UHPLC-MS/MS method for sensitive, specific and simultaneous detection of bovine blood meal, blood products and milk products in compound feed

Feed availability is one of the biggest challenges for the future. Solutions will be found by increasing the production efficiency and finding new sources without jeopardizing feed quality and safety.

Animal by-products are an interesting source of feed materials. These materials are rich in proteins of high nutritional value and have an economic interest since their non-use results to a logical loss of gains.
However, since the mad cow disease crisis, their use has been strictly regulated. In 2013, non-ruminant processed animal proteins (PAPs) were reauthorised in aquafeed but ruminant PAPs remain forbidden. Official controls are based on a combination of light microscopy and PCR. But sometimes these methods are unable to distinguish some feed materials.

The objective of this work was to develop a sensitive method using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) for the specific detection of bovine blood-derived products and milk powder in feed. This method has the advantage to be species and tissue specific. Peptide biomarkers identified in previous studies were used. The sample preparation and the analytical method were designed to provide a fast, simple and powerful method suitable for routine.
Proteins were extracted in a buffer containing 200 mM TRIS-HCl pH 9.2, 2 M urea followed by trypsin digestion and purification with tC18 SPE (Waters). Analyses were performed by liquid chromatography (Acquity UHPLC system, Waters) coupled with a triple quadrupole mass spectrometer (Xevo TQS, Waters). Labelled peptides were used as internal standard in order to compare the results independently of the retention time variation due to matrix effect.

Various commercial aquafeed batches artificially adulterated at levels of 0.1 % to 1 % (w/w) with bovine blood meal, bovine blood products or milk powder were analysed in order to assess the influence of matrix and to evaluate sensitivity and specificity of the method.

The method was able to detect all adulterants at 0.1 % level in all matrices. This makes the method suitable for application in feed control and offers an innovative and complementary solution for the simultaneously identification of authorised and unauthorised animal by-products such as PAPs.