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Analytical Chemistry

Silver Lining In Melamine Crisis

Deadly adulteration of Chinese milk drives development of analytical methods to detect contaminant in food products

by Sophie L. Rovner
May 25, 2009 | A version of this story appeared in Volume 87, Issue 21

Credit: Stephen Shaver/UPI
Chinese families whose children died after consuming dairy products laced with melamine protested in front of a government building in Beijing on May 8.
Credit: Stephen Shaver/UPI
Chinese families whose children died after consuming dairy products laced with melamine protested in front of a government building in Beijing on May 8.

LAST YEAR'S SCANDAL involving the deliberate contamination of Chinese milk products with melamine continues to have heartbreaking consequences. Yet this unfolding tragedy has also had a positive impact, prompting international food safety reforms and advances in analytical techniques.

Melamine is used to make plastics, laminates, fertilizers, and other products but is not approved as an ingredient in food. Milk dealers in China added the nitrogen-rich compound to boost the apparent protein content of diluted or substandard milk, according to the country's official news agency, Xinhua. The adulterated milk was then used to make powdered milk and infant formula, as well as candy, cookies, flavored milk drinks, and other foods.

In all, the contaminated milk products killed at least six Chinese infants and sickened nearly 300,000 other children last year, Xinhua reported. This year, Chinese courts handed down death sentences for two people who sold melamine-laced products, and a dairy-food company executive was sent to prison for life for her role in the scandal. Several other government officials and dairy industry workers have resigned or been fired or imprisoned.

China is taking action on the regulatory front as well. The nation adopted a regulation in September 2008 that requires producers to list additives on food labels, Xinhua reports. The Chinese legislature also passed a food safety law that will take effect this June. It will "enhance monitoring and supervision, toughen safety standards, recall substandard products, severely punish offenders," and establish a food safety commission, according to the news agency. The U.S. Food & Drug Administration has opened three offices in China to work with local officials and manufacturers on the quality and safety of foods and drugs.

Credit: JEOL
JEOL's DART-MS technique rapidly analyzes food without sample preparation.
Credit: JEOL
JEOL's DART-MS technique rapidly analyzes food without sample preparation.

This tragic episode wasn't China's first encounter with melamine adulteration. In 2007, approximately 1,000 cats and dogs in the U.S. died and many other animals became ill after eating pet food contaminated with melamine and significant quantities of compound analogs, including cyanuric acid, ammeline, and ammelide. The contaminants were traced to ingredients provided by Chinese suppliers that were adulterated to artificially inflate apparent protein content (C&EN, May 12, 2008, page 41).

At the time, animal studies indicated that melamine by itself wasn't particularly toxic. But researchers discovered that melamine, in combination with cyanuric acid, produced crystals that clogged the kidneys of pets that consumed the adulterated food. In some pets the crystals led to kidney failure.

However, the contaminant in the Chinese milk products was fairly pure melamine, according to the World Health Organization (WHO), so researchers took another look at the behavior of melamine in the body. They now believe that uric acid, which is found in all human urine but is present at highest concentration in infants, can also complex with melamine, says David G. Hattan, a toxicologist at FDA's Center for Food Safety & Applied Nutrition (CFSAN). The resulting complex can precipitate out of the urine to form kidney stones made of melamine urate that, if present in high enough concentrations, apparently cause kidney failure.

Adulteration of food products with melamine to boost apparent protein content has eluded detection in the past because the conventional analytical tests for protein don't check for protein itself; instead, they measure nitrogen content as a surrogate.

Chinese dairy producers exploited the nonspecificity of these methods by diluting their milk and then adding nitrogen-rich melamine to make up for the missing protein. Likewise, in the earlier pet-food incident, suppliers substituted less nutritious ingredients for wheat gluten and rice protein concentrate and then doctored the mixture with melamine.

When the pet-food scandal broke in 2007, no analytical technique had been officially established for detecting melamine in food or tissue samples. After all, "no one expected to find melamine in consumer products," says Robert B. (Chip) Cody, product manager for mass spectrometry at the instrument company JEOL USA, in Peabody, Mass. Chemists and food scientists responded by devising new analytical techniques that continue to be refined and expanded to address contamination of food intended for humans.

Credit: Andy Hancock/Purdue U
Purdue's Zheng Ouyang (from left), an assistant biomedical engineering professor; Cooks; and postdoc Guangming Huang devised a method for detecting melamine in milk products.
Credit: Andy Hancock/Purdue U
Purdue's Zheng Ouyang (from left), an assistant biomedical engineering professor; Cooks; and postdoc Guangming Huang devised a method for detecting melamine in milk products.

FDA, for instance, developed a technique that combines gas chromatography with mass spectrometry (GC-MS) to detect melamine and its analogs. The technique is described in FDA Laboratory Information Bulletin No. 4423. The agency also developed two other methods, numbered 4421 and 4422, that combine high-performance liquid chromatography with triple-quadrupole tandem MS (LC-MS/MS).

Method 4421 was originally developed for grain-based animal feed during the 2007 pet-food contamination incident but can also be used for dry-milk-based products such as infant formula. FDA describes the technique as a "dilute and shoot method with no sample cleanup." That relative simplicity has a downside with respect to detection of melamine and cyanuric acid. Compared with 4422, the method "is more subject to potential interferences from other chemical components in the sample," so it's less suitable for complex samples such as candy or baked goods, says Greg Diachenko, director of CFSAN's Analytical Chemistry Division.

The 4422 method, on the other hand, incorporates sample cleanup to separate melamine and cyanuric acid from most of the other components in food that could interfere with the results, so it can be used with a wider range of food and tissue samples, says CFSAN chemist Alexander J. Krynitsky, who developed the method.

IN A DRY SAMPLE such as infant formula, method 4421 can detect and quantify as little as 250 ppb of melamine and cyanuric acid; method 4422 can take that limit down to 100 ppb. Both of these tests are capable of assessing risk to human health because concentrations of melamine or one of its analogs below 1 ppm in infant formula and below 2.5 ppm in other foods don't pose a health risk, FDA says. Melamine levels in some of the Chinese dairy products topped 6,000 ppm, according to WHO.

Both methods require a fair amount of labor and time. Sample prep for both methods takes about 15 minutes. Run time is about 20 minutes for the 4421 analysis, Krynitsky says, whereas the 4422 analysis takes about 60 minutes because melamine and cyanuric acid have to be detected in two separate runs.

In an effort to reduce the time and labor required, researchers continue to seek alternative melamine-detection methods that are "rapid, sensitive, high-throughput, widely available, and cost-effective," according to Purdue University food scientist Lisa J. Mauer.

FDA itself evaluated a commercial enzyme-linked immunosorbent assay (ELISA) as a potentially cheaper and faster screening tool this past December, says Selen Stromgren, a chemist in FDA's Field Science Division. But the technique had some shortcomings. It could only detect melamine, not its analogs. And in several types of samples, the food matrix interfered with antibodies on the ELISA plates, so the assay couldn't give an accurate result when melamine contamination was low.

Other approaches have proven more fruitful. For instance, Mauer and her colleagues have found that near- and mid-infrared (IR) spectroscopy can be used as a simple, quick, and sensitive method to detect melamine (J. Agric. Food Chem., DOI: 10.1021/jf900587m).

The Fourier transform mid-IR methods take five minutes or less, and the near-IR method takes just two minutes. All three methods can detect as little as 1 ppm of melamine. But the techniques have a significant drawback: The statistical tools used to analyze the spectra and correlate them with melamine concentration have to be recalibrated for different brands or formulations of infant formula or for other food products, Mauer notes.

Other rapid methods that can be used for detecting melamine include several "ambient ionization" MS techniques, which permit a sample to be ionized under ambient conditions rather than in the vacuum chamber required by conventional mass spectrometers. R. Graham Cooks, an analytical chemist at Purdue, published the first paper describing one of these methods—desorption electrospray ionization (DESI)—in 2004. His method uses an ionized stream of solvent produced by an electrospray source to dislodge ions from the surface of a sample. The ions are then analyzed by MS. Cooks has been testing this DESI method for use in melamine detection, which can be done in five seconds at a sub-parts-per-million detection limit, and the results look promising, he says.

Cooks's group recently reported a new ambient ionization tandem MS technique in which a heated sample is ionized by low-temperature plasma. His group has adapted the method for detecting melamine in infant formula and other foods. The technique requires no sample prep, takes just 25 seconds, and can detect as little as 10 ppb of melamine (Chem. Commun. 2009, 556).

Renato Zenobi, an analytical chemist at the Swiss Federal Institute of Technology, in Zurich, and his colleagues have adapted their own extractive electrospray ionization (EESI) tandem MS method to be used for melamine detection (Chem. Commun. 2009, 559). The technique requires little sample prep. Ultrasound converts a drop of milk into a fine spray that is fed into a solvent plume from an electrospray device, which ionizes the sample for analysis by a mass spectrometer. Analysis takes less than 30 seconds, Zenobi says. The technique can be used for many food types and has a detection limit of 500 ppb.

The first commercially available ambient ionization method compatible with MS was JEOL's direct analysis in real time (DART) technique, which Cody coinvented.

The DART method, which requires no sample preparation, ionizes the sample with a spray of plasma formed from helium or nitrogen gas. University of the Pacific chemistry professor O. David Sparkman and graduate student Teresa M. Vail were the first to use DART for melamine analysis when they adapted the technique to examine pet food in 2007. And when the milk scandal broke, John Dane, an applications chemist for MS at JEOL, evaluated the DART-MS method and found that it would be an effective screening tool for dairy products as well as many other foods. The analysis for melamine in milk powder takes five seconds, and the detection limit is about 1 ppm.

SOME RESEARCHERS have taken a lower tech approach in the battle against melamine. For instance, a team of chemists and chemical engineers in China recently developed an extremely simple method to detect melamine in milk products such as milk powder, liquid milk, and yogurt. Their chemiluminescence technique makes use of luminol, which emits a blue glow when mixed with an oxidizing agent in an alkaline solution. Zhenghua Song and colleagues at Northwest University, in Xi'an, had previously found that myoglobin, a heme protein that transports oxygen in muscle tissue, reacts with luminol to produce a strong glow.

In their new work, the researchers showed that melamine complexes with myoglobin and reduces the glow to an extent proportional to the concentration of melamine. Song's team showed that this behavior can be used to analyze melamine content in milk products in 20 seconds with a detection limit below the parts-per-billion range (J. Agric. Food Chem. 2009, 57, 3464). The technique uses a simple apparatus, including a photomultiplier tube and luminometer, to measure light levels. Some other researchers caution that this method's simplicity might make it vulnerable to false positives.

No matter what technique is used, the intense focus currently on melamine appears to have paid off. "Right now, we're seeing very few melamine-positive samples," FDA's Stromgren says. Yet regulators and food producers know that they will have to remain vigilant. And melamine certainly isn't the only possible adulterant to monitor. Publications including the newspaper Shanghai Daily are now reporting that Chinese officials have detected hydrolyzed leather protein in milk.


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