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Mass Spectrometry-based Immunoassay for the Quantification of Banned Ruminant Processed Animal Proteins in Vegetal Feeds Andreas E. Steinhilber, Felix F Schmidt, Wael Naboulsi, Hannes Planatscher, Alicia Niedzwiecka, Jutta Zagon, Albert Braeuning, Alfonso Lampen, Thomas O. Joos, and Oliver Poetz Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00120 • Publication Date (Web): 22 Feb 2018 Downloaded from http://pubs.acs.org on February 23, 2018
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Analytical Chemistry
Mass Spectrometry-based Immunoassay for the Quantification of Banned Ruminant Processed Animal Proteins in Vegetal Feeds Andreas E. Steinhilber1, Felix F. Schmidt1, Wael Naboulsi3, Hannes Planatscher3, Alicia Niedzwiecka2, Jutta Zagon2, Albert Braeuning2, Alfonso Lampen2, Thomas O. Joos1, and Oliver Poetz*1,3 1
NMI Natural and Medical Sciences Institute at the University of Tuebingen, 72770 Reutlingen, Germany Federal Institute for Risk Assessment, 10609 Berlin, Germany 3 SIGNATOPE GmbH, 72770 Reutlingen, Germany 2
ABSTRACT: The ban of processed animal proteins (PAPs) in feed for farmed animals introduced in 2001 was one of the main EU measures to control the bovine spongiform encephalopathy (BSE) crisis. Currently, microscopy and polymerase chain reaction (PCR), are the official methods for the detection of illegal PAPs in feed. However, the progressive release of the feed ban, recently with the legalization of non-ruminant PAPs for the use in aquaculture, requires the development of alternative methods to determine the species origin and the source (legal or not). Additionally, discussions about the need for quantitative tests came up, particularly if the zero-tolerance-concept is replaced by introducing PAP thresholds. To address this issue, we developed and partially validated a multiplex mass spectrometry-based immunoassay to quantify ruminant specific peptides in vegetal cattle feed. The workflow comprises a new sample preparation procedure based on a tryptic digestion of PAPs in suspension, a subsequent immunoaffinity enrichment of the released peptides and a LC-MS/MS based analysis for peptide quantification using isotope labelled standard peptides. For the very first time, a mass spectrometry-based method is capable of detecting and quantifying illegal PAPs in animal feed over a concentration range of four orders of magnitude with a detection limit in the range of 0.1 % to 1 % (w/w).
The bovine spongiform encephalopathy (BSE) crisis in the European Union has led to several new regulations concerning the use of processed animal proteins (PAPs) in feed for farmed animals. One important regulation was the feed ban for mammalian derived proteins introduced with Commission Regulation (EC) No. 999/2001 and later expanded to all animal proteins by the amendment No. 1234/2003 1,2. The regulation prohibited the feeding of PAPs, such as meat and bone meal (MBM), to animals that are intended for human consumption. Prion contaminated cattle MBM, which was supposed as the main vector of disease, was successfully excluded from animal feed which has led to a decreasing number of registered BSE cases nearly down to zero in Europe including UK, the former hotspot of the BSE pandemic 3,4. PAPs are produced from animal by-products that are not intended for human consumption. In addition fishmeal produced from water animals excluding mammals is a separately defined type of PAP 5,6. PAPs are known to provide high levels of fat, proteins, minerals, and essential vitamins, and therefore can be considered to improve the nutritional value of feed for monogastric animals like chicken and pigs or in aquaculture 7-9. Particularly blood meals (BM), representing a special category of PAP, are used to replace expensive fish meal as protein source in fish feed 9. Furthermore, lower processed blood products such as spray-dried hemoglobin meal (SDHM) and spraydried plasma (SDP) in the sense of the Regulation (EU) No. 142/2001 10 are two types of animal feed that are gaining popularity due to their high digestibility 11,12. For economic and ethical reasons, the European Commission is currently working on the reintroduction of non-ruminant PAPs in feed. The first steps without by-passing the ban for intra-species feeding 13 were
done with the allowance of fishmeal in milk replacer for weaning calves in 2008 14 and for non-ruminant PAPs in feed for aquaculture in 2013 15, amended by insects in 2017 16. Future EU plans comprise legalizing PAPs from pig and poultry origin (see Tab. S1) 17. The progressive lifting of the feed ban demands for methods with high sensitivity and specificity to detect and to prove the origin of PAPs to ensure the absence of ruminant material. In this regard the risk assessment as well as the analytical methodology should cope with the real-life situation in the production chain. Minor cross contamination due to allowed animal proteins (e.g. derived from milk powder) or transport between slaughter houses, rendering plants and feed producers can never be ruled out. Therefore producers, represented by the European Fat Processors and Renderers Association (EFPRA), but also control laboratories call for quantitative accurate thresholds to replace the pending zero-tolerance-concept. However, any decision on threshold levels of the European Commission will depend on a thorough risk assessment. A risk study of the European Food Safety Agency (EFSA) in the year 2011 18 concludes that a contamination level of 0.1 % of non-ruminant PAP in feed would lead to less than one BSE infected cattle in the EU per year with an upper 95 % confidence level 19,20. EFSA currently works on an updated risk assessment as well as the implementation of a threshROG µWHFKQLFDO ]HUR¶ IRU PLQRU UX minant cross contamination on demand of the European Commission 21. If and at which tolerance level finally thresholds for ruminant and non-ruminant PAPs will be implemented is not yet clear but it can be strongly assumed that quantitative methods will be required with a sensitivity in the range of
