2. Prospective study on five commercial aquafeeds
• Five aquafeeds containing porcine hemoglobin powder as feed material were
analysed to assess the potential of the method in explaining the origin of the
PCR positive result for ruminant DNA for AQF#1.
• The same MRM transitions as for point 1. were used.
AQF#1 AQF#2 AQF#3 AQF#4 AQF#5
Rumin . biomark er (E FT P VL QADF QK ) P or cine biomark er (VL QSF SDG LK)
1. LOD evaluation
• LODs were evaluated for two biomarkers (one ruminant + one porcine) on
two feed matrices adulterated with a mixed ruminant/porcine blood meal.
• Signal-to-noise ratio (S/N) was calculated for the two most intense multiple
reaction monitoring (MRM) transition of each biomarker.
Context: Blood derived products are valuable animal products used in
feed for their nutritional value and their positive effects on growth and
health. Nevertheless, since the BSE crisis, the use of animal by-products
is strictly regulated. The identification of the species of origin and the
protein type is therefore crucial to ensure feed safety.
The objective was to set-up a routine mass spectrometry method
[1]for
the specific detection of blood meal and hemoglobin powder. This
method has to reach the limit of detection (LOD) of 0.1 % w/w imposed
by the European Commission (EC) for the detection of processed animal
proteins.
Rumin. biomarker (EFTPVLQADFQK) Porcine biomarker (VLQSFSDGLK) Rumin. biomarker (EFTPVLQADFQK) Porcine biomarker (VLQSFSDGLK)0
%
0.05
%
0.1
%
Walloon Agricultural Research Centre
Valorisation of Agricultural Products Department
Food and Feed Quality Unit
www.cra.wallonie.be
Specific detection of blood derived products
in animal feed using UPLC-MS/MS
Lecrenier M.C.
1, Dieu M.
2, Veys P.
1, Planque M.
3& Baeten V.
11
Food and Feed Quality Unit of Agricultural Product Valorisation Department - CRA-W, Belgium;
2
MaSUN Mass Spectrometry facility - University of Namur, Belgium;
3Health Department – CER Groupe, Belgium.
m.lecrenier@cra.wallonie.be; FoodFeedQuality@cra.wallonie.be
[1] Marbaix, H. et al. (2016). Identification of Proteins and Peptide Biomarkers for Detecting Banned Processed Animal Proteins (PAPs) in Meat and Bone Meal by Mass Spectrometry. Journal of
Agricultural and Food Chemistry, 11, 2405-2414.
[2] Lecrenier, M. C. et al. (2016). Identification of specific bovine blood biomarkers with a non-targeted approach using HPLC ESI tandem mass spectrometry. Food Chemistry, 213, 417-424.
2. Prospective study on five commercial aquafeeds (AQF)
Aquafeeds containing porcine hemoglobin powder. Their analysis using official PCR method gave: positive result for ruminant DNA for AQF#1
negative results for ruminant DNA for the other AQF (AQF#2, AQF#3, AQF#4 and AQF#5)
By official methods (PCR and light microscopy), it is impossible to determine whether ruminant DNA originates from authorised or non-authorised by-products.
Materials & study design
Method
Introduction
1. LOD evaluation
Feed matrices: Pig feed and aquafeed
Adulterant: mixed blood meal (80 % ruminant, 20 % porcine blood)
Results
• The method is highly specific: no signal was obtained in blank feeds.
• LODs were defined as the minimum concentration giving a S/N ratio > 3 for the most
intense MRM transition.
• The method is sensitive: 0.05 % (w/w) of a mixed ruminant/porcine blood meal was
detected. This level corresponds to ~ 0.04 % ruminant blood meal and ~ 0.01 % porcine blood meal.
• S/N varied depending of the feed. Matrix effect can affect the LOD.
• Porcine blood derived products are allowed to be used in aquafeed.
• By using UHPLC/MS-MS, blood derived products were easily detected in feeds at
the concentration level usually used in feed for fish ( ~ 10 % w/w). This shows the applicability of the method on real commercial samples.
• Ruminant blood derived products are not allowed to be used in animal feed. • Ruminant blood was detected in AQF#1 while it was absent in the other AQFs.
• UHPLC/MS-MS could be an efficient method to confirm positive results obtained by
PCR, indicating in addition the type of by-product used and therefore the presence or not of non-authorised animal by-products.
Conclusion and perspectives
Adulteration levels : 0.05 % to 1 %
Biomarkers:
8 ruminant + 1 porcine hemoglobin peptides identified by high resolution MS [2]
3 – 4 transitions were selected for each biomarker
Sample preparation protocol:
Extraction: TCA/aceton precipitation, Dige labelling buffer (DLA) extraction Purification: 2-D Clean-Up Kit (GE Healthcare, USA)
Digestion: trypsin
UHPLC-MS/MS:
Waters Acquity system (C18 BEH130 Column; 2.1 x 150 mm) Waters Xevo TQS triple quadrupole
I = 1.1E+6 S/N = 554 I = 5.7E+5 S/N = 236 I = 1.7E+6 S/N = 584 I = 7.8E+5 S/N = 341 I = 8.3E+5 S/N = 54 I = 7.2E+5 S/N = 11 I = 1.3E+6 S/N = 81 I = 1.1E+6 S/N = 9 I = 2.1E+5 S/N = 52 I = 8.5E+4 S/N = 45 I = 4.0E+5 S/N = 73 I = 1.7E+5 S/N = 68 I = 7.0E+4 S/N = 27 I = 8.0E+4 S/N = 3 I = 1.9E+5 S/N = 36 I = 1.8E+5 S/N = 5 I = 1.5E+4 S/N = 31 I = 1.2E+4 S/N = 7 I = 1.0E+8 S/N = 37 I = 1.1E+8 S/N = 49 I = 1.2E+8 S/N = 63 I = 1.1E+8 S/N = 79 I = 8.5E+7 S/N = 31 I = 7.0E+7 S/N = 39 I = 9.1E+7 S/N = 50 I = 7.3E+7 S/N = 65 I = 1.2E+8 S/N = 46 I = 1.0E+8 S/N = 60
References
In first estimation, the method would be able to detect ruminant and porcine blood at levels of 0.05 % w/w which is below the 0.1 % LOD required
by the EC for animal proteins detection methods. The combination of these biomarkers could give an accurate answer about the feed material used
and the species origin of the proteins in support to the official methods.
Figure 1: Chromatograms of the 2 higher MRM transitions of ruminant biomarker EFTPVLQADFQK (711.9 > 1041.6 & 711.9 > 736.4) and porcine biomarker VLQSFSDGLK (547.3 > 753.4 & 547.3 > 881.4) in pig or aqua- feed.Figure 2: Chromatograms of the 2 higher MRM transitions of ruminant biomarker EFTPVLQADFQK (711.9 > 1041.6 & 711.9 > 736.4) and porcine biomarker VLQSFSDGLK (547.3 > 753.4 & 547.3 > 881.4) in 5 aquafeeds.
