Rapid detection of economic adulterants in fresh milk by liquid chromatography–tandem mass spectrometry

A method to aid in the detection of the economically driven adulteration of fresh milk with a range of small, nitrogen containing compounds, including melamine, ammeline, ammelide, cyanuric acid, allantoin, thiourea, urea, biuret, triuret, semicarbazide, aminotriazine, 3- and 4-aminotriazole, cyanamide, dicyandiamide, guanidine, choline, hydroxyproline, nitrate, and a range of amino acids, has been developed. 15N2-Urea is used as an internal standard. The adulteration of milk with exogenous urea has previously been difficult to detect because of the variation in the naturally occurring levels of urea in milk. However, by monitoring the contaminants biuret and triuret, which comprise up to 1% of synthetic urea, the adulteration of milk with urea-based fertilizer can be detected. We estimate that to be economically viable, adulteration of the order of 90–4000 ppm of the above adulterants would need to be added to fresh milk. For most of the compounds, an arbitrary detection threshold of 2 ppm is therefore more than sufficient. For biuret, a lower detection threshold, better than 0.5 ppm, is desirable and the sensitivity for biuret and triuret can be improved by the post-column addition of lithium to create lithium adducts under electrospray ionisation. Sample handling involves a two-step solvent precipitation method that is deployed in a 96-well plate format, and the hydrophilic interaction liquid chromatography uses a rapid gradient (1.2 min). Three separate injections, to detect the positively charged compounds, the negatively charged compounds and amino acids and finally the lithium adducts, are used. This rapid and qualitative survey method may be deployed as a second tier screening method to quickly reduce sample numbers indicated as irregular by an FTIR based screening system, and to direct analysis to appropriate quantification methods.

► A rapid LCMS means to spot 36 N-compounds that could be used to adulterate milk.
► Biuret may be used as a marker for synthetic (fertilizer) urea adulteration in milk.
► We threshold our interest to those levels where economic adulteration is plausible.
► The method provides a presence/absence indication for each analyte in ppm range.
► The method screens samples abnormal by FTIR to reduce numbers sent to evidential tests.