Seeing Red: Detecting Illegal Sudan Dyes in Foodstuffs

Making foods look fresh, edible and acceptable to consumers is common sense within the food industry. Who wants to buy pallid bakery products, wrinkled veggies or shriveled meat, for example? However, in using certain additives such as prohibited synthetic dyes, some unscrupulous producers take extreme measures to enhance their product, endangering consumer health in the process.

Sudan Dyes and Food Fraud
Sudan dyes are widely used in commercial manufacturing to impart a rich red, red-orange or yellow-orange color to plastics and textiles. They are cheap and easy to obtain; for this reason, they have also been used for enhancing presentation of spices such as chili powder, paprika and curry pastes. However, large amounts (mg per kg) are required for effect. These fat-soluble Azo dyes, more commonly known in laboratory staining protocols or as constituents of hair dye, are Group 3 carcinogens. They also have potential for genotoxic activity through formation of mutation-inducing DNA adducts, as well as allergenic activity. For these reasons, many countries ban their use in foods. Starting in 2003, the European Union took steps to eliminate foodstuffs containing Sudan dyes from entering the marketplace (see EU Regulations listed below).

Food Fraud Detection
There are various methods available to food safety laboratories for detecting Sudan dye adulteration in foodstuffs, ranging from HPLC plus UV diode array detection (HPLC-DAD) to enzyme-linked immunosorbent assay (ELISA). Most of these techniques are laborious and time-consuming, requiring extensive sample preparation and analysis however, there are new methods in development.

DiAnibal et al. (2012) reported using Raman spectroscopy for detecting Sudan I dye in adulterated paprika powder¹. The researchers compared three types of Raman spectroscopy, including fourier-transformed (FT)-Raman using the Nicolet 5700 FT-IR (Thermo Scientific), analyzing prepared samples on aluminium foils. Their results showed that surface-enhanced Raman spectroscopy (SERS) gave the best results for rapid and reliable detection.

In 2013, Dar et al. explored thin layer chromatography (TLC) to detect Sudan III and IV in red chili powder, which they spiked with commercial dye standards². After testing various solvents, they used acetonitrile to extract the samples prior to TLC. The researchers reported that this method discriminated effectively between natural colorants such as carotenoids and the spiked Sudan dye standards, with an effective visual limit of detection found at 5mg/kg.

Meanwhile, Haughey et al. (2015) examined vibrational spectroscopy techniques, near infrared (NIR) spectroscopy and Raman spectroscopy to detect Sudan dyes in foodstuffs³. In their feasibility study, the Antaris II FT-NIR (Thermo Scientific) achieved lower limits of detection (LOD) and better calibration results for chili powders spiked with Sudan I standards analyzed than Raman spectroscopy. The authors did report however, that both techniques were rapid, easy to use and involved minimal or no sample preparation.

Another practical and efficient method uses two-dimensional high-pressure liquid chromatography in conjunction with solid phase extraction (2D-HPLC-SPE) prior to mass spectrometry⁴. Once the extraction is completed offline, separation and MS detection are automated using the Dionex Ultimate 3000 system controlled with Chromeleon software (both Thermo Scientific). Using a UV spectral standards library for reference, this method gives good results detecting Sudan I, II, II and IV in chili oil and curry paste.

Dynamic Detection for Evolution of Fraud
It is worth noting, however, that as detection methods for one fraudulent additive improve, unscrupulous producers will shift to using other adulterants, as shown in work by Ruf et al.⁵ In a 2012 paper, the researchers reported detecting Azo dye, Basic Red 46, masquerading as a Sudan dye in sumac spice.

Using standard methods for detection (HPLC-DAD), the team could not identify any Sudan dye content but were perplexed by the intense red staining retained in the fractionation column. It was only when they turned to mass spectrometry that the illegal dye was spotted. Ruf et al. analyzed their spice sample first by liquid chromatography coupled with tandem mass spectrometer (LC-MS/MS) TSQ Quantum Discovery, and then by high resolution LC-MS using an Orbitrap Exactive HCD mass spectrometer (both Thermo Scientific).

Once they collected spectral data, the researchers then turned to searching online databases for information on the structure isolated from the spice. Finally, they confirmed its identity by comparing spectral data with LC-MS analysis of a commercial hair product containing Basic Red 46.

Regulatory Issues
In 2003, the European Commission (EC) released an emergency measures decision (2003/460/EC) regarding Sudan dye adulteration of hot chili and chili products, requiring producers to test raw materials, releasing another measure in 2005 due to high levels of adulteration and food fraud involving this dye group. The importance of the measures in maintaining food chain safety became apparent during 2005, when the United Kingdom recalled Worcester Sauce contaminated with Sudan I, tracing the source back to a 2003 shipment of chili powder.

The EC Decision 2005/402/EC implementing emergency measures regarding chili, chili products, curcuma and palm, requiring monitoring and reporting by member states. With the success of this measure and reduction in reported cases, European Commission Regulation (EC) No 669/2009 relaxed the monitoring requirements in 2009.

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References 

1. DiAnibal, C.V. et al. (2012) “Surface Enhanced Raman Spectroscopy (SERS) and multivariate analysis as a screening tool for detecting Sudan I dye in culinary spices“, Spectrochimica Acta Part A 87 (pp. 135– 141)

2. Dar, M.M. et al. (2013) “Detection of Sudan Dyes in Red Chilli Powder by Thin Layer Chromatography“, 2:586 doi:10.4172/ scientificreports.586

3. Haughey, S.A. “The feasibility of using near infrared and Raman spectroscopic techniques to detect fraudulent adulteration of chili powders with Sudan dye“, Food Control 48 (2015) 75e83

4. Tukkeeree, S. and Rohrer, J. (2012) “Determination of Sudan Dyes I–IV in Curry Paste”, Thermo Scientific App Note http://www.dionex.com/en-us/webdocs/113854-AN1023-LC-2D-Sudan-Dyes-Curry-Paste-AN70212_E.pdf

5. Ruf, J. et al. (2012) “Discovery and structural elucidation of the illegal azo dye Basic Red 46 in sumac spice“, Food Additives and Contaminants Part A 29(pp.897-907) 

 

EU Regulations

• European Union Rapid Alerts System for Food and Feed (EU RASFF). (2012). Annual Report, 21
• EUROPEAN COMMISSION DECISION 2003/460/EC on emergency measures regarding hot chili and chili products. Official Journal of the European Union, L154/114.
• EUROPEAN COMMISSION DECISION 2005/402/EC on emergency measures regarding chili, chili products, curcuma and palm. Official Journal of the European Union, L135/34.
• EUROPEAN COMMISSION REGULATION (EC) No 669/2009 implementing regulation (EC) No 882/2004 of the European Parliament and of the Council as regards the increased level of official controls on imports of certain feed and food of nonanimal origin and amending Decision 2006/504/EC. Official Journal of the European Union (EC 669/2009) L194/11.