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Laboratory tools can quickly and accurately analyze melamine and dioxin
by Yolanda Fintschenko, PhD, and Guiping Lu, PhD
Numerous food contamination incidents in recent years have put food safety in the spotlight as never before. The obvious challenges of policing a global, interdependent food production network are prompting consumers to question food safety, governments to increase regulation, and food producers to search for new testing solutions. In light of public concern over food safety, heightened in particular by dioxins found in Irish pork and melamine found in infant formula made in China, government agencies and food processors are selecting instruments that can accurately identify contaminants in the global food supply.
A growing list of new testing mandates has made many traditional testing solutions and workflows obsolete. In 2006, Japan’s Positive List for agricultural chemical residues in foods increased the number of chemicals with minimum residue limits (MRLs) from 238 to 758; now the number is nearing 900.1 For lettuce alone, the European Union (EU) raised the number of chemical compounds with MRLs from 102 in 1999 to 435 in 2008. As a result, food producers have a growing list of requirements their workflows must provide: lower limits of detection (LOD), high-throughput screening capabilities, ease of use for quick adoption, and robust reporting and data management capabilities, to name a few.
Melamine and Dioxin Contamination
Two topical food safety issues that have recently hit the headlines are melamine and dioxins, both of which have been found in finished food products. Melamine is an industrial chemical that is used as a binding agent and flame retardant in the manufacturing of cooking utensils and plates. It has a high nitrogen content (66% by weight) and is used as a fertilizer in some parts of the world. It was added to animal feed as a cheap protein source in China before October 2008, and it was intentionally added to pet food and infant formula to mimic protein, despite never having been approved for pet and human food. Because humans and animals cannot metabolize melamine, a variety of illnesses result from its consumption. Melamine contamination has been linked to several deaths in China.
The toxicity of melamine results from the formation of insoluble crystals between melamine and cyanuric acid (a byproduct of melamine), which can cause the development of kidney stones in pets and babies. Besides melamine and cyanuric acid, two melamine-related compounds, ammeline and ammelide, were also found in adulterated pet food.
Due to the severe consequences of melamine adulteration in milk products, government food safety agencies around the world, including the General Administration of Quality Supervision, Inspection, and Quarantine of the People’s Republic of China, the U.S. Food and Drug Administration (FDA), and the U.K. Food Standards Agency have established limits of 1 mg/kg and 2.5 mg/kg MRL for melamine in baby formula and milk products, respectively.
Because it has been used as a fertilizer and was added to animal feed, other products may also have been contaminated by melamine. For example, eggs exported from China to Hong Kong were found to be contaminated by this compound. The testing list for melamine now extends beyond milk products or products with milk powder to meat, poultry, eggs, and vegetables.
Dioxins are another cause for concern in the food safety industry. Long-term exposure to dioxins, a group of chemicals formed during combustion processes like waste incineration, is known to increase the likelihood of cancer. In December 2008, the Irish government recalled all pork products originating in Ireland after the discovery of dioxins in slaughtered pigs. This recall was soon followed by an announcement from the Chinese government banning all imports of pork from Ireland after some of the meat was found to be contaminated with elevated levels of the chemicals.
The European Commission and the U.S. Environmental Protection Agency (EPA) have set maximum levels for dioxins in food. Tests on some of the Irish pork products showed up to 200 times more dioxins than the recognized safety limit. Because the directives require limits of quantitation (LOQ) that are 80% lower than the lowest reported level in the US EPA Method 1613 Rev.B[3-7], laboratories require more demanding detection limits, selectivity, and sensitivity to confirm their presence, along with tools that manage data and can detect problems earlier in the process.
Selecting the right method is critical to getting the right result. For a laboratory testing a large number of samples, throughput is a priority, and a fast screening method is ideal. If detecting a toxic or potentially lethal chemical is the goal, the selectivity of the method is paramount. Below are two timely examples, using melamine and dioxins, to illustrate the numerous variables involved in method selection and the important role the right technology can play.
The FDA has published six methods for melamine analysis using:
- Liquid chromatography-ultraviolet (LC-UV, FCC high-pressure liquid chromatography-ultraviolet [HPLC-UV] method);
- Gas chromatography-mass spectrometry (GC-MS) or GC-MS/MS (FDA LIB 4423);
- LC-MS/MS (FDA LIB 4396, 4421 and 4422); and
- Enzyme-linked immunosorbent assay (ELISA) using antigen-antibody reaction, a method developed for screening melamine in milk and milk products.
The LC-MS/MS method provides the lowest LOQ of all the FDA methods, 10-50 µg/kg. The superior LOQ is a result of highly selective reaction monitoring (H-SRM) technology, which significantly reduces matrix effects, producing cleaner mass spectrometry data and unmatched analyte specificity. The LC-MS/MS method for melamine involves solid phase extraction cleanup, which removes interfering compounds in dirty matrices such as seafood and meat. LC-MS/MS solutions can yield accuracy and precision values for FDA methods that are well within the guidelines of the FDA for analytical method development and analysis.
The GC-MS method requires a derivatization step to convert melamine and its related compounds, cyanuric acid, ammeline, and ammelide, to volatile derivatives. In selected ion monitoring mode, the GC-MS method can obtain LOQ of about 100 µg/kg for melamine. Using SRM of GC-MS/MS, the method can reach a lower LOQ of 10 µg/kg.
The LC-UV method is fast and does not need sample cleanup for melamine in milk products. If upgraded to ultra-high pressure liquid chromatography (UHPLC), the LC-UV method can detect melamine within two minutes in an LC run.
The ELISA method is fast and easy to use. Up to 96 samples can be run within two hours in a microplate using ELISA, though there is the possibility of cross-reaction with related compounds. If there is a non-detect, no further analysis is necessary. If melamine is detected, other tests may have to be run in order to meet regulatory requirements and ensure that a cross-reacting compound did not cause the response.
Dioxins, also called polychlorinated dibenzodioxins (PCDDs), are a group of organic compounds with one to 10 chlorines attached to biphenyl (see Figure 1, above). Theoretically, there are 209 congeners of dioxins, with TCDD (2,3,7,8-tetrachlorinated dibenzo-p-dioxin) being the most toxic dioxin to humans. Dioxins are environmental pollutants generated by the incineration of chlorinated compounds, as in trash burning, for example. Dioxins can cause cancer and severe reproductive and developmental problems in humans. Due to the toxicity of dioxins, the EPA has established MRLs for dioxins at 1 to 5 µg/kg in solids and 0.5 to 5 µg/kg in extract.
There are three analytical methods that have an LOD in the 10-12 g range, the performance criteria specified for dioxins by EU Commission Directive 2002/70/EC:
- High-resolution GC/MS (HRGC/HRMS);
- Low-resolution MS (LRMS) such as ion-trap and triple-quadrupole MS; and
- Cell-based bioassay (EPA method 4425).
For confirmation, HRGC/HRMS (EPA 1613B) is the only choice that meets the requirements of both EPA and EU regulations due to the fact that LRMS is unable to separate isomers of dioxin congeners and dioxin-like polychlorinated biphenyls (PCBs). For confirmation of dioxins, EPA method 1613B (requiring HR GC-HRMS) or its equivalent will be the choice.
The cell-based bioassay utilizes a human cell line (101L) with a reporter gene, firefly luciferase. In the presence of dioxins, the enzyme luciferase is produced, and its reaction with luciferin can be detected by measuring relative light units on a luminometer. This method requires several hours, both to prepare the human cell line 101L culture and for a 16-hour incubation time of sample extract with the cell line culture in a culture plate.
It is important for food safety laboratories to select the right method in order to meet the goal of the analysis and the operating requirements of the laboratory for productivity and cost efficiency. Often several methods are available; selection is dependent on each particular problem and the final goal of the analysis. For example, when large numbers of samples have to be monitored for potential contamination, sample throughput is the highest priority, and a fast screening method should be chosen.
When a sample is suspected to be contaminated with an illegal compound such as malachite green and accuracy is the top requirement, however, the method’s selectivity will be the key criterion. In this case, a method that can be used to confirm the identity of malachite green will be used. In most cases, screening and confirmation will be combined.
For all these methods, the analytical figures of merit and results can be stored in a workflow-oriented laboratory information management system to automate alerts for food quality and safety, method performance, laboratory performance, and quality of analysis. Any results that fall outside the specifications are automatically flagged, and data is centralized for easy access and reporting.
A Public Responsibility
While the food supply network and food safety regulations are increasingly global, the ultimate responsibility for food safety will always fall on the company that manufactured the final product. Many solutions are emerging that are ideal for streamlining sample preparation, data analysis/reporting, and method selection, areas that are critical for meeting these growing regulatory challenges.
The issues of melamine and dioxin contamination have highlighted the need for food safety testing to protect public health. With currently available technologies, careful selection of the right tools will enable laboratories to reach the goal of the analysis efficiently. Driven by the demand in food safety testing, technology and techniques will continue to be developed to enhance accuracy, productivity, and consumer safety.
Dr. Fintschenko is manager, food safety technologies, at Thermo Fisher Scientific in San Jose, Calif.; reach her at firstname.lastname@example.org. Dr. Lu is a food safety application specialist at Thermo Fisher Scientific in Beijing; reach her at email@example.com.
- The Japan Food Chemical Research Foundation. The Japanese Positive List System for Agricultural Chemical Residues in Foods. May 29, 2006. Available at: www.ffcr.or.jp/zaidan/FFCRHOME. nsf/pages/MRLs-p. Accessed June 10, 2009.