Analytical and Bioanalytical Chemistry

, Volume 410, Issue 22, pp 5641–5651 | Cite as

Analysis of unauthorized Sudan dyes in food by high-performance thin-layer chromatography

  • Wolfgang SchwackEmail author
  • Elodie Pellissier
  • Gertrud Morlock
Research Paper
Part of the following topical collections:
  1. Food Safety Analysis


Food authenticity and food safety are of high importance to organizations as well as to the food industry to ensure an accurate labeling of food products. Respective analytical methods should provide a fast screening and a reliable cost-efficient quantitation. HPTLC was pointed out as key analytical technique in this field. A new HPTLC method applying caffeine-impregnated silica gel plates was developed for eight most frequently found fat-soluble azo dyes unauthorizedly added to spices, spice mixtures, pastes, sauces, and palm oils. A simple post-chromatographic UV irradiation provided an effective sample cleanup, which took 4 min for up to 46 samples in parallel. The method was trimmed to enable 23 simultaneous separations within 20 min for quantitation or 46 separations within 5 min for screening. Linear (4–40 ng/band) or polynomial (10–200 ng/band) calibrations of the eight azo dyes revealed high correlation coefficients and low standard deviations. Limits of detection and quantification were determined to be 2–3 and 6–9 ng/zone, respectively. After an easy sample extraction, recoveries of 70–120% were obtained from chili, paprika, and curcuma powder as well as from chili sauce, curry paste, and palm oil spiked at low (mainly 25–50 mg/kg) and high levels (150–300 mg/kg). For unequivocal identification, the compound in a suspect zone was eluted via a column into the mass spectrometer. This resulted in the hyphenation HPTLC-vis-HPLC-DAD-ESI-MS.

Graphical abstract

Simplified clean-up by UV irradiation for Sudan dye analysis in food by HPTLC-vis-HPLC-DAD-ESI-MS.


Sudan dyes High-performance thin-layer chromatography (HPTLC) HPTLC–mass spectrometry 



Thank is owed to Merck (Darmstadt, Germany) and Macherey-Nagel (Düren, Germany) for providing HPTLC plates, and to CAMAG (Berlin, Germany) with regard to HPTLC instruments, and to Dr. Claudia Oellig (University of Hohenheim, Germany) for repeating a couple of MS experiments.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_945_MOESM1_ESM.pdf (1022 kb)
ESM 1 (PDF 0.99 mb)


  1. 1.
    European Commission. RASFF - Food and Feed Safety Alerts.
  2. 2.
    The European Parliament and the Council of the European Union. Regulation (EC) no 178/2002 of 28 January 2002 laying down the general principles and requirements of food law, establishing the European food safety authority and laying down procedures in matters of food safety. Off J Eur Union. 2002;L 31:1–24.Google Scholar
  3. 3.
    The Comission of the European Communities. Comission decision of 20 June 2003 on emergency measures regarding hot chilli and hot chilli products (2003/460/EC). Off J Eur Union. 2003;L 154:114–5.Google Scholar
  4. 4.
    The Comission of the European Communities. Commission decision of 23 May 2005 on emergency measures regarding chilli, chilli products, curcuma and palm oil (2005/402/EC). Off J Eur Union. 2005;L 135:34–6.Google Scholar
  5. 5.
    The Comission of the European Communities. Commission decision of 21 January 2004 on emergency measures regarding chilli, chilli products, curcuma and palm oil (2004/92/EC). Off J Eur Union. 2004;L 27:52–4.Google Scholar
  6. 6.
    Rebane R, Leito I, Yurchenko S, Herodes K. A review of analytical techniques for determination of Sudan I-IV dyes in food matrixes. J Chromatogr A. 2010;1217:2747–57.CrossRefPubMedGoogle Scholar
  7. 7.
    Ferrer Amate C, Unterluggauer H, Fischer RJ, Fernandez-Alba AR, Masselter S. Development and validation of a LC-MS/MS method for the simultaneous determination of aflatoxins, dyes and pesticides in spices. Anal Bioanal Chem. 2010;397:93–107.CrossRefPubMedGoogle Scholar
  8. 8.
    Zheng M-M, Wu J-H, Feng Y-Q, Huang F-H. Rapid and sensitive determination of Sudan dyes in hot chilli products by solid-phase extraction directly combined with time-of-flight mass spectrometry. Anal Methods. 2011;3:1851–8.CrossRefGoogle Scholar
  9. 9.
    Erdemir US, Izgi B, Gucer S. An alternative method for screening of Sudan dyes in red paprika paste by gas chromatography-mass spectrometry. Anal Methods. 2013;5:1790–8.CrossRefGoogle Scholar
  10. 10.
    Siangproh W, Sonamit K, Chaiyo S, Chailapakul O. Fast determination of Sudan I-IV in chili products using automated on-line solid phase extraction coupled with liquid chromatography-mass spectrometry. Anal Lett. 2013;46:1705–17.CrossRefGoogle Scholar
  11. 11.
    Genualdi S, MacMahon S, Robbins K, Farris S, Shyong N, DeJager L. Method development and survey of Sudan I-IV in palm oil and chilli spices in the Washington, DC, area. Food Addit Contam Part A. 2016;33:583–91.Google Scholar
  12. 12.
    Qi P, Zeng T, Wen Z, Liang X, Zhang X. Interference-free simultaneous determination of Sudan dyes in chili foods using solid phase extraction coupled with HPLC-DAD. Food Chem. 2011;125:1462–7.CrossRefGoogle Scholar
  13. 13.
    Enriquez-Gabeiras L, Gallego A, Garcinuno RM, Fernandez-Hernando P, Durand JS. Interference-free determination of illegal dyes in sauces and condiments by matrix solid phase dispersion (MSPD) and liquid chromatography (HPLC-DAD). Food Chem. 2012;135:193–8.CrossRefGoogle Scholar
  14. 14.
    Zhu Y, Wu Y, Zhou C, Zhao B, Yun W, Huang S, et al. A screening method of oil-soluble synthetic dyes in chilli products based on multi-wavelength chromatographic fingerprints comparison. Food Chem. 2016;192:441–51.CrossRefPubMedGoogle Scholar
  15. 15.
    Schummer C, Sassel J, Bonenberger P, Moris G. Low-level detections of Sudan I, II, III and IV in spices and chili-containing foodstuffs using UPLC-ESI-MS/MS. J Agric Food Chem. 2013;61:2284–9.CrossRefPubMedGoogle Scholar
  16. 16.
    Li J, Ding X-M, Liu D-D, Guo F, Chen Y, Zhang Y-B, et al. Simultaneous determination of eight illegal dyes in chili products by liquid chromatography-tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci. 2013;942-943:46–52.CrossRefGoogle Scholar
  17. 17.
    Tang B, Xi C, Zou Y, Wang G, Li X, Zhang L, et al. Simultaneous determination of 16 synthetic colorants in hotpot condiment by high performance liquid chromatography. J Chromatogr B Anal Technol Biomed Life Sci. 2014;960:87–91.CrossRefGoogle Scholar
  18. 18.
    Fukuji TS, Castro-Puyana M, Tavares MFM, Cifuentes A. Sensitive and fast determination of Sudan dyes in chilli powder by partial-filling micellar electrokinetic chromatography-tandem mass spectrometry. Electrophoresis. 2012;33:705–12.CrossRefPubMedGoogle Scholar
  19. 19.
    Khalikova MA, Satinsky D, Solich P, Novakova L. Development and validation of ultra-high performance supercritical fluid chromatography method for determination of illegal dyes and comparison to ultra-high performance liquid chromatography method. Anal Chim Acta. 2015;874:84–96.CrossRefPubMedGoogle Scholar
  20. 20.
    Li Z, Zhang Y-W, Zhang Y-D, Bai Y, Liu H-W. Rapid analysis of four Sudan dyes using direct analysis in real time-mass spectrometry. Anal Methods. 2015;7:86–90.CrossRefGoogle Scholar
  21. 21.
    Cao Y, Fang Z, Yang D, Gao Y, Li H. Voltammetric sensor for Sudan I based on glassy carbon electrode modified by SWCNT/β-Cyclodextrin conjugate. Nano. 2015;10:1550026.CrossRefGoogle Scholar
  22. 22.
    Elyasi M, Khalilzadeh MA, Karimi-Maleh H. High sensitive voltammetric sensor based on Pt/CNTs nanocomposite modified ionic liquid carbon paste electrode for determination of Sudan I in food samples. Food Chem. 2013;141:4311–7.CrossRefPubMedGoogle Scholar
  23. 23.
    Chang XC, Hu XZ, Li YQ, Shang YJ, Liu YZ, Feng G, et al. Multi-determination of Para red and Sudan dyes in egg by a broad specific antibody based enzyme linked immunosorbent assay. Food Control. 2011;22:1770–5.CrossRefGoogle Scholar
  24. 24.
    Qi YH, Shan WC, Liu YZ, Zhang YJ, Wang JP. Production of the polyclonal antibody against Sudan 3 and immunoassay of Sudan dyes in food samples. J Agric Food Chem. 2012;60:2116–22.CrossRefPubMedGoogle Scholar
  25. 25.
    Zvereva EA, Zaichik BT, Eremin SA, Zherdev AV, Dzantiev BB. Enzyme immunoassay for detection of Sudan I dye and its application to the control of foodstuffs. J Anal Chem. 2016;71:944–8.CrossRefGoogle Scholar
  26. 26.
    Li C, Wu Y-L, Shen J-Z. UPLC-ESI-MS/MS analysis of Sudan dyes and Para red in food. Food Addit Contam Part A. 2010;27:1215–20.CrossRefGoogle Scholar
  27. 27.
    Zhu Y, Zhao B, Xiao R, Yun W, Xiao Z, Tu D, et al. Simultaneous determination of 14 oil-soluble synthetic dyes in chilli products by high performance liquid chromatography with a gel permeation chromatography clean-up procedure. Food Chem. 2014;145:956–62.CrossRefPubMedGoogle Scholar
  28. 28.
    Li Y, Wang Y, Yang H, Gao Y, Zhao H, Deng A. Establishment of an immunoaffinity chromatography for simultaneously selective extraction of Sudan I, II, III and IV from food samples. J Chromatogr A. 2010;1217:7840–7.CrossRefPubMedGoogle Scholar
  29. 29.
    Piao C, Chen L. Separation of Sudan dyes from chilli powder by magnetic molecularly imprinted polymer. J Chromatogr A. 2012;1268:185–90.CrossRefPubMedGoogle Scholar
  30. 30.
    Hu X, Cai Q, Fan Y, Ye T, Cao Y, Guo C. Molecularly imprinted polymer coated solid-phase microextraction fibers for determination of Sudan I-IV dyes in hot chili powder and poultry feed samples. J Chromatogr A. 2012;1219:39–46.CrossRefPubMedGoogle Scholar
  31. 31.
    Rajabi M, Sabzalian S, Barfi B, Arghavani-Beydokhti S, Asghari A. In-line micro-matrix solid-phase dispersion extraction for simultaneous separation and extraction of Sudan dyes in different spices. J Chromatogr A. 2015;1425:42–50.CrossRefPubMedGoogle Scholar
  32. 32.
    Xu Z, Wang S, Fang G, Song J, Yan Z. On-line SPE coupled with LC for analysis of traces of Sudan dyes in foods. Chromatographia. 2010;71:397–403.CrossRefGoogle Scholar
  33. 33.
    Morlock GE, Brett N. Correct assignment of lipophilic dye mixtures? A case study for high-performance thin-layer chromatography–mass spectrometry and performance data for the TLC–MS Interface. J Chromatogr. 2015;1390:103–11.CrossRefGoogle Scholar
  34. 34.
    Yüce I, Morlock GE. Streamlined structure elucidation of an unknown compound in a pigment formulation. J Chromatogr. 2016;1469:120–7.CrossRefGoogle Scholar
  35. 35.
    Marshall PN. Thin-layer chromatography of Sudan dyes. J Chromatogr. 1977;136:353–7.CrossRefPubMedGoogle Scholar
  36. 36.
    Dixit S, Khanna SK, Das M. A simple 2-directional high-performance thin-layer chromatographic method for the simultaneous determination of curcumin, metanil yellow, and sudan dyes in turmeric, chili, and curry powders. J AOAC Int. 2008;91:1387–96.PubMedGoogle Scholar
  37. 37.
    Guffog S, Brwon PA, Stangroom SG, Sutherland CA. The detection of sudan I, II, III and IV in palm oil by thin layer chromatography. Food Standards Agency Information Bulletin on Methods of Analysis and Sampling for Foodstuffs. 2004; (No. 52).Google Scholar
  38. 38.
    Kandler H, Bleisch M, Widmer V, Reich E. A validated HPTLC method for the determination of illegal dyes in spices and spice mixtures. J Liq Chromatogr Relat Technol. 2009;32:1273–88.CrossRefGoogle Scholar
  39. 39.
    Morlock G, Kopacz S. Fast and precise SBSE-HPTLC/FLD method for quantification of six polycyclic aromatic hydrocarbons frequently found in water. J Liq Chromatogr Relat Technol. 2008;31:1925–42.CrossRefGoogle Scholar
  40. 40.
    Snyder LR. Classification of the solvent properties of common liquids. J Chromatogr Sci. 1978;16:223–34.CrossRefGoogle Scholar
  41. 41.
    Kowalska T, Kaczmarski K, Prus W. Theory and mechanism of thin-layer chromatography. In: Sherma J, Fried B, editors. Handbook of thin-layer chromatography, 3rd edition (chromatographic science series volume 89). New York: Marcel Dekker; 2003. p. 47–80.Google Scholar
  42. 42.
    Tsai C-F, Kuo C-H, Shih DY-C. Determination of 20 synthetic dyes in chili powders and syrup-preserved fruits by liquid chromatography/tandem mass spectrometry. J Food Drug Anal. 2015;23:453–62.CrossRefPubMedGoogle Scholar
  43. 43.
    Pellissier E. HPTLC-Bestimmung von unerlaubt zugesetzten fettlöslichen Azofarbstoffen in Lebensmitteln: Diploma thesis, University of Applied Science, Western Switzerland/University of Hohenheim, Germany. 2009.Google Scholar
  44. 44.
    The American Oil Chemists‘ Society Lipid Library, AOCS Lipid Library.
  45. 45.
    Morlock G, Schwack W. Hyphenations in planar chromatography. Journal of Chromatography A. 2010;1217:6600-6609Google Scholar
  46. 46.
    Oellig C, Schwack W. Strategies of coupling planar chromatography to HPLC–MS. In: Kowalska T, Sajewicz M, Sherma J, editors. Planar chromatography—mass spectrometry (chromatographic science series volume 110). Boca Raton, London, New York: CRC Press; 2016. p. 169–94.Google Scholar
  47. 47.
    Wang Y, Gu M. The concept of spectral accuracy for MS. Anal Chem. 2010;82:7055–62.CrossRefPubMedGoogle Scholar
  48. 48.
    Ruf J, Walter P, Kandler H, Kaufmann A. Discovery and structural elucidation of the illegal azo dye Basic Red 46 in sumac spice. Food Additives & Contaminants: Part A 2012;29:897-907Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Wolfgang Schwack
    • 1
    Email author
  • Elodie Pellissier
    • 1
  • Gertrud Morlock
    • 1
    • 2
  1. 1.Institute of Food ChemistryUniversity of HohenheimStuttgartGermany
  2. 2.Chair of Food Science, Institute for Nutritional Science, and Interdisciplinary Research Center (IFZ)Justus Liebig University GiessenGiessenGermany

Personalised recommendations