When Bad Things Happen to Good Food: HPLC Application to Detect Food Adulteration

Although it has been happening to some degree for centuries, food adulteration is increasingly becoming a worldwide epidemic, as evidenced by the melamine scandal of 2008 and recent meat and fish substitutions at major food chains. Analysts are applying more sophisticated chromatographic, spectroscopic, and enzymatic analytical techniques to monitor and measure food adulterants, which are often economically motivated. In this installment, guest authors Jeff Hurst and Kendra Pfeifer from Hershey Foods explore high performance liquid chromatography (HPLC), ultrahigh-pressure liquid chromatography (UHPLC), and mass spectrometry (MS) approaches being adopted to keep ahead of the food adulteration game.

Food adulteration, sometimes called food contamination, is an intriguing topic that can sometimes seem daunting because it is in everyday commerce and not just the analytical laboratory. First, let's discuss a few general thoughts on food adulteration.

The focus of this installment is on detecting food adulteration with high performance liquid chromatography (HPLC), but a veritable arsenal of techniques are currently being used. These include HPLC coupled with a variety of detection methods ranging from UV to mass spectrometry (MS) and other nonchromatographic techniques such as immunoassay and mid-infrared (IR), near infrared (NIR), and Raman spectroscopy.

Another interesting point of contention is that there are some who perceive scientists that are involved in food adulteration from the contaminations side as second-rate scientists. However, that seems to be far from the truth because those scientists exhibit not only a certain level of scientific expertise, but also ingenuity. One obviously doesn't condone these food adulteration activities, but we should be aware.

Food fraud is seemingly large and growing, with articles on the topic appearing in publications ranging from Chemical and Engineering News to The Economist. In March 2014, The Economist published an article titled "A la Cartel," indicating that organized crime is diversifying into food and alcoholic beverages (1). Several examples are given in the article, including the horsemeat scandal in 2013 and another case in which nearly 2500 jars of honey were filled with sugar syrup. There was also a report on the seizure of 17,000 L of fake vodka worth $1.7 million. Finally, this article contained information from Europol that in the United Kingdom crooks have switched from drugs to food since everyone buys food and drink.

Despite the recent news coverage, food fraud is nothing new (2). The Romans had laws that focused on the adulteration of wines because wine back then tended to become bad rather rapidly and a number of items were added to improve the flavor. One of the compounds happened to be lead salts, which sweetened the wine and likely added to the lead load in the Roman population. In the early 19th century, Frederick Accum wrote a book titled Treatise on the Adulteration of Food and Culinary Poisons Exhibiting the Fraudulent Sophistications of Bread, Beer, Wine, Spirituous Liquors, Tea, Coffee, Crème. Confectionary, Vinegar, Mustard, Pepper, Cheese, Olive Oils, Pickles and other Articles Employed in Human Commerce (3). In that time period, used tea and coffee grounds could be purchased inexpensively. The tea grounds were then boiled with sheep dung and ferrous sulfate and colored with a mixture of tannin, copper acetate, and ferrrocyanide while coffee grounds were mixed with other roasted bean, gravel, sand, and chicory. Burnt sugar was used to add color to coffee. In the case of confectionary, lead, copper, and mercury salts were used to make bright colors that were eye catching for children, but toxic. Green vitrol, alum, and salt were added to give beer a good head because beer was sometimes diluted.

The most likely event that brought the topic of food adulteration to the forefront today was the melamine incident in 2008, which killed six Chinese infants and sickened more than 30,000. Another recent incident occurred in the past month, in which meats used by two very visible restaurant chains was found to be tainted — meat more than a year old that was mixed with fresh meat (5,6).

Top Five Food Targets for Adulteration

According to Chemical and Engineering News, the top five foods targeted for adulteration are milk, olive oil, honey, saffron, and seafood (4). The adulterants can be divided into three categories: targeted, nontargeted, and economically motivated (EMA). This division recognizes that there is a crossover effect and that HPLC plays a key role in the detection of adulterants. Although there is no exact definition for economically motivated adulteration, the United States Food and Drug Administration (FDA) adopted a working definition for EMA as the "Fraudulent, intentional substitution or addition of a substance in a product for the purpose of increasing the apparent value of the product or reducing the cost of its production, that is, for economic gain" (7). Common types of EMAs include substitution or dilution of an authentic ingredient with a cheaper product (for example, replacing extra virgin olive oil with a cheaper oil), flavor or color enhancement using illicit or unapproved substances (such as unapproved dyes), and substitution of one species with another (such as fish species fraud).

Figure 1: Chromatogram of the separation of cyanuric acid and melamine and their C13 analogs on a 50 mm × 2.1 mm, 2.6-µm dp 100-Å Kinetex HILIC LC column.

Milk

In the case of milk, many individuals tend to focus on melamine as the "poster compound" for milk adulteration. Figure 1 shows a chromatogram of a melamine-contaminated milk with the compounds cyanuric acid and melamine identified by MS using an isocratic mobile phase consisting of acetonitrile and 100 mM ammonium acetate. Peaks 1 and 2 are cyanuric acid and its ¹³C analog and peaks 3 and 4 are melamine and its ¹³C analog. While melamine is a high visibility target, it is being monitored and anecdotal information indicates that it is now being replaced by urea and even amino acids because they are more difficult to identify. In addition to melamine, soy protein, corn syrup, whey, leather, and even shampoo have been reported as potential adulterants in milk. When leather is added to milk, it can be hydrolyzed to improve its solubility. The use of this material can be detected by the determination of the amino acid hydroxyproline from the hydrolysis of leather protein that is not seen in milk protein (8). There is an active group at the United States Pharmacopeia called the Skim Milk Advisory Group that is investigating potential adulterants in skim milk, one of which is soy. Figure 2 shows an example chromatogram of various samples ranging from skim milk powder to soy (9). Other potential contaminants could be different types of milk such as goat's milk, which can be detected at the 1% level by the determination of beta-lactoglobulin.

Figure 2: UHPLC chromatograms in Masslynx (Waters) format: red = pure skim milk powder (SMP), black = 99:1 (w/w) SMP–soy protein isolate (SPI), purple = 90:10 (w/w) SMP–SPI, and green = pure SPI. Detection: UV absorbance at 215 nm.

Olive Oil

The contamination of olive oil with other oils including corn, sunflower, safflower, and sesame requires continual testing to verify it is pure olive oil with both polyphenols and triglycerides used as marker compounds. Furthermore, olive oil contains a higher concentration of oleic acid than other oils, but less linoleic and linolenic.

In a similar vein, wines are being adulterated by the addition of polyphenols. Other things that have been added to wine include pigments and glycerol to give a wine "body." In a paper published in Food Chemistry (10), the authors described an HPLC method for the anthocyanins in red wine, in which elderberry extracts were added to improve the color. The method determined that wine adulterated with the elderberry contained an extra peak attributed to cyanidin-3-bubioside-5-glucoside.

Honey

The third food on the top five list from Chemical and Engineering News was honey. The dilution of honey with less expensive materials, such as corn syrup, has been around for decades. The initial work on this topic was done by White of the United States Department of Agriculture using nuclear magnetic resonance (NMR) spectroscopy (11), but as technology evolved there have been a number of HPLC applications to monitor this phenomenon. In addition to the EMA activities, there is also concern about honey being contaminated with the antibiotic chloramphenicol used by beekeepers to treat their hives against the crippling foulbrood disease. Figure 3 provides a chromatogram from a liquid chromatography–mass spectrometry (LC–MS) method developed for this application (12) with either a 100 mm × 4.6 mm RP-18e column (EMD Millipore) or a 250 mm × 4.6 mm, 5-µm d_p Zorbax XDB C18 (Agilent Technologies). For both columns, the mobile phase was 45:55 (v/v) methanol–0.2% aqueous ammonia acetate at a flow rate of 1 mL/min.

Figure 3: (a) Total ion chromatogram (TIC) and (b) extracted ion chromatogram (EIC) obtained from chloramphenicol with detection by MS in negative ESI mode.

Saffron and Other Spices

Because of its cost, saffron is fourth on the list (according to Chemical and Engineering News), but other literature seems to be more inclusive by indicating that spices, in general, are targets for adulteration. Epicurean Digest indicated seven spices of concern: cayenne pepper, cumin, coriander, pepper, saffron, turmeric, and salt (13). Unscrupulous suppliers can adulterate cayenne pepper with sawdust and colors, cumin with sawdust, turmeric with sawdust and yellow colors, and saffron with corn silk. Figure 4 provides a chromatogram of Sudan Red I, II, III, IIB, and IV dyes corresponding to peaks 1–5, respectively, which can be used to adulterate spices by enhancing color. The separation was performed on a 150 mm × 4.6 mm, 4-µm Synergi Polar-RP 80A LC column (Phenomenex) using a 65:20:15 (v/v/v) methanol–acetonitrile–water mobile phase. Detection was UV absorbance at 480 nm.

Figure 4: Chromatogram of sudan dyes (see text for details).

Meat and Fish

As was indicated in the introduction, just this month there was an incident in China where tainted meat was mixed with fresh meat and provided to unsuspecting organizations including KFC and McDonald's (6). Last year in Europe, it was determined that horsemeat was mixed with beef. In addition, there is a lot of data indicating that a large percentage of fish is mislabeled, leading to a cheaper fish being sold as a more expensive one (14). This does not include the issue with farmed and wild caught salmon. Although DNA-based techniques have been widely used, a number of HPLC techniques including proteomics, or "foodomics" as it is now called, have been used to help solve this quandary. Furthermore, information about fish fraud indicates that a substantial amount of fish sold in outlets ranging from sushi bars to the local fish market is not as advertised. A recent report found that fish samples purchased at grocery stores, restaurants, and sushi bars in major cities were often mislabeled, including red snapper (actually tilefish); white tuna and butterfish (actually escolar); wild Alaskan salmon (actually farmed Atlantic salmon); caviar (actually catfish roe); and monkfish (actually puffer fish) (14).

A paper by Giaretta (15) described an ultrahigh-pressure liquid chromatography (UHPLC) method using myoglobin as a marker for meat adulteration with an example given on detecting pork in beef. Figure 5 is an example chromatogram of a variety of meat types. This method used a Protein-Pak Hi Res Q column (Waters) with photodiode-array detection and a mobile-phase system consisting of three buffers in a discontinuous gradient. A final example can be seen in a recent study in which Chou and coworkers (16) used HPLC with electrochemical detection using copper nanoparticle–plated electrodes to differentiate meat from 15 animal species.

Figure 5: Chromatogram of myoglobin from different sources.

Conclusion

This column installment has provided a modest snapshot on the use of HPLC techniques to address challenges in EMA of the food supply. This issue will continue to be a moving target with a need to be vigilant to monitor developments of EMA in parallel with the food industry, instrument vendors, and government labs. There is still the need for simpler sample preparation protocols and it would seem that an expansion of the various "ambient" LC–MS techniques in this area could be helpful. One of the techniques that is being touted by HPLC–MS vendors is the application of exact-mass LC–MS, but, in our opinion, one must ensure that peak identifications are appropriate since sometimes standards are not available.

Finally, as the adulterers become more sophisticated, it seems like we will need more sophisticated HPLC-based techniques such as foodomics paired with other instrumental techniques like immunoassay and Fourier transform infrared (FTIR), NIR, and Raman spectroscopy.

References

(1) "A la Cartel," The Economist, March 2014.

(2) B. Wilson, Swindled: The Dark History of Food Fraud, from Poisoned Candy to Counterfeit Coffee (Princeton University Press, Princeton, New Jersey, 2008).

(3) F.C. Accum, Treatise on the Adulteration of Food and Culinary Poisons Exhibiting the Fraudulent Sophistications of Bread, Beer, Wine, Spirituous Liquors, Tea, Coffee, Crème. Confectionary, Vinegar, Mustard, Pepper, Cheese, Olive Oils, Pickles and other Articles Employed in Human Commerce (Longman, Hurst, Rees, Orme and Brown, London, UK, 1820).

(4) Chem. Eng. News Online, "Food Fraud," August 25, 2014.

(5) http://www.nbcnews.com/id/28787126/ns/world_news-asia_pacific/t/face-execution-over-china-poison-milk-scandal/.

(6) http://money.cnn.com/2014/07/21/news/companies/kfc-mcdonalds-china/.

(7) http://foodfraud.msu.edu/wp-content/uploads/2014/01/CRS-Food-Fraud-and-EMA-2014-R43358.pdf.

(8) "Dionex Solutions: Methods for Detecting Leather Protein Adulteration in Milk," Dionex Corporation, http://www.dionex.com/en-us/markets/food-beverage/news-articles/lp-110606.html.

(9) J.E. Jablonski, C. Pardo, L.S. Jackson, B. Rohrback, J. Moore, and M. Han, "Chemometrics and UPLC-UV to Detect Adulteration of Skim Milk Powder with Soy Protein Isolate," poster from United States Pharmacopeia Skim Milk Powder Advisory Group.

(10) P. Brindle and C. García-Viguera, Food Chemistry 55, 111 (1996).

(11) J.W. White, Jr., J. Assoc. Off. Anal. Chem. 63, 11 (1980).

(12) C. Pan et al., Acta Chromatographia 16, 320 (2006).

(13) Epicurean Digest, epicureandigest.com.

(14) http://oceana.org/en.

(15) N. Giaretta et al., Food Chemistry 141, 1814 (2013).

(16) C.-C. Chou et al., J. Chromatog. B 846, 203 (2007).

This month's guest coauthors:

Kendra C. Pfeifer is a manager of regulatory affairs in the quality and regulatory affairs department at the Hershey Company. She has a bachelor's degree in chemistry and a master's degree in food science. Kendra has a wide variety of experiences within the corporation with a tenure in the analytical research and services organization before joining the regulatory affairs group. She was active in the Association of Analytical Communities (AOAC) methods committee and implemented robotics in the lab environment.

Kendra C. Pfeifer

Jeff Hurst is a principal scientist with the Hershey Company. He is the author of a substantial number of papers on HPLC and food analysis and is a member of numerous scientific organizations including the American Chemical Society (ACS), the Institute of Food Technologists (IFT), the American Society for Mass Spectrometry (ASMS), and the American Association for Integrative Medicine (AAIM). He indicates that Hershey was his first "real job."

Jeff Hurst

Editor of Column Watch:

Ronald E. Majors is the editor of "Column Watch," an analytical consultant, and a member of LCGC's editorial advisory board. Direct correspondence about this column to lcgcedit@lcgcmag.com

Ronald E. Majors