Lactose semicarbazone as a marker for semicarbazide adulteration in milk

• Semicarbazide is a potential adulterant, used to increase nitrogen in milk.
• We found that spiked semicarbazide partially disappeared from milks.
• In milk, a semicarbazone is formed with lactose under ambient conditions.
• We have confirmed the semicarbazone in milk, and developed the assay.
• Semicarbazide adulteration can be detected by assay of the semicarbazone.

A liquid chromatography–mass spectrometry method to detect semicarbazide and lactose semicarbazone 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. The detection of semicarbazide was hampered by that fact that this compound tended to give broad, poor intensity peaks in the hydrophobic interaction chromatographic method employed. When spiked into milk at levels of 20–200 ppm, semicarbazide either partially or completely reacted with the matrix, which both increased the limit of detection of the method and made the setting of a threshold by using low level spikes almost impossible. Thus using lactose semicarbazone as a marker for semicarbazide addition to milk was investigated. Lactose semicarbazone was detected in semicarbazide-spiked milk, and its identity was confirmed by fragmentation analysis and comparison with the synthesised compound. The level of lactose semicarbazone correlated with the amount of semicarbazide added to the milk, and the acidic conditions employed in the extraction method appeared to enhance the sensitivity of detection by driving the semicarbazone-forming reaction towards completion. Thus lactose semicarbazone can be used as a marker for the addition of semicarbazide to milk; however, both compounds should be monitored during surveys looking for the semicarbazide adulteration of milk.