Elisabeth Schneider

Improved enzyme immunoassay for group-specific determination of penicillins in milk

Polyclonal antibodies against penicillin antibiotics were raised in rabbits after immunization with an ampicillin-bovine serum albumine conjugate. The specificity and sensitivity of these antibodies were tested in a competitive direct enzyme immunoassay (EIA), using an ampicillin-horseradish peroxidase conjugate as the labelled antigen, and penicillin G as the reference antibiotic. The 50% binding inhibition concentration of penicillin G standard curves was at 2–4 ng ml−1, with a detection limit (20% binding inhibition) at 0.5–1 ng ml−1. The antiserum showed strong relative cross-reactivities with all penicillin-type betalactam antibiotics tested, including those regulated by maximum residue limits (MRLs) within the European Union (ampicillin 64%, amoxicillin 24%, oxacillin 100%, cloxacillin 29%, dicloxacillin 27%, and nafcillin 120%). After alkaline hydrolysis of the betalactam ring, no cross-reactivity of the respective penicillins was observed anymore. Cephalosporin-type betalactam antibiotics did not react in this test system. The recovery rates for penicillin G in artificially contaminated milk samples (2–32 ng ml−1) were at 89–97%. Penicillin G, ampicillin, amoxicillin, oxacillin, cloxacillin, dicloxacillin and nafcillin were all detectable in spiked milk samples at or below the respective MRL level. Comparison analysis of the EIA and a microbiological inhibition assay using violative milk samples obtained from routine milk testing scheme in Bavaria showed excellent agreement between both methods.

Deoxynivalenol in food

Within a joint research project entitled “Analysis and occurrence of importantFusarium toxins (deoxynivalenol and zearalenone) and dietary intake of these toxins by the German consumer”, supported by the German Federal Ministry of Consumer Protection, Food and Agriculture (BMVEL), representative analytical data are generated on the contamination level of foods withFusarium mycotoxins. This paper gives a comprehensive summary concerning the contamination of foods from the German market with deoxynivalenol (DON) in the period from August 2001 to April 2004. More than 4700 food samples (mostly cereals and cereal-containing foods) were purchased from food shops in Germany and analysed for DON by enzyme immunoassay, HPLC, and LC-MS/MS, respectively. All analytical methods were validated through intra- and interlaboratory studies and gave mean recoveries of >80% for each matrix. Although DON was detected with high frequency in all cerealcontaining samples, the mean and median levels were in most products well below the recently established maximum permitted limits in Germany.

Deoxynivalenol in Lebensmitteln

Within a joint research project entitled “Analysis and occurrence of importantFusarium toxins (deoxynivalenol and zearalenone) and dietary intake of these toxins by the German consumer”, supported by the German Federal Ministry of Consumer Protection, Food and Agriculture (BMVEL), representative analytical data are generated on the contamination level of foods withFusarium mycotoxins. This paper gives a comprehensive summary concerning the contamination of foods from the German market with deoxynivalenol (DON) in the period from August 2001 to April 2004. More than 4700 food samples (mostly cereals and cereal-containing foods) were purchased from food shops in Germany and analysed for DON by enzyme immunoassay, HPLC, and LC-MS/MS, respectively. All analytical methods were validated through intra- and interlaboratory studies and gave mean recoveries of >80% for each matrix. Although DON was detected with high frequency in all cerealcontaining samples, the mean and median levels were in most products well below the recently established maximum permitted limits in Germany.

Mycotoxins in Food in Germany

Mycotoxins are worldwide contaminants in food and feed, and they generate an increasing concern for human and animal health (Pittet, 1998). Cases of acute primary mycotoxicosis in humans have been reported, although with low frequency, while acute intoxication of animals is relatively common. However, chronic disease caused by low but constant dietary intake of mycotoxins seems to be the most important factor in aspects of public health. Long–term effects are well documented in animals, and epidemiological studies indicate that a correlation between chronic intake of some mycotoxins and the incidence of specific diseases may exist.

Determination of deoxynivalenol in bread and beer

Analytical procedures for the determination of deoxynivalenol (DON) in bread and beer, using enzyme immunoassay (EIA) and HPLC methods, were developed. For determination of DON by EIA, aqueous raw extracts of bread or degassed beer were extracted by liquid-liquid partitioning with ethyl acetate, the organic solvent evaporated, and the residue redissolved in phosphate buffered saline (PBS) for analysis. For determination by HPLC (UV detection at 218 nm), DON in bread extracts or beer was purified on immunoaffinity chromatographic columns. In bread, detection limits for DON of 15 µg/kg (EIA) and 7 µg/kg (HPLC) were achieved, with mean recoveries of 81%. In beer, the detection limit for DON was 2 µg/l both in EIA and HPLC, with recoveries of 91-93%. Both methods showed good agreement of the results for naturally contaminated sample materials, with r(2)=0.993 for bread and r(2)=0.823 for beer, respectively.Analytical procedures for the determination of deoxynivalenol (DON) in bread and beer, using enzyme immunoassay (EIA) and HPLC methods, were developed. For determination of DON by EIA, aqueous raw extracts of bread or degassed beer were extracted by liquid-liquid partitioning with ethyl acetate, the organic solvent evaporated, and the residue redissolved in phosphate buffered saline (PBS) for analysis. For determination by HPLC (UV detection at 218 nm), DON in bread extracts or beer was purified on immunoaffinity chromatographic columns. In bread, detection limits for DON of 15 µg/kg (EIA) and 7 µg/kg (HPLC) were achieved, with mean recoveries of 81%. In beer, the detection limit for DON was 2 µg/l both in EIA and HPLC, with recoveries of 91–93%. Both methods showed good agreement of the results for naturally contaminated sample materials, with r2=0.993 for bread and r2=0.823 for beer, respectively.

Bestimmung von Deoxynivalenol in Brot und Bier

Analytical procedures for the determination of deoxynivalenol (DON) in bread and beer, using enzyme immunoassay (EIA) and HPLC methods, were developed. For determination of DON by EIA, aqueous raw extracts of bread or degassed beer were extracted by liquid-liquid partitioning with ethyl acetate, the organic solvent evaporated, and the residue redissolved in phosphate buffered saline (PBS) for analysis. For determination by HPLC (UV detection at 218 nm), DON in bread extracts or beer was purified on immunoaffinity chromatographic columns. In bread, detection limits for DON of 15 µg/kg (EIA) and 7 µg/kg (HPLC) were achieved, with mean recoveries of 81%. In beer, the detection limit for DON was 2 µg/l both in EIA and HPLC, with recoveries of 91-93%. Both methods showed good agreement of the results for naturally contaminated sample materials, with r(2)=0.993 for bread and r(2)=0.823 for beer, respectively.Analytical procedures for the determination of deoxynivalenol (DON) in bread and beer, using enzyme immunoassay (EIA) and HPLC methods, were developed. For determination of DON by EIA, aqueous raw extracts of bread or degassed beer were extracted by liquid-liquid partitioning with ethyl acetate, the organic solvent evaporated, and the residue redissolved in phosphate buffered saline (PBS) for analysis. For determination by HPLC (UV detection at 218 nm), DON in bread extracts or beer was purified on immunoaffinity chromatographic columns. In bread, detection limits for DON of 15 µg/kg (EIA) and 7 µg/kg (HPLC) were achieved, with mean recoveries of 81%. In beer, the detection limit for DON was 2 µg/l both in EIA and HPLC, with recoveries of 91–93%. Both methods showed good agreement of the results for naturally contaminated sample materials, with r2=0.993 for bread and r2=0.823 for beer, respectively.

Zearalenon in Lebensmitteln

The research project “Methods of Analysis and occurrence of important Fusarium toxins (deoxynivalenol and zearalenone) as well as the intake of these toxins by the German consumer” supported by the Federal Ministry of Consumer Protection, Food and Agriculture (BMVEL) is processed by the institutions mentioned above. This work represents a comprehensive summary of the contamination of food by zearalenone (ZEA).ZusammenfassungIm Rahmen eines Fusarienforschungvorhabens wurden Analyseverfahren für Zearalenon auf der Basis von ELISA, HPLC-FLD und LC-MS/MS MS/MS validiert. Bislang wurden 4556 Lebensmittel auf den Gehalt an Zearalenon untersucht. 84% der Maismehle und 53% Maisgrieße weisen eine mittlere und 95 Maiskeimöl eine hohe Zearalenonbelastung auf. Mit Maiskeimöl hergestellte Säuglings- und Kleinkindernahrung weisen eine erhöhte Belastung auf. Alle anderen aus Zerealien hergestellten Lebensmittel weisen eine niedrige Belastung auf.

Zearalenone in food

The research project “Methods of Analysis and occurrence of important Fusarium toxins (deoxynivalenol and zearalenone) as well as the intake of these toxins by the German consumer” supported by the Federal Ministry of Consumer Protection, Food and Agriculture (BMVEL) is processed by the institutions mentioned above. This work represents a comprehensive summary of the contamination of food by zearalenone (ZEA).ZusammenfassungIm Rahmen eines Fusarienforschungvorhabens wurden Analyseverfahren für Zearalenon auf der Basis von ELISA, HPLC-FLD und LC-MS/MS MS/MS validiert. Bislang wurden 4556 Lebensmittel auf den Gehalt an Zearalenon untersucht. 84% der Maismehle und 53% Maisgrieße weisen eine mittlere und 95 Maiskeimöl eine hohe Zearalenonbelastung auf. Mit Maiskeimöl hergestellte Säuglings- und Kleinkindernahrung weisen eine erhöhte Belastung auf. Alle anderen aus Zerealien hergestellten Lebensmittel weisen eine niedrige Belastung auf.

Bestimmung von Deoxynivalenol und Deepoxy-Deoxynivalenol in Milch

A HPLC method with UV/diode array detection for the determination of deoxynivalenol (DON) and deepoxy-deoxynivalenol (DOM-1) in milk was developed. Milk was incubated with β-glucuronidase and then defatted. After purification by immunoaffinity chromatography, DON and DOM-1 were separated on a C18 reversed phase column with acetonitril/water (10/90) as the mobile phase and detected at 218 nm. Limits of quantification were 1 μg/l for both toxins, with mean recoveries (1–10 μg/l) of 97% (DON) and 84% (DOM-1), respectively. Milk samples (pasteurized, UHT; n=32) from German retail shops were analysed by this method. Neither DON/DOM-1 nor their glucuronides were found in any sample. These results are consistent with published studies indicating that in lactating cows, DON and DOM-1 are mostly eliminated through urine, and that the carry-over into milk is negligible.

[Determination of deoxynivalenol and deepoxy-deoxynivalenol in milk].

A HPLC method with UV/diode array detection for the determination of deoxynivalenol (DON) and deepoxy-deoxynivalenol (DOM-1) in milk was developed. Milk was incubated with β-glucuronidase and then defatted. After purification by immunoaffinity chromatography, DON and DOM-1 were separated on a C18 reversed phase column with acetonitril/water (10/90) as the mobile phase and detected at 218 nm. Limits of quantification were 1 μg/l for both toxins, with mean recoveries (1–10 μg/l) of 97% (DON) and 84% (DOM-1), respectively. Milk samples (pasteurized, UHT; n=32) from German retail shops were analysed by this method. Neither DON/DOM-1 nor their glucuronides were found in any sample. These results are consistent with published studies indicating that in lactating cows, DON and DOM-1 are mostly eliminated through urine, and that the carry-over into milk is negligible.