NUTRITIONAL AND FOOD SAFETY ANALYTICAL SUPPORT

Information on Nitrofurans Metabolites in Foods

Nitrofurans are a class of drugs that have the ability to kill micro-organisms. The group consists of four drugs. Since 1989, nitrofurazone has been discontinued from being classified as a drug by the US Food and Drug Administration.  It has been reported that most nitrofurans are mutagenic and carcinogenic. Nitrofurantion, for example, has been observed to damage the building unit of the cell, namely the deoxyribonucleic acid of bacterial and human cells.  The damage caused to these cells resembled that of ultraviolet irradiation. The DNA damage, however, was noted to be reparable. Since these drugs have been shown to damage DNA synthesis, particular care should be exercised in using these drugs on humans or using foodstuff contaminated by these drugs. Besides DNA damage, nitrofurans have been reported to cause hemolysis of red blood cells in individuals lacking in glucose-6 phosphate dehydrogenase (G-6-PD) enzyme.  The intake of such drugs or foodstuff such as poultry contaminated with these drugs will result in the oxidation of the red blood cells, leading to the rupture of the cells. Once the cells rupture, and individual may experience dizziness, lethargy, headache, vomiting, chills, fever and abdominal pain.  In extreme cases, the presence of red blood cell remnants may be detected in urine and renal failure may occur. Hence, these drugs should be avoided by G-6-PD patients. Nitrofurans have been found in honey, meat and seafood.

1. Is there a USA, Canada or an EU-reference method for nitrofuran drugs?
There are no formal reference methods in the  EU.  There is a formal method in Canada.  In the USA, the FDA has published in their web site an LC-MS/MS method for the analysis of nitrofurans metabolites in seafood.  In Europe, no official reference methods are available, this is because there is a limited number of laboratories involved in this research but it is also a consequence of the European approach to residue analysis. In Europe, instead of prescribing a method, there are criteria established that analytical results need to comply with. 

2. How can the analysis for nitrofuran drugs be performed?
The analysis of residues of nitrofuran drugs needs to be based on the detection of the (tissue-bound) metabolites of the nitrofuran parent drugs. There are no immuno-chemical or microbiological screening methods presently available for the four metabolites. ELISA methods are only available for two metabolites, which is not acceptable in the US, EU or other countries.  Most commonly the analysis is carried out by LC-MS or LC-MS/MS techniques.  Metabolites are derivatized to their corresponding nitrophenyl derivatives prior to analysis. 

3. How is the analysis of nitrofurans performed at ADPEN?
At ADPEN we developed methods to analyze various substrate samples using the extremely selective LC-MS/MS technique. This technique excludes the possibility of false positive results or in other words a positive result obtained by this technique has the highest degree of certainty that can be obtained. Therefore this technique is suitable for confirmatory analysis and LC-MS/MS results in general are accepted in legal procedures as reliable and unequivocal proof.  ADPEN has been using this procedure for several years for various substrates.  Currently an FDA LC-MS/MS procedure is available which is similar to ADPEN methodology.

4. Is there a risk of contamination of samples leading to erroneous results?
At ADPEN ample measures are taken to avoid contamination. First of all as much as possible disposable single-use labware are used, only unequivocal results are reported. 

5. Which matrices can ADPEN analyze?
The methods used at ADPEN are able to analyze honey, porcine muscle and liver, poultry muscle, shrimp meat, fish and other animal tissue.

6. Is it sufficient to monitor the parent drugs
The analysis of residues of nitrofuran drugs needs to be based on the detection of the (tissue-bound) metabolites of the nitrofuran parent drugs. Since the parent drugs are very rapidly metabolized, they are not detectable shortly after treatment. In contrast, the (tissue-bound) residues are detectable for several weeks after administration and hence are a much better marker residue for the detection of abuse of nitrofurans.

7. Is it sufficient to monitor only residues of furazolidone
No there is abundant evidence that three nitrofurans other than furazolidone are also used and hence it is necessary to measure the four metabolites of these nitrofurans. 

8. Which are the metabolites to analyze for?
The metabolites of furazolidone, furaltadone, nitrofurantoine and nitrofurazone are respectively 3-amino-2-oxazolidinone (AOZ), 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD) and semicarbazide (SEM) 

9. Are there any simple screening methods for nitrofurans or their metabolites?
For the four metabolites there are no microbiological or immuno-chemical screening methods available. The simplest method suitable is LC-UV. This however may suffer from considerable interference from matrix compounds, thus false positives or negatives. Furthermore it does not comply with US or EU regulations regarding the compulsory confirmatory analysis of residues of banned substances. LC-UV so far has proven insufficiently sensitive to achieve the required detection limits.   LC-MS as an alternative to the more expensive LC-MS/MS technique has been used, however, while this technique with carefully optimization of separating conditions may be suitable for screening at 1 ug/kg (ppb) concentrations, could suffer from interferences and it is not suitable for identification according to EU requirements (Decision 2002/657/EC). 

10. What is the Limit of Quantitation (LOQ) for the metabolites of the nitrofuran drugs
At ADPEN the LOQ for the metabolites of the nitrofuran drugs is well below 1 ug/kg (ppb), 0.3 ppb for several matrices. Currently FDA tests at an LOQ of 1 ppb. 

11. What are the FDA testing requirements for detained seafood such as shrimps? FDA requires analysis of 12 shrimp subsamples per lot at an LOQ of 1 ppb per subsample.  The reason for this is that residues are not uniform throughout a particular lot and if only one subsample tests positive at 1 ppb and the others are clean, FDA indicates that this is sufficient for the lot to be violative.  According to the FDA, during their testing they have never found consistent residues in subsamples from any particular lot. 

12. Is the analysis of 12 subsamples per lot by LC-MS/MS required by FDA?             That is what FDA has specified, to avoid sample dilution of the residues, this would be very expensive.  Compositing of various subsamples into a smaller amount of samples per lot reduces the number of analyses and the cost of analysis.  To compensate for the sample dilution effect a lower LOQ would be required.   ADPEN uses state-of-the-art LC-MS/MS equipment in its research and development facilities, therefore it can reach the lower LOQ that would be required to compensate for the sample dilution and has consulted with the FDA and obtained their acceptance. 

12. Is there a tolerance level for nitrofuran metabolites?
Nitrofurans are banned substances and hence should be completely absent from food products. Regulatory laboratories are therefore obliged to try and find residues of these substances at the lowest technically possible concentration. Currently FDA is testing at a 1.0 ppb LOQ.

13. Are there any alternative methods for the analysis of nitrofuran residues?
At ADPEN LC/MS/MS is used. So far only LC-MS/MS offers a reliable procedure taking the limit of detection and the selectivity into account.

14. Is there a health risk in consuming products containing a residue of nitrofurans?
Toxicologists calculated that there is no acute health risk if occasionally a product containing residue of a nitrofuran is consumed. However, since there is reasonable doubt with respect to the health risk involved with nitrofurans and their carcinogenic and mutagenic effects, the substances were banned for veterinary use and should be completely absent from food products. 

15. Are the residues of nitrofurans stable in common food preparation procedures like cooking and baking?
The stability of tissue-bound residues of nitrofuran drugs has been extensively studied. It has been found that upon common food preparation techniques like cooking, baking, grilling and microwaving, the tissue-bound residues of nitrofurans were stable and did not degrade to a significant extent. 

16. Are the residues of nitrofurans that are ingested by consumption of a contaminated product, bio-available in the consumer?
The bio-availability of tissue-bound residues of nitrofurans has been a subject of study. The overall conclusion from these studies is that tissue-bound residues of furazolidone are bio-available, are absorbed by the consumer and again form tissue-bound residue in the consumer as well. 

17. What are likely causes of nitrofuran residues in animals?
Contamination of honey appears to be related to the use of banned antibiotic products in some cases. For shrimp there are more and more indications that the origin of residues is treatment of shrimp in a very early stage of the life cycle.  In shrimp fry from treated tanks the corresponding nitrofuran tissue-bound residues were detected. Similarly, in model experiments using cold-water shrimp, tissue-bound residues were detected in exposed shrimp. For poultry the most likely cause of residues are either administration via feed or drinking water or exposure via contaminated feed. From feeding experiments at therapeutic levels, high residue levels in broilers were detected. These results indicate that even low level contamination of feed is likely to give rise to measurable residue levels in broilers. 

18. What about the analysis of the parent drugs?
The analysis of parent drug is not very useful in animal tissue due to the rapid depletion and metabolism. For feed and water however, the analysis of parent drugs should be performed.

19. Can the metabolite molecules that are considered markers of nitrofurans, originate from other sources than nitrofuran use?
AOZ is specifically indicated as the marker residue for furazolidone (see FAQ6), other markers for other nitrofurans are indicated in literature and have been derived from the parent drug molecular structure in analogy with furazolidone. Before using SEM, which is a very small molecule, as a marker for nitrofurazone, an extensive literature survey was conducted, this survey did not reveal any other known naturally occurring source or precursor of SEM.  There is currently no doubt concerning the other marker metabolites

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