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Biomedical subjects

H P van Egmond

Publications and source records attributed to H P van Egmond.

At least 19 recordsLinked to original sources

Processing and purity assessment of standards for the analysis of type-B trichothecene mycotoxins.

The lack of reliable, certified calibrant solutions for the Fusarium mycotoxins deoxynivalenol (DON), 3-acetyl-DON (3-Ac-DON), 15-acetyl-DON (15-Ac-DON) and nivalenol (NIV) is a serious drawback in the already problematic area of trichothecene analysis. For this reason, purified DON, 3-Ac-DON, 15-Ac-DON and NIV standards were processed, the conditions required for their isolation and purification were optimised, and the crystalline toxins were thoroughly characterised. Several complimentary analytical methods were used to evaluate the identities of the mycotoxins and the types and amounts of impurities; results obtained from 1H and 13C NMR spectra, as well as from IR-spectra, were in agreement with the literature. Elemental analysis revealed that the isolated NIV occurs as monohydrate. If this is not known it results in a weighing error of approximately 5%. Differential scanning calorimetry (DSC) was only successful for 15-Ac-DON, as the other trichothecenes decomposed during measurements. No traces of chloride, nitrate and sulphate were found by means of ion chromatography (IC). As expected UV absorption spectra for DON, NIV, 3-Ac-DON and 15-Ac-DON yielded lambda(max) values of 216, 217, 217 and 219 nm, respectively. Minor peaks due to impurities were observed by high performance liquid chromatography (HPLC) with UV detection. The main impurity peak in the DON sample was identified by LC-tandem mass spectroscopy (LC-MS/MS) as 4,7-dideoxy-NIV (7-deoxy-DON), which occurs at levels of approximately 1.4%. Gas chromatography (GC) was performed, coupled with either an electron capture detector (ECD), a flame ionisation detector (FID), or a mass spectrometric detector (MS); however, derivatisation prior to GC analysis makes the estimation of impurities difficult. LC-MS/MS was found to be unsuitable for quantifying levels of impurities. It can be concluded that high-purity (>97%) B-trichothecene standards were successfully processed and fully characterised for the first time.

Chromatography, High Pressure Liquid↗

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

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

Animals↗

Subacute toxicity of alpha-ergocryptine in Sprague-Dawley rats. 1: general toxicological effects.

The dietary subacute toxicity of the ergot alkaloid alpha-ergocryptine was studied in Sprague-Dawley rats. Rats were fed 0, 4, 20, 100 or 500 mg ergocryptine/kg diet for 28-32 days (equal to 0, 0.36, 1.7, 8.9 and 60 mg ergocryptine/kg body weight/day for females and 0, 0.34, 1.4, 6.6 and 44 mg ergocryptine/kg body weight/day for males). The present study describes the general toxicological effects; the effects on metabolic and hormonal parameters will be reported separately. Body weight, body weight gain, food intake and food efficiency were all decreased with a U-shaped dose-response curve, as in both sexes the ranking severity of effects was in the order 100-20-500 and 4 mg/kg diet. Other changes with a U-shaped dose-response relationship included: hematological parameters (decreased MCV and MCH), serum enzyme activities (slightly increased/decreased ALAT, ASAT, GGT), increased serum urea concentrations, decreased glomular filtration (creatinine and urea clearances), decreased absolute organ weights, increased and decreased relative organ weights, atrophy of thymus and in females atrophy of ovary and uterus with in the mid-dose groups no detectable morphological features of an oestric cycle in the uterus. Other parameters, including increased relative liver, heart and ovarian weights and necrosis of the tail, were influenced in a dose-related manner or only in the high dose group. The U-shaped changes for the parameters mentioned above might be caused by the U-shaped dose-response relationship for food intake, which may be explained by the dopaminergic properties of alpha-ergocryptine. It is concluded that in rats fed ergocryptine for 28 days the dose-effect curve is rather steep and that the NOAEL is 4 mg/kg diet.

Animals↗

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

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

Animals↗

Assessment of human exposure to fumonisin B1.

Fumonisin B1 is currently regarded as the most significant mycotoxin produced by Fusarium spp. It has carcinogenic properties and may play a role in the etiology of human esophageal cancer. The human population is exposed to fumonisin B1 primarily by intake of fumonisin B1-contaminated maize. Maize consumed in the Netherlands is imported from all parts of the world. Since processing will not affect the overall toxic effect, the fumonisin B1 intake is directly related to the quantity of maize consumed. Literature results concerning the occurrence of fumonisin B1 in a total of 349 samples of maize from 18 countries worldwide demonstrated the presence of this mycotoxin in 93% of the samples. The median fumonisin B1 contamination of all samples was 420 ng of fumonisin B1 per g of maize, and the average contamination level was 1,359 ng of fumonisin B1 per g of maize. Human intake of fumonisin B1 was estimated based on the maize consumption of all people in the Netherlands in 1992. A probability distribution was derived to allow estimation of the exposure of the population to fumonisin B1 intake in relation to maize intake. It showed that among those in the group considered to be at risk, people with gluten intolerance such as people with celiac or Dühring's disease, 37% are estimated to be exposed to an intake of at least 10(5) ng and 97% to an intake of at least 10(3) ng of fumonisin B1 per person per day. For all people in the Netherlands these percentages would be 1% and 49%, respectively.

Carboxylic Acids↗

Worldwide regulations for mycotoxins in 1994.

Since the discovery of the aflatoxins in the 1960s, regulations have been established in many countries to protect the consumer from harmful effects of mycotoxins that may contaminate foodstuffs. Various factors play a role in the decision-making process of setting limits for mycotoxins. These include scientific factors such as availability of survey data, toxicological data, analytical methodology, and knowledge about the distribution of mycotoxins in contaminated commodities. Economical and political factors such as commercial interests and sufficiency of food supply have their impact as well. International enquiries on existing mycotoxin regulation in foodstuffs and animal feedstuffs have been carried out several times in the 1980s and details about tolerances, legal bases, responsible authorities, official protocols of analysis and sampling have been published. The most recent enquiry is currently taking place as part of a contract with the Food and Agriculture Organization. At least 77 countries now have regulations for mycotoxins. An overview is presented of the tentative results of this enquiry, including frequency distributions of existing tolerances for aflatoxins and current trends.

Food Analysis↗

Worldwide regulations for ochratoxin A.

Currently, some 60 countries have enacted or proposed regulations for levels of mycotoxins in food and animal feed. Various factors may influence the establishment of tolerances for certain mycotoxins, such as the availability of data on dietary exposure and on toxicology, the distribution of the mycotoxins over commodities, the availability of analytical methodology, legislation in countries with which trading occurs and a sufficient food supply. Most of the existing regulations for mycotoxins concern aflatoxins, but the number of countries that regulate other mycotoxins as well is growing. Of the nephrotoxic mycotoxins, ochratoxin A was the only one for which regulations existed in 1990. At least 11 countries have proposed or official limits for ochratoxin A. The acceptable levels range from 1 to 50 micrograms/kg for food and from 100 to 1000 micrograms/kg for animal feed. The scientific basis for the established regulations appears to be weak, and a rationalization of tolerance levels for ochratoxin A would be highly desirable.

Animal Feed↗

Methods for determining ochratoxin A and other nephrotoxic mycotoxins.

Chemical assays are of major importance for the determination of mycotoxins. Generally, all chemical methods for the analysis of mycotoxins include the basic steps of extraction, clean-up, separation, detection, quantification and confirmation of identity. The various approaches that exist for the determination of nephrotoxic mycotoxins, and in particular the ochratoxins, are discussed below. In conventional procedures, clean-up is usually achieved by liquid-liquid extraction or adsorption column chromatography, followed by thin-layer chromatography or high-performance liquid chromatography and ultraviolet or fluorescence detection. The recent introduction of methods based on immunochemical principles has had a large impact on analytical methodology for mycotoxins, including the ochratoxins, in both the purification and determination steps. The enzyme-linked immunosorbent assay approach for screening and (semi-)quantitative determination and the immunoaffinity column approach for rapid clean-up followed by conventional instrumental analysis are rapidly gaining ground. These techniques also offer possibilities for automated systems. To assist analysts in improving their data in mycotoxin research, the Community Bureau of Reference of the European Commission has produced several reference materials for mycotoxins; others, such as a reference material for ochratoxin A in grains, are in development.

Chromatography↗

[Developments in research on mycotoxins].

Since the early 1960's attention has been paid to mycotoxins by various disciplines of the National Institute of Public Health and Environmental Hygiene (RIVM) (Analytical Chemistry, Microbiology, Toxicology). A major part of the work is carried out at the request of the Chief Medical Office. The activities undertaken on behalf of the Chief Veterinary Officer concern investigations on the causes of fungal growth and toxin production as well as on the presence of aflatoxin B1 and the analytical possibilities of determining aflatoxin B1 in feeds and aflatoxin M1 in milk. Because of its structural relationship with aflatoxin B1, aflatoxin M1 is suspected of having carcinogenic properties. In order to be able to do chronic toxicity studies in rats, efforts are being made to synthesize approximately 25 g. of aflatoxin M1 in co-operation with the State University of Utrecht, which was found to be a time-consuming and extremely difficult job. From the point of view of the Chief Veterinary Officer, it is expected that the analytical and toxicological features of aflatoxin M1 will continue to be of interest in the near future.

Aflatoxin B1↗

Improved method for confirmation of identity of aflatoxins B1 and M1 in dairy products and animal tissue extracts.

A method is described for confirming the identity of aflatoxins B1 and M1 in dairy products and liver extracts on a thin layer plate. Extracts and standards containing aflatoxins B1 and M1 are spotted on 10 x 10 cm plates, which are developed 2-dimensionally in mixtures of isopropanol-acetone-chloroform. After the first development, trifluoroacetic acid-hexane (1 + 4) is sprayed on that part of the plate containing the separated extract components and the underdeveloped standard spots of B1 and M1, and the plate is heated 6-8 min at 75 degrees C. Then the plate is developed in a second direction, and the reaction products of B1 and M1 with trifluoroacetic acid from the extract are compared with the same derivatives of the respective standards. The method has been used successfully on extracts of milk, cheese, and liver containing 0.1 ng B1 or M1/g and can be completed in 35-45 min.

Aflatoxin B1↗

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

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

Cheese↗

Fungal growth and the presence of sterigmatocystin in hard cheese.

Molds isolated from visibly molded cheeses in shops, households, and warehouses have been identified. Mold flora of cheeses in shops and households consisted mainly of Penicillium verrucosum var. cyclopium. On cheeses ripening in warehouses, Penicillium verrucosum var. cyclopium, Aspergillus versicolor, Aspergillus repens, and Enicillium verrucosum var. verrucosum were the dominant mold species. Cheese ripening in warehouses and molded with A. versicolor were examined for sterigmatocystin. Nine of 39 cheese samples contained sterigmatocystin in the surface layer in concentrations ranging from 5 to 600 micrograms/kg.

Animals↗

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

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

Animals↗

Mycotoxins: regulations, quality assurance and reference materials.

Some 60 countries have currently enacted or proposed regulations for control of mycotoxins (primarily the aflatoxins) in food and animal feed. Various factors influence the establishment of limits for certain mycotoxins, but there is no consistent rationale for setting limits or for enforcement control. The enforcement of the regulations requires monitoring of suspected commodities. Many laboratories perform large numbers of determinations of mycotoxins, in particular aflatoxins, and consider their results to be reliable. Nevertheless, it often happens that laboratories find quite different analytical results on samples that have been especially homogenized for interlaboratory studies. The application of Quality Assurance principles contributes to the reliability of mycotoxin measurements. Quality Assurance is focused on the organizational process and the conditions under which laboratory studies are planned, performed, monitored, recorded, reported and archived. This is, in itself, an important contribution to the scientific value of a study. A Quality Assurance programme should include various elements, including (certified) reference materials, if available. Certified reference materials are stable, homogeneous products with certified values of the analyte(s) of interest. Various certified reference materials for mycotoxins have been prepared by the European Commission's Community Bureau of Reference, in co-operation with several European laboratories (aflatoxin M1 in milk powder, aflatoxins B1 B2, G1, and G2 in peanut butter, aflatoxin B1 in peanut meal and compound feed, deoxynivalenol in wheat and maize flour). Other reference materials are in a well-advanced development stage (ochratoxin A in wheat flour and pig kidney, fumonisins B1 and B2 in maize).

Food Contamination↗

The occurrence of fumonisin B1 in maize-containing foods in The Netherlands.

Seventy-eight maize-containing foods obtained from retail stores in The Netherlands were analysed for fumonisin B1 contamination. Thirty-six per cent of the samples were contaminated with fumonisin B1 in the range of 8 micrograms kg-1 (limit of detection) to 1430 micrograms/kg-1. Forty-six per cent of the minimally treated maize samples (n = 39; maize for bread production, maize for popcorn, maize flour and polenta) were contaminated with fumonisin B1 in the range of 8-380 micrograms kg-1. Twenty-six per cent of the maize-containing processed foods (n = 39; tostada, canned maize, maize starch, maize bread, popped maize, flour mixes, maize chips and cornflakes) were contaminated with fumonisin B1 in the range of 8-1430 micrograms/kg-1. This survey shows that maize-containing foods in The Netherlands frequently can be contaminated with fumonisin B1.

Bread↗

Fumonisin B1 in maize for food production imported in The Netherlands.

Sixty-two samples of maize imported in The Netherlands and intended for human consumption were screened for the presence and concentration of fumonisin B1. Sixty-one of those samples contained fumonisin B1 with concentrations ranging from 30 to 3350 micrograms kg-1, 11 maize samples contained > 1000 micrograms kg-1. The average fumonisin B1 concentration was 640 micrograms kg-1 for the positive samples and 620 micrograms kg-1 for all samples. Medians were 600 micrograms kg-1 and 550 micrograms kg-1 for positive and all samples, respectively. The results obtained were comparable to results from other studies in maize from various countries.

Carboxylic Acids↗