Screening for Adulterants

Adulteration of foods and food ingredients has been a problem for centuries. Treatises on adulteration of food were published in England and the United States as far back as the early 19th century. Concern regarding the adulteration of food in the United States led to the passage of the Food and Drugs Act of 1906 and the eventual establishment of the U.S. Food and Drug Administration (FDA).

Attention to the subject increased dramatically in 2007 and 2008 with the discovery of adulteration in China of foods and food ingredients with the nitrogen-based compound melamine to inflate the reported protein content. Its addition to wheat gluten and rice protein concentrate used in pet food led to the sickness and deaths of many pets in the United States (see “Testing for Adulteration,” Food Technology 62(7):72), and its addition to infant formula led to the deaths of six children in China and the sickening of nearly 300,000 others there (see “Analyzing for Melamine,” Food Technology 63(2):70). Since then, laboratories have vigilantly analyzed foods and ingredients for the presence of melamine and other nitrogen-based adulterants.

Melamine is not the only adulterant of concern. Papers, symposia, and a short course at the 2011 IFT Annual Meeting and Food Expo covered a variety of topics related to adulteration of foods and ingredients. Here are highlights as well as information on analytical methods and instruments.

Targeted Approaches
Several symposia covered targeted approaches, analyzing directly for specific compounds in specific foods.

• Melamine. Speakers in the symposium “Analyzing Melamine in Multiple Food Matrices: Challenges and Opportunities” discussed various methods to detect melamine and similar compounds in foods. Shaun MacMahon of the FDA said that targeted methods have been developed to accurately detect and quantify melamine and cyanuric acid in various food matrices but that other adulterants with a high percentage of nitrogen might not be detectable by these targeted methods. He reported on the use of liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to reveal the presence of six compounds having the potential to be used to artificially enhance the protein content in various food matrices, including milk, powdered milk, soy protein, wheat gluten, wheat flour, and corn gluten meal. The method detected the compounds at concentrations as low as 1 ppm.

Per Waaben Hansen of Foss Analytical A/S illustrated how most organic compounds have unique fingerprints in the mid-infrared region, allowing differentiation between nitrogen-containing compounds that other analytical methods such as Kjeldahl and Dumas are unable to separate. He stated that the limit of detection for melamine and other nitrogen-containing compounds is typically 50 ppm – 100 ppm, using a Fourier-transform infrared (FTIR) milk analyzer.

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Galina Holloway of U.S. Pharmacopeia Convention (USP) indicated that foods could be contaminated with many other substances besides melamine, such as ammonium salts, urea, uracil, and lower-grade proteins. She reported on the USP’s investigation of chromatographic techniques such as strong-cation-exchange high-performance liquid chromatography (HPLC) and reverse-phase HPLC with ultraviolet (UV) detection and spectrophotometric techniques such as UV and Raman for identifying melamine and other contaminants in different food matrices.

• Acrylamide. Formed between carbohydrates and amino acids in fried and oven-baked foods, this product of Maillard reaction is a potential human carcinogen and genotoxicant whose effects in rodents have been studied by the U.S. National Toxicology Program (NTP). In the symposium “The Long-Awaited NTP Acrylamide Bioassay: Where Do We Go from Here?” James R. Coughlin of Coughlin & Associates stated that the NTP’s study results must be evaluated for their relevance in predicting possible effects in humans. Then a carefully considered risk-management evaluation must be developed by health and regulatory bodies to guide industry in acrylamide reduction strategies, and risk-communication programs must be developed to guide consumer behavior, taking into account safe human consumption levels of the contaminant and the overall risks and benefits of each food containing acrylamide.

Nega Beru of the FDA reported that after the discovery of acrylamide in foods in 2002, the agency initiated a broad range of activities. It developed an analytical method; analyzed acrylamide levels in food samples; assessed the quantity and primary sources of exposure to acrylamide; conducted research on acrylamide formation, mitigation, and toxicology; and participated in international efforts on acrylamide. He said that much work has been done internationally on potential ways to reduce acrylamide in food, such as developing alternative cooking profiles, changing ingredients, and using asparaginase to break down the acrylamide precursor asparagine.

Ron P. Guirguis of Fleishman-Hillard said that government regulators have been looking at different risk-management options to deal with dietary acrylamide and that the food industry has been working to better understand how acrylamide is formed and how it can be reduced in the factory and the restaurant. This takes place, he said, against a communications and media backdrop that tends to look at these issues as opportunities to scare the public and vilify companies and food safety authorities.

• Colors. In the symposium “Food Colors: Various Aspects Affecting Their Quality and Risk for Adulteration,” speakers provided an overview of U.S. and European Union (EU) regulations regarding natural and synthetic colors and standards in place to help reduce adulteration of natural colors. Jordi Serratosa of the European Food Safety Authority described how the organization conducts risk assessment in the EU, and Janet L. Balson of Chr. Hansen Inc. provided an industry perspective on the relationship of food safety and color regulation and discussed the effects the Food Safety Modernization Act will have on color regulation in the United States.

Markus Lipp of the USP reported that natural food colors have been the target for adulteration through the addition of synthetic colors to raise the apparent quality or the addition of inert material to increase available quantities through dilution. It is necessary, he said, to update quality standards to include robust specifications to help prevent these adulterations. He described the USP’s efforts to modernize the Food Chemical Codex, the compendium of quality standards for food ingredients and test methods, and discussed challenges in the development of methods to identify all potential adulterants.

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Non-Targeted Approaches
In contrast to targeted approaches, there is increasing interest among industry and government laboratories and instrument manufacturers in developing methods for screening foods and ingredients for unknown adulterants. In the symposium “Non-Targeted Analytical Approaches for Detecting Economically Motivated Adulteration of Food and Food Ingredients,” Jonathan DeVries of Medallion Laboratories pointed out that non-targeted approaches use a priori knowledge of the profile of normal or authentic materials and combine the results of an assay with chemometrics—mathematical analysis via multivariate techniques such as partial-least squares regression, principal component regression, and others—to construct a mathematical model that can be used to discriminate authentic from adulterated samples. The ultimate goal, he said, would be an authenticating system having a sufficiently rapid response for use in routine inspections.

DeVries reported on the use of high-pressure liquid chromatography coupled with high-resolution tandem mass spectrometry (LC/MS/MS) and chemometrics to distinguish authentic from adulterant-spiked foods and ingredients. The approach taken was to collect representative samples of an ingredient, analyze them by the chromatographic mass spectrometry technique to establish a baseline fingerprint and the range of acceptable variability about that baseline, then analyze new samples (some of which are adulterated), determine the degree of difference from the baseline fingerprint, and predict whether the particular sample was adulterated or not.

Using Thermo Scientific Inc.’s Exactive system, the LC/MS/MS analyses were performed on wheat gluten with and without melamine or urea as adulterant, red wine with and without ethylene glycol or apple juice, orange juice with and without high-fructose corn syrup, sodium ascorbate with and without starch or iso-ascorbic acid, corn flour with and without wheat flour, and inulin with and without maltodextrose. All potential adulterants were added at expected economically relevant levels. The approach taken was able to predict adulteration in each case except where the adulterant had a mass of less than 100 m/z. The lower-molecular-weight adulterants were likely not predicted since the program was set to run at m/z of 100 or greater, but DeVries believes that accurate predictions will also be made in those cases if the program is set for lower m/z ranges.

Jack C. Cappozzo of the Institute for Food Safety & Health said that the food industry generally analyzes incoming raw materials for known substances that could be harmful, such as pesticides and mycotoxins in vegetables. The melamine problem should have been caught earlier, he said, since instruments capable of detecting it were available and the spectra for it were available in databases, but no one was targeting it. Now instrument companies are coming up with strategies for non-targeted screening.

The general approach is to utilize LC/MS or LC/MS/MS to build databases of foods for all compounds and their fragments normally present at a signal-to-noise ratio about three times the baseline level. Instrument manufacturers are developing software that can scan all data points to find something different (i.e., compounds and fragments present in a scan above the s/n ratio but not found in normal samples). When found, the compound or its fragment can be further fragmented under controlled conditions to identify it by LC/MS/MS or GC/MS/MS, using databases of mass spectra currently available. Instrument manufacturers are working to expand the databases.

Cappozzo concluded that the combination of liquid chromatography with time-of-flight mass spectrometry (LC/Q-TOF MS) and accurate MS and MS/MS spectra is a powerful and practical method for screening samples for unknown compounds.

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Timothy R. Croley of the FDA discussed development and application of software tools to enable non-targeted screening using LC/MS alone or in combination with other techniques. LC/MS is a powerful tool, he said, but new software tools are required to identify chromatographic components and their associated accurate masses, discard uncorrelated chemical noise, compare mass lists to control samples, and search the identity of unknown components utilizing the exact mass information.

Jon Wong of the FDA showed how ultra-HPLC coupled with high-resolution/high-accuracy mass spectrometry (UHPLC/HR/HA/MS) offers sensitivity to detect changes in a matrix, selectivity to elucidate the differences among a number of samples of the same matrix, and speed and precision to aid in the molecular identification of differences in component makeup of samples. He said that investigations are in progress using UHPLC/HR/HA/MS and conventional LC/MS techniques to screen and identify pesticides in food samples.

Per Waaben Hansen pointed out that infrared spectroscopy is used worldwide for routine milk analysis and may also be employed as a rapid screening technique for detecting adulterants in milk. However, he added, the presence of adulterants must subsequently be confirmed by a reference method to establish whether a sample is truly adulterated or a false positive. An important challenge when introducing screening methods for food adulteration is how to balance false-positive results against the limit of detection of the adulterants.

Michèle Lees of Eurofins Analytics said that high-throughput profiling using 1H nuclear magnetic resonance (1H-NMR) spectroscopy has become the accepted method for the rapid screening of fruit juice raw materials and that Spin-Generated Fingerprint Profiling (SGFP) is an example of its use for non-targeted screening in industry. She described how Bruker BioSpin’s JuiceScreener NMR instrument, which uses the SGFP technique, can deliver huge amounts of information from a single experiment instead of multiple individual analysis steps.

Rohit Schroff of the Nestlé Research Center reported on the use of matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) combined with chemometrics to screen for adulteration of milk. The combination generated characteristic fingerprints of milk and milk adulterated with various foreign proteins, small nitrogen-rich molecules, and inorganic salts. The statistical models developed allowed the detection of adulterants at levels as low as 0.03% and indicated that the approach could be routinely applied for detecting adulteration.

Other Papers on Food Adulteration
Various technical papers presented during the IFT annual meeting also addressed food adulteration. John J. Johnston of the U.S. Dept. of Agriculture (USDA) reported that in response to the 2007–2008 melamine problem, the agency conducted a risk evaluation and concluded that consumption of meat from animals that consumed melamine-adulterated feed did not warrant further action but that consumption of meat products prepared with adulterated milk products could present unacceptable risks to consumers. Based on these analyses, the USDA initiated a monitoring program to quantify melamine in potentially hazardous food products and to quantify risk to consumers. He said that the approaches used to respond to these food safety incidents provide a template for responding to future accidental and/or intentional food contamination events.

Laila H. Ali of the FDA reported on the use of ChromaDex Inc.’s Bioluminex system to detect toxins in milk products spiked with strychnine. The system, which uses thin-layer chromatography and a luminescence biosensor, was able to detect strychnine in milk products at a concentration of 500 μg/mL.

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Yiqun Ou of Jinan University explained that since the Kjeldahl method for determining protein content measures total organic nitrogen, it includes non-protein-nitrogen compounds such as urea and melamine. He compared a rapid dye-binding method for protein using CEM Corp.’s Sprint Rapid Protein Analyzer to Kjeldahl and near-infrared methods for determining protein contents in soy milk with and without melamine or urea and concluded that the dyebinding method would be a good method for detecting non-protein nitrogen adulterants when used in conjunction with the Kjeldahl method.

Saskia van Ruth of Wageningen University looked at cyanide as a potential contaminant in milk powders. She evaluated the infrared spectra obtained during routine quality control for detection of chemical industry waste components and constructed classification models to predict the presence and concentration of these adulterants, including cyanide. She also calibrated the spectra against cyanide concentrations directly. Both approaches, she said, showed promising results for detection of waste compounds and cyanide in milk powders at non-lethal concentrations.

Information Available
Many companies and organizations offer information on instruments and applications for detection of adulteration. AB SCIEX (www.absciex.com) offers its TripleTOF ™ 5600 System, which screens, quantifies, and confirms the presence of known adulterants and identifies unknown (non-targeted) contaminants in a single analysis by LC/MS/MS. The instrument identifies compounds using accurate mass spectrometry and empirical formula calculation or accurate MS/MS spectra and library searching. Software allows users to perform background subtraction, conduct statistical comparisons, engage in library searching, and carry out empirical formula calculation for the detection and identification of unknown compounds without prior knowledge.

Agilent Technologies (www.agilent.com) offers a number of application notes on analysis of melamine in foods as well as a webinar titled “Detect and Identify Non-targeted and Unknown Compounds in Our Food.” In the webinar, Jerry Zweigenbaum says that an overall strategy for the analysis of unknown adulterants in food must encompass all types of contaminants from heavy metals to organic compounds and must consider sampling, sample preparation, and analytical methodology. He describes use of liquid hromatography/quadrupole time-of-flight mass spectroscopy (LC/Q-TOF MS) and data-mining tools for the detection and identification of unknown compounds.

AOAC International (www.aoac.org) in late June approved as Official Method 2011.04 a protein-tagging and colorimetric technique for the determination of protein content in raw and processed meat and meat products. Used in the collaborative study conducted as part of the approval process was the Sprint Rapid Protein Analyzer from CEM Corp. (www.cem.com).

The International Dairy Federation (www.fil-idf.org) reported that guidelines developed in collaboration with the International Organization for Standardization for the determination of melamine and cyanuric acid in milk products by LC/MS/MS were expected to be adopted by the Codex Alimentarius Committee in July. If adopted, the internationally harmonized procedure would allow authorities to check the level of melamine in powdered infant formula against the recently adopted Codex maximum level of 1 mg/kg.

Gerstel Inc. (www.gerstelus.com) has application notes (ANs) available describing use of its automated QuEChERS extraction method for sample preparation in the determination of pesticide residues in foods using GC/MS (AN 5/2011) and LC/MS/MS (AN4/2010).

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PerkinElmer Inc. (www.perkinelmer.com) offers ANs on use of its Clarus® 600 GC/MS for detection of melamine adulteration of dairy products and other protein-based foods.

Picarro Inc. (www.picarro.com) in its AN023 describes use of its Combustion Module-Cavity Ring-Down Spectroscopy (CM-CRDS) system to detect adulteration of honey with corn or cane sugar. The instrument uses the AOAC 998.12 Internal Standard Isotope Ratio Analysis (ISCIRA) method. The system quickly tests for adulteration by measuring both the 13C/12C isotope ratio of the honey sample itself and that of the protein content isolated from honey. Since the protein in pure honey originates from the bee, the stable carbon isotope value of the protein will be unchanged even if corn or cane sugar is added. The system can detect adulteration with high-fructose corn syrup at concentrations as low as 5%.

Polarmetrics Corp. (www.polarmetrics-corp.com) offers its vIRtuous Adulteration Analyzer for rapid determination of adulteration of honey and maple syrup with lower-cost syrups such as cane, corn, beet, rice, and tapioca. The analyzer uses infrared spectroscopy to accurately, rapidly, and easily measure the purity of honey along with the concentration of the adulterant syrup.

Thermo Fisher Scientific Inc. (www.thermoscientific.com) in its AN 43060 describes the use of the company’s iCE 3500 atomic absorption spectrometer for analysis of trace elements in honey. The instrument’s dualatomizer design provides automatic switching between flame and graphite furnace, allowing users to conduct both high-level and low-level analyses on the same instrument.

Waters Corp. (www.waters.com) in June introduced its new Synapt G2-S mass spectrometer for qualitative and quantitative high-definition mass spectometry. It incorporates the company’s’ StepWave™ ion-transfer optics and Triwave® ion-mobility technologies along with new informatics tools. It is said to provide a 30x improvement in signal intensity for mass spectral peaks, greater than 5x improvement in s/n ratio, and up to a 10-fold improvement in limits of quantitation over previous-generation mass spectrometers. The company also has available a webinar titled “The Science of Food Profiling: Determining the Adulteration, Authenticity, and Origin of Foods.”

 

Neil H. Mermelstein , a Fellow of IFT, is Editor Emeritus of Food Technology 
[email protected]