Melamine has caused a great deal of anxiety in the past two years for parents and pet owners alike. First of all, it was found in pet food in the United States that was prepared with imported wheat gluten and rice protein concentrate from China, leading to the deaths of many cats and dogs due to kidney failure. Then in 2008, the same chemical was discovered as an adulterant in infant formula and related dairy foods in China. In this case, more than 51,000 children were hospitalised, according to the WHO, and 6 deaths were confirmed.

The cyclic compound melamine, also known as cyanuramide, caused urinary problems in the children such as kidney stones, which are usually exceptionally rare in infants. Renal tube blockages also occurred and, in the extreme cases, kidney failure. Non-fatal cases were also reported in neighbouring countries that imported contaminated products, including Hong Kong, Macao and Taiwan.

So, what was melamine doing in the infant formula in the first place? In China, where the adulteration originated, raw milk was diluted to increase its volume but this lowered the protein concentration. The addition of melamine, with its considerable nitrogen content, fools the subsequent checks for protein levels, which measure the total nitrogen content in the milk. Needless to say, melamine addition to food is not approved by any national authorities.

Although several analytical methods exist for measuring the melamine content of foods and feeds, a team of Taiwanese scientists has identified a gap in urinary analysis. They reported that only one method exists, but it requires derivatisation before the GC/MS analysis. So, the team has devised a non-derivatisation method based on ultra-performance LC (UPLC) linked to tandem mass spectrometry on a triple quadrupole instrument.

In the first instance, I-Jen Wang and colleagues from Taipei Hospital, the Taipei Department of Health, Taipei Medical University and the China Medical University in Taichung optimised the method with standard solutions of melamine. A hydrophilic interaction chromatography (HILIC) column was employed. The material had a small particle size (1.7 µm) that allows higher pressure to be applied to the column, increasing sensitivity.

A stable isotope-labelled internal standard with ring-¹³C₃ and amine-¹⁵N₃ substitution was added and the mobile phase was an isocratic mixture of acetonitrile containing 20 mM ammonium acetate (95:5, v/v). At a flow rate of 0.4 mL/min, the melamine retention time was just 3.1 minutes.

The mass spectrometer was operated in multiple reaction monitoring mode, following the fragmentation of the protonated molecular ions of melamine and the internal standard to ions at m/z 85 and 89, respectively, corresponding to the loss of a CH2N2 molecule. Calibration was linear from 0.01-0.5 µg/mL.

The optimised method was applied to urine samples from 10 affected children who were diagnosed with kidney stones and who might have consumed melamine-contaminated dairy products. In addition, urine from 20 age- and gender-matched controls was tested. Samples were acidified and subjected to a simple SPE step using a mixed-mode ion exchange cartridge to produce concentrated melamine solutions for analysis. The recoveries were 96-99% and the urinary detection and quantitation limits were 0.006 and 0.01 µg/mL, respectively.

All of the controls showed melamine levels below the detection limit apart from two, both of which contained 0.02 µg/mL, or 2.3 and 2.6 µg/mmol creatinine. These two gender-matched girls were 9 years old, so must have been exposed to melamine by a different route than infant food, perhaps different dairy products or environmental exposure. So, 0.02 µg/mL was set as the melamine background level for Chinese children.

Of the 10 kidney stone cases, three had elevated levels of melamine of 0.03, 0.08 and 0.30 µg/mL, or 9, 50 and 71 µg/mmol creatinine. The other 7 were below the detection limit. So, it is not yet clear whether melamine is associated with kidney stones in all of the cases tested.

The researchers argued that, because only spot urine samples were collected, the melamine could have been eliminated before sampling since its half-life is only 3 h. In addition, some samples had not been collected immediately after exposure. They recommended that repetitive urine samples be taken on visits to clinics in an attempt to confirm melamine as the cause of kidney stones.

Future studies should include a larger number of subjects to help to establish a cut-off point of clinical relevance and to confirm the background levels of melamine in children's urine.

Related links:

• Rapid Communications in Mass Spectrometry 2009, 23, 1776-1782: "Determination of urine melamine by validated isotopic ultra-performance liquid chromatography/tandem mass spectrometry"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.