The discovery of the toxic combination of melamine and cyanuric acid in food supplies worldwide continues to spread. From the announcement in September of melamine in Chinese baby formula, to the most recent reports of its presence in a wide range of food products containing milk or milk-derived ingredients, the melamine scare grows daily (see IBO 10/15/08). Earlier this month, the Chinese government reported that 300,000 babies were poisoned by contaminated infant formula and six infants died. In November, the US FDA issued an import alert, requiring that Chinese food products that contain milk to be tested for melamine before entering the supply chain. This month, the US Department of Agriculture announced that it will begin random-testing of meat products on market shelves that contain milk-derived ingredients. The EU recently banned the import of children’s food products from China containing more than 2.5 mg/kg of soya. And last week, China announced that it is investigating reports of melamine-contaminated animal feed, after it took action in October to limit the practice following the reports of melamine in eggs.

The scandal has increased demand for testing for melamine and cyanuric acid, as well as two additional melamine analogues, ammeline and ammelide, in food products. Such testing has driven demand from the government, food industry and private-testing sectors for GC, LC and MS systems. Chromatography and MS companies have met these demands with solutions that include sample preparation products, instrumentation, columns and methods. The melamine incidents illustrate not only the instrument solutions that are available for food testing, but also how companies differentiate their offerings.

Melamine and its analogues can be tested using several techniques, including GC/MS, GC/MS/MS, LC-UV, LC-DAD, LC/MS, LC/MS/MS and ELISA. The choice of technique depends on the type of testing (screening, confirmation, quantification) and the product that is being tested. In an October publication based on comments from analytical experts, the World Health Organization (WHO) characterized triple quadrupole LC/MS/MS and GC/MS/MS as among the most reliable methods and capable of a low level of quantification in a variety of matrices. Compared to them, LC/MS and GC/MS offer selectivity that is slightly lower, according to the WHO. For the analysis of complex samples, such as candies, the WHO noted that the use of HPLC-UV is complicated by the “interfering compounds that also will absorb UV at 240 nm.” The WHO reported that ELISA can be useful for screening, but detection limits may be insufficiently sensitive.

Detection and quantification limits for each technique vary by method and the type of product tested. In a document released in November compiling available analytical methods for melamine testing in food and feed, the WHO lists an FDA method for the detection of melamine, cyanuric acid, ammeline and ammelide in various matrices at 2 ppm using GC/MS. A method developed by the French Ministry of Agriculture and Fishing’s LABERCA laboratory specifies a detection limit of 50 ppb for all four compounds in food samples with milk using GC/MS/MS. The only HPLC-UV method referenced in the document for detection of all four metabolites is for cereal flour with detection limits of 5 ppm for melamine, ammeline and ammelide, and 90 ppm for cyanuric acid.

The WHO document lists only one LC/MS/MS method capable of detecting or quantifying melamine and all three analogues. This method is for the detection of all four metabolites in kidney tissue. In October, the FDA published an LC/MS/MS method for the quantification of melamine and cyanuric acid down to 250 ppb in dry infant formula.

As Sandra Rasmussen, vice president of Applied Analytix for PerkinElmer Analytical Sciences, pointed out, GC/MS is one of the few analytical techniques able to detect melamine and all three target analogues. “Based on the extraction methods used for doing LC/MS/MS, two of the metabolites, ammeline and ammelide, can’t be detected because they don’t get extracted efficiently,” she told IBO. According to Mark Kuracina, senior marketing manager for Applied Biosystems Food and Environmental Solutions, LC/MS/MS’s benefits include its sensitivity, ability to screen for a wider range of food contaminants than other techniques and speed. “With GC/MS and LC-UV, you have to do a lot of sample preparation and you also have to derivatize,” he stated, noting that this adds cost and complexity. “And even doing that, you’re only getting detection limits around the ppm range. . . . With LC/MS/MS, you can go down to the low ppb range. ”

Applied Biosystems, PerkinElmer, Thermo Fisher Scientific and Waters are among the instrument providers that have supplied instrument for melamine detection domestically and abroad. The melamine incidents have increased sales for each company. In addressing this application, the companies each emphasize a solutions-based approach, while tailoring their particular product lines to melamine testing.

Thermo Fisher Scientific and Waters’ offerings for the detection of melamine and its metabolites include solid phase extraction products and LC columns, as well as GC, LC and MS instruments. Stuart Cram, vice president of Strategic Marketing at Thermo Fisher Scientific, told IBO that Thermo’s broad range of products and method-development support differentiate its offerings. “I think the major difference is that: 1) we’re the world’s largest supplier of solvents and consumables through Fisher, [and] 2) we’ve spent so much time and effort with methods development and supporting laboratories in the public and private sector to implement these methods.” He emphasized the importance of method development to melamine detection, as each time a new product-type is tested, a new method must be created. “The challenge here is that every time you change the matrix, you’ve got to change the method,” he stated.

James Willis, Waters’s Managing Director for Chemical Analysis, told IBO that the use of the company’s solid phase extraction products with its instrumentation provide rapid and accurate results. Waters offers a LC/MS/MS method for the detection of melamine, cyanuric acid and ammeline in pet food in less than two minutes. Its LC/MS/MS method for the detection and quantitation of melamine in infant formula and liquid milk down to the low ppbs employs the company’s Condition Oasis MCX cartridges, BEH HILIC column, ACQUITY UPLC and TQ MS Detector with Multiple Reaction Monitoring. Waters also offers GC/MS and GC/MS/MS solutions.

For Applied Biosystems, part of Life Technologies, and PerkinElmer, the choice of products are less exhaustive, but no less compelling, according to the companies, which have crafted their own distinctive approaches to the market. Applied Biosystems offers LC/MS/MS systems. According to Mr. Kuracina, Applied Biosystems typically suggests the API 3200 LC/MS/MS for applications involving melamine detection, but customers have purchased the API 4000, 5000 and 5500 systems for melamine applications. Also among Applied Biosystems’ offerings are its Cliquid and iMethod software, which improve ease of use and facilitate the sharing of methods between labs. “[Cliquid] is very simple software that sits on top of the operating system that was designed to be able to have someone whose never seen LC/MS/MS before be able to run the instrument in half a day,” explained Mr. Kuracina. “On top of that, we’ve created what’s called an iMethod, which people can download and put into the system that loads up all the parameters so they can start validation immediately.” The company currently offers an iMethod for melamine testing.

PerkinElmer’s product range for melamine analysis includes GC/MS products and LC with UV, DAD or fluorescence detectors. Among its offerings is the Melamine Analyzer, a dedicated solution based on its Clarus GC. “Through our EcoAnalytix initiative, what we’re dong is taking our generic technologies, in a sense, and tailoring them for specific markets and applications. And that is exactly what we did with our Melamine Analyzer a year ago when we introduced it in response to the pet food scare,” said Ms. Rasmussen. The Analyzer comes with a method, SOP and spectral libraries, making it easy to use for inexperienced technicians. One example of such users cited by Ms. Rasmussen is dairy factories in Asia. For more experienced end-users, PerkinElmer offers standard GC/MS systems.

Each company agreed that the food safety is a growing issue. “There have been more than 150 brands of pet food that have been found to be contaminated. And that started a year ago,” explained Mr. Cram. “If it to took us, the whole food safety community, a year to find 150 different brands, you can imagine where this melamine thing is leading. Milk and milk products are in so many things that we eat.” Mr. Kuracina believes that increased concern by food companies and greater oversight by regulatory agencies has led to a heightened awareness about food safety. “This melamine issue is just the tip of the iceberg that’s opened people’s eyes over the last year to the need for more food testing.”

Chinese National Standard for the Determination of Melamine in Raw Milk and Dairy Products

Analytical Method Quantitation Limit Type of Analysis

HPLC-UV or HPLC-DAD 2 mg/kg Quantitiative

LC/MS/MS 0.01 mg/kg Qauntitative and Qualitative

GC/MS 0.05 mg/kg Qauntitative and Qualitative

GC/MS/MS 0.005 mg/kg Qauntitative and Qualitative