What kind of dye is sudan iv

Chemistry Practice Questions

Analysis of Carcinogenic Sudan Azo Dyes in Food Products

Tejkiron Gottapu

Southeast Missouri State University

Submitted To

Dr. David Cunningham

ANALYSIS OF CARCINOGENIC SUDAN AZO DYES IN FOOD PRODUCTS

Abstract

Sudan azo dyes, categorized as I, II, III and IV are some of the most appealing coloring agents in the field of food industry. The use of these dyes as colorants and additives enhances the uniformity of color in foods, improves the appearance of foods and thereby promoting their salability. Use of these dyes, however, presents harmful health effects of mutagenicity, carcinogenicity and toxicity. They have since been banned from use although a few cases of their illegal are still being reported. In the present study, the adulteration of selected red palm oil and palm cream in Ghana was investigated by quantifying the amount of Sudan IV present in the spiked and non-spiked samples. The samples were prepared by Solid-Phase extraction and analyzed by HPLC.

The results obtained indicated no observed presence of Sudan IV dye in both the neat oil palm oil and palm cream. Even though the method was not validated by determining the limit of detection, the correlation factor for the calibration curves of 0.99 suggests the possibility of the results being reproducible. It was therefore concluded that both the red palm oil and the palm cream considered in the study might not have Sudan IV dye.

1.0 Introduction

Color is one of the most distinguished features of food products in the food industry. Many coloring agents are applied to various foods to improve the appearance and therefore promote the salability of the products. Coloring agents are also added to reinforce and enhance uniformity of color in foods that have color already present in them. One of such groups of dyes used as colorants in the food industry is Sudan Azo dyes.

Azo dyes are synthetic compounds containing diazotized amine (-N=N-) bonded to hydrocarbons. Generally, azo dyes are characterized by one or more azo bonds. The conjugation of their bonds make the azo dyes shine with characteristic colors, one that gives them a greater appeal to consumers. Azo dyes are grouped into several categories defined by the fibers for which they have affinity, which depends on the other variations in chemical features. Sudan azo dyes (I, II, III, and IV) are fat-soluble azo dyes, widely applied for coloring food products, triglycerides and lipoproteins (Hunger et al. 2000). However, the use of Sudan dyes has been banned worldwide and is now classified by the International Agency for Research on Cancer (IARC) as a Class-III carcinogen. Even with the ban on the use of azo dyes, Sudan azo dyes continue being use used illegally and have been reported in cases of adulteration.

Fig.A : Structures of Sudan I, II, III and IV

The first case of adulteration was identified in chilli powder in 2003, in which the contaminant was Sudan I. It was established that the contaminated chili powder, with brand name genus, had its origins from India. Analytical studies on chili powder showed Sudan I adulteration concentration of 4000 mg kg−1 (RASFF 2003; ASTA 2005). The shocking revelations forced governments to impose rules on the quality requirements of imported chili powder to ensure that the said product did not contain any Sudan I dye (European Commission 2003). In 2004, this requirement was extended for Sudan II, III and IV. More products were also incorporated for testing including palm oil and the genus Curcuma in 2004 (turmeric) (European Commission 2005). Unauthorised colours continue to be reported in the Rapid Alert System for Food and Feed (RASFF) portal, with a total of 16 notifications in 2014 and 2015 (RASFF 2015).

Studies on animal models have shown that Sudan dyes are carcinogenic, mutagenic and toxic substances. The listed harmful effects of these azo dyes are reported to stem from the metabolism of these dyes once the animals are exposed to them. Metabolism of azo dyes starts with an enzyme catalyzed reduction of azo bonds by azoreductase. This reduction process of azo bonds is believed to be responsible for the harmful effects manifested by the dyes. Although many studies on the harmful effects of these dyes has been done on Sudan I, many researchers seem to concur that the other forms of Sudan dyes are potentially carcinogenic since all their chemical structures resemble each other.

The continued use of Sudan azo dyes is not only a threat to human life due to their harmful health effects, but also a potential hazard to the environment especially when released to the water system. Humans get exposed to these dyes through ingestion, skin contact and/or inhalation. Exposure through ingestion, as food additives and colorants, is one of the most ways in which these dyes get into the human system. Although the use of these dyes has since been banned in most countries, they somehow get into the food market illegally. As a result, these dyes have been reported in foods such as palm oils, chili powders, eggs, Worcestershire sauce, garlic curry sauce, among others as contaminants and adulterant (Arora & Bharti 2005; Mishra et al. 2007). Still the food products are adulterating with Sudan dyes to enhance their color and make huge market. In this research we have received some food products from Nutrition department with different brand names to identify Sudan dye adulterants in them by using a suitable chromatographic method (Gesualdi 2016). In Spring 20 research, we had analysed 2 different food manufacturers samples and one other manufacturer sample in Fall 20 research to identify Sudan I and IV dye adulterants if any. Same HPLC method was used for analysing all the samples, but Sample preparations were changed slightly. method was used Before the HPLC technique, Tthe samples were pre-treated using solid-phase extraction (SPE). The SPE procedure is mostly employed for extraction, concentration, and fractioning of organic compounds (Andrade-Eir8oa et al. 641; Rocha et al. 803. In the present study we report studies on quantification of Sudan IV in potentially adulterated red palm oils and palm soup cream. Sample bottles were not heated in a 66°C oven for until the oil liquefied as mentioned in method (Gesualdi 2016). In current study, method recovery was not satisfied with lower LOD’s as previous methods (Gesualdi 2016). Comment by David Cunningham: Multiple phrases and sentences. Remove capital letters within the sentence. Please revise. Comment by David Cunningham: Your introduction only includes one sentence on what was done on the project. Expand this to include information of what was done in Spring and Fall. Include the goal(s) of your work. Indicate that the method you used was based on (Genualdi 2016) and indicate how your work was different than work reported in this earlier publication. Comment by David Cunningham: Comment by Gottapu, Tej: Included more information

1.1 Materials and Methods

1.1.1 Chemicals and Reagents

The solvents used in this study include methanol (HPLC grade), hexane, diethyl ether, and ethyl acetate.

1.1.2 Standards

Sudan I and IV analytical standards of purity greater than 96% were purchased from Sigma Aldrich. Stock solutions of these standards were prepared in methanol to ensure complete solubility of the Sudan dyes. Samples and prepared standards were stored at Room temperature.

Preparation of Sudan IV Stock Std Solution:

S20: Accurately weighed 1.17 mg of Sudan IV and transferred to a vial and dissolved in 20.0 ml methanol.

F20: Accurately weighed 2.12 mg of Sudan IV and transferred to a vial and dissolved in 20.0 ml methanol.

Preparation of Sudan I Stock Std Solution:

S20: Accurately weighed 2.25 mg of Sudan I and transferred to a vial and dissolved in 20.0 ml methanol.

F20: Accurately weighed 2.08 mg of Sudan I and transferred to a vial and dissolved in 20.0 ml methanol.

Mixed Standard: Preparation:

Mixed 5ml of each Sudan std stock I&IV then made further dilutions to get 50%, 25% of mixed standard solution. Comment by David Cunningham: Also include S2020 preps: Worksheet Soln Prep and Cal Curves Comment by Gottapu, Tej: Excel graph included with cal curve and preparations

1.1.2 Samples and Preparation

The samples for this study included fresh palm fruit extract from Neat Food Company in Ghana, and Praise Palm Cream from Praise Export Services Limited in Ghana, Praise samples well. The red palm oil samples were unrefined and displayed a red color was due to originally existing carotenes. In their unrefined form, adulteration with Sudan dyes would enhance the red color make the palm oil appear better and nutritionally richer.

Sample Preparation 1:

Weighed and transferred 5.182 g of sample to a vial and dissolved in 5.252 g of Hexane. Comment by David Cunningham: F20 prep. Please include Spring 20 prep. Comment by Gottapu, Tej: Included

Sample Preparation 2 (S20):

Weighed and transferred 0.95 g of sample to a vial and dissolved in 8.55 g of Hexane. Comment by David Cunningham: F20 prep. Please include Spring 20 prep. Comment by Gottapu, Tej: Included

Spiked Sample Preparation 1:

For sample preparation, 5.138 g of red palm oil sample was weighed and dissolved in 5.252g of hexane. The sample was then spiked with 100 µl of Sudan I and 100 µl of Sudan IV and mixed well and allowed to settle down. Comment by David Cunningham: Include S20 prep Comment by Gottapu, Tej: Included

Spiked Sample Preparation 2 (S20):

For sample preparation, 1.05 g of red palm oil sample was weighed and dissolved in 9.5 g of hexane. The sample was then spiked with 100 µl of Sudan IV and mixed well and allowed to settle down. Comment by David Cunningham: Include S20 prep Comment by Gottapu, Tej: Included

Spiked sample also extracted from SPE by using Ethyl Ether and Ethyl Acetate wash.

1.1.3 Solid-Phase Extraction (SPE) Clean-up and Preparation of Extracts

Solid Phase Extraction was performed using LC-Alumina-B SPE tubes (1 g/3 ml) with Cartridge being conditioned with 6mL methanol, 6mL ethyl acetate and 6mL hexane solvents. Sample and Spiked samples were extracted by using SPE Ethyl Acetate and Ethyl Ether washes. After conditioning, the 3mL of clear supernatant prepared samples were introduced into the SPE column and washed with 6mL hexane and 6mL ethyl ether added in 2 ml increments and finally by 2mL ethyl acetate. Hexane filtrate discarded, and last 2 ml of ethyl ether and 2 ml of ethyl acetate was collected for HPLC analysis. These fractions were set aside, and the final eluent of Sudan dyes was collected from the column with ethyl acetate/methanol (90:10) solvent system.4-2 elution volumes for this system were collected. Comment by David Cunningham: Samples tested should probably be after 1st sentence in this paragraph. Also, include S20 sample Comment by Gottapu, Tej: Changed Comment by David Cunningham: Indicate this was the (clear?) hexane layer. Please include sample added S20 (see worksheet: Soln Prep and Cal Curves) Comment by Gottapu, Tej: included Comment by David Cunningham: Added in 2-mL increments? Comment by Gottapu, Tej: Elobarated Comment by David Cunningham: These fractions – this is unclear. Please describe fractions clearly; maybe a table would help. Comment by Gottapu, Tej: Explained with table

Elution solvent

Collected fractions for the Analysis

Hexane (6ml) added as 2ml increments

Discorded

Ehtyl ether (6ml) added as 2 ml increments

2ml- Discarded

2ml- Discorded

2ml- collected for HPLC analysis (T1)

Ethyl Acetate (2ml)

2ml- collected for HPLC analysis (T2)

Ethyl acetate: Methanol (90:10), added 2ml solvent each wash for 4 times.

Named each fraction as T3, T4, T5, T6

Table 1 : SPE elution solvents and their collected samples for HPLC analysis

The elution solvent was evaporated until it dried fully and topped up to a mark of 1 ml with methanol (S20 preparation). The eluents were filtered via a 0.2μm PTFE filter prior to inject into the HPLC Comment by David Cunningham: Please describe S20 and F20 prep. You told me that the F20 samples were not dried. Also drying was performed with a Meyer N-Evap Model 111. Comment by Gottapu, Tej: We have collected T2,T3,T4,T5 eluents and kept in wooden test tube holder. Samples were dried when we came and see next day. We did not dilute with 1ml methanol and injected due to trouble with the HPLC. Comment by David Cunningham: Add “…prior to injection into the HPLC.” Comment by Gottapu, Tej: added

1.1.4 High Performance Liquid Chromatography (HPLC) Analysis

HPLC (Shimadzu) was employed in the quantification of the Sudan I and IV dyes in the red palm oil. The equipment comprised degasser, a gradient pump, Injection valve and dual wavelength UV detector (Esen et al. 73; Moldoveanu and Victor 9; Hu et al. 2126; Weisz et al. 1835). The chromatographic conditions involved are Comment by David Cunningham: This is background information, more suitable for the introduction. Comment by Gottapu, Tej: Moved to introduction Comment by David Cunningham: Correct: Injection valve Comment by Gottapu, Tej: Corrected Comment by David Cunningham: Change to : dual wavelength UV detector Comment by Gottapu, Tej: Corrected Comment by David Cunningham: I’m not sure why these references are here Comment by Gottapu, Tej: about HPLC and chromatographic techniques Comment by David Cunningham: Include all HPLC cionditions: detector wavelength, injection volume and flow rate. Also, brand and dimensions of CCC-18 column. And analysis time. Comment by Gottapu, Tej: Included HPLC conditions

Mobile Phase: 95% methanol: 5% pH 5 buffer

Column: C18, 250mm×4.6×mm,5um

Detector Wavelength: 340 nm

Injection Volume: 10 µl

Flow Rate: 0.8 ml/min

Run Time: 15 min

The use of high organic HPLC mobile phase (95% methanol, 5% pH 5 buffer) and C-18 column, wavelength 340 nm, flow 0.8 ml/min. 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. Following samples were injected into HPLC Comment by David Cunningham: Calibration curves are a result. Perhaps include a table with all samples and standards which were injected in S20 and F20. For standards that were injected, the table would include the concentration of dye(s). The concentrations should be in units of mass/volume, for example ug/mL or ng/mL. Comment by Gottapu, Tej: Detailed table has included

1

RUNNING HEAD: Analysis of Carcinogenic Sudan Azo Dyes In Food Products

6

ANALYSIS OF CARCINOGENIC SUDAN AZO DYES IN FOOD PRODUCTS

Fall20 Samples

Spring20 Samples

Sudan I std

Methanol blank

Sudan IV std

Methanol blank 2

Sudan std I&IV 25%

250 S4 S1 Carotene

Sudan std I&IV 50%

500 S4 S1 Carotene

Sudan std I&IV 100%

1000 S4 S1 Carotene

Spiked sample collected from Ethyl Ether wash

5000 S4 S1 Carotene

Spiked sample collected from Ethyl Acetate wash

2500 S4 1/2 S1 1/2Carotene

Sample collected from Ethyl Ether wash

Mix SI & SIV

Sample collected from Ethyl Acetate wash

Mix SI & SIV carotene

NA

10 ng/mL SIV 180 S1

25 ng/mL SIV 180 S1

50 ng/mL SIV 180 S1

100 ng/mL SIV 180 S1

250 ng/mL SIV 450 S1

Spike 1 sample

Unspiked1 sample

Spike 2 Sample

Unspike 2 Sample

Spike 3 Sample

Unspike 3 Sample

Spike 4 Sample

Unspike 4 Sample

Table 2 : List of Samples injected into HPLC analysis

1.2 Results and Discussion

Even though Sudan dyes have different chemical structures from carotenoids, they have similar solubility features and spectral absorption characteristic range. This makes it difficult to discriminate and detect them when present in natural matrices unless selective separation methods such as SPE and HPLC are applied. This is what informed the rigorous sample preparation that was done prior to analysis.

The calibration of Sudan I and Sudan IV azo dyes was successful with results showing calibration graphs with correlation factors of 0.9969 and 0.9952(S20), 0.9792 and 0.9984(F20) respectively. This suggests that the method could be accurate and that results are reproducible. Usually, results with a correlation factor of less than 0.95 are not accepted since it implies that they cannot be reproduced. The calibration outcome therefore shows that the outcome of the present study can be trusted. Comment by David Cunningham: Also include cal curve from F20 data Comment by Gottapu, Tej: Calibration curve was plotted

a: Sudan I (S20) b: Sudan IV (S20)

c: Sudan I (F20) d: Sudan IV (F20)

Fig.B : Calibration curves for the standards; a for Sudan I and b for Sudan IV

Upon calibration, the results of the Liquid Chromatography (LC) analysis show that Sudan I and IV elute at retention times of between 5.38-5.41 min and 11.61-11.92 min respectively (Table 3& Fig. 3). In the present study, the variation range was narrow implying that the method was consistent and accurate. Comment by David Cunningham: Include a table with retention times for all standards S20, samples S20 and samples and standards F20. I would also include the peak areas of Sudan I and IV in this table. Comment by Gottapu, Tej: Detailed table was included

Fig. C : Chromatograms showing retention times for elution of Sudan I and IV

A summary of the retention times and peak areas of the standards and red palm oil sample in a number of trials is shown in table 3 below. All chromatograms are included in appendix. Comment by David Cunningham: It isn’t clear what these samples are since these names are not in the text. As per above, tables with all samples seems better. Or, include additional information on why data from some samples is grouped together. For the red palm oil sample, additional information is needed on the fraction tested. Are these S20 or F20 samples? Comment by Gottapu, Tej: This is for S20 std & samples. Now I added table for injected samples and another table for their results.

Standard/Sample

Retention Time

Peak Area

250 S4 S1 Carotene (Figure 3)

5.400&11.722

48464&6919

500 S4 S1 Carotene (Figure 4)

5.366&11.585

25229&15046

1000 S4 S1 Carotene (Figure 5)

3.374&11.646

50477&27299

5000 S4 S1 Carotene (Figure 6)

5.372&11.565

44355&146681

2500 S4 1/2 S1 1/2Carotene (Figure 7)

5.372&11.539

22850&63430

Mix SI& SIV (Figure 8)

5.362&11.506

35258&29252

Mix SI& SIV Carotene (Figure 9)

5.632&11.833

23697&13365

10 ng/mL SIV 180 S1 (Figure 10)

ND

NA

25 ng/mL SIV 180 S1 (Figure 11)

ND

NA

50 ng/mL SIV 180 S1 (Figure 12)

5.386&11.695

3555&1341

100 ng/mL SIV 180 S1 (Figure 13)

5.361&11.508

5651&2313

250 ng/mL SIV 450 S1 (Figure 14)

5.301&11.077

13307&7122

Spike 1 Sample (Figure 15)

ND

NA

Unspiked1 Sample (Figure 16)

ND

NA

Spike 2 Sample (Figure 17)

ND

NA

Un spike 2 Sample (Figure 18)

ND

NA

Spike 3 Sample (Figure 19)

ND

NA

Un spike 3 Sample (Figure 20)

ND

NA

Spike 4 Sample (Figure 21)

ND

NA

Unspike 4 Sample (Figure 22)

ND

NA

Table 3 : Retention times and peak areas for various red palm oil sample preparations and

standards.

From the results, it’s clear that the neat red palm oil sample obtained did not show presence of either Sudan I or IV dyes. However, the absence Sudan IV dye in the spiked sample of the ethyl acetate wash casts aspersions as to whether the method was able to accurately quantify Sudan IV dye. Limit of detection was 50ng/ml, hence there was no Sudan IV adulterant with the limit of 50ng/ml of sample. It could not be confirmed whether the oil had zero quantities of Sudan IV because the Limit of detection (LOD) was not established for the chosen method of quantification. Comment by David Cunningham: Better if also include peak areas in data and analysis to demonstrate your points. Comment by Gottapu, Tej: Added peak areas Comment by David Cunningham: Not correct; see question in email. Comment by David Cunningham: Could a limit of detection be determined from the standards? Comment by Gottapu, Tej: Comment by Gottapu, Tej: No because LOD level samples were not prepared.

The chromatograms for unspiked palm cream and that of the Palm cream spiked at 1000ng Sudan IV/g are shown in Fig. 4 below. The developed HPLC method provided excellent separation of the individual components in the palm cream. Some of the individual components that were clearly separated along with the Sudan azo dyes are the carotenoid pigments (Mingzhu, et al., 2016). From the obtained results, the palm cream did not contain Sudan IV azo dyes. It was therefore concluded that the palm cream considered in the present study might not have Sudan I and IV azo dyes. This was, however, not validated by determining the limit of detection.

Fig. D : The Chromatogram for the Palm Cream

Standards/Samples

Retention Time

Peak Area

Sudan I std (Figure 23)

Sudan IV peak at 5.41 min

2931042

Sudan IV std (Figure 24)

Sudan IV peak at 11.92 min

12265713

100% Sudan std I & IV (Figure 25)

5.38 & 11.66 min

1083100 & 2030131

50% Sudan std I & IV (Figure 26)

5.42 & 11.79 min

678256 & 1076363

25% Sudan std I & IV (Figure 27)

5.41 & 11.72

268342 & 486254

Sudan I and IV from spiked Sample

preparation- Ethyl ether wash (Figure 28)

5.38 & 11.61 min

700 & 36451

Sudan I and IV from spiked Sample preparation- Ethyl Acetate wash (Figure 29)

Sudan IV at 11.718 min, Sudan I not detected

4104

Sample Ethyl Ether wash (Figure 30)

11.70 min

1663

Sample Ethyl Acetate wash (Figure 31)

ND

NA

Table 4 : The results from the analysis of red palm oil sample (tested in Fall)

From the above tabulated results, it is evident that both Sudan I and IV had extracted from palm oil. The different concentrations of Sudan I and IV used for calibration curve. There is a slight change in retention times with good precision( RSD of Sudan I is 0.60 and Sudan IV is 0.65). The spiked ethyl ether sample showed the presence of Sudan I and IV corresponding to the retention times of 5.41 and 11.72 minutes respectively. The spiked sample prepared by ethyl acetate showed the presence of Sudan IV at a retention time of 11.78 minutes, although Sudan I was absent from that sample. The sample from ethyl ether wash thus established the presence of Sudan IV, illustrated by a peak at 11.70 minutes, whereas there was no peak with ethyl acetate wash. Comment by David Cunningham: Please reword; meaning is unclear Comment by David Cunningham: Should a change in concentration result in a change in retention time? Comment by Gottapu, Tej: Change in concentration would not affect the RT, but peak shape may affect with higher concentrations. Comment by David Cunningham: Precision is generally indicated by the relative standard deviation of the results. Please include the relative standard deviations.

Figure E: The Sample Chromatogram Figure F: The Sample Chromatogram

from Ethyl Acetate wash from Ethyl Ether wash

Thus, the above results demonstrated the detection of Sudan I and IV using ethyl ether wash, and Sudan IV was detected solely in ethyl acetate wash.

Peak area of Sudan IV std in spiked sample = 36451

Peak area of Sudan IV in sample from Ethyl ether wash = 1663

Concentration of Sudan IV in spiked sample = 0.106 mg/ml Comment by Gottapu, Tej: I am not sure this calculations, Please help me with this

Concentration of Sudan IV in sample = 0.106 × 1663 / 39451 = 0.0044682 mg/ml

1.3 Conclusion and Recommendation

The analysis of Sudan I and IV azo dyes was successful with calibration graphs giving a correlation factor of 0.99 which indicated a high degree of correlation. This implies that even though the method was not validated by determining the limit of detection, the results reported herein can be reproduced. The results of the present study indicated no observed presence of Sudan IV dye in both the neat oil palm oil and palm cream. It was therefore concluded that both the red palm oil and the palm cream considered in the study might not have Sudan IV dye.

We recommend the use of more accurate methods, such as liquid chromatography-mass spectrometry (LC-MS)/mass spectrometry (MS), Fourier transform infrared spectroscopy (FTIR), and mid-infrared spectroscopy (MIR) to provide further proof or contrary outcome of the same. The use of these techniques would yield accurate results with higher precision since they are more efficient. When FTIR instrumentation combined with the powerful multivariate data analysis methods make this technology ideal for large volume, rapid screening, and minor food components characterization down to parts per billion levels. The MIR is beneficial for simple structural investigation and raw material ingredient or additive identification by library comparison. Comment by David Cunningham: Please revise. IR methods detect all vibrational bands in organic molecules, thus, they perform poorly on mixtures. The palm oil food samples contain many components so IR is not able to measure small amounts of analyte in these samples

Works Cited

Ahmed, F., et al. "Non-destructive FT-IR Analysis of Mono Azo Dyes." Bulg. Chem. Commun, Vol. 48, No.1, 2016: pp. 71-77.

Al Tamim, Abdullah, et al. "Fast and Simple Method for the Detection and Quantification of 15 Synthetic Dyes in Sauce, Cotton Candy, and Pickle by Liquid Chromatography/Tandem Mass Spectrometry." Arabian Journal of Chemistry Vol. 13, No.2 (2020: pp, 3882-3888.

Andoh, Sampson Saj, et al. "Qualitative Analysis of Sudan IV in Edible Palm Oil." Journal of the European Optical Society-Rapid Publications, vol. 15, No.1, 2019: pp. 1-5.

Andrade-Eiroa, Auréa, et al. "Solid-Phase Extraction of Organic Compounds: A Critical Review (Part I)." TrAC Trends in Analytical Chemistry, Vol. 80, No. 1, 2016: pp. 641-654.

Aresta, Antonella, Nicoletta De Vietro, and Carlo Zambonin. "Ultra-Trace Determination of Sudan I, II, III, and IV in Wastewater by Solid-Phase Microextraction (SPME) and on-Line Solid-Phase Extraction (SPE) with High-Performance Liquid Chromatography (HPLC)." Analytical Letters, Vol. 3, No. 1, 2020: pp. 1-12.

Arora S, Bharti S. Effect of mechanical drying on quality of chilli varieties. J Food Sci Technol. 2005; 42:179–182.

ASTA. Sudan Red and related dyes - white paper [Internet]. 2005. [cited 2015 Mar 5].

Dhakal, Sagar, et al. "Detection of Additives and Chemical Contaminants in Turmeric Powder Using FT-IR Spectroscopy." Foods, Vol. 8, No.5, 2019: pp.143-158.

Esen, Betül, Tülay Oymak, and Emrah Dural. "Determination of Food Colorings in Pharmaceutical Preparations and Food Additives by a Validated HPLC Method." International Journal of Scientific & Engineering Research, Vol. 9, No. 8, 2018: pp. 72-76.

European Commission. Commission decision of 20 June 2003 on emergency measures regarding hot chilli and hot chilli products. Off J Eur Commun L. 2003:114–115.

European Commission. Commission decision of 23 May 2005 on emergency measures regarding chilli, chilli products, curcuma and palm oil. 2005/402/EC. Off J Eur Commun L. 2005:34–35.

Hu, Mingzhu, et al. "Determination of Sudan Dyes in Juice Samples via Solidification of Ionic Liquid in Microwave-Assisted Liquid-Liquid Microextraction Followed by High-Performance Liquid Chromatography." Food analytical methods, Vol. 9, No.7, 2016: pp. 2124-2132.

Hunger, K.; Mischke, P.; Rieper, W. Ullmann’s encyclopedia of industrial chemistry. Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA; 2000. Azo dyes, 1. General.

Iarc Monographs on the Evaluation of the Carcinogenic Risks to Humans. International Agency for Research on Cancer. Lyon: World Health Organization; 1987.

Lohumi, Santosh, et al. "Quantitative Analysis of Sudan Dye Adulteration in Paprika Powder Using FTIR Spectroscopy." Food Additives & Contaminants: Part A, Vol. 34, No.5. 2017: pp. 678-686.

Moldoveanu, Serban C., and Victor David. Selection of the HPLC method in chemical analysis. Elsevier, 2016.

Ntrallou, Konstantina, Helen Gika, and Emmanouil Tsochatzis. "Analytical and Sample Preparation Techniques for the Determination of Food Colorants in Food Matrices." Foods, Vol. 9, No.1, 2020: pp. 58-82.

Oliveira, Marciano M., J. P. Cruz‐Tirado, and Douglas F. Barbin. "Nontargeted Analytical Methods as a Powerful Tool for the Authentication of Spices and Herbs: A Review." Comprehensive Reviews in Food Science and Food Safety 18.3 (2019): 670-689.

Otero, Paz, et al. "Simultaneous Determination of 23 Azo Dyes in Paprika by Gas Chromatography-Mass Spectrometry." Food Analytical Methods, Vol. 10, No. 4, 2017: pp. 876-884.

RASFF. Contamination of Worcester sauce by the unauthorised colour Sudan I [Internet]. 2005. [cited 2015 Mar 5].

RASFF. Rapid Alert System for Food and Feed (RASFF), 2003 [Internet]. Luxembourg: Publications Office of the European Union; 2003. Annual report 2003 ed. [cited 2015 Mar 5]

RASFF. Rapid alert system for food and feed (RASFF) – RASFF portal, 2015 [Internet]. Online Searchable Database. 2015. [cited 2016 Jan 4].

Rocha, Fabio RP, et al. "Solid-Phase Extractions in Flow Analysis." Anais da Academia Brasileira de Ciências, Vol. 90, No.1, 2018, pp. 803-824.

Sudan I consolidated product list from February 2005 recall [Internet]. 2015. [cited 2014 Mar 4].

Susie Genualdi, Shaun MacMahon, Katherine Robbins, Samantha Farris, Nicole Shyong, and Lowri DeJager Method development and survey of Sudan I–IV in palm oil and chilli spices in the Washington, DC, area.

Tran, K.; Young, M.; Neue, U. Effective SPE strategies for LC-MS determination of Sudan dyes in chili products [Internet]. 2005. [cited 2015 Mar 5].

Weisz, Adrian, et al. "Determination of Sudan I and a Newly Synthesized Sudan III Positional Isomer in the Color Additive D&C Red No. 17 Using High-Performance Liquid Chromatography." Food Additives & Contaminants, Vol. 34, No.11, 2017: pp. 1831-1841.

Appendix

Figure 1 Methanol Blank

Chart Description automatically generated

Figure 2 Methanol Blank

Diagram Description automatically generated

Figure 3 250 S4 S1 Carotene

A picture containing schematic Description automatically generated

Figure 4 500 S4 S1 Carotene

Diagram Description automatically generated

Figure 5 1000 S4 S1 Carotene

A picture containing diagram Description automatically generated

Figure 6 5000 S4 S1 Carotene

A picture containing table Description automatically generated

Figure 7 2500 S4 1/2 S1 1/2Carotene

Figure 8 Mix SI& SIV

A picture containing engineering drawing Description automatically generated

Figure 9 Mix SI & SIV

Diagram, schematic Description automatically generated

Figure 10 10 ng/mL SIV 180 S1

Diagram Description automatically generated

Figure 11 25 ng/mL SIV 180 S1

Diagram Description automatically generated

Figure 12 50 ng/mL SIV 180 S1

A picture containing diagram Description automatically generated

Figure 13 100 ng/mL SIV 180 S1

Diagram Description automatically generated

Figure 14 250 ng/mL SIV 450 S1

A picture containing diagram Description automatically generated

Figure 15 Spike 1 sample

Diagram Description automatically generated

Figure 16 Unspiked1 sample

Diagram Description automatically generated

Figure 17 Spike 2 Sample

Diagram Description automatically generated

Figure 18 Unspike 2 Sample

Figure 19 Spike 3 Sample

Diagram Description automatically generated

Figure 20 Unspike 3 Sample

Figure 21 Spike 4 Sample

A picture containing graphical user interface Description automatically generated

Figure 22 Unspike 4 Sample

Diagram Description automatically generated

A picture containing graphical user interface Description automatically generated

Figure 23 Sudan I std

Diagram Description automatically generated

A picture containing background pattern Description automatically generated

Figure 24 Sudan IV std

Diagram Description automatically generated

A picture containing graphical user interface Description automatically generated

Figure 25 100% Sudan std I & IV

Diagram Description automatically generated

A picture containing graphical user interface Description automatically generated

Figure 26 50% Sudan std I & IV

Diagram Description automatically generated

A picture containing graphical user interface Description automatically generated

Figure 27 25% Sudan std I & IV

A picture containing graphical user interface Description automatically generated

Figure 28 Sudan I and IV from spiked Sample

preparation- Ethyl ether wash

A picture containing chart Description automatically generated

A picture containing shape Description automatically generated

Figure 29 Sudan I and IV from spiked Sample preparation- Ethyl Acetate wash

A picture containing chart Description automatically generated

A picture containing graphical user interface Description automatically generated

Figure 30 Sample Ethyl Ether wash

Diagram Description automatically generated A picture containing graphical user interface Description automatically generated

Figure 31 Sample Ethyl Acetate wash

Calibration Curve, Sudan I

Peak area

0 2.5999999999999999E-2 5.1999999999999998E-2 0.104 0 268342 678256 1083100

Sudan I, mg/ml

Peak Area

Calibration Curve, Sudan IV

Peak area

0 2.6499999999999999E-2 5.2999999999999999E-2 0.106 0 486254 1076363 2030131

Sudn IV, mg/ml

Peak Area

N

N

OH

Sudan I

N

N

OH

N

N

Sudan III

N

N

OH

H

3

C

CH

3

Sudan II

N

N

OH

N

N

H

3

C

H

3

C