Analyzing for Melamine
LABORATORY
There have always been unscrupulous people who think more of making profits through adulteration of foods than the health of the consumers of those foods. The most recent examples have occurred in China, where products have been adulterated with melamine to increase their apparent protein content.
In 2007, many dogs and cats in the United States were harmed or killed by eating pet food manufactured with melamine-adulterated wheat gluten and rice protein concentrate obtained from China (see “Testing for Adulteration” in the July 2008 issue of Food Technology). And in 2008, adulteration of infant formula led to the deaths of six children in China and sickened nearly 300,000 others there.
Melamine is not approved for direct addition to human or animal foods, and no manufacturer is allowed to deliberately add it to any food for U.S. consumers. In November 2008, the Food and Drug Administration expanded its import controls on dairy products and other food and feed products manufactured in China that contain dairy ingredients to help ensure that only those products not contaminated with melamine and related compounds reach U.S. consumers. FDA is also examining other protein-containing products besides dairy and dairy-containing products for contamination with melamine and related compounds. In mid-December, the government of China blacklisted melamine and other nonfood substances that could be added to food.
FDA’s Analytical Approaches
FDA is using both liquid chromatography and gas chromatography methods with mass spectrometry in its analyses:
• LC-MS/MS. The liquid chromatography triple quadrupole tandem mass spectrometry method for measuring melamine and cyanuric acid in infant formula consists of extraction with aqueous formic acid, followed by filtration, centrifugation, and dilution, then analysis using a zwitterionic HILIC (hydrophilic liquid chromatography) LC column. The method is described in Laboratory Information Bulletin 4421, “Determination of Melamine and Cyanuric Acid Residues in Infant Formula using LC-MS/MS” (www.cfsan.fda.gov/~frf/lib4421.html) and Bulletin 4422, “Interim Method for Determination of Melamine and Cyanuric Acid Residues in Foods Using LC-MS/ MS” (www.cfsan.fda.gov/~frf/lib4422.html).
• GC/MS. The gas chromatography/mass spectrometry method for measuring melamine and analogs in a variety of matrices consists of extraction with a mixture of acetonitrile/water/diethylamine, conversion of analytes to trimethylsilyl derivatives, then analysis. The method has been evaluated using dry protein materials (wheat gluten, rice protein, corn gluten, and soy protein), wet and dry pet foods, and dry animal feeds and is also applicable to a variety of other matrices. The method is described in Bulletin 4423, “GC-MS Screen for the Presence of Melamine, Ammeline, Ammelide, and Cyanuric Acid” (www.cfsan.fda.gov/~frf/lib4423.html).
Other Approaches
Other approaches for analysis of melamine are also being developed and applied.
• Amino Acid Analysis. Waters Corp., Milford, Mass. (www.waters.com), is using amino acid analysis to directly measure protein content. According to Tom Wheat ([email protected]), Principal Scientist and Life Science Laboratory Manager, since proteins are composed of amino acids, we can hydrolyze the sample to its constituents and add them up. This gives a direct measurement of protein. The only way to fool the results would be to add amino acids.
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In the Waters® UPLC® Amino Acid Analysis Solution, the amino acids in the sample are separated by reversed-phase ultra-performance liquid chromatography (UPLC), and the peaks are identified by retention time and quantified by peak area. It’s a very sensitive method and is very robust with complex samples, Wheat said. UPLC is a new generation of HPLC that employs very small particles of packing material and an optimized instrument, the Acquity UPLC®, to use them, he explained; this provides better resolution, better sensitivity, and increased speed.
However, he said, preparation of a sample for amino acid analysis takes about 24 hr, and the analysis itself can take up to several hours. So Waters is collaborating with CEM Corp., Matthews,N.C. (www.cem.com), to make sample preparation more efficient by using a microwave-assisted hydrolysis system that reduces the hydrolysis process from 18–24 hr to less than 30 min.
Grace Vanier ([email protected]), Product Manager, Bioscience Div., at CEM Corp., said that the Discover® Protein Hydrolysis System, which can be used for both liquid- and vapor-phase hydrolysis, allows for the hydrolysis to be performed under inert, anaerobic conditions. The hydrolysis is performed by adding 6 N HCl and then heating the mixture with microwave irradiation to break the peptide bonds in the protein and liberate the amino acids. Microwaving the contents of the vessel accelerates the rate of reaction without altering the fundamental chemistry of amino acid analysis, she said, and reduces the time required for cleavage of difficult-to-hydrolyze hydrophobic peptide linkages without excessively degrading the labile amino acids serine and threonine.
The goal of the collaboration, Wheat said, is to reduce a 2- to 3-day turnaround to 8 hr by accelerating both parts of the process and making the process robust so analyses need not be repeated.
• Protein Tagging. CEM Corp. is also using tagging technology to measure protein directly, instead of nitrogen. According to John Urh ([email protected]), Product Manager, Process Products, the company’s new Sprint™ Rapid Protein Analyzer uses iTAG™ protein tagging technology to avoid the problem of false protein readings from nitrogen-containing contaminants such as melamine. The instrument complies with existing AOAC Official Methods for major dairy applications and AACC Methods for grain testing, and studies are under way for meat applications.
The proprietary iTAG tagging agent uses an azo dye, Crocein Orange G, that features an acid group that binds with protein. A solution of the tagging dye is added to a sample, and a portion of the tag binds, by acid–base reaction, to the histidine, arginine, and lysine in the protein. A colorimeter measures how much of the tag remains in solution and determines the percent protein in the sample by difference.
The instrument automatically homogenizes the sample, adds the tagging solution, and reads the results at the touch of a button. The system is compact and safer, faster, and more environmentally friendly than the Kjeldahl and Dumas methods, Urh said.
The great benefit of the system, he added, is that it yields accurate test results in as little as 2 min and avoids the problems inherent in the Kjeldahl and Dumas methods, namely, that a well-trained operator is needed and the tests must be conducted in a laboratory environment. In contrast, the Sprint system can be located in a lab or on or near the production floor; the latter allows machine operators to run their own samples.
Development of the analyzer earned CEM Corp. the 2008 IFT Food Expo Innovation Award.
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• SERS. Researchers at the University of Missouri, Columbia, have developed a novel method for detecting melamine and its analogs in grain-based foods using surface-enhanced Raman spectroscopy (SERS) coupled with various nanosubstrates. Mengshi Lin ([email protected]), Assistant Professor, Food Systems & Bioengineering, and his coworkers tested wheat gluten, chicken feed, cake, and noodles and found that SERS was faster and simpler than HPLC-MS for detecting melamine.
SERS is a sensitive technique based on a phenomenon that Raman scattering signals of probed molecules on nanostructures can be enhanced by more than a million times as a result of the effects of electromagnetic field and chemical enhancement. Most recent data obtained in Lin’s lab show that SERS is able to rapidly detect 2 ppm of melamine in milk using zinc oxide nanosubstrates. The major advantage of SERS is that melamine contamination can be accurately detected on-site and in real-time (less than 15 min/sample).
The researchers suggested that both SERS and HPLC methods could be used in combination to provide an even more rapid and cost-effective way to detect melamine in food. This would involve using SERS to screen foods, eliminating presumptive negative samples of melamine contamination from the sample population, then verifying presumptive positive samples using HPLC protocols. They reported part of their findings in the October 2008 issue of Journal of Food Science. Partnering with Nanova Inc., Columbia, Mo., the University of Missouri researchers are developing a variety of nanosubstrates for potential commercialization of SERS for melamine detection and other food safety applications.
• ELISA. Romer Labs Inc., Union, Mo. (www.romerlabs.com), has introduced its AgraQuant® Melamine Sensitive Test Kit , a direct competitive enzyme-linked immunosorbent assay for the analysis of melamine in dairy products. The ELISA utilizes horseradish peroxidase conjugate as the competing, measurable entity. The 48- or 96-well microtiter-plate test kit provides quantitative results comparable with results obtained by HPLC in 15–20 min. Each kit comes complete with melamine standards, antibody-coated 48- and 96-well microtiter plates, enzyme conjugate, substrate, and stop solution. The test can be run with 50 min of total incubation time. The immunoassay has been validated for dairy products such as milk, milk powder, yogurt, and yogurt drinks.
• GC/MS. In October 2007, PerkinElmer Life and Analytical Sciences, Waltham, Mass. (www.perkinelmer.com), introduced a GC/MS analyzer for the determination of melamine adulteration in protein-based foods. The EcoAnalytix™ Melamine Analyzer includes a Clarus® 600 T GC/MS with autosampler and TurboMass™ GC/MS software, all consumables needed for analysis, and an application CD featuring methods and sample data, a library of derivatized compounds spectra of melamine and related compounds, a melamine application guidebook, and an application note. Details are available at www.erkinelmer.com/melamine.
Additional Industry Responses
In response to the China incidents, instrumentation companies are developing methods for the determination of melamine in dairy products and working with the Chinese government on methods development.
• Applied Biosystems (www.appliedbiosystems.com), part of Life Technologies Corp., Carlsbad, Calif., announced in December that the Chinese government and two leading food manufacturers in China recently deployed five API 3200 mass spectrometry systems for food analysis as part of efforts to identify and contain the spread of melamine in the food supply. Through its joint venture with MDS Analytical Technologies, the company offers mass spectrometry systems, including the ABSCIEX 5500 systems and the API 3200 system, that are capable of identifying melamine at the lowest possible levels of detection.
• Waters Corp. (www.waters.com), Milford, Mass., in October published a rapid method for the detection of melamine in infant formula and liquid milk. The new method uses the Acquity TQD System, a combination of UPLC and tandem quadrupole mass spectrometry (UPLC/MS/MS), which can detect melamine contamination as low as 1 ppb at analysis speeds greater than one sample/15 min.
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• Thermo Fisher Scientific Inc. (www.thermo.com/foodsafety), San Jose, Calif., announced in December that it has launched a program focused on assisting government and commercial food testing laboratories in the development and implementation of methods for the analysis of melamine in food products imported from China. The company has developed a testing method based on its TSQ Quantum LC-MS/MS system, which is being used as a reference by government labs in China for the detection of melamine in milk and infant formula. The company can also provide screening and confirmation techniques using its ITQ™ Series quadrupole ion trap GC/MSn or its TSQ Quantum triple quadrupole GC/MS.
The company also recently introduced an application note that outlines use of the Flash 4000 N/Protein Analyzer to determine the protein composition in meat products. The instrument is based on the Dumas combustion method of analysis. Because different meats have unique levels of water and fat content, combustion temperatures and oxygen concentrations vary in the protein determination process. The associated Eager Xperience software automatically calculates the amount of oxygen necessary for sample combustion and automatically calculates the protein content according to the food type. Application Note 42131, “Nitrogen/Protein Determination in Meat Derivative Products Using the Thermo Scientific Flash 4000,” is available at www.thermo.com/oea.
FSIS Tests Retail Products for Melamine
The U.S. Dept. of Agriculture’s Food Safety & Inspection Service in mid-December issued a notice (www.fsis.usda.gov/OPPDE/rdad/FSISNotices/97-08.pdf) instructing its investigators to collect certain types of products at retail stores for testing for the presence of melamine and its three analogs—cyanuric acid, ammelide, and ammeline.
Sampling focuses on those meat and poultry products that contain milk-derived ingredients, namely, baby food containing a significant amount of meat or poultry products; cooked sausages (including hot dogs or frankfurters with and without cheese products); breaded chicken (bite-sized morsels or nuggets with and without cheese products); meatballs; and meat and poultry wrapped in dough and pizza (including calzones).
The agency intends to collect and test 45 samples/week for 12 weeks and send them to the agency’s Food Emergency Response Network (FERN) Laboratory at the Russell Research Center in Athens, Ga. If any of the laboratory results are above the guidance level identified by the Food and Drug Administration, USDA will convene its Emergency Management Committee, as appropriate.
FDA’s Center for Food Safety and Applied Nutrition considers that levels of melamine or an analog below 1.0 ppm in infant formula and below 2.5 ppm in other foods do not raise a public health concern.
Standard Methods for Protein Analysis
The standard methods for determination of protein are the Kjeldahl method and the Dumas method.
In the Kjeldahl method, concentrated sulfuric acid is added to the sample in the presence of potassium sulfate and a metal catalyst. The mixture is heated to the boiling point, and sodium hydroxide is added to liberate ammonia. The ammonia is then distilled and titrated with hydrochloric acid to determine the amount of nitrogen in the sample.
In the Dumas method, a solid or liquid sample is burned at high temperature in an oxygen-rich atmosphere in the presence of a metal catalyst. The resulting nitrogen oxides are reduced to elemental nitrogen, and the nitrogen content is determined by a thermal conductivity detector calibrated against EDTA.
Both methods measure nitrogen content, which is then generally back-calculated to protein content by multiplying by 6.25. Thus, as occurred in the China incidents, adding a less-expensive nitrogen-containing compound such as melamine to a sample such as milk can inflate the results and falsely indicate a higher protein content.
Neil H. Mermelstein
Editor Emeritus of Food Technology
[email protected]
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