Food QC and nutritional testing

Protecting the food supply: rapid, specific analysis of melamine by UPLC/MS/MS

Incidences of kidney stones and renal failure in infants have been reported in China since July 2008 and are believed to be associated with the ingestion of infant formula intentionally contaminated with melamine.  Allegedly, nitrogen-rich melamine was added to diluted, raw milk to boost the apparent protein content. Over 300,000 people became ill after ingesting the milk, with more than 13,000 hospitalised and a number of deaths reported. Following this outbreak, the Government of China has taken measures to eliminate the practice of food and drink adulteration. Court trials have been held for a number of people linked to the scandal and, in January 2009, two people were sentenced to death¹ .

Melamine has many industrial uses, including the production of laminates, adhesives and melamine resins, some of which may contact foods, leaving a trace level of melamine residue. Common items such as cleaning products, countertops, dry erase boards and glues often contain melamine. Additionally, melamine has been reported to be a metabolite of the pesticide cryomazine².

The tolerable daily intake (TDI) for melamine has been established by the US FDA at 0.63 mg/kg body weight³ per day in food other than infant formula. The TDI quoted by the European Food Safety Authority (EFSA) is in broad agreement at 0.5 mg/kg body weight⁴. More recently, the FDA has applied an additional 10-fold safety factor for infant formula. Infants may be more susceptible than adults to melamine exposure because infant formula may be the sole source of nutrition for some infants. Underdeveloped renal function in infants, compared to adults, increases the exposure and sensitivity to melamine.

Maximum permitted concentrations for melamine in adult foods are typically around 2.5 μg/kg (EU, US and Hong Kong); however, Hong Kong has set a tolerance at 1 μg/kg in infant foods. Additionally, member states of the European Union are required under Commission Decision 2008/757/EC to ensure that all products containing at least 15% milk product ingredient and originating from China, are tested before import and that all products which are shown to contain melamine in excess of 2.5 mg/kg are disposed of⁵.

During the 2007 pet food recall in North America, melamine-tainted pet foods caused significant renal damage in dogs and cats. The cause was traced to the presence of melamine and cyanuric acid, a structural analogue of melamine. While individually melamine and cyanuric acid post low acute toxicity, a mixture of these two compounds forms an insoluble precipitate in renal tubules leading to progressive tubular blockage and degeneration⁶ . This recall promoted the initiation of melamine monitoring in food, which had not been routinely done due to the previously assumed low toxicity of the compound.

There is, therefore, a need to develop methods for the detection of both melamine and cyanuric acids capable of quantifying both compounds in infant formula milk products from ppm (mg/kg) levels down to very low ppb (ug/kg) levels.  The FDA issued an interim method for determining residual melamine and cyanuric acids in foods using LC/MS/MS.  In this method, melamine and cyanuric acids are extracted from infant formula with a 50/50 acetonitrile-water solution.  After centrifugation, two aliquots of each extract are individually cleaned up using two different mixed-mode ion-exchange solid-phase extraction (SPE) procedures.  Oasis MCX is used to selectively extract melamine from the sample and Oasis MAX is used to selectively extract cyanuric acid.  Following extraction, the FDA suggests using a HILIC HPLC method for analysis.

HPLC analysis requires the use of matrix-matched calibration in order to minimise signal suppression.  Stable isotope labelled internal standards were also utilised in order to increase the accuracy of the quantitative analysis. When utilising the Waters Atlantis HILIC column, the results that were obtained for melamine and cyanuric acid were consistent with the FDA method, demonstrating good linearity across a wide range of concentrations.

Following HPLC analysis, the tainted infant formula samples were analysed with an ACQUITY UPLC system to increase method optimisation.  The ACQUITY system utilises sub 2 μm particle LC columns, providing greater separation speed, sensitivity and resolution.   For this analysis, the ACQUITY was combined with a TQD MS detector.  The application of the UPLC for this analysis provided significant advantages in speed and sensitivity. Results were obtained in less than two minutes for melamine and less than 0.75 minutes for cyanuric acid.  For samples containing melamine in liquid infant formula, the LOQ of 20 ppb on the HPLC HILIC column was reduced to 3 ppb using the ACQUITY UPLC BEH HILIC colum⁷. 

With the desire of food producers worldwide to demonstrate due diligence regarding the safety and quality of milk products, the analysis of melamine and its metabolites using the ACQUITY UPLC system offers significant business advantages in meeting the challenge of timely, uninterrupted supply of product, while simultaneously ensuring the safety of consumers.

¹ British Broadcasting Corporation 2009. Chinese Milk Scam Duo Face Death [Online]. Available from: http://news.bbc.co.uk/2/hi/asia-pacific/7843972.stm [Accessed 22 January 2009].

² Tomlin, C.D.S. (Editor). The Pesticide Manual, Eleventh Edition, The British Crop Protection Council. 1997; 321-322.

³ US FDA: Interim Melamine and Analogues Safety/Risk Assessment.  May 25, 2007.  US FDA/CFSAN – Interim Melamine and Analogues Safety/Risk Assessment.

⁴ EFSA:  EFSA’s provisional statement on a request from the European Commission related to melamine and structurally related compounds such as cyanuric acid in protein-risk ingredients used for feed and food.  June 7, 2007.

⁵ European Commission decision (2008/798/EC) imposing special conditions governing the import of products containing milk or milk products originating from China.

⁶ Dobson, R.L.M. et al. Identification and Characterization of Toxicity of Contaminants in Pet Food Leading to an Outbreak of Renal Toxicity in Cats and Dogs. Toxicological Sciences 2008;106(1):251-262.

⁷ Shia, J. and Diehl, D. Protecting the Food Supply:  Rapid, Specific Analysis of Melamine and Cyanuric Acid in Infant Formula by LC/MS/MS. Chemistry Applied Technology, Waters Corporation.  Application Note 720002865EN.  December 2008.

Article by Waters Corporation

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