Recent food safety scares, such as the discovery of melamine in milk, have sharpened the global awareness of the links between profit, the food chain and the global food supply. Melamine contamination has now caused illness in more than 300,000 infants and is linked to a growing number of infant deaths. A cheap industrial chemical, melamine has been used to artificially increase the amount of protein content in diluted milk, thereby increasing the price for the milk.

The combination of melamine and a degradation product, cyanuric acid, results in crystals that can create blockages in the kidneys. In March 2007, public awareness of melamine contamination was heightened when the contaminant was found in pet food ingredients imported from China, causing the death of many animals. Following this scare, it was revealed that melamine-tainted fodder may have been used to feed animals, including chickens, swine and catfish intended for human consumption. As melamine is still being found in eggs, fish and a variety of processed foods imported from China, the scrutiny of products for melamine is intense.

Melamine contamination can also have an economic impact. Voluntary and mandatory product recalls and detentions involve a significant cost to food companies. In November 2008, the FDA issued a detention order for milk products imported from China.[1] As a result, companies exporting food into the US must prove that products are free of contaminants including melamine through independent third-party analysis in order to avoid the detention of products.

As a result of food safety scares such as the discovery of melamine, both consumers and food producers are seeking assurance that food products and ingredients are not contaminated with industrial chemicals. The official quality control methods for measuring the total protein content in food, the Kjeldahl or Dumas methods, are not specific enough to differentiate between industrial chemicals and proteins. Both methods measure the total nitrogen content in a sample and correlate the result to protein. Melamine is high in nitrogen but not protein, meaning that it can be used to imitate protein in food.

In response to the recent melamine discoveries, the FDA and USDA have published several methods that are capable of screening samples and confirming the presence of melamine, cyanuric acid and melamine metabolites.[2] Additionally, maximum residue levels (MRLs) for acceptable amounts of these contaminants have been set worldwide. In China and the US, the melamine MRL for infant formula has been set at 1 ppm and at 2.5 ppm for milk and other milk products, while in Europe, the Food Safety Authority has set the limit to 2.5 ppm for all products containing greater than 15% milk.

Critical Issues for Melamine Analysis
It is possible to analyze food samples from raw ingredients to the finished product, depending on the aim of the analysis. Thermo Fisher Scientific can provide all the required solutions for a laboratory’s total integrated workflow for melamine detection as well as the instrument, method support and consumables that are necessary for screening and confirmation.

Table 1 provides an overview of analytical techniques describing the advantages, limitations, and efficiency of each in the melamine analysis workflow. The producer and consumer alike are challenged by bottlenecks in the integrated workflow regardless of the technique selected. This can be particularly challenging during the sample preparation stage, which can account for 60% of the time of analysis. Certain approaches are less susceptible to the wide variety of matrices that must be analyzed for melamine to regain consumer confidence, reducing sample preparation time and speeding up the analysis.

Melamine Detection Tools
Enzyme-Linked Immunosorbent Assay (ELISA)
Enzyme-linked immunoassay, or ELISA, is a high-throughput technique for screening food for melamine. ELISA tests can be performed in 96-well microplates, enabling the simultaneous analysis of several samples and standards. The general principle of these competitive ELISA assays is shown in Figure 1.

Figure 1: General principle of the competitive ELISA assay. Enzyme-conjugated melamine competes with the melamine from the sample for binding to melamine antibody. This enzyme activity and absorbance values decrease according to increasing amount of the unlabeled melamine from the unknown sample.

Ultra High Performance Chromatography-Ultraviolet Absorption (UHPLC-UV)
Using UHPLC for melamine analysis, separation times can be reduced to less than two minutes. Analysts can rapidly and economically screen samples for melamine at the threshold levels set by EU, USA, and Chinese governments using instrumentation suitable for other types of food analysis by using a photodiode array (PDA) UV detector. Figure 2 shows the UHPLC-PDA analysis workflow. The same system can be coupled to a Thermo Scientific triple quadrupole mass spectrometer for performance as a confirmation tool.

Figure 2: UHPLC-PDA Analysis Workflow

Gas Chromatography –Tandem Mass Spectrometry (GC-MS/MS)
The FDA method for GC-MS/MS first requires the derivatization of cyanuric acid and melamine and its metabolites, which is followed by the analysis of the compounds in a single injection. This technique offers significant benefits as it is rapid and capable of providing confirmed identification and low limits of detection. However, as this method required the additional step for derivatization, it can be more time-consuming than other techniques. Nonetheless, it meets the FDA and Chinese requirements and allows many labs to use existing equipment or purchase equipment they will find useful for other applications in food analysis such as the analysis of pesticides, persistent organic pollutants, flavor agents and other volatile food analysis targets. Figure 3 shows the FDA GC-MS/MS workflow in diagram form.

Figure 3: FDA GC-MS/MS Workflow for the Thermo Scientific ITQ Ion Trap GC-MS/MS 700

Thermo Fisher provides several GC-MS/MS solutions for melamine analysis, including the ITQ™ series and TSQ Quantum GC. Figure 3 shows a method developed for the Thermo Scientific ITQ ion trap GC-MS/MS 700, providing highly sensitive quantitation down to the low ppb level with more than four orders of magnitude quantitation range. This cost-effective approach was selected to minimize the effect of the different matrices in the food samples on the analysis while maintaining low limits of detection and cost performance. By using the ITQ GC-MS/MS scan function confirmation and quantification are accomplished in a single injection.

Liquid Chromatography – Tandem Mass Spectrometry (LC-MS/MS)
The FDA has published a number of methods for the detection of melamine and cyanuric acid using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This is a versatile technique that can be used to analyze samples from feed, animals and, most recently, milk and milk products. Thermo Fisher offers solutions for the analysis of melamine and cyanuric acid using LC-MS/MS, including the TSQ Quantum™ Ultra triple stage quadrupole system coupled with the Thermo Scientific Accela high-speed chromatography system. This system has been demonstrated on the FDA LC-MS/MS method to monitor melamine and its hydrolytic products in processed foods. The TSQ Quantum triple stage quadrupole system is the only instrument that enables Highly Selective Reaction Monitoring (H-SRM) performance, facilitating the fast and efficient analysis of complex samples such as animal tissue and processed foods. The Thermo Scientific LC-MS/MS solution has yielded accuracy and precision values for FDA methods that are well within the guidelines of the FDA for analytical method development and analysis.

Conclusion
The discovery of melamine in food products highlights the need for rapid and accurate analysis of food including feed, livestock, livestock products (eggs, milk) and processed food. Selecting the right analytical tool and workflow is an important consideration for food manufacturers, processers and exporters and analytical solutions can be implemented throughout the lifecycle of the food, from the factory stage to the export. In order to establish confidence in the quality of food provided, it is essential that analysts have access to tools that allow for fast and accurate screening and confirmation. Thermo Fisher offers the essential tools for melamine detection and works with customers to extend their applicability into different food matrices, providing the necessary assurance that the final food product is high-quality and free from potentially harmful contaminants.

For the latest information about Thermo Fisher Scientific solutions and the wide range of applications for food safety, please call 800.532.4752, e-mail analyze@thermofisher.com or visit www.thermo.com/foodsafety.

References:
1. http://www.cfsan.fda.gov/~comm/labmeth.html#melamine
2. FDA LIB 4423, FDA LIB 4396, FDA LIB 4421, USDA FERN-CHE.0003.00