Analytical Chemistry Research Paper

Analysis of Carcinogenic Sudan Azo Dyes in Food Products


T. Gottapu1 and D. Cunningham1, 1Department of Chemistry and Physics, 2Department of Kinesiology Nutrition Recreation, Southeast Missouri State University. ANALYSIS OF CARCINOGENIC AZO DYES IN FOOD PRODUCTS. Previous studies have found carcinogenic Sudan azo dyes in food products including red palm oil. Our initial work showed the addition of red Sudan IV to light yellow inexpensive oils at levels of 1-5 ppm produced a red colour similar to the natural red palm oil. To quantitate the Sudan IV level in adulterated oils and palm soup cream, samples were extracted with hexane and pre-treated using solid phase extraction (SPE). Sample addition, washing and elution from the SPE cartridges were characterized by HPLC analysis of fractions from the cartridges. Due to the hydrophobic nature of Sudan IV, a very high organic HPLC mobile phase (95% methanol; 5% pH 5 buffer) was used with a C-18 column. Linear calibration curves were obtained in the 50-5000 ppb range at 340 nm with an analysis time of 15 minutes.


Sudan I–IV are azo dyes used for colouring Food products, triglycerides and lipoproteins (Hunger et al. 2000). They are labelled category 3 carcinogens by the IARC (IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans 1987) and are not permitted to be used in foods in most countries. Because these dyes are looks bright in colour and not expensive, they have been used intentionally to adulterate palm oil and spices to enhance the adulterated quality of the product. Following Dyes are widely using in Food.

Sudan I (limit of limit of 0.5 ug per g of Sunset Yellow FCF)Sudan II
Sudan IIISudan IV

Adulteration with dyes has been used to cover up products in which the natural colour has been lost due to improper drying, storage or fungal infestations where pigment is degraded (Arora & Bharti 2005; Mishra et al. 2007). The first adulteration of chilli powder with Sudan I was found in 2003, and the source of contamination was identified that chilli powder imported from India named with the brand name genus with a concentration of 4000 mg kg−1 (RASFF 2003; ASTA 2005). Government was imposed rule to quality analytical test for all the products imported with the brand name genus to ensure adulteration with sudan I. (European Commission 2003). In 2004, this requirement was expanded to test Sudans II–IV also. Testing was expanded to include palm oil and the genus Curcuma in 2004 (turmeric) (European Commission 2005). In the UK in 2005, Sudan I was found in a sauce (3 μg ml−1) that was found to contain adulterated chilli powder (80 mg kg−1) (RASFF 2005). This sauce had been used to prepare a lot of foods including soups, salad dressings, sausage ready meals, pate, seafood, mince. (Sudan I Consolidated Product List from February 2005). As a result, almost 500 food products were recalled, and it is the largest recall in the UK history. Unauthorised colours continue to be reported in the RASFF portal, with a total of 16 notifications in 2014 and 2015 (RASFF 2015).


Chemicals and Reagents:Some of the chemicals and reagents utilized in the experiment included fresh palm fruit extract from Neat Food Company from Ghana, Praise Palm Cream from Praise Export Services Limited from Ghana, HPLC methanol, hexane, ethyl ether, and ethyl acetate.

Solid-phase extraction (SPE) clean-up and preparation of extracts: SPE was performed using LC-Alumina-B SPE tubes (1 g/3 ml) from Supelco (Tran et al. 2005). The cartridge was conditioned with 6 ml of methanol, 6 ml of ethyl acetate and 6 ml of hexane. Then, 1 ml of the sample solution was added to the SPE column. The sample was washed with 6 ml of hexane and 6 ml wash of ethyl ether (palm oil only), followed by 2 ml of ethyl acetate The Sudan dyes were then eluted from the column using 8 ml of 90:10 ethyl acetate: methanol solution. The extract was filtered through a 0.2 μm PTFE filter and internal standard was added (10 μl of 1 ng/μl d6-Sudan III solution) prior to LC-MS/MS analysis.

Procedure: Shimadzu conducted the separation and quantification of Sudan IV levels in the spiked oil and palm soup cream in Japan. A high-performance liquid chromatography (HPLC) was employed for the process. Before the HPLC technique, the samples were pre-treated using solid-phase extraction (SPE). The SPE procedure is mostly employed for extraction, concentration, and fractioning of organic compounds (Andrade-Eiroa et al. 641; Rocha et al. 803). The equipment compresses a degasser, a gradient pump, a liquid sampler, a column oven, and a diode array detector (Esen et al. 73; Moldoveanu and Victor 9; Hu et al. 2126; Weisz et al. 1835). The chromatographic conditions involved the use of high organic HPLC mobile phase (95% methanol, 5% pH 5 buffer) and C-18 column. After the chromatographic specifications, the calibration curves were plotted in the 50-5000 ppb range at 340 nm with an analysis time of 15 minutes. Finally, the presence of Sudan IV in different quantities of the prepared palm oil and cream were determined from the generated chromatograms.

Figure 1: The Prepared Samples


The study started with the calibration of the equipment using different samples of Praise and Neat. The data obtained from the first experiment was recorded, as presented in Table 1. Besides,the data obtained for red palm oil is recorded in Table 1, whereas the results for the palm cream are illustrated in Figure 2.

Table 1: The Calibration Data

2500 S4 1/2 S1 1/2CaroteneSudan IV peak at 11.54 min
5000 S4 S1 CaroteneSudan IV peak at 11.57 min
500 S4 S1 CaroteneSudan IV peak at 11.59 min
1000 S4 S1 CaroteneSudan IV peak at 11.65 min
250 S4 S1 CaroteneSudan IV peak at 11.72 min

Table 2: The results from the analysis of red palm oil samples

250 ng/mL SIV 450 S1Sudan IV peak at 11.07 min
100 ng/mL SIV 180 S1Sudan IV peak at 11.51 min
50 ng/mL SIV 180 S1Sudan IV peak at 11.70 min
25 ng/mL SIV 180 S1No Sudan IV peak
10 ng/mL SIV 180 S1No Sudan IV peak
Unspiked1 red palm oilNo Sudan IV peak

Figure 2: The Chromatogram for the Palm Cream


It was observed that there was no presence of Sudan IV dye in the neat oil palm oil. However, it could not be confirmed whether the oil had zero quantities of Sudan IV because the technique used for quantification began detecting the dye when 50 ng/mL and above volumes were added. It was also observed the time taken to separate the dye from the mobile phase decreases with the increasing quantities of the compound. Similarly, the results for palm cream indicate that there was no presence of Sudan IV for the unspiked sample. It was concluded that both the red palm oil and the palm cream considered in the study might not have Sudan IV dye. However, the accuracy of the process used was in doubt. Therefore, for future analysis, more accurate methods, such as liquid chromatography-mass spectrometry (LC-MS)/mass spectrometry (MS), Fourier transform infrared spectroscopy (FTIR), and mid-infrared spectroscopy (MIR), should be utilized. Overall, the experiment was successfully conducted.

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