A Stable Isotope and Chemometric Framework to Distinguish Fresh Milk from Reconstituted Milk Powder and Detect Potential Extraneous Nitrogen Additives

Stable isotopes (delta H-2, delta C-13, delta N-15 and delta O-18) of 232 milk powders and 88 fresh milk samples collected globally were used to distinguish fresh milk from reconstituted milk and qualitatively detect extraneous nitrogen in milk products. Nitrogen isotopes of six nitrogen-containing milk additives, including animal protein (peptone), soy protein, and four inorganic nitrogen additives (urea, melamine, ammonium chloride and biuret), were characterised to identify potential extraneous nitrogen (PENs) that could be fraudulently used to increase the protein N content of milk. Results showed that fresh milk and reconstituted milk can be distinguished by using stable isotopes combined with chemometrics (PCA, LDA and ANN) methods. LDA achieved accuracy rates of 94.9 % and 94.9 % for the testing and training sets respectively, while ANN had higher accuracy rates, up to 99.6 % for the training set, and 96.6 % for the testing set. Nitrogen stable isotopes were also found to be a useful tool to identify the addition of PENs in milk and could accurately detect the addition of inorganic nitrogen and soybean protein to less than 20 % of the total nitrogen in milk. The methods established in this paper are simple, efficient and fast, and can be used for the regulatory supervision of domestic and imported milk products by food safety testing agencies.