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Determination of emerging nitrogenous economic adulterants in milk proteins by high‐performance liquid chromatography/compact mass spectrometry


Milk‐derived ingredients are widely used around the world in the manufacturing of nutritional products. They are prone to economically motivated adulteration with nitrogenous compounds such as melamine and its analogs in order to increase the nitrogen content of these ingredients. The need to rapidly screen milk‐derived ingredients to detect adulteration is of paramount public health concern. A liquid chromatography/mass spectrometry (LC/MS)‐based method using a single quadrupole mass spectrometer has been developed for the rapid frontline analysis of six nitrogenous protein adulterants, i.e. melamine, ammeline, ammelide, amidinourea, cyromazine and cyanuric acid, in three key milk‐derived ingredients, i.e. whole milk powder, nonfat milk powder and whey protein concentrate.


The sample preparation scheme involves both 'dilute and shoot' as well as solid‐phase extraction (SPE)‐based methods. The 'dilute and shoot' scheme uses a tenfold dilution of sample with water followed by protein precipitation using 2% formic acid in acetonitrile. The SPE scheme involves tenfold dilution of sample with water, followed by protein precipitation using acetonitrile, and further cleanup through Strata Melamine SPE cartridges. Sample extracts were analyzed by hydrophilic interaction chromatography/single quadrupole mass spectrometry (HILIC/MS) in both positive and negative electrospray ionization mode. Accurate quantitation was achieved using stable isotope labeled internal standards.


A multi‐day method validation study was conducted using three different milk‐derived ingredients. Average accuracies, relative standard deviations (RSD) and method detection limits (MDL) for all analytes in whole milk powder were 65–118%, 7–11% and 0.9–30 mg/kg, using the 'dilute and shoot' extraction procedure. The SPE procedure results were 102‐111%, 5–13%, and 0.4–2.5 mg/kg, respectively, for melamine, ammeline, ammelide and cyromazine only.


A rugged and simple to use analytical method to screen for the presence of nitrogenous economic adulterants in milk‐derived ingredients has been developed for routine frontline use. Copyright © 2016 John Wiley & Sons, Ltd.

Authors:   Jonathan Draher, Stefan Ehling, Nick Cellar, Todime Reddy, Jack Henion, Nigel Sousou
Journal:   Rapid Communications in Mass Spectrometry
Volume:   30
edition:   11
Year:   2016
Pages:   1265
DOI:   10.1002/rcm.7568
Publication date:   27-Apr-2016
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