¹³C NMR detection of non-protein nitrogen substance adulteration in animal feed

Illegal adulteration of melamine in animal feed and food has been widely studied. However, the risk of using substitute non-protein nitrogen substances still exists. In this study, we developed the ¹³C NMR method for the detection of non-protein nitrogen substance adulteration in animal feed. Three compounds, i.e., urea, melamine, and biuret, were used for method development. We found that the chemical shifts of the characteristic peaks in the carbon spectra of high-nitrogen adulterants were all between 150 and 170 ppm, whereas the chemical shifts of real protein peptide bonds (-CO-NH-) were between 170 and 180 ppm, demonstrating a good distinction between non-protein nitrogen and authentic protein. The method for analyzing melamine, urea, and biuret was validated. The R² values were all above 0.99 within the calibration range of 0.05-2% (w/w). The limits of quantification of urea, melamine, and biuret were 0.0120%, 0.0660%, and 0.0806%, respectively. This method involves simple sample pretreatment and rapid detection while also providing high accuracy. All the sample information obtained by NMR detection does not require strict impurity removal. Compared with a previously reported ¹H NMR method, the developed ¹³C NMR method does not require strict moisture removal to avoid active hydrogen exchange, and the interfering peak overlap is mitigated.