Innovative Laboratory Technologies Facilitate Rapid Responses to Public Health Threats Due to Food Adulteration

It was late summer 2008 in China, and Beijing was abuzz as it prepared to host the Summer Olympic Games. However, just as the nation was gearing up to take center stage in one of the brightest moments in its history, behind the scenes an event was unfolding that would soon steal the spotlight.

The ordeal began on July 16, when 16 infants in the Gansu province were diagnosed with kidney stones. Over the next four months, six Chinese infants died and nearly 300,000 more became ill. The culprit: baby formula, sold by 22 Chinese companies, made with milk powder contaminated with melamine,1 a compound also implicated in a string of pet food recalls tied to Chinese wheat gluten, rice protein and corn gluten that had sickened pets in North America, Europe and South Africa just a year earlier.2 As a trimer of cyanamide, melamine is rich in nitrogen, making it appealing for those who wish to artificially increase the apparent protein content of a substance. Because basic test methods for deciphering protein content, such as Kjeldahl and Dumas, are based on determining total nitrogen content, materials contaminated with melamine display artificially high protein levels. Thus, by intentionally adding melamine to liquid milk, Chinese milk collection station operators (who were acting as middlemen between Chinese dairy farmers and large processors, and were ultimately identified as the offenders) were able to dilute the milk, maintain the apparent protein content and thereby increase their revenue. However, in doing so, they sickened thousands of infants with the toxic substance, caused worldwide food product recalls and dealt a huge economic blow to Chinese dairy farmers.

In general, foods, beverages and dietary supplements are considered adulterated if they are unwholesome, impure, unsafe or otherwise unfit for consumption. However, regulatory agencies around the world have established specific criteria to define adulterated products, and these vary from one country to another. These criteria are constantly evolving as new ingredient sources are utilized, new processing technologies are implemented and new contaminants are discovered. For instance, before 2007, few would have considered melamine contamination to be a food adulteration issue. Essentially no one was testing for melamine and its related compounds (cyanuric acid, ammelide, ammeline and dicyandiamide) in food prior to these events. However, as soon as the issues arose, methods had to be quickly developed and validated to test for the contaminant in implicated products. Fortunately, with the availability of robust laboratory technologies, a method was rapidly developed by groups at FDA to help laboratories respond to the need for testing.3

In its Food Safety Modernization Act Proposed Rule for Focused Mitigation Strategies to Protect Food Against Intentional Adulteration,4 the FDA has taken a stance against acts of adulteration that are intended to cause public health harm. Even acts of intentional adulteration that are not intended to cause public health harm have the potential to do so. Economically motivated adulteration (EMA) events, like the 2008 Chinese melamine scandal, are generally focused on obtaining a financial advantage, not intentionally harming public health. Most events involve a benign ingredient and are noticed only when the adulterant happens to be toxic or allergenic. However, EMA events have been pervasive throughout history.5 After performing a literature search for EMA events that mainly impacted U.S. consumers since 1980, researchers from the University of Minnesota and Michigan State University identified 137 distinct EMA events, 47 of which originated in the U.S.6  Table 1 lists the food categories implicated in these EMA events.

Table 1 – Total number of economically motivated adulteration incidents and number of incidents with adulteration originating in the United States (adapted from Ref. 6)

Methods to detect food adulteration

Just as food products are diverse, so, too, are the means by which they are adulterated for economic gain. Thus, laboratories must continue to develop methods that detect intentional adulteration. Likewise, method development must also encompass unintentional adulterants such as incidental microbiological contamination of food products.

LC-MS/MS

When powdered infant formula contaminated with melamine first emerged, FDA laboratories that were involved in the method development looked to liquid chromatography with triple quadrupole tandem mass spectrometry (LC-MS/MS).3 After researchers subjected samples to formic acid/acetonitrile extraction, they ran melamine and cyanuric acid on LC-MS/MS using a zwitterionic hydrophilic interaction liquid chromatography column to separate the two compounds. They detected and quantified melamine using the instrument’s positive ion mode, and cyanuric acid in the negative ion mode. Numerous derivatives of the FDA method exist, but it is still one of the benchmarks for testing melamine and related compounds in the laboratory.

FRET, SERS, UHPLC/HR-MS

Since 2008, myriad other methods have been developed to detect melamine in    milk and milk products. Some of the most recent have used technologies such as        1) a fluorimetric nanosensor based on fluorescence resonance energy transfer (FRET) between nanoparticles that are disassociated by melamine,7  2) a    biosensor that combines molecularly imprinted polymers and surface-enhanced Raman spectroscopy (SERS)8 and 3) dispersive microsolid-phase extraction cleanup with PCX sorbent material prior to analysis on ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC/HR-MS).9 Innovative methods such as these can be adopted in the future to make melamine detection more efficient, rapid and economical, and to help counteract matrix effects that are inherent to older methodologies.

GC/LC, HPLC, NIR

Honey is another food product that has historically been plagued by intentional adulteration.6 Honey adulteration typically occurs via the addition of cheaper carbohydrate syrups to extend the volume of the product. One of the oldest reports for detecting honey adulteration with high fructose corn syrup used gas-liquid chromatography with an OV-17 column as the detection method.10 Recently a group of researchers used HPLC to analyze honey samples and discovered a peak at the 15.25-min retention time on their chromatograms, which allowed them to distinguish between honey samples adulterated with starch syrups and pure honey samples.11 Another group used three-dimensional fluorescence spectra to determine the concentration of rice syrup in different honey samples.12 Still others have employed near-infrared (NIR) spectroscopy in combination with chemometric methods to distinguish between authentic honey samples and those adulterated with beet syrup, a common adulterant of honey.13

Reversed-phase LC-MS/MS with electrospray, electrochemical detection, nanomicelles

Spices have also been adulterated for economic gain. In 2005, chili powder adulterated with Sudan I dye (an industrial dye classified as a category 3 carcinogen) initiated hundreds of recalls around the globe.14 One of the first methods developed to detect the dye in chili powder was based on reversed-phase LC-MS/MS interfaced with electrospray.15 However, more recent research has highlighted the use of electrochemical detection based on silver nanoparticle-decorated graphene oxide modified glassy carbon electrodes16 and water-soluble nanomicelles containing fluorescent conjugated polymers17 as a means to detect the dye in spice products.

PCR

While economic motives typically involve the addition of a chemical to a food product, detecting unintentional adulteration due to the presence of microbiologicals also requires lab method development. The advent of real-time polymerase chain reaction (PCR)-based technologies allowed methods to be developed to detect the presence of microbiological adulterants more rapidly and with often greater sensitivity than cultural methods. However, one of the greatest limitations is the number of targets that can be detected in one reaction.

The ability to detect numerous serotypes or other subtypes of foodborne pathogens via detection of multiple genetic targets in one reaction has long been of interest to those in the food industry. Tagging oligonucleotide capture and extension (TOCE) technology (Seegene, Gaithersburg, Md.) helps meet this demand.18 The technology utilizes tagging nucleotides, known as “pitchers,” whose cleaved tagging portions can be designed to have different lengths and sequences that alter their melting temperatures, and fluorescently labeled artificial templates called “catchers,” which are able to hybridize with the cleaved tagging portions of the pitchers. When these are coupled with melting temperature analysis at different cycles in the PCR process, TOCE technology enables the detection of up to 20 targets in a single reaction on a conventional four-channel, real-time PCR unit. The technology therefore allows for many more targets to be detected without the need for laboratories to update their existing real-time PCR instrumentation.

Isothermal amplification

There has also been significant movement toward non-PCR-based technologies for the detection of foodborne pathogens. One such method that has been harnessed for assay development is loop-mediated isothermal amplification.19 With this technique, the thermal cycling steps involved in PCR are replaced with a single temperature setting, and results can be obtained in as little as 15 minutes. The method also helps to counteract the action of matrix-derived inhibitors that can plague conventional PCR. Methods currently on the market that utilize this technology have been successful competitors with real-time PCRbased detection techniques.

Conclusion

In the event of another adulteration event like the melamine scandal, laboratories will have to respond quickly. Continuing to drive innovation and improve detection methods will facilitate the rapid response to public health threats due to food adulteration.

References

  1. http://news.bbc.co.uk/2/hi/7720404.stm. Accessed 2/27/15.
  2. http://www.fda.gov/animalveterinary/safetyhealth/recallswithdrawals/ucm129932.htm. Accessed 2/23/15.
  3. Turnipseed, S.; Casey, C. et al. Determination of melamine and cyanuric acid residues in infant formula using LC-MS/MS. Lab. Info. Bull. 2008, 24, 4421.
  4. www.fda.gov/Food/GuidanceRegulation/FSMA/ucm378628.htm. Accessed 2/23/15.
  5. Wilson, B. Swindled—The Dark History of Food Fraud, From Poisoned Candy to Counterfeit Coffee. Princeton University Press, Princeton, N.J., 2008.
  6. Everstine, K.; Spink, J. et al. Economically motivated adulteration (EMA) of food: common characteristics of EMA incidents. J. Food Prot. 2013, 76, 723–35.
  7. Wu, Q.; Long, Q. et al. An upconversion fluorescence resonance energy transfer nanosensor for one step detection of melamine in raw milk. Talanta 2015, 136, 47–53.
  8. Hu, Y.; Feng, S. et al. Detection of melamine in milk using molecularly imprinted polymers-surface enhanced Raman spectroscopy. Food Chem. 2015, 176, 123–9.
  9. Chen, D.; Zhao, Y. et al. A novel dispersive micro solid phase extraction using PCX as the sorbent for the determination of melamine and cyromazine in milk and milk powder using UHPLC-HRMS/MS. Talanta  2015, 134, 144–52.
  10. Doner, L.W.; White, J.W. et al. Gas-liquid chromatographic test for honey adulteration by high fructose corn syrup. J. Assoc. Off. Anal. Chem. 1979, 62, 186–9.
  11. Wang, S.; Guo, Q. et al. Detection of honey adulteration with starch syrup by high performance liquid chromatography. Food Chem. 2015, 172, 669–74.
  12. Chen, Q.; Qi, S. et al. Determination of rice syrup adulterant concentration in honey using three-dimensional fluorescence spectra and multivariate calibrations. Spectrochim. Acta. A Mol. Biomol. Spectrosc. 2014, 131, 177–82.
  13. Li, S.F.; Wen, R.Z. et al. Qualitative and quantitative detection of beet syrup adulteration of honey by near-infrared spectroscopy: a feasibility study. Guang. Pu. Xue. Yu. Guang. Pu. Fen. Xi. 2013, 2637–41.
  14. http://news.bbc.co.uk/2/hi/health/4285285.stm. Accessed 2/27/15.
  15. Calbiani, F.; Careri, M. et al. Development and in-house validation of liquid chromatography-electrospray-tandem mass spectrometry method for the simultaneous determination of Sudan I, Sudan II, Sudan III, and Sudan IV in hot chili products. J. Chromatogr. A 2004, 1042, 123–30.
  16. Prabakaran, E.; Pandian, K. Amperometric detection of Sudan I in red chili powders using Ag nanoparticles decorated graphene oxide modified glassy carbon electrode. Food Chem. 2015, 166, 198–205.
  17. Ye, X.; Zhang, J. et al. Fluorescent nanomicelles for selective detection of Sudan dye in pluronic F127 aqueous media. ACS Appl. Mater. Interfaces. 2014, 6, 5113–21.
  18. www.seegene.com/neo/en/introduction/core_toce.php. Accessed 2/27/15.
  19. Notomi, T.; Okayama, H. et al. Loop-mediated isothermal amplification of DNA. Nucl. Acid Res. 2000, 28, e63.

Alex L. Brandt, Ph.D., is Director of Technical Services, Food Safety Net Services, Ltd., 199 W. Rhapsody, San Antonio, Texas 78216, U.S.A.; tel.: 888-525-9788; fax: 210-525-1702; e-mail: [email protected]www.fsns.com

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