Phycotoxins: Plant Toxins

442 GENERAL REFEREE REPORTS: JOURNAL OF AOAC INTERNATIONAL, VOL. 83, NO. 2, 2000 GENERAL REFEREE REPORTS

GENERAL REFEREE REPORTS: JOURNAL OF AOAC INTERNATIONAL, VOL. 83, NO. 2, 2000

Committee on Natural Toxins 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 B1, B2, and B3 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).

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 M1 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 B1 in animal feed by immunoaffinity column, liquid chromatographic method (Jörg Stroka, Joint Research Centre, Ispra (VA), Italy). (4) Aflatoxin B1 in baby food by immunoaffinity column, liquid chromatographic method (Jörg Stroka). Selected Associate Referee Topics Sampling and Subsampling

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.

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

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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 B1 and 15 ng/g total aflatoxin. For consumer-ready peanut products, the maximum limit is 2 ng/g B1 and 4 ng/g total aflatoxin. The sample design is currently under discussion among member nations. Aflatoxin M1 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 M1 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 RSDR 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 M1 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 M1 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 M1/L. All laboratories used methods based on immunoaffinity cleanup except one,

that used ELISA. RSDR values were at 20 and 16% for the 2 studied levels, respectively, indicating good analytical competency for the determination of aflatoxin M1 at the EU regulation level (9). The results of the ELISA method were comparable to the results obtained with the HPLC methods. Aflatoxin M1 standard solutions can now be obtained from the Institute for Reference Materials and Measurements (IRMM) in Geel, Belgium. The IRMM supplies aflatoxin M1 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 M1 standard solutions. A very sensitive method using column-switching LC technique for aflatoxin M1 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 M1 in milk and milk products in several countries have been reported (12–16). In Brazil (12), Italy (15) and Poland (16) the aflatoxin M1 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 M1 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 B1 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 B1 (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 Q1, P1, and B1 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 B1–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 C18 reversed phase column eluted with a methanol-water gradient and fractions (0.5 ml each) were collected and then analyzed by ELISA. Fumonisin B1 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 B2 and B3 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 B1 (1–16 µg/kg; 43). Analysis of 45 strains of Aspergillus flavus isolated from the feed samples showed that 42.2% produced


CPA, 51.1% produced aflatoxin B1, 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 B1 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 applica-

tion 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.


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 > 50 µg/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-


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. References (1) Whitaker, T.B., Trucksess, M.W., Johansson, A.S., Giesbrecht, F.G., Hagler, Jr, W.M., & Bowman, D.T. (1998) J. AOAC Int. 81, 1162–1168 (2) Whitaker, T.B., Hagler, Jr, W.M., & Giesbrecht, F.G. (1999) J. AOAC Int. 82, 264–270 (3) Hart, L.P., & Schabenberger, O. (1998) Plant Disease 82, 625–630 (4) Food and Agriculture Organization (1993) Sampling Peanuts and Corn for Aflatoxin, Food and Nutrition Paper 55, FAO, Rome, Italy, p. 75 (5) Dragacci, S., & Grosso, F. (1999) European Commission, Directorate-General Science, Research and Development, Luxembourg, EUR Report 18856 EN (6) Dragacci, S., Grosso, F., & Gilbert, J. (in press) J. AOAC Int. (7) Commission of the European Communities (1998) Offic. J. Eur. Communities L 201/93–101 (8) Commission of the European Communities (1998) Offic. J. Eur. Communities L 201/43–46 (9) Grosso, F., Dragacci, S., & Lombart, B. (1999) Agence Française de Securité des Aliments, Paris, France, Report No. AFFSA/TOMI/FG9901 (10) van Egmond, H.P., Boenke, A., Paulsch, W.E., Grosso, F., Dragacci, S., & Pittet, A. (1999) European Commission, Directorate-General Science, Research and Development, Luxembourg, EUR Report 18876 EN (11) Simon, P., Delsaut, P., Lafontaine, M., Morele, Y., & Nicot, T. (1998) J. Chromatogr. B. Biomed. Sci. Appl. 712, 95–104 (12) Correa, B., Galhardo, M., Costa, E.O., & Sabino, M. (1997) Revista de Microbiologia 28, 279–283 (13) Amra, H.A. (1998) Rev. Méd. Vét. 149, 695 (14) Dhand, N.K., Joshi, D.Y., & Jand, S.K. (1998) Ind. J. Dairy Sc. 51, 129–131 (15) Finoli, C., & Vecchio, A. (1997) Microbiol., Alim. Nutri. 15, 47–52

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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 LD50 for estimating toxicity by using serial


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 ([email protected]). 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


Committee. New topics to be considered for future study are: (a) Development of an immunological assay for palytoxin in marine products, including fish; and (b) development of a sensitive assay for analysis of ciguatoxin and related polyethers in human serum. Diarrhetic Shellfish Poisoning Toxins, Assay Methods Associate Referee J. Marc Fremy (Agence Française de Sécurité Sanitaire des Aliments, Laboratoire Central d’Hygiène Alimentaire, Maisons Alfort, France). Hexane soluble derivatives of okadaic acid (OA) and dinophysistoxin-2 (DTX2) were found in clams harvested from the southern coast of Portugal and were implicated in a DSP outbreak. These apolar acyl derivatives, globally known as dinophysistoxin-3 (DTX3), surpassed 50% of the total amount of DSP toxins (59). These findings pose a problem for the current methods used to detect DSP toxins which include a hexane washing step in their protocols. Such methods are able to detect the parent toxins but not the acyl derivatives, which are highly soluble in hexane. An ELISA, commercially available as DSP Check test kit, was tested against LC analysis for detecting DSP toxins in the above-mentioned toxic Portuguese clams. The ELISA was capable of accurately quantitating OA in samples. No problems were observed when using hydrolyzed semi-purified extracts of shellfish in order to detect ester derivatives of OA. A high correlation was found between the 2 methods when appropriate dilutions were performed. This ELISA test kit appeared to be more sensitive, specific and rapid than the LC procedure for detecting DSP toxins in total shellfish meat extracts (60). An application of immunoaffinity cleanup using columns packed with specific antibodies anti-OA, was developed by Puech et al. (61) as a purification step for subsequent LC determination. The monoclonal antibody (mAb) used for gel binding into the column was purchased from Calbiochem. The coupling yield and the stability as well as capacity to remove interfering compounds were satisfactory. The bound antibody showed good reactivity to all three toxins tested (OA, DTX1, and DTX2), thus allowing the determination of multiple toxins in a single analysis. The detection limit for OA in mussel digestive gland by combining immunoaffinity columns and LC can be as low as 100 ng/g. Another assay system that has been investigated is the fluorometric protein phosphatase inhibition assay, which was compared with the mouse bioassay, immunoassay, and LC/MS in a recent study (62). Diarrhetic Shellfish Poisoning Toxins, Instrumental Methods Associate Referee Kevin James (Dept. Chemistry, Cork RTC, Bishopstown, Cork, Ireland) could not provide a report this year. Microcystins Associate Referee Geoffrey A. Codd (Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, United Kingdom) has been appointed Associate Referee for

this newly adopted topic. Understanding of the health hazards presented by this family of over 60 cyclic heptapeptide hepatotoxins produced by cyanobacteria and their occurrence in aquatic environments is increasing apace with progress being advanced by investigations into adverse health outcomes and the development of physico-chemical, enzyme-based, and immunodetection methods. Reviews of current and developing analytical methods are in progress and requirements will be identified in the coming year. Neurotoxic Shellfish Poisoning Toxins Associate Referee Daniel G. Baden (Rosenthal School of Marine and Atmospheric Sciences, University of Miami, Miami, FL) has moved to University of North Carolina and Chiral Corp. (producer of brevetoxin standards) has been closed. Baden has decided to resign from this position. Okadaic Acid in Mussels, 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. Paralytic Shellfish Poisoning Toxins, Immunological Methods Associate Referee Ewald Usleber (Institute for Hygeine and Technology of Food of Animal Origin, University of Munich, Munich, Germany) reports no progress. Paralytic Methods

Shellfish

Poisoning

Toxins,

Instrumental

Associate Referee James F. Lawrence (Health Canada, Ottawa, ON, Canada) reports no progress. Paralytic Shellfish Poisoning Neuroblastoma Cell Bioassay

Toxins

in

Mussels,

Associate Referee Joanne Jellett (Jellett Biotek Ltd., PO Box 790, Dartmouth, NS, Canada) reports an AOAC interlaboratory collaborative study has been completed for the quantitative MIST™ cell bioassay method (63, 64). In total, 14 participants from 10 countries were recruited to take part in the study. Long delays were experienced in the study after April 30, 1997, when saxitoxin, the standard used in the cell bioassay kit, became a heavily controlled substance under its U.N. classification as a chemical weapon. Under this international treaty, permits for both shippers and receivers were required. More recently, there have been some exemptions, but during the course of the collaborative study, the permit process was in full force and some participants waited up to a year to receive import permits from their home governments. After an initial practice round, data analysis pointed toward some weaknesses in the packaging. This data, along with trial data


from the UK and Alaska, led to upgrading of packaging significantly in late 1998, prior to the commencement of the actual AOAC trial. Nine different samples of mussel homogenate, including controls, spiked, and naturally contaminated tissue were provided to participants. Three different profiles were used for the naturally contaminated samples. The profiles and toxin levels were also tested using LC and mouse bioassay in two independent laboratories. To their credit, all 14 participants remained involved throughout the delays and difficulties. The last of the data was received in June 1999, and analysis of the data has been underway since. The results of this trial will be presented to the Methods Committee for examination.

Recommendations (1) (2) (3) (4) (5) (6) (7) (8)

Receptor Assays for Phycotoxins Associate Referee Frances Van Dolah (NOAA National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC). AOAC Peer-Verified Method trials of microplate format assays for 3 toxin classes (PSP, NSP, and ASP) are either currently underway or are in the planning stages. Progress towards AOAC trials and research published this year on receptor assays are summarized as follows: (a) PSP.—A major impediment to progress in validation trails of receptor assays for PSP is the classification of saxitoxin (STX) as a class 1 chemical weapon by the U.N. Chemical Weapons Convention, which has severely hampered the availability of 3H-STX. However, a ruling this year that recognizes salts of STX-diacetate as different chemical forms has recently eased restrictions. Amersham now offers its 3H-STX in the diacetate form, making it more readily available. An AOAC Peer-Verified Method trial of a microplate receptor assay for PSP toxins (65) is in the planning stages. A microplate receptor assay using commercially available 3H-tetrodotoxin (TTX) (66) as a heterologous ligand offers an alternative to 3H-STX. The expansion of PSP in southeast Asia in recent years has created interest in the application of receptor assays for regulatory purposes, and a U.N.-sponsored red tide program has been established to provide training on phycotoxin receptor assays in that region. This increases the need to complete formal interlaboratory calibration of the assay performance, and peer-verified trials are planned for this year. (b) NSP.—An AOAC Peer-Verified Method trial on a microplate receptor assay (67) for brevetoxins in oysters is in progress. Lack of availability of tritium-labeled standard (3H-PbTx3), due to the recent closure of Chiral Corp., may delay the trial a few more months, but renewed supplies should be available from U.N.C. Wilmington by spring 2000. (c) ASP.—The receptor assay for domoic acid utilizing a cloned glutamate receptor (GLUR6; 68) has been further refined this year for determining domoic acid in field samples of Pseudo-nitzschia and results of the receptor assay have been compared quantitatively with LC–UV and LC–fluorescence methods (69, 70). This method was used to confirm domoic acid as the causative agent in the mass mortality of California sea lions in 1998 (23).

(9) (10) (11) (12) (13) (14) (15)

(16) (17)

Amnesic Shellfish Poisoning Toxins: Continue study. Anatoxins: Continue study. Bioassays for Phycotoxins: Continue study. Cell Bioassays for Phycotoxins: Continue study. Ciguatoxins, Instrumental Methods: Continue study. Ciguatoxins in Fish, Solid-Phase Immunobead Assay: Discontinue topic. Ciguatoxins in Fish, Membrane Immunobead Assay: Complete collaborative study. Diarrhetic Shellfish Poisoning Toxins, Assay Methods: Continue study. Diarrhetic Shellfish Poisoning Toxins, Instrumental Methods: Continue study. Microcystins: Continue study. Neurotoxic Shellfish Poisoning Toxins: Find a new AR. Okadaic Acid in Mussels, Solid-Phase Immunobead Assay: Discontinue topic. Paralytic Shellfish Poisoning Toxins, Immunological Methods: Continue study. Paralytic Shellfish Poisoning Toxins, Instrumental Methods: Continue study. Paralytic Shellfish Poisoning Toxins in Mussels, Neuroblastoma Cell Bioassay: Complete collaborative study. Receptor Assays for Phycotoxins: Continue study. Pfiesteria Toxins: Establish as new topic. Find an AR.

References (1) (2) (3) (4) (5) (6) (7)

(8)

(9) (10) (11) (12)

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454 GENERAL REFEREE REPORTS: JOURNAL OF AOAC INTERNATIONAL, VOL. 83, NO. 2, 2000 (62) Mountfort, D.O., Kennedy, G., Garthwaite, I., Quilliam, M., Truman, P., & Hannah, D.J. (1999) Toxicon 37, 909–922 (63) Jellett, J.F.L. Marks, L.J., Stewart, J.E., Dorey, M.L., Watson-Wright, W., & Lawrence, J. F. (1992) Toxicon 30, 1143–1156 (64) Jellett, J.F., Doucette, L.I., & Belland, E.R. (1998) J. Shellfish Research 17, 1653–1655 (65) Doucette, G.J., Logan, M.M., Ramsdell, J.S., & Van Dolah, F.M. (1997) Toxicon 35, 625–636 (66) Doucette, G.J., Powell, C.L., Do, E.U., Byon, C.Y., Cleves, F., & McClain, S.G. Toxicon (in review) (67) Van Dolah, F.M, Finley, E.L., Haynes, B.L., Doucette, G.J., Moeller, D.P., & Ramsdell, J.S. (1994) Nat. Toxins 2, 189–196 (68) Van Dolah, F.M., Leighfield, T.A., Haynes, B.L., Hampson, D.R., & Ramsdell, J.S. (1997) Anal. Biochem. 245, 102–105 (69) Scholin, C.A., Marin, R., Miller, P.E., Doucette, G.D., Powell, C.L., Haydock, P., Howard, J., & Ray, J. J. Phycology, in press (70) Parsons, M.L., Scholin, C.A., Doucette, G.J., Fryxell, G.A., Dortch, Q., & Soniat, T.M. (1998) J. Phycology Supplement 34, 45

Plant Toxins TAM GARLAND Texas A & M University, College Station, Texas 77843-4466, USA Plant poisonings continue to be a problem and there are numerous reports in the veterinary literature. Plant poisonings have an economic impact on the livestock raiser when animals succumb, therefore, much learning with regard to toxicity of plants has come from the observations, study, and clinical reports of animals being poisoned by a variety of plants. Furthermore, as many herbal products are plant based and ‘natural’ approaches to medicine continue to be popular and ever more prevalent in our society, it would behoove scientists to look at information in the veterinary literature as guides to potential toxicities. Metabolism may vary slightly between the species, but animals are routinely used in drug trials before pharmaceutical agents are placed into clinical trials with human beings. Therefore, it seems wisdom would dictate a look at the experience of veterinary medicine when evaluating safety of products intended for human consumption. Indeed, there are reports, and experimental and field accounts documenting intoxications in cattle from Dichapetalum mossambicens Engl. and D. stuhlmanni Engl. (1), Myoporum laetum (2), Gutierrezia spp. (3), Quercus calliprinos (4), Cestrum diurnum (5), and Senecio alpinus (6). Poisonings in sheep have occurred from Tylecodon (7), in sheep and goats from Ornithogalum nanodes (Leighton; 8), while goats suffered intoxications from Ipomoea carnea (9). A miniature Shetland pony was a casualty following Digitalis purpurea ingestion while the second pony was successfully treated (10). Poisonings suffered by wildlife are more frequently reported in the literature. Free-ranging moose were naturally poisoned by consumption of Narthecium ossifragum

and/or Quercus spp. (11) while moose, red deer, reindeer, and fallow deer suffered from Narthecium ossifragum intoxication (12). Intoxications in deer from endophytic fungus have also been reported (13). Oleander (Nerium spp.) continues to be a problem, but this year in dogs there may be other treatments (14). Intoxications in rodents occurred from Rhazya stricta (15) and rodents and humans were the subject of an investigation involving pyrrolizidine alkaloid-containing plants (15). Intoxication in rabbits occurred from ingestion of endophyte-infected fescue seed (16). A brine shrimp bioassay was used as a monitor of the bioactivity of reversed-phase HPLC (RP-LC) fractions leading to the isolation of ptaquiloside Z, a new illudane-type sesquiterpene glucoside (17), and ptaquiloside, the known bracken carcinogen, from an aqueous extract of the neotropical bracken fern Pteridium aquilinum var. caudatum (17). Chemical degradation and spectroscopic analyses confirmed the structure of ptaquiloside Z (17). Brine shrimp had similar toxic reactions to both compounds. Plants are no less a problem in human beings as they represent a diagnostic challenge for physicians and a confusing trial for epidemiologists. Pharmacologically active ingredients of many plants are often unknown, or may not be helpful (18) as treatment is still based on the clinical signs exhibited by the victim(s). Analytical methods such as HPLC, GC/MS and immunoassays can provide identification of the toxin, but biological screening methods remain to be developed (18). Hallucinogenic properties of some plants continue to intrigue individuals who often find themselves victims of their own vices. Jimson weed (Datura spp.) has previously been in the news this year, with a single incident of 11 patients being investigated. The clinical signs of asymptomatic mydriasis and tachycardia, severe agitation, disorientation, and hallucinations were due to ingestion of an atropine-containing alkaloid found in concentrated form in the seed pods (19). While many human cases are solved by taking histories of the victims (something not possible with animals), others are not so fortunate. Such was the case in Burkina Faso (Africa) where preschool children suffered a fatal encephalopathy from the consumption of the unripe ackee (Blighia sapida) fruit. Gas chromatography was used to measure the dicarboxylic acid concentration in blood and urine samples from the victims and their families. Poisoning by the unripe ackee fruits was suggested by urine concentrations of dicarboxylic acids 4–200 times greater in cases than in controls. Plants and their toxins influence our lives disguised as dietary supplements. These various plants and plant products, widely consumed and often used for medicinal purposes, have been scarcely studied in terms of their interaction with conventional drug therapy. However, Zhu did a study with regard to the influence of Sanguisorba officinalis L. on the pharmacokinetics of ciprofloxacin in the rat. Blood and urine samples were analyzed by HPLC for ciprofloxacin following administration of Sanguisorba officinalis L. The presence of the dietary supplement decreased the plasm concentration and the urinary recovery of ciprofloxacin (20).


There are 5 major biologically active ingredients in saffron that were measured by HPLC with ultraviolet (UV) detection. Those compounds are crocin-1, crocin-2, crocin-3, crocin-4 and crocetin. Calibration curves were derived by spiking authentic compounds and internal standard, 12-cis-retinoic acid, into herbal samples prior to extraction. The extraction was conducted by stirring dried herb with 90% aqueous methanol at ambient temperature in the dark. The HPLC assay was performed on a reversed-phase C18 column with linear gradient elution using methanol and 1% aqueous acetic acid. Calibrations were reported as linear for all 5 analytes. The assay was successfully applied to the determination of 4 crocins and crocetin in 3 saffron samples and 2 Zhizi (another crocein-containing herb). Results indicate the developed HPLC assay can be readily utilized as a quality control method for crocin-containing medicinal herbs (21). The role of 1,8-dihydroxyanthraquinones in human carcinogens is currently debated, and these compounds are present in laxatives, fungi imperfecti, Chinese herbs and perhaps some vegetables. A variety of vegetables and some herbs and herbal-flavored liquors were screened for their content of the “free” anthraquinones, emodin, chrysophanol, and physcion. RP-LC, GS/MS, and RP-LC/MS were used for qualitative and quantitative analyses. The vegetables showed a batch-to-batch variability of total anthraquinones per kg fresh weight. Physcion predominated in all vegetables tested. In the herbs, anthraquinones were above the limit of detection. All 3 anthraquinones were found in 7 of 11 herbal-flavored liquors. The genotoxicity of the analyzed anthraquinones was investigated in the comet assay, the micronucleus test, and the mutation assay in mouse lymphoma L5178Y tk+/- cells. Emodin was genotoxic but chrysophanol and physcion showed no effects. Complete vegetable extract on its own did not show any effect in the micronucleus test. Taking into consideration the measured concentrations of anthraquinones, estimated daily intakes, the genotoxic potency, as well as protective effects of the food matrix, the analyzed constituents do not represent a high priority genotoxic risk in a balanced human diet (22). There were numerous cases in 1999 of poisoning in people. Cases were from adolescents attempting to achieve hallucinogenic responses through consumption of Angel trumpet lily (23), favism in nursing neonates deficient in glucose-6-phosphate dehydrogenase whose mothers had consumed fava beans, and assessments of neurolathyrism in a community in northern Ethiopia (24). These are only a few of the clinical cases that have been reported. Selected Associate Referee Topics Aristolochic Acid in Traditional Chinese Medicines Associate Referee Catharina Ang (U.S. Food and Drug Administration, Jefferson, AR) has nothing new to report this year, although she is hopeful the collaborative study protocol for evaluation of the method will produce results. Recommend continued study.

Glucosinolates No associate referee has been found. Recommend continued study. Hypoglycine Associate Referee G. William Chase, Jr (U.S. Food and Drug Administration, Atlanta, GA). Nothing new to report. Phytoestrogens Associate Referee Patricia Murphy (Iowa State University, Ames, IA). Phytoestrogens in foods has been a very hot topic during 1999 with over 680 citations for genistein. However, the analytical methods for genistein and related isoflavone compounds are still rather limited. Most reported methods in the past 12 months are RP-LC, but a few by capillary electrophoresis have appeared. An ELISA method for daidzein and equol and a radioimmunoassay for genistein have been reported. The web publication of USDA-Iowa State University Isoflavones Database as downloadable files is the major addition to the food composition field this year, at www.nal.usda.gov/fnic/foodcomp/data/isoflav/isoflav.html. The main source of isoflavones in the diet are from soybeans and soy foods. Other food legumes contain very small amounts of isoflavones. Data for isoflavone contents were collected from scientific articles published in refereed journals. In addition, isoflavone data was generated by extensive sample collection and subsequent analysis by Iowa State University. Data for only the most predominant isoflavones, daidzein, genistein and glycitein, and their glucoside forms were evaluated by using the expert system described by Mangels et al. (25) for 5 categories: analytical method; analytical quality control; number of samples; sample handling; and sampling plan. The analytical methods of Murphy et al. (26) were used as the reference for evaluating analytical methodologies. Since this is the first database on isoflavones, the methodology criteria for inclusion in the database were relaxed so as to include as many foods as possible. The database contains values for 128 foods. The glucoside forms of the isoflavones are converted to aglycones to be absorbed in the gut and exert their effects. Therefore, all values in the database are converted to moles and reported as mass of aglycone forms. Simple addition of aglycone and glucoside forms will overestimate the true isoflavone concentration by almost a factor of 2 (27, 28). Each mean is assigned a Confidence Code of a, b, or c. The Confidence Code is an indicator of the relative quality of the data and the reliability of the given mean value. A Confidence Code of “a” indicates considerable reliability, due either to a few exemplary studies or to a large number of studies of varying quality. Besides soy, alfalfa and clover sprouts, and garbanzo beans, a few other food items are reported to contain isoflavones, although at concentrations usually about 1000 times lower than soy, including hops in beer (29) and dry cherries (30). Other non-food herbal items containing


isoflavones are reported in the literature with the largest number containing Kudzu root (daidzin and puerarin, 31–34). Eleven refereed publications have appeared on analysis of soy isoflavones in foods by HPLC and 2 reports ON an isotope dilution-GC method. One report compared capillary zone electrophoresis (CZE) with HPLC. Two reports evaluated the effect of processing on isoflavone contents. Barnes et al (35) reported analysis of isoflavones and their metabolites utilizing HPLC/MS. No derivatization is required. Optimization of ion energies in formation and collision of parent ions was essential for detection. Setchell et al. (36) reported isoflavone levels in soy-based infant formula as measured by HPLC and reported total mass of isoflavone without adjustment for molecular weight differences. Song et al. (37) reported some quality control measures for soyfood isoflavone analysis and use of 2,4,4′-trihydroxydeoxybenzoin (THB) as an internal standard for HPLC analysis of foods. Murphy et al. (38) reported precision and accuracy measurements for isoflavone database development over a 9-month sampling period. Recoveries of daidzein and genistein averaged 92%, while genistin and THB recoveries were 99%. Coefficients of variation (CVs) were evaluated for 15 isoflavone forms in 2 food matrixes stored under 2 conditions for 9 months for within-day and between-day precision. Franke et al. (39) reported isoflavone levels in soy foods in Singapore and Hawaii for 25 food groups. An external standard (as extract of soy flour) and flavone as internal standard were used. Jeong et al. (40) used isoflavone levels as quality parameter for fermented soy pastes. King et al. (41) used HPLC to evaluate isoflavone levels in cow’s milk with detection levels of 2 ng/mL. Seasonal variations in levels were observed with peaks of 293 ng/mL equol occurring in summer months. Krishnan (42) reported genistein in Apois Americana medikus, the American groundnut, an indigenous North American tuber, by HPLC analysis with ultraviolet absorbance detection and MS confirmation.

Coward et al. (43) monitored effects of baking and frying on soy isoflavone glucoside interconversion by reversed-phase HPLC/MS. Extraction at 4°C resulted in highest concentrations of malonylglucoside forms. Mahungu et al. (44) reported effects of extrusion processing on isoflavone distribution using 80% methanol for extractions. They reported lower extraction rates in extruded foods unless rehydrated initially. This solvent has previously been reported to incompletely extract the acetylglucosides (45). Mazur et al. (46) and Liggins et al. (47) reported an isotope dilution GC method for evaluation of isoflavones in foods and non-food seeds. Liggins et al. (47) reported interassay CVs for genistein and daidzein of 4.7 and 2.7%, respectively. Both methods require derivatization. Aussenac et al. (48) reported a CZE method for soybean isoflavones. Mellenthin and Galensa (49) reported a CZE method to detect soy and lupin protein in meat products by measuring isoflavone content. The same authors (50) compared HPLC and CZE for isoflavone detection. CZE was recommended for rapid screening but HPLC was less dependent on matrix effects and more sensitive. A radioimmunoassay was reported by Lapcik et al. (51) for genistein in serum with intra-assay CV of 3.5–9.3% and interassay CV of 6.7–19.7%. Instances of cross reactivity with daidzein (5.8%) and formononetin (2.2%) were reported. An ELISA was reported for daidzein and equol in human plasma with detection limits of 21 pg daidzein per well and 70 pg equol per well by Creeke et al. (52). Standards for isoflavone analysis are still a difficult issue. Several chemical suppliers have genistein and daidzein. Sigma Chemical Company (St. Louis, MO) has genistin. Recently, LC Laboratories (Woburn, MA) began selling genistein, genistin, daidzein, daidzin, glycitein, and glycitin. The malonylglucosides are quite unstable. The acetylglucosides are not usually commercially available, although one firm does advertise them. If analysts want to stan-

Table 1. Ultraviolet absorbance maximum and extinction coefficients of soy isoflavones Compounda

Source

λMax, nm

Molecular weight

Extinction coefficient, ε

Working range, µg/g

Dein

Synthesized by T. Song

249

254

26915

5 to 200

Din

Purified by H.J. Wang

249

416

26915

5 to 200

ADin

Adjusted from Din

256

458

31622

MDin

Adjusted from Din

258

522

18197

Gein

Synthesized by T. Song

263

270

31622

Gin


263

432

31622

5 to 200

AGin

Adjusted from Gin

261

474

32358

MGin


260

518

16331

5 to 100

Glein

Purified by T. Song

256

284

22387

5 to 100

Glin

Purified by T. Song

259

446

26303

5 to 100

AGlin

Adjusted from Glin

260

488

26303

MGlin

Adjusted from Glin

260

532

26303

a

Abbreviations: Dein = daidzein, Din = daidzin, ADin = acetyldaidzin, MDin = malonyldaidzin, Gein = genistein, Gin = genistin, AGin = acetylgenistin, MGin = malonylgenistin, Glein = glycitein, Glin = acetylglycitin, MGlin = malonylglycitin.


dardize with these compounds, they must be willing to isolate them in their laboratories. There is some small variance in extinction coefficients for these compounds based on work in the General Referee’s laboratory (Table 1). Rasku and Wahala (53) reported synthesis of deuterium labeled isoflavones. Pyrrolizidine Alkaloids Association Referee Helmut Wiedenfeld, (Pharmazeutisches Institut der Universitat, Bonn, Germany). Leaves and rhizomes of in situ grown Petasites hybridus were investigated for their PA content using already described methods such as densitometric measurement and ELISA (54). In the rhizomes, values of 1 to 80 mg/kg were found, whereas in leaves less than 1 mg/kg could be detected. These data clearly differ from those reported earlier, where high PA doses in leaves were found (55). A monoclonal antibody against retrorsine was generated and characterized (56). The specificity was tested with 20 different PAs using competitive ELISA. The PAs acetylgynuramine, gynuramine, integerrimine, neoplatyphylline, platyphylline, rosmarinine, senecionine, and seneciphylline were able to bind the antibody. The other PAs showed no cross-reactivity. A standard curve was established in the concentration range from 10 to 10 000 ng/mL. A more specific HPLC/MS method for the detection and quantitation of PAs was reported (57). The collision-induced dissociation (CID) HPLC/MS using electrospray ionization techniques leads to specific MS fragments which can be used for a rapid identification of different types of PAs. Both reported CID methods (CID-HPLC/MS and HPLC/MS/MS) are able to analyze a possible PA toxin in plants as well as in herbal medicines. A comparison of the degradation power of rumen fluids on the PA jacobine and sneciphylline (from tansy ragwort) lead to the finding that sheep rumen fluid has a higher degradation power than that of calves (58). A study of prenatal and postnatal effects of monocrotaline verified the toxicity in lungs, liver and kidney, using the rat as a model (59). The known toxicity of PAs on the placenta and through the milk was confirmed. A toxicity study of the effect of monocrotaline in chicken eggs was reported (60). The toxic response was 20 mg/kg egg and the livers of chicken embryos showed megalocytosis and atrophy. Some reports can be found on the identification of PAs in different plants. In some Boraginaceae from Egypt (Sinai region) 40 PAs were detected by an already known and described GLC and GLC/MS method (61). The structures were determined by specific retention indexes and mass fragmentation from the literature. The plants investigated were Paracaryum rugulosum, P. intermedium, Anchusa milleri, Gastrocotyle hispida (syn. A. hispida), A. arvensis, Lappula spinocarpos, Trichodesma africanum, Alkanna orientalis, A. tuberculata (syn. A. tinctoria). These PAs are mainly open chain diesters with retronecine as well as heliotridine as the necine moiety. The same method was used to investigate Ipomoea hederifolia and related species (Convolvulacea; 62).

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