Detection of 3-amino-1,2,4-triazine adulteration in milk using an oxidation product 3-amino-1,2,4-triazin-5(2H)-one

A rapid liquid chromatography–mass spectrometry method to detect 3-amino-1,2,4-triazine (ATZ) in milk was developed as part of a programme to set up methods for detecting the economically motivated adulteration of raw milk with nitrogen-containing compounds. When ATZ was added to unpasteurised or pasteurised milk at levels of 10–1000 ppm, the levels declined over a period of a few days and in some cases declined below the limit of detection of the analytical method (1 ppm). ATZ did not degrade in deproteinised milk extracts, in aqueous standards or in aqueous (non-milk) controls, suggesting that degradation was mediated by a pasteurisation-resistant enzymatic or microbial process. An oxidation product of ATZ was detected by mass spectrometry, and a tentative structure for this compound (3-amino-1,2,4-triazin-5-one, ATZO) was determined by fragmentation analysis and high resolution mass spectrometry. The accumulation of this oxidation product correlated with the loss of ATZ in milk samples. It was concluded that the detection of ATZO could be used as a marker for the addition of ATZ and that both compounds should be monitored during surveys looking for the ATZ adulteration of milk.