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Determination of seven illegal dyes in Egyptian spices by HPLC with gel permeation chromatography clean up
Journal of Food Composition and Analysis 84 (2019) 103304
Contents lists available at ScienceDirect
Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original Research Article
Determination of seven illegal dyes in Egyptian spices by HPLC with gel permeation chromatography clean up
T
Ahmed Salem Sebaeia,⁎, Manar Ibrahim Youssifb, Ahmed Abdel-Maksoud Ghazia a b
Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Foods, Agricultural Research Center, Ministry of Agriculture, Giza, 12311, Egypt Faculty of Biotechnology, October University for Modern Sciences and Arts University (MSA), Giza, 12566, Egypt
ARTICLE INFO
ABSTRACT
Keywords: Hot chilli Sudan dyes Method validation Food spices
Sudan I, II, III, IV, Para Red, Orange G and Red 7B are synthetic regularly dyes utilized to dye plastics and other manufactured materials. In recent years, concerns about the genotoxic possibilities of Sudan colors have been raised. A few national bodies and food experts consider Sudan dyes to be genotoxic cancer-causing agents while others view these dyes as possible cancer-causing agents. Therefore, it is of the outmost importance to assess the risk and presence of Sudan dyes in Egyptian spices, including; sumac, hot chilli, cumin, paprika, curry and turmeric. A total of 83 samples were collected randomly from different supermarkets and spice shop in various municipalities of Egypt (Cairo, Giza, Qalyubia, Faiyum and Alexandria). For Sudan dyes determination, gel permeation chromatography (GPC) and high-performance liquid chromatography (HPLC) were used. Dyes were extracted from spices with acetonitrile and selectively initial separated by GPC. Fractions were collected from 12 min to 24 min and then determined by HPLC with diode array detection (HPLC-DAD). The method performance was validated on hot chilli samples, and the limit of quantification (LOQ) was 0.1 mg/kg for the seven Sudan dyes. Moreover, other method validation parameters were determined in this work, including linearity, accuracy, recovery, limit of detection (LOD), repeatability, reproducibility and expanded uncertainty. Finally, a market survey was conducted to assess the incidence of such illegal dyes and enlighten buyers, almost 50% of the tested samples were contaminated with Sudan dyes. Sudan Red 7B and Para Red were not detected in the samples, and the mean contamination level varied from 0.01 mg/kg for Sudan Ⅱ to 50.1 mg/kg for Sudan Ӏ. It is recommended to buy raw whole spices – not in powder form- and blend them at home. In addition, spices should be purchased from established retailers, and buyers should avoid spices with abnormally red shading.
1. Introduction Sudan dyes are synthetic lipophilic red-coloured azo dyes that do not occur naturally; however, such dyes are currently used in various applications as a colouring agents in several matrixes, including spices (Petrakis et al., 2017). Azo dyes are a large class of synthetic organic dyes that contain nitrogen in an azo group (-N = N-) as part of the molecular structures (Otero et al., 2017). In 2003, the Federal Institute for Risk Assessment stated that the European Union (EU) does not permit the use of these dyes as food additives, citing the degradation products which are considered to be carcinogens and teratogens. The azo dyes Sudan I-IV are classified as carcinogenic (category II-III) due to their ability to be separated into amines after administration into the human body (Alim-Un-Nisa and Akhlaq, 2015). Sudan dyes belong to the azo dyes, and the most common types are Sudan I, Sudan II, Sudan III, Sudan IV, Orange G, Red
⁎
7B and Para Red (Fig. 1) (Ertaş et al., 2007) Sudan dyes are illegal in many industries. However, these dyes have the advantages of colour fastness and low price. Despite the fact that they are banned from use by the Food Standards Agency (FSA) and the EU (Yan et al., 2011), some industries use Sudan dyes in manufactured products, including; spices, polishes, waxes, textiles, and cosmetics, and in scientific applications, furthermore, it has been proven that Sudan dyes are present in egg yolk (Bazregar et al., 2017). In Africa, particularly in Nigeria, traditional and homemade delicacies are prepared with traditional and indigenous spices. These spices are used as additives or curing agents to add taste or flavour to food (Ene-Obong et al., 2018). Since 1999, azo dyes have been used to treat textiles. It has been estimated that over ten thousand dyes are used in industial fields with an equivalent number of products (Chequer et al., 2013). As mentioned by Carmen and Daniela (2012), consumers/buyers typically seek certain basic product characteristics, including non-
Corresponding author at: QCAP Laboratory, Agriculture Research Center, Ministry of Agriculture, Giza, 12311, Egypt. E-mail address: [email protected] (A.S. Sebaei).
https://doi.org/10.1016/j.jfca.2019.103304 Received 6 March 2019; Received in revised form 28 June 2019; Accepted 29 August 2019 Available online 30 August 2019 0889-1575/ © 2019 Elsevier Inc. All rights reserved.
Journal of Food Composition and Analysis 84 (2019) 103304
A.S. Sebaei, et al.
Fig. 1. Chemical Structures of Sudan dyes.
themselves (Di Anibal et al., 2011). These instruments include; HPLC with electrochemical detection (Chailapakul et al., 2008), pressurized capillary electrochromatography (CEC) with amperometric detection (Fukuji et al., 2011), tandem mass spectrometry coupled with isotope dilution (Di Donna et al., 2004), multi-wall carbon nanotube-based electrochemical sensing (Gan et al., 2008), and enzyme-linked immunosorbent assay (ELISA) (Ju et al., 2008). In most electrochemical methods, a single Sudan dye compound is quantified; otherwise, analytical sensitivity sacrificed. Many relevant chromatography methods, require the use of isotopic internal standards, tandem mass techniques, many pre-separation steps or time-consuming and tedious preparation methods to achieve the required selectivity, and sensitivity and also to overcome the matrix effect of spices. Herein, a selective, robust, sensitive and simple test method is proposed that takes advantage of the large gap in molecular size between Sudan dyes and the heavy matrix of spice components to selectively separate multiple-dyes by size exclusion chromatography and then determine the analytes by through their UV spectra through liquid chromatography with diode array detection. Overall, the main objective of this proposed work is the determination of seven Sudan dyes in the Egyptian spice market. In addition, a selective, reliable and accurate gel permeation chromatography (GPC) method is developed, including; optimization, validation and quality control assessments. Furthermore, a survey is administered regarding spices sold in different municipalities in Egypt and their adulteration with Sudan dyes. The levels of contamination found in this overview are noteworthy. The results show that it is important to monitor Sudan dyes in foodstuffs.
fading colours in response to light, washing, heating, and perspiration, both initially and after prolonged use of the material. People have the right to presume that the food they eat is safe and suitable for consumption. Foodborne illness and injury are disagreeable at best; at worst, they can be fatal. Eruptions of foodborne illness can damage trade and tourism, and lead to loss of earnings, unemployment and litigation. Food spoilage is wasteful and, costly and can adversely affect trade and consumer confidence (Baş et al., 2007). Since 2001, Sudan dyes have been in spices in EU. A February 2017 search of Europe'sRaid Alert System for Food and Feed (RASFF) for "unapproved dyes" and "Sudan" in the "herbs and spices" product category retrieved approximately 429 notifications. In a search of the FDA Import Alert 45-02 (Detention Without Physical Examination and Guidance of Foods Containing Illegal and/or Undeclared Colors) the discovered no record of Sudan dye adulteration. Referring to the (RASFF); in 2003, the European Commission established Decision 2003/460/EC, the first community measure to control the unlawful use of the colouring substance Sudan dye in adulterated chilli and chilli products. A second decision extending the scope of the measures was adopted on 21 January 2004 and is still in force. In addition, a report issued in 2016 found 5 notifications detailing Sudan dyes in spices (1 in palm oil) and three revealing Rhodamine dye. Given the fewer instances of non-compliance; in 2017 there were 8 notifications associated with the adulteration of palm oil with Sudan I. In 2015, the Minister of Health (MOH) issued Decree 204/2015 regarding food additives that are acceptable for use by the food industry. Exporters should check with the MOH to confirm the satisfactoriness of all food colourings. Moreover, food dyes are continuously reviewed and updated according to Codex Alimentarius standards. There are no exceptions to the regulations governing food colourings. Egyptian authorities will not allow a product to be imported if it contains an unauthorized dye, even if the use of the dye is acceptable in another country. The targeted spices that are suspected to contain Sudan dye and its include; hot chilli, cumin, curry, paprika, sumac and turmeric. Moreover, these spices are characterized by a colourful appearance. As mentioned in several previous papers, multiple methods are used worldwide in the detection and analysis of Sudan azo dyes in foodstuffs, depending on the availability and financial stability of the laboratories
2. Materials and methods 2.1. Chemicals and materials All chemicals and reagents were of HPLC or analytical grade. Deionized water used throughout the experiments was obtained by using the Integral Water Purification System for Ultrapure Water (Milli-Q A10) from Merck, and sodium sulfate was activated at 150 °C overnight. Bio-Beads –SX3 (200–400 mesh) was purchased from Bio-Red Laboratories for GPC column packing.
2
Journal of Food Composition and Analysis 84 (2019) 103304
A.S. Sebaei, et al.
Table 1 GPC profile of eluted fractions in mg/kg for the seven colors vs fraction time in min. Analyte
Orange G Para Red Sudan I Sudan II Sudan III Red 7B Sudan IV
Time (min) 0-3
3-6
6-9
9-12
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
12-15 15-18 Eluted fractions in mg/kg 0.88 7.93 6.87 7.28 2.63 4.25 1.65
7.00 1.08 2.75 1.28 5.94 3.95 3.31
18-21
21-24
24-27
27-30
1.20 0.73 1.69 0.13 0.49 0.34 2.91
0.9 0.6 1.96 0.085 0.33 0 1.64
0 0 0 0 0 0 0
0 0 0 0 0 0 0
Table 2 Sudan dyes adulteration profile of the 41 samples. Sample type
No.
No. of detected samples
chili turmeric paprika cumin sumac curry total
18 13 17 15 10 10 83
11 6 10 6 5 3 41
Percentage (%) 61.1 46.2 58.8 40.0 50.0 30.0 49.4
% % % % % % %
Fig. 4. Sudan I calibration curve and accepted correlation for HPLC standard linearity.
2.2. Sample collection A total of 83 spice samples (Table 1) were randomly purchased from different commercial supermarkets and spice shops in various municipalities of Egypt (Cairo, Giza, Qalyubia, Faiyum and Alexandria) during the period from October 2017 to September 2018. 2.3. Standard preparation The 7 compounds Orange G., Para Red, Sudan Ӏ, Sudan Ⅱ, Sudan Ⅲ, Sudan Red 7B and Sudan Ⅳ were purchased as certified reference standards from LGC. Stock solutions for individual compounds were prepared at 1000 mg/kg in acetonitrile: dichloromethane (1:1 v/v) and a working solution was prepared as a mixture of all dyes at 100 mg/kg for spiking and HPLC intermediate check tests. For GPCfraction,
Fig. 2. GPC separation profile and the fraction elution time of the seven Sudan dyes.
Fig. 3. Para Red and Sudan dyes HPLC-DAD chromatogram standard of 1 mg/L. 3
Journal of Food Composition and Analysis 84 (2019) 103304
A.S. Sebaei, et al.
Table 3 Repeatability of a contaminated hot chili sample and reproducibility as inter-day repeatability at 0.1 mg/kg spiking level, (n = 6).
Repeatability Inter-day Repeatability
RSD% RSD%
Sudan І
Sudan Ⅱ
Sudan Ⅲ
Sudan Ⅳ
Sudan red 7b
Orange G
3 6
2 6
3 8
5 10
2 9
2 8
Table 4 Average violated concentration mg/kg of the seven illegal dyes in 6 spices commodities. Spices
Orange G
Para red
Sudan І
Sudan Ⅱ
Sudan Ⅲ
Red 7b
Sudan Ⅳ
chili cumin paprika turmeric curry sumac Total average
3.9 n.d 6.2 n.d n.d n.d 2.1
n.d n.d n.d n.d n.d n.d n.d
58.7 60.9 22.5 86.2 60.4 0.2 50.4
0.01 n.d n.d n.d n.d n.d 0.01
0.5 n.d n.d n.d n.d n.d 0.1
n.d n.d n.d n.d n.d n.d n.d
64.0 0.1 0.1 9.2 4.5 0.2 12.9
n.d: not detected.
acetate: n-hexane (1: 1, v/v)). Each sample was transferred to a new labelled vial, centrifuged for 1–2 min, and then placed sequentially into the GPC tray. For each sample, 1 ml was passed through the GPC system at a flow rate of 2 ml/min; thus, analysis took 24 min per sample. After clean up, the eluted fractions were evaporated to dryness using a rotary evaporator and then 2 ml of acetonitrile was added to each sample to dissolve the residue. Finally, the samples were filtered through 0.45 μm Acrodiscs into autosampler vials, at which point they were ready for injection. Diode array detector parameters: The absorbance was measured at 230 nm Sudan I, II, III, IV and Red 7B while measured at 380 nm for Sudan Orange G. The HPLC injected volume was 25 μl and the mobile phase was acetonitrile: water (95: 5) in isocratic mode with a 1 ml/min flow rate. The above sequential process was repeated until all spice samples had been completely analysed.
Fig. 5. Contamination percentage of each spice cheated with Sudan dyes.
2.5. GPC and HPLC analysis The GPC clean-up system (Waters, USA) consists of a built-in binary pump (Waters 1525), 100 vial autosampler tray (4 ml) (Waters 717 plus) with a 5000 μl sample loop, and a fraction collector (Waters III). The system was equipped a 10 mm i.d., 500 mm long chromatographic glass column (Omnifit, 006cc-25-25-AF, Diba industries Ltd., Cambridge, England), which was prepared by soaking 10 g Bio-Beads –SX3 more for more than 2 h in GPC mobile phase hexane: ethyl acetate (1:1) and loaded into the column with slight flow. The column lifetime allows the injection of up to 2000 samples. The 1100 series HPLC is from (Agilent, Germany) was equipped with a quaternary pump (G1311A), vacuum degasser (G1322), autosampler (G1313A), and diode array detector (Agilent, (G1321B)). The analytical column was an Agilent Eclipse® plus C18 (250 mm × 4.6 × 5 μm).
Fig. 6. Sudan dyes mean concentrations in spices in Egypt.
validation was performed with a 100 μg/ml Sudan dye mixture prepared by diluting 10 ml of the stock standard solution of Sudan dyes in 4 ml of ethyl acetate: n-hexane this mixture was then used as a control for various fractionation steps essential for method development.
2.6. LOQ Ten grams of cumin was weighed, and 10 μl of each of the seven Sudan mixtures was added to the weighed cumin; the mixture was then dissolved in 100 ml of acetonitrile solution in a conical flask. The sample was filtered through glass funnels into another volumetric flask with 15 g of activated sodium sulfate as a separating agent. After filtration, an aliquot of 50 ml of the filtrate was pipetted into another 100 ml flask. The collected solvent was then evaporated to dryness using a rotary evaporator at approximately 50 °C and then diluted with 4 ml GPC mobile phase (1: 1 acetonitrile: hexane) into a vial. After 30 min of GPC processing, the samples were evaporated to dryness and then diluted with 1 ml of acetonitrile in HPLC vials. The recovery and coefficient o variation (CV%) at the LOQ were determined after completing injection.
2.4. Sample preparation The samples to be analysed by HPLC were subjected to an extraction process: ten grams of sample was weighed and 100 mL of acetonitrile was added. The samples were shaken for 20 min at 200 rpm and then underwent filtration by gravity through 15 g of activated sodium sulfate as a separating agent over a glass funnel. After filtration, an aliquot of 50 ml of the filtrate was pipetted into another 100 ml flask. The collected solvent was then evaporated to dryness using a rotary evaporator at approximately 40 °C and diluted with 4 ml GPC mobile phase (ethyl 4
Journal of Food Composition and Analysis 84 (2019) 103304
A.S. Sebaei, et al.
3. Results and discussion
linearity for Sudan I is was 0.99975 (Fig. 4).
3.1. GPC initial separation method development Four hundred microliters of the 100 mg/kg Sudan mixture was evaporated and reconstituted in 4 ml of GPC mobile phase. One millilitre of the standard mixture was injected directly into the GPC, and a 2 ml/min flow rate was applied. The standard sample was fractionated into 10 separate flasks. The GPC clean-up process took 30 min; thus, the eluate was separated every 3 min was collected in a distinct flask; and labelled as follows; (0–3 min, 3–6 min, 6–9 min, 9–12 min, 12–15 min, 15–18 min, 18–21 min, 21–24 min, 24–27 min, and 27–30 min). Then, the samples were evaporated, 1 ml of acetonitrile was added to each flask, and the contents were transferred to HPLC vials. The above step was performed to collect 30 min of GPC eluate into 10 flasks instead of the typical 1 flask to observe the GPC separation stages.
3.5.3. Precision Precision is the level of agreement between replicate estimations under determined conditions. Precision is represented by measurable quantities, for example, a standard deviation or certainty limit; and lower precision is reflected by a larger standard deviation. The precision was identified in terms of repeatability and reproducibility. (Table 3) indicates the estimated repeatability and inter-day repeatability from 6 replicates of a spiked sample at 1 mg/kg. Trueness is the level of agreement between the mean value of a series of successive measurements and the true value or an accepted reference value and is related to systematic error (bias). Bias is calculated from the absolute relative difference (RD%), which must not surpass 20% in a sample of choice, which in this case was all the hot chilli samples. Thus, RD% reached a maximum of 11%, which is below the maximum level.
3.2. Extraction solvent efficiency
3.6. Survey of Sudan dyes in spices
Different combinations of extraction solvents were tested. Ultimately, acetonitrile was better for extraction than ethanol, acetone and methanol due to its higher recovery. Thus, acetonitrile was chosen as the extraction solvent for the different types of spices. The elution recovery using acetonitrile showed a significant percentage of 98% in contrast to the recoveries for the other tested solvents.
The average sum (Table 4) of the Sudan dyes was determined for hot chilli, cumin, turmeric, curry, sumac and paprika. The results were analysed to determine the Sudan contamination levels, the proportion of polluted samples and levels of contamination above specific and general limits. Each sample was broken down as indicated in the method. Sudan adulteration was found in 41 of the 83 collected and tested spice samples, as tabulated below (Fig. 5), so 49% of the investigated Egyptian market spices were adulterated with Sudan dyes. This survey was the first to be adopted in Egypt (Fig. 6). The highest average contamination level was found for Sudan Ӏ at 50.4 mg/kg and the lowest average concentration was found for Sudan Ⅱ at 0.01 mg/kg. Para Red and Sudan Red 7B were not detected in any samples.
3.3. GPC clean up The seven Sudan dyes were detected by known signals in the sample fractions from minutes 12–15 to 21–24 (Table 2). Moreover, the elution of the seven Sudan dyes varied during the 30 min of fractionation. No Sudan dyes were detected in the first 12 min or in the last 9 min which were considered the washing period, thus, the fraction times in the GPC separation profile of the seven Sudan dyes are explained in Fig. 2. GPC is an excellent method for isolating Sudan dyes and related azo dyes from normal dyes and other fat soluble substances in coloured spices. Thus, GPC allows the efficient extraction of Sudan dyes from spices with the simple application of size-exclusion clean-up which avoids crosscontamination.
4. Conclusion The fundamental goal of this work was the assessment of seven Sudan dyes in the Egyptian spice market. In addition, a solid and precise GPC strategy was developed and evaluated in terms of the improvement, optimization, validation and quality control of the technique utilized. Furthermore, a LOQ of 0.1 mg/kg was established. To meet the proposed objective; a series of method validation operational parameters were fully addressed in this study. Finally, this study showed 50% adulteration in six types of spices by five Sudan dyes.
3.4. HPLC analysis After HPLC analysis, it was determined that the mobile phase acetonitrile: water in a ratio of 95: 5 was the most effective mobile phase for the determination of the seven Sudan dyes, yielding fast retention times for the dyes (Fig. 3).
References Alim-Un-Nisa, N.Z., Akhlaq, F., 2015. Detection of Sudan dyes in different spices. Pak. J. Food Sci. 25 (3), 144–149. Baş, M., Ersun, A.Ş., Kıvanç, G., 2007. The evaluation of food hygiene knowledge, attitudes, and practices of food handlers’ in food businesses in Turkey. Food Control 17 (4), 317–322. Bazregar, M., Rajabi, M., Yamini, Y., Arghavani-Beydokhti, S., Asghari, A., 2017. Centrifugeless dispersive liquid-liquid microextraction based on salting-out phenomenon followed by high performance liquid chromatography for determination of Sudan dyes in different species. Food Chem. 244, 1–6. Carmen, Z., Daniela, S., 2012. Textile organic dyes—characteristics, polluting effects and separation/elimination procedures from industrial effluents—a critical overview. In: Puzyn, T. (Ed.), Organic Pollutants Ten Years after the Stockholm Convention—Environmental and Analytical Update. InTech Press, Croatia, pp. 55–86. Chailapakul, O., Wonsawat, W., Siangproh, W., Grudpan, K., Zhao, Y., Zhu, Z., 2008. Analysis of sudan I, sudan II, sudan III, and sudan IV in food by HPLC with electrochemical detection: comparison of glassy carbon electrode with carbon nanotubeionic liquid gel modified electrode. Food Chem. 109 (4), 876–882. Chequer, F.M.D., de Oliveira, G.A.R., Ferraz, E.R.A., Cardoso, J.C., Zanoni, M.V.B., de Oliveira, D.P., 2013. Textile Dyes: Dyeing Process and Environmental Impact. Di Anibal, C.V., Ruisánchez, I., Callao, M.P., 2011. High-resolution 1H nuclear magnetic resonance spectrometry combined with chemometric treatment to identify adulteration of culinary spices with Sudan dyes. Food Chem. 124 (3), 1139–1145. Di Donna, L., Maiuolo, L., Mazzotti, F., De Luca, D., Sindona, G., 2004. Assay of Sudan I contamination of foodstuff by atmospheric pressure chemical ionization tandem mass spectrometry and isotope dilution. Anal. Chem. 76 (17), 5104–5108. Ene-Obong, H., Onuoha, N., Aburime, L., Mbah, O., 2018. Chemical composition and
3.5. Method validation (fit for intended use) 3.5.1. LOQ and LOD The LOQ is the minimum concentration of analyte in a test sample that can be determined with acceptable precision, repeatability and recovery under the stated test conditions. The lowest practical LOQ was estimated by repeatedly analyzing a cumin sample at the expected lowest quantification level of 0.1 mg/kg for the seven Sudan dyes. The LOD is the lowest amount of analyte that can be reliably determined but is not quantifiable with adequate accuracy, the LOD was defined as the standard deviation of blank samples at minimum resolution and was found to be 0.01 mg/kg for Sudan Ӏ, Ⅱ, Ⅲ and Ⅳ, 0.03 mg/kg for Sudan Red 7B and 0.02 mg/kg for Sudan Orange G. The estimation criterion for the LOD is a signal-to-noise ratio (S/N) ≥ 3. 3.5.2. Linearity The lowest calibration level for each dye was 0.1 μg/L and the highest level was 50 μg/L. Thus the correlation coefficient was greater than 0.999 for the seven compounds, and the accepted standard 5
Journal of Food Composition and Analysis 84 (2019) 103304
A.S. Sebaei, et al. antioxidant activities of some indigenous spices consumed in Nigeria. Food Chem. 238, 58–64. Ertaş, E., Özer, H., Alasalvar, C., 2007. A rapid HPLC method for determination of Sudan dyes and Para Red in red chili pepper. Food Chem. 105 (2), 756–760. Fukuji, T.S., Castro-Puyana, M., Tavares, M.F., Cifuentes, A., 2011. Fast determination of Sudan dyes in chili tomato sauces using partial filling micellar electrokinetic chromatography. J. Agric. Food Chem. 59 (22), 11903–11909. Gan, T., Li, K., Wu, K., 2008. Multi-wall carbon nanotube-based electrochemical sensor for sensitive determination of Sudan I. Sens. Actuators B Chem. 132 (1), 134–139. Ju, C., Tang, Y., Fan, H., Chen, J., 2008. Enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody as a new tool to detect Sudan dyes and Para red.
Anal. Chim. Acta 621 (2), 200–206. Otero, P., Saha, S.K., Hussein, A., Barron, J., Murray, P., 2017. Simultaneous determination of 23 azo dyes in paprika by gas chromatography-mass spectrometry. Food Anal. Methods 10 (4), 876–884. Petrakis, E.A., Cagliani, L.R., Tarantilis, P.A., Polissiou, M.G., Consonni, R., 2017. Sudan dyes in adulterated saffron (Crocus sativus L.): identification and quantification by 1H NMR. Food Chem. 217, 418–424. Yan, H., Wang, H., Qiao, J., Yang, G., 2011. Molecularly imprinted matrix solid-phase dispersion combined with dispersive liquid-liquid microextraction for the determination of four Sudan dyes in egg yolk. J. Chromatogr. A 1218 (16), 2182–2188.
6
Contents lists available at ScienceDirect
Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original Research Article
Determination of seven illegal dyes in Egyptian spices by HPLC with gel permeation chromatography clean up
T
Ahmed Salem Sebaeia,⁎, Manar Ibrahim Youssifb, Ahmed Abdel-Maksoud Ghazia a b
Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Foods, Agricultural Research Center, Ministry of Agriculture, Giza, 12311, Egypt Faculty of Biotechnology, October University for Modern Sciences and Arts University (MSA), Giza, 12566, Egypt
ARTICLE INFO
ABSTRACT
Keywords: Hot chilli Sudan dyes Method validation Food spices
Sudan I, II, III, IV, Para Red, Orange G and Red 7B are synthetic regularly dyes utilized to dye plastics and other manufactured materials. In recent years, concerns about the genotoxic possibilities of Sudan colors have been raised. A few national bodies and food experts consider Sudan dyes to be genotoxic cancer-causing agents while others view these dyes as possible cancer-causing agents. Therefore, it is of the outmost importance to assess the risk and presence of Sudan dyes in Egyptian spices, including; sumac, hot chilli, cumin, paprika, curry and turmeric. A total of 83 samples were collected randomly from different supermarkets and spice shop in various municipalities of Egypt (Cairo, Giza, Qalyubia, Faiyum and Alexandria). For Sudan dyes determination, gel permeation chromatography (GPC) and high-performance liquid chromatography (HPLC) were used. Dyes were extracted from spices with acetonitrile and selectively initial separated by GPC. Fractions were collected from 12 min to 24 min and then determined by HPLC with diode array detection (HPLC-DAD). The method performance was validated on hot chilli samples, and the limit of quantification (LOQ) was 0.1 mg/kg for the seven Sudan dyes. Moreover, other method validation parameters were determined in this work, including linearity, accuracy, recovery, limit of detection (LOD), repeatability, reproducibility and expanded uncertainty. Finally, a market survey was conducted to assess the incidence of such illegal dyes and enlighten buyers, almost 50% of the tested samples were contaminated with Sudan dyes. Sudan Red 7B and Para Red were not detected in the samples, and the mean contamination level varied from 0.01 mg/kg for Sudan Ⅱ to 50.1 mg/kg for Sudan Ӏ. It is recommended to buy raw whole spices – not in powder form- and blend them at home. In addition, spices should be purchased from established retailers, and buyers should avoid spices with abnormally red shading.
1. Introduction Sudan dyes are synthetic lipophilic red-coloured azo dyes that do not occur naturally; however, such dyes are currently used in various applications as a colouring agents in several matrixes, including spices (Petrakis et al., 2017). Azo dyes are a large class of synthetic organic dyes that contain nitrogen in an azo group (-N = N-) as part of the molecular structures (Otero et al., 2017). In 2003, the Federal Institute for Risk Assessment stated that the European Union (EU) does not permit the use of these dyes as food additives, citing the degradation products which are considered to be carcinogens and teratogens. The azo dyes Sudan I-IV are classified as carcinogenic (category II-III) due to their ability to be separated into amines after administration into the human body (Alim-Un-Nisa and Akhlaq, 2015). Sudan dyes belong to the azo dyes, and the most common types are Sudan I, Sudan II, Sudan III, Sudan IV, Orange G, Red
⁎
7B and Para Red (Fig. 1) (Ertaş et al., 2007) Sudan dyes are illegal in many industries. However, these dyes have the advantages of colour fastness and low price. Despite the fact that they are banned from use by the Food Standards Agency (FSA) and the EU (Yan et al., 2011), some industries use Sudan dyes in manufactured products, including; spices, polishes, waxes, textiles, and cosmetics, and in scientific applications, furthermore, it has been proven that Sudan dyes are present in egg yolk (Bazregar et al., 2017). In Africa, particularly in Nigeria, traditional and homemade delicacies are prepared with traditional and indigenous spices. These spices are used as additives or curing agents to add taste or flavour to food (Ene-Obong et al., 2018). Since 1999, azo dyes have been used to treat textiles. It has been estimated that over ten thousand dyes are used in industial fields with an equivalent number of products (Chequer et al., 2013). As mentioned by Carmen and Daniela (2012), consumers/buyers typically seek certain basic product characteristics, including non-
Corresponding author at: QCAP Laboratory, Agriculture Research Center, Ministry of Agriculture, Giza, 12311, Egypt. E-mail address: [email protected] (A.S. Sebaei).
https://doi.org/10.1016/j.jfca.2019.103304 Received 6 March 2019; Received in revised form 28 June 2019; Accepted 29 August 2019 Available online 30 August 2019 0889-1575/ © 2019 Elsevier Inc. All rights reserved.
Journal of Food Composition and Analysis 84 (2019) 103304
A.S. Sebaei, et al.
Fig. 1. Chemical Structures of Sudan dyes.
themselves (Di Anibal et al., 2011). These instruments include; HPLC with electrochemical detection (Chailapakul et al., 2008), pressurized capillary electrochromatography (CEC) with amperometric detection (Fukuji et al., 2011), tandem mass spectrometry coupled with isotope dilution (Di Donna et al., 2004), multi-wall carbon nanotube-based electrochemical sensing (Gan et al., 2008), and enzyme-linked immunosorbent assay (ELISA) (Ju et al., 2008). In most electrochemical methods, a single Sudan dye compound is quantified; otherwise, analytical sensitivity sacrificed. Many relevant chromatography methods, require the use of isotopic internal standards, tandem mass techniques, many pre-separation steps or time-consuming and tedious preparation methods to achieve the required selectivity, and sensitivity and also to overcome the matrix effect of spices. Herein, a selective, robust, sensitive and simple test method is proposed that takes advantage of the large gap in molecular size between Sudan dyes and the heavy matrix of spice components to selectively separate multiple-dyes by size exclusion chromatography and then determine the analytes by through their UV spectra through liquid chromatography with diode array detection. Overall, the main objective of this proposed work is the determination of seven Sudan dyes in the Egyptian spice market. In addition, a selective, reliable and accurate gel permeation chromatography (GPC) method is developed, including; optimization, validation and quality control assessments. Furthermore, a survey is administered regarding spices sold in different municipalities in Egypt and their adulteration with Sudan dyes. The levels of contamination found in this overview are noteworthy. The results show that it is important to monitor Sudan dyes in foodstuffs.
fading colours in response to light, washing, heating, and perspiration, both initially and after prolonged use of the material. People have the right to presume that the food they eat is safe and suitable for consumption. Foodborne illness and injury are disagreeable at best; at worst, they can be fatal. Eruptions of foodborne illness can damage trade and tourism, and lead to loss of earnings, unemployment and litigation. Food spoilage is wasteful and, costly and can adversely affect trade and consumer confidence (Baş et al., 2007). Since 2001, Sudan dyes have been in spices in EU. A February 2017 search of Europe'sRaid Alert System for Food and Feed (RASFF) for "unapproved dyes" and "Sudan" in the "herbs and spices" product category retrieved approximately 429 notifications. In a search of the FDA Import Alert 45-02 (Detention Without Physical Examination and Guidance of Foods Containing Illegal and/or Undeclared Colors) the discovered no record of Sudan dye adulteration. Referring to the (RASFF); in 2003, the European Commission established Decision 2003/460/EC, the first community measure to control the unlawful use of the colouring substance Sudan dye in adulterated chilli and chilli products. A second decision extending the scope of the measures was adopted on 21 January 2004 and is still in force. In addition, a report issued in 2016 found 5 notifications detailing Sudan dyes in spices (1 in palm oil) and three revealing Rhodamine dye. Given the fewer instances of non-compliance; in 2017 there were 8 notifications associated with the adulteration of palm oil with Sudan I. In 2015, the Minister of Health (MOH) issued Decree 204/2015 regarding food additives that are acceptable for use by the food industry. Exporters should check with the MOH to confirm the satisfactoriness of all food colourings. Moreover, food dyes are continuously reviewed and updated according to Codex Alimentarius standards. There are no exceptions to the regulations governing food colourings. Egyptian authorities will not allow a product to be imported if it contains an unauthorized dye, even if the use of the dye is acceptable in another country. The targeted spices that are suspected to contain Sudan dye and its include; hot chilli, cumin, curry, paprika, sumac and turmeric. Moreover, these spices are characterized by a colourful appearance. As mentioned in several previous papers, multiple methods are used worldwide in the detection and analysis of Sudan azo dyes in foodstuffs, depending on the availability and financial stability of the laboratories
2. Materials and methods 2.1. Chemicals and materials All chemicals and reagents were of HPLC or analytical grade. Deionized water used throughout the experiments was obtained by using the Integral Water Purification System for Ultrapure Water (Milli-Q A10) from Merck, and sodium sulfate was activated at 150 °C overnight. Bio-Beads –SX3 (200–400 mesh) was purchased from Bio-Red Laboratories for GPC column packing.
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Table 1 GPC profile of eluted fractions in mg/kg for the seven colors vs fraction time in min. Analyte
Orange G Para Red Sudan I Sudan II Sudan III Red 7B Sudan IV
Time (min) 0-3
3-6
6-9
9-12
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
12-15 15-18 Eluted fractions in mg/kg 0.88 7.93 6.87 7.28 2.63 4.25 1.65
7.00 1.08 2.75 1.28 5.94 3.95 3.31
18-21
21-24
24-27
27-30
1.20 0.73 1.69 0.13 0.49 0.34 2.91
0.9 0.6 1.96 0.085 0.33 0 1.64
0 0 0 0 0 0 0
0 0 0 0 0 0 0
Table 2 Sudan dyes adulteration profile of the 41 samples. Sample type
No.
No. of detected samples
chili turmeric paprika cumin sumac curry total
18 13 17 15 10 10 83
11 6 10 6 5 3 41
Percentage (%) 61.1 46.2 58.8 40.0 50.0 30.0 49.4
% % % % % % %
Fig. 4. Sudan I calibration curve and accepted correlation for HPLC standard linearity.
2.2. Sample collection A total of 83 spice samples (Table 1) were randomly purchased from different commercial supermarkets and spice shops in various municipalities of Egypt (Cairo, Giza, Qalyubia, Faiyum and Alexandria) during the period from October 2017 to September 2018. 2.3. Standard preparation The 7 compounds Orange G., Para Red, Sudan Ӏ, Sudan Ⅱ, Sudan Ⅲ, Sudan Red 7B and Sudan Ⅳ were purchased as certified reference standards from LGC. Stock solutions for individual compounds were prepared at 1000 mg/kg in acetonitrile: dichloromethane (1:1 v/v) and a working solution was prepared as a mixture of all dyes at 100 mg/kg for spiking and HPLC intermediate check tests. For GPCfraction,
Fig. 2. GPC separation profile and the fraction elution time of the seven Sudan dyes.
Fig. 3. Para Red and Sudan dyes HPLC-DAD chromatogram standard of 1 mg/L. 3
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Table 3 Repeatability of a contaminated hot chili sample and reproducibility as inter-day repeatability at 0.1 mg/kg spiking level, (n = 6).
Repeatability Inter-day Repeatability
RSD% RSD%
Sudan І
Sudan Ⅱ
Sudan Ⅲ
Sudan Ⅳ
Sudan red 7b
Orange G
3 6
2 6
3 8
5 10
2 9
2 8
Table 4 Average violated concentration mg/kg of the seven illegal dyes in 6 spices commodities. Spices
Orange G
Para red
Sudan І
Sudan Ⅱ
Sudan Ⅲ
Red 7b
Sudan Ⅳ
chili cumin paprika turmeric curry sumac Total average
3.9 n.d 6.2 n.d n.d n.d 2.1
n.d n.d n.d n.d n.d n.d n.d
58.7 60.9 22.5 86.2 60.4 0.2 50.4
0.01 n.d n.d n.d n.d n.d 0.01
0.5 n.d n.d n.d n.d n.d 0.1
n.d n.d n.d n.d n.d n.d n.d
64.0 0.1 0.1 9.2 4.5 0.2 12.9
n.d: not detected.
acetate: n-hexane (1: 1, v/v)). Each sample was transferred to a new labelled vial, centrifuged for 1–2 min, and then placed sequentially into the GPC tray. For each sample, 1 ml was passed through the GPC system at a flow rate of 2 ml/min; thus, analysis took 24 min per sample. After clean up, the eluted fractions were evaporated to dryness using a rotary evaporator and then 2 ml of acetonitrile was added to each sample to dissolve the residue. Finally, the samples were filtered through 0.45 μm Acrodiscs into autosampler vials, at which point they were ready for injection. Diode array detector parameters: The absorbance was measured at 230 nm Sudan I, II, III, IV and Red 7B while measured at 380 nm for Sudan Orange G. The HPLC injected volume was 25 μl and the mobile phase was acetonitrile: water (95: 5) in isocratic mode with a 1 ml/min flow rate. The above sequential process was repeated until all spice samples had been completely analysed.
Fig. 5. Contamination percentage of each spice cheated with Sudan dyes.
2.5. GPC and HPLC analysis The GPC clean-up system (Waters, USA) consists of a built-in binary pump (Waters 1525), 100 vial autosampler tray (4 ml) (Waters 717 plus) with a 5000 μl sample loop, and a fraction collector (Waters III). The system was equipped a 10 mm i.d., 500 mm long chromatographic glass column (Omnifit, 006cc-25-25-AF, Diba industries Ltd., Cambridge, England), which was prepared by soaking 10 g Bio-Beads –SX3 more for more than 2 h in GPC mobile phase hexane: ethyl acetate (1:1) and loaded into the column with slight flow. The column lifetime allows the injection of up to 2000 samples. The 1100 series HPLC is from (Agilent, Germany) was equipped with a quaternary pump (G1311A), vacuum degasser (G1322), autosampler (G1313A), and diode array detector (Agilent, (G1321B)). The analytical column was an Agilent Eclipse® plus C18 (250 mm × 4.6 × 5 μm).
Fig. 6. Sudan dyes mean concentrations in spices in Egypt.
validation was performed with a 100 μg/ml Sudan dye mixture prepared by diluting 10 ml of the stock standard solution of Sudan dyes in 4 ml of ethyl acetate: n-hexane this mixture was then used as a control for various fractionation steps essential for method development.
2.6. LOQ Ten grams of cumin was weighed, and 10 μl of each of the seven Sudan mixtures was added to the weighed cumin; the mixture was then dissolved in 100 ml of acetonitrile solution in a conical flask. The sample was filtered through glass funnels into another volumetric flask with 15 g of activated sodium sulfate as a separating agent. After filtration, an aliquot of 50 ml of the filtrate was pipetted into another 100 ml flask. The collected solvent was then evaporated to dryness using a rotary evaporator at approximately 50 °C and then diluted with 4 ml GPC mobile phase (1: 1 acetonitrile: hexane) into a vial. After 30 min of GPC processing, the samples were evaporated to dryness and then diluted with 1 ml of acetonitrile in HPLC vials. The recovery and coefficient o variation (CV%) at the LOQ were determined after completing injection.
2.4. Sample preparation The samples to be analysed by HPLC were subjected to an extraction process: ten grams of sample was weighed and 100 mL of acetonitrile was added. The samples were shaken for 20 min at 200 rpm and then underwent filtration by gravity through 15 g of activated sodium sulfate as a separating agent over a glass funnel. After filtration, an aliquot of 50 ml of the filtrate was pipetted into another 100 ml flask. The collected solvent was then evaporated to dryness using a rotary evaporator at approximately 40 °C and diluted with 4 ml GPC mobile phase (ethyl 4
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3. Results and discussion
linearity for Sudan I is was 0.99975 (Fig. 4).
3.1. GPC initial separation method development Four hundred microliters of the 100 mg/kg Sudan mixture was evaporated and reconstituted in 4 ml of GPC mobile phase. One millilitre of the standard mixture was injected directly into the GPC, and a 2 ml/min flow rate was applied. The standard sample was fractionated into 10 separate flasks. The GPC clean-up process took 30 min; thus, the eluate was separated every 3 min was collected in a distinct flask; and labelled as follows; (0–3 min, 3–6 min, 6–9 min, 9–12 min, 12–15 min, 15–18 min, 18–21 min, 21–24 min, 24–27 min, and 27–30 min). Then, the samples were evaporated, 1 ml of acetonitrile was added to each flask, and the contents were transferred to HPLC vials. The above step was performed to collect 30 min of GPC eluate into 10 flasks instead of the typical 1 flask to observe the GPC separation stages.
3.5.3. Precision Precision is the level of agreement between replicate estimations under determined conditions. Precision is represented by measurable quantities, for example, a standard deviation or certainty limit; and lower precision is reflected by a larger standard deviation. The precision was identified in terms of repeatability and reproducibility. (Table 3) indicates the estimated repeatability and inter-day repeatability from 6 replicates of a spiked sample at 1 mg/kg. Trueness is the level of agreement between the mean value of a series of successive measurements and the true value or an accepted reference value and is related to systematic error (bias). Bias is calculated from the absolute relative difference (RD%), which must not surpass 20% in a sample of choice, which in this case was all the hot chilli samples. Thus, RD% reached a maximum of 11%, which is below the maximum level.
3.2. Extraction solvent efficiency
3.6. Survey of Sudan dyes in spices
Different combinations of extraction solvents were tested. Ultimately, acetonitrile was better for extraction than ethanol, acetone and methanol due to its higher recovery. Thus, acetonitrile was chosen as the extraction solvent for the different types of spices. The elution recovery using acetonitrile showed a significant percentage of 98% in contrast to the recoveries for the other tested solvents.
The average sum (Table 4) of the Sudan dyes was determined for hot chilli, cumin, turmeric, curry, sumac and paprika. The results were analysed to determine the Sudan contamination levels, the proportion of polluted samples and levels of contamination above specific and general limits. Each sample was broken down as indicated in the method. Sudan adulteration was found in 41 of the 83 collected and tested spice samples, as tabulated below (Fig. 5), so 49% of the investigated Egyptian market spices were adulterated with Sudan dyes. This survey was the first to be adopted in Egypt (Fig. 6). The highest average contamination level was found for Sudan Ӏ at 50.4 mg/kg and the lowest average concentration was found for Sudan Ⅱ at 0.01 mg/kg. Para Red and Sudan Red 7B were not detected in any samples.
3.3. GPC clean up The seven Sudan dyes were detected by known signals in the sample fractions from minutes 12–15 to 21–24 (Table 2). Moreover, the elution of the seven Sudan dyes varied during the 30 min of fractionation. No Sudan dyes were detected in the first 12 min or in the last 9 min which were considered the washing period, thus, the fraction times in the GPC separation profile of the seven Sudan dyes are explained in Fig. 2. GPC is an excellent method for isolating Sudan dyes and related azo dyes from normal dyes and other fat soluble substances in coloured spices. Thus, GPC allows the efficient extraction of Sudan dyes from spices with the simple application of size-exclusion clean-up which avoids crosscontamination.
4. Conclusion The fundamental goal of this work was the assessment of seven Sudan dyes in the Egyptian spice market. In addition, a solid and precise GPC strategy was developed and evaluated in terms of the improvement, optimization, validation and quality control of the technique utilized. Furthermore, a LOQ of 0.1 mg/kg was established. To meet the proposed objective; a series of method validation operational parameters were fully addressed in this study. Finally, this study showed 50% adulteration in six types of spices by five Sudan dyes.
3.4. HPLC analysis After HPLC analysis, it was determined that the mobile phase acetonitrile: water in a ratio of 95: 5 was the most effective mobile phase for the determination of the seven Sudan dyes, yielding fast retention times for the dyes (Fig. 3).
References Alim-Un-Nisa, N.Z., Akhlaq, F., 2015. Detection of Sudan dyes in different spices. Pak. J. Food Sci. 25 (3), 144–149. Baş, M., Ersun, A.Ş., Kıvanç, G., 2007. The evaluation of food hygiene knowledge, attitudes, and practices of food handlers’ in food businesses in Turkey. Food Control 17 (4), 317–322. Bazregar, M., Rajabi, M., Yamini, Y., Arghavani-Beydokhti, S., Asghari, A., 2017. Centrifugeless dispersive liquid-liquid microextraction based on salting-out phenomenon followed by high performance liquid chromatography for determination of Sudan dyes in different species. Food Chem. 244, 1–6. Carmen, Z., Daniela, S., 2012. Textile organic dyes—characteristics, polluting effects and separation/elimination procedures from industrial effluents—a critical overview. In: Puzyn, T. (Ed.), Organic Pollutants Ten Years after the Stockholm Convention—Environmental and Analytical Update. InTech Press, Croatia, pp. 55–86. Chailapakul, O., Wonsawat, W., Siangproh, W., Grudpan, K., Zhao, Y., Zhu, Z., 2008. Analysis of sudan I, sudan II, sudan III, and sudan IV in food by HPLC with electrochemical detection: comparison of glassy carbon electrode with carbon nanotubeionic liquid gel modified electrode. Food Chem. 109 (4), 876–882. Chequer, F.M.D., de Oliveira, G.A.R., Ferraz, E.R.A., Cardoso, J.C., Zanoni, M.V.B., de Oliveira, D.P., 2013. Textile Dyes: Dyeing Process and Environmental Impact. Di Anibal, C.V., Ruisánchez, I., Callao, M.P., 2011. High-resolution 1H nuclear magnetic resonance spectrometry combined with chemometric treatment to identify adulteration of culinary spices with Sudan dyes. Food Chem. 124 (3), 1139–1145. Di Donna, L., Maiuolo, L., Mazzotti, F., De Luca, D., Sindona, G., 2004. Assay of Sudan I contamination of foodstuff by atmospheric pressure chemical ionization tandem mass spectrometry and isotope dilution. Anal. Chem. 76 (17), 5104–5108. Ene-Obong, H., Onuoha, N., Aburime, L., Mbah, O., 2018. Chemical composition and
3.5. Method validation (fit for intended use) 3.5.1. LOQ and LOD The LOQ is the minimum concentration of analyte in a test sample that can be determined with acceptable precision, repeatability and recovery under the stated test conditions. The lowest practical LOQ was estimated by repeatedly analyzing a cumin sample at the expected lowest quantification level of 0.1 mg/kg for the seven Sudan dyes. The LOD is the lowest amount of analyte that can be reliably determined but is not quantifiable with adequate accuracy, the LOD was defined as the standard deviation of blank samples at minimum resolution and was found to be 0.01 mg/kg for Sudan Ӏ, Ⅱ, Ⅲ and Ⅳ, 0.03 mg/kg for Sudan Red 7B and 0.02 mg/kg for Sudan Orange G. The estimation criterion for the LOD is a signal-to-noise ratio (S/N) ≥ 3. 3.5.2. Linearity The lowest calibration level for each dye was 0.1 μg/L and the highest level was 50 μg/L. Thus the correlation coefficient was greater than 0.999 for the seven compounds, and the accepted standard 5
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