Melamine analysis at the forefront

By Catherine Watkins

In This Section

September 2009

Recent food safety scares involving melamine have put the development of new standards and methods for protein detection on the fast track.

The deaths of thousands of pets in 2007 and of at least six babies in China the following year had a food adulterant in common: the industrial chemical melamine. Used properly, it is a component of countertops, dry-erase boards, fabrics, glues, housewares, fertilizers, and flame retardants. Used improperly, melamine powder-which is 66% nitrogen by weight-adds economic value by raising the apparent protein level of foods and food ingredients. To date, melamine adulteration has been confirmed in wheat gluten, infant formula, milk and milk products, eggs, vegetables, and fish and livestock feed.

It is against this backdrop that the US Pharmacopeia (USP; Rockville, Maryland, USA) convened a Food Protein Workshop for representatives from academia, government, and industry. Held June 16-17, 2009, at USP headquarters, the two-day meeting was attended by AOCS Technical Director Richard Cantrill and Project Manager Amy Johnson. The aim of the workshop was to review current analytical techniques for protein quantification and explore better ways to detect deliberately falsified protein content in food ingredients.


The test most often used to ascertain protein content for trade purposes is the Kjeldahl assay. Developed in 1883 by Johan G.C.T. Kjeldahl, the test determines the total nitrogen in a sample from both protein and nonprotein sources by measuring organic nitrogen and ammonia. The protein content is then estimated by multiplying the nitrogen content by a factor that takes into account the nitrogen content of a known or average amino acid composition. (For example, the soybean industry uses a factor of 6.25 to estimate soy protein content.)

Another classical approach that is gaining in use is the Dumas method. First described by Jean-Baptiste Dumas in 1848, the method also determines total nitrogen in a sample but takes only a few minutes per measurement as compared with the hour or more for the Kjeldahl method. One major disadvantage, however, is the high initial cost of equipment.

The US Food and Drug Administration has published six methods for melamine analysis using liquid chromatography (LC)-ultraviolet spectroscopy, gas chromatography-mass spectrometry (GC-MS) or GC-MS/MS, LC-MS/MS, and enzyme-linked immunosorbent assay using antigen-antibody reaction. Workshop participants reviewed those techniques as well as chemometrics; Fourier-transform infrared spectroscopy; and near-infrared, mid-infrared, and Raman spectroscopy for characterization of total protein.

Two new rapid techniques have been developed by researchers in Switzerland and the United States. The first, based on work by Renato Zenobi and colleagues at ETH Zürich, uses ultrasound to nebulize liquid milk samples. The spray is then ionized by extractive electrospray ionization and analyzed by tandem mass spectrometry. The technique takes 30 seconds per sample, the researchers say, and the lower limit of detection is in the range of a few nanograms of melamine per gram of milk (Chemical Communications 5:559, 2009).

In other work, scientists at Purdue University, West Lafayette, Indiana, USA, used a low-temperature plasma probe to ionize liquid samples. The researchers, led by Graham Cooks, also used tandem mass spectrometry and achieved a similar speed and limit of detection (Chemical Communications 5:556, 2009).

A third technology devised by researchers at the BioTechnology Institute (BTI) of the University of Minnesota in Minneapolis (USA) simplifies the detection of melamine in liquids. The BTI researchers, led by Larry Wackett and Michael Sadowsky, developed melamine deaminase, which breaks one of the carbon-nitrogen bonds in melamine to release ammonia. The ammonia is then reacted to produce a blue color that is quantified by measuring the absorbance in the sample wells at 620 nm. The detection limit of the kit is 0.25 parts per million.


In the end, the consensus opinion among the USP workshop participants was that two methods-one general (Kjeldahl or Dumas) and one specific (amino acid analysis)-will be required to ensure protein quality in trade. This two-pronged approach could also be useful in detecting adulteration of other commodities, they noted.

In a parallel project funded by the United Soybean Board, AOCS is acting to improve amino acid analysis of whole seeds by setting performance criteria for the most popular methods of amino acid analysis. Although current methods of amino acid analysis are time consuming, laboratories are committed to whichever method they use for many reasons, including the high cost of equipment. Therefore, instead of developing an official method, AOCS is working with government, industry, and academic collaborators to set performance criteria for the methods currently in use. An initial study took place in 2008 and a second study with additional collaborators is set for October 2009. Contact Amy Johnson at  for more information about participating in this or future studies.

For its part, USP is working on making final a 100-page article on the history of protein methodology detailing the pros and cons of each method. The article will be submitted soon to a peer-reviewed journal; USP will then publish pieces of the article in the trade press. In addition, the USP Food Ingredients Intentional Adulterants Advisory Panel "is looking for ways to fast-track analytical changes to USP monographs," Jim Griffiths said. Griffiths is USP's vice president of food, dietary supplement, and excipient standards. The panel also is developing a comprehensive list of potential adulterants covering all subclasses of proteins. "Protein issues cut across many sectors," he noted, "including cereal, oilseed, meat, and dairy." Analytical methodology, however, remains USP's top priority.

"Protein adulteration is a complex problem," said Markus Lipp, director of food standards for USP, adding that all national and international regulations regarding protein in trade make reference to specific methods (generally Kjeldahl). "To change all of these will require the collaboration and cooperation of many different stakeholders as well as a detailed understanding of what the changes would entail and how they would impact the supply chain," he noted.

"Many AOCS constituents produce and use protein concentrates in their daily business activities," AOCS Technical Director Richard Cantrill emphasized. "The assurance of quality and safety relies on the frequent use of appropriate methods of analysis."   

Catherine Watkins is associate editor of inform and can be reached at .