of GC/EC with an ELISA was made for DON in wheat (87). The correlation was close enough for the authors to recommend the ELISA for sample screening. The remaining reports were related to methodologies for several of the Fusarium mycotoxins. An interlaboratory collaborative study of an LC method for the determination of DON in wheat flour and bran was conducted (88). The method was accepted as a Peer-Verified Method for DON concentrations at 1.0 µg/g or greater. One procedure uses a combination of LC diode array detection and GC ECD for the determination of nivalenol, DON, 3- and 15-acetylDON in wheat flour (89). In another report, 8 trichothecenes are determined by GC/MS using a 2-stage cleanup for various cereals and cereal-based products (90). A direct on-column injection method for determining 7 trichothecenes and zearalenone in extract from barley was reported (91). A report on a cleanup procedure for DON and zearalenone recommended the multi-functional Mycosep SPE column for DON (92). The final report is a review of instrumental methods for the determination of nonmacrocylic trichothecenes in cereals and foodstuffs (93). The Associate Referee recommends that studies for the determination of the naturally occurring trichothecenes be continued. Zearalenone Associate Referee Winston M. Hagler, Jr. (North Carolina State University, College of Agriculture and Life Sciences, Department of Poultry Science, Mycotoxin Laboratory, Raleigh, NC) summarizes some of the research and natural occurrence information published over the past 12 months. An interesting report on in vitro binding of zearalenone using modified montmorillonite clay was published (97). The clay was modified by exchange with several organic pyridinium and ammonium cations. The use of bentonite clays to bind aflatoxin and other mycotoxins is receiving increased research emphasis. A review of grains and feeds contaminated with Fusarium toxins worldwide was published (98). Zearalenone, trichothecenes, and fumonisins were very common and at rather high levels. It was concluded that surveys in the tropics revealed contamination with several Fusarium mycotoxins co-occurring with aflatoxins. This type of co-contamination is also common in the southeastern United States and its importance is probably under-appreciated. A novel method for zearalenone determination by LC/MS using an atmospheric-pressure chemical ionization interface was developed (99). After solid phase extraction or immunoaffinity cleanup, the detection limit was 120 ng/kg. A new ELISA method for detection of zearalenone using antibodies isolated from egg yolks of immunized hens was devel-

GENERAL REFEREE REPORTS: JOURNAL OF AOAC INTERNATIONAL, VOL. 83, NO. 2, 2000 447 oped (100). A titer of 1:76 000 was obtained; zearalenone was detected at concentrations as low as 10 ng/ml. The Associate Referee recommends continued development and refinement of methods, natural occurrence surveys, and further research on biological activity of zearalenone. Recommendations (1) Investigate total diet study for mycotoxins and commodities as suggested by the Food Safety Programme of the World Health Organization. (2) Conduct combined mycotoxin toxicological studies. (3) Conduct a collaborative study of an LC method for ergot alkaloid in grains. (4) Develop thin-layer chromatographic method for fumonisins. (5) Conduct a collaborative study of a LC method for deoxynivalenol in wheat, corn, and barley. (6) Develop isolation procedure of toxins from ELISA device and confirmation of identity procedure of the toxins. (7) Continue study on all Associate Referee topics. 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GENERAL REFEREE REPORTS: JOURNAL OF AOAC INTERNATIONAL, VOL. 83, NO. 2, 2000 449 Phycotoxins MICHAEL A. QUILLIAM Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford St, Halifax, Nova Scotia B3H 3Z1, Canada Summary Since the last general referee report (1), several review articles have been published. Yasumoto described bioactive compounds from marine microalgae (2) and reviewed fish poisoning due to toxins of microalgal origins in the Pacific (3). Shellfish poisoning problems in the Asia Pacific region were reviewed by Mair (4). An overview of marine food poisoning in Mexico was given by Sierra-Beltran and co-workers (5). Park (6) described design and implementation of seafood safety monitoring programs for aquatic biotoxins. There have been a number of developments in areas not presently covered under Associate Referee topics. Aune et al. (7) reported that zinc accumulation in Norwegian oysters resulted in mouse deaths in paralytic shellfish poisoning bioassays, which confirmed an earlier report in 1989 that reported the problem of zinc giving false positives in this assay (8). A number of studies have recently been conducted to determine the extent of thermal degradation of PSP and ASP toxins in shellfish during cooking and canning processes (9 12). Three new yessotoxin analogs have been reported: 45-hydroxyyessotoxin from mussels of the Adriatic Sea (13); 1-desulfoyessotoxin from mussels from Norway (14); and adriatoxin from mussels of the Adriatic Sea (15). Analytical studies of yessotoxins using LC and LC/MS have also been reported (16, 17). Advances in the analytical chemistry of specific toxin categories and the progress of collaborative studies are presented in the following Associate Referee reports. Selected Associate Referee Topics Amnesic Shellfish Poisoning Toxins Associate Referee Michael A. Quilliam (Institute for Marine Biosciences, National Research Council, Halifax, NS, Canada). A semi-quantitative thin layer chromatography (TLC) method for the detection of domoic acid in shellfish has been published (18). This method should prove useful for those laboratories not equipped with instrumentation. Further examination of capillary electrophoresis as a method for domoic acid has been published by Gago-Martinez and co-workers (19). Enzyme-linked immunosorbent assay (ELISA) methods for domoic acid have been developed by Garthwaite et al. (20) and Kawatsu et al. (21). These look very promising for rapid assay of shellfish samples if they can be commercialized. Domoic acid was proved to be the causative agent in the mass mortality of California Sea Lions in 1998 using various analyses, including LC, LC/MS and a glutamate receptor assay (22, 23). The earlier planned collaborative study on the cleanup and LC method for domoic acid could not be conducted in 1999, but it is expected that funding will be made available through the Asian-Pacific Economic Cooperation to move this ahead in 2000. Anatoxins Associate Referee Kevin James (Dept. Chemistry, Cork RTC, Bishopstown, Cork, Ireland). Cyanobacteria produce a number of toxins of great concern regarding the safety of drinking water supplies (24). The anatoxins are neurotoxins produced by various species of Anabaena. Anatoxin-a is a potent nicotinic agonist that has been dramatically lethal to various animals (25). Recent toxicological studies of anatoxin-a have confirmed that the WHO guideline limit of 1 µg/l in drinking water should provide an adequate margin of safety (26). A methylene analog, homoanatoxin-a, was isolated from Oscillatoria formosa (27). These anatoxins degrade rapidly, especially in sunlight and at elevated ph, to produce non-toxic, dihydro- and epoxydegradation products (28, 29). A number of chromatographic methods are available for the analysis of anatoxin-a in cyanobacterial bloom materials, these include TLC (30, 31) and LC with ultra-violet (LC UV) (32,33) or mass spectrometric (LC MS) detection (34). To improve both the chromatography and limit of detection, derivatization of analytes, followed by gas chromatography with electron capture (GC EC) (35) or mass spectrometric (GC/MS) detection (36, 37), has been used to determine anatoxin-a. One study compares the techniques of LC, GC/MS and capillary electrophoresis (38). A sensitive isocratic fluorimetric LC method has been developed and was applied to the determination of 6 anatoxins, anatoxin-a, homoanatoxin-a, and their dihydro- and epoxy-analogs, in reservoirs and in cyanobacteria (39 41). This method can be applied to the routine monitoring of water supplies as well as for the forensic investigation of toxic incidents. Anatoxin-a(s) is an unusual neurotoxin since it is a naturally occurring organophosphate. Anatoxin-a(s) is unrelated, both toxicologically and chemically, to anatoxin-a and is an irreversible inhibitor of acetyl cholinesterase (42). Detection of anatoxin-a(s) in water and algae has been possible using assays that exploit its anti-cholinesterase activity but no sensitive chromatographic method for determining this toxin has yet been published. Bird deaths in Denmark were reported to be due to an anti-cholinesterase toxin (43) and this was confirmed by the production of anatoxin-a(s) by the cyanobacterium Anabaena lemmermannii (44). Clearly there is a need for the development of analytical methods for anatoxin-a(s). Bioassays for Phycotoxins Co-Associate Referees Donald Richard and Edmond Arsenault (Canadian Food Inspection Agency, Biotoxins Unit, Moncton, New Brunswick, Canada). The mouse bioassay is the oldest and most widely used method of detecting and quantitating paralytic shellfish toxins. Developed in the 1930s by Sommer and Meyer (45), the method originally applied a modified LD 50 for estimating toxicity by using serial

450 GENERAL REFEREE REPORTS: JOURNAL OF AOAC INTERNATIONAL, VOL. 83, NO. 2, 2000 dilutions of alcoholic extracts. The method was time-consuming, inaccurate, and lacked precision. The extraction and bioassay procedures were modified by the Department of National Health and Welfare, Ottawa, Canada, and the Fisheries Research Board of Canada (46, 47), and after an AOAC collaborative study in 1957, was granted Official First Action status the following year (48). Although successfully used for the past 40 years, AOAC Official Method 959.08 suffers from several poorly defined parameters in both the extraction and bioassay procedures. It is envisioned that both in-house and outside laboratory evaluations of a substantial number of parameters relating to both the extraction and bioassay procedures will be made, followed by an AOAC INTERNATIONAL collaborative study. As of August 1999, Associate Referees have a) assessed the possibility of using larger mice than the 23 g specified in Method 959.08; b) compared present day false negative results to those reported in the literature and evaluated possible causes; c) reconfirmed the validity of Sommer s table for purified saxitoxin; d) investigated different proficiency criteria which could be used to determine analyst competency, accuracy, and precision; e) investigated the possible reuse of animals; f) investigated the effects of time on the ph of shellfish extracts before boiling as specified in Method 959.08; g)investigated the suitability of using gravimetric as opposed to volumetric measurements during the extraction procedure; h) assessed the suitability of homogenizers versus blenders for tissue preparation, i) measured the effect of extract ph on the bioassay; and j) assessed the implications of using a 2-mouse screen as opposed to the 3-mouse test presently used in most labs. The following research initiatives will be undertaken prior to a full collaborative study: a) the use of male versus female mice and corresponding conversion values; b) effect of acid concentration on extraction efficiency; c) relative standard deviations (RSDs) of death times using extracts at different PSP concentrations; d) confirmation of the mouse weight correction table, especially < 18 g and > 23 g and the RSDs for each weight interval; e) number of shellfish required for a representative sample; f) method of measuring ph; and g) sample integrity during collection, shipping, freezing, cleaning, and processing. This list is by no means all-inclusive. The mouse bioassay is also used extensively for other phycotoxins, especially the lipophilic suite comprised of diarrhetic shellfish toxins (49), ciguatoxins (50), spirolides (51), azaspiracid (52) and gymnodimine (53). Although the principal goal is to initially target the PSP bioassay, mouse bioassay problems involving lipophilic extracts will also be investigated on a collaborative and specific as-required basis. Some of the issues associated with the lipophilic toxin assays are often analogous to the PSP problems, e.g., mouse weight correction factors. Interfering co-extractives such as free fatty acids (54, 55) and zinc (8), which can cause false-positive results in the bioassay, are usually specific to the extraction methodology. Standardized extraction procedures suitable for both biological and analytical methods remain an elusive but primary objective. Cell Bioassays for Phycotoxins Associate Referee Ronald Manger (Fred Hutchinson Cancer Research Center, Seattle, WA; and U.S. Food and Drug Administration, Bothell, WA). In the past year 3 additional laboratories began performing the cell-based assay for detection of sodium-channel active marine toxins. These laboratories include the Department of Biomedical Food Research at the National Institute of Infectious Diseases (Fumiko Kasuga, Tokyo, Japan), the Fishery Industrial Technology Center of the University of Alaska Fairbanks School of Fisheries Center (Brian Himmelbloom, Kodiak), and the California Health Department (Greg Inami, Berkeley). On-site training was provided at the U.S. FDA Seafood Products Research Center (SPRC). Additionally, collaboration between the Fred Hutchinson Cancer Research Center, Seattle Washington, and SPRC will facilitate technology transfer and provide a cell banking resource for target cell lines. This collaboration will establish both master and working cell banks for target cell lines for use by SPRC and collaborators, and provide a common base for future comparative studies. Copies of the current protocol and associated reprints (56, 57) can be obtained from Jim Hungerford at FDA, Bothell, WA (jhungerford@ora.fda.gov). Ciguatoxins, Instrumental Methods Associate Referee Robert W. Dickey (Gulf Coast Seafood Laboratory, U.S. Food and Drug Administration, Dauphin Island, AL) reports no progress in the development of collaborative studies. A recent study presented an LC/MS method for the determination of sub-ppb levels of Pacific and Caribbean ciguatoxins in crude extracts of fish (58). Ciguatoxin in Fish, Solid-Phase Immunobead Assay Co-Associate Referees Douglas L. Park (Dept. Food Science, Louisiana State University, Baton Rouge, LA) and J. Marc Fremy (Agence Française de Sécurité Sanitaire des Aliments, Laboratoire Central d Hygiène Alimentaire, Maisons Alfort, France) have decided to resign from their positions until current work on a new antibody purification and characterization has been completed. Ciguatoxin in Fish, Membrane Immunobead Assay Yoshitsugi Hokama (John A. Burns Sch. Med., University of Hawaii, Honolulu, HI) reports that the study protocol for the membrane immunobead assay (MIA) of ciguatoxin and related polyethers in fish tissue was submitted and approved in May, 1999. The collaborative study was initiated in May with 16 laboratories after the practice run. The study was completed in the latter part of June with 14 of the 16 laboratories submitting their results. The results from the collaborative group were examined by the blind duplicates design with balance or unbalance replicates. The qualitative data was transformed to numerical values with positives = 1, borderline response = 0.5, and negative = 0. The results of this study appear to be promising and are being examined by the Methods