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Volume: 9, Issue: 7, July, 2019
DOI: 10.7324/JAPS.2019.90714

Short Communication

Validation and application of reversed-phase high-performance liquid chromatography for quantitative analysis of acid orange 7 and Sudan II in blusher products

Novalina B. R. Purba1 & 2, Abdul Rohman1 & 3, Sudibyo Martono1

  Author Affiliations


The objective of this study was to develop and to validate reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of acid orange 7 (AO7) and Sudan II (SII) in blusher products. Separation of AO7 and SII was performed using C18 column (Thermo Synergy Gold 250 mm × 4.6 mm i.d., 5 μm) with mobile phase consisting of acetonitrile-water as a solvent (1:1 v/v) and delivered in gradient manner. The mobile phase was delivered at flow rate of 0.9 ml/minute, using column temperature of 40°C. RP-HPLC was validated by assessing several performance characteristics which included selectivity, linearity and range, sensitivity, precision, accuracy, and robustness. The developed RP-HPLC was selective for the separation of AO7 and SII. Over concentration of 10.326– 41.304 μg/ml (AO7) and 9.967–39.869 (SII), the method was linear with coefficient of correlation (r) >0.999. The method was sensitive as indicated by low limit of detection and limit of quantification. The relative standard deviation (RSD) values during intra-assay and inter-assay were lower than those required by RSD Horwitz. The percentage of recovery was within acceptable ranges as required by Association of Official Analytical Chemists method. The validated RP-HPLC method has been successfully applied for the analysis of AO7 and SII in commercial blusher products, and both AO7 and SII were not detected in the tested samples.


Acid orange 7, Sudan II, RP-HPLC, blusher products.

Citation: Purba NBR, Rohman A, Martono S. Validation and application of reversed phase high performance liquid chromatography for quantitative analysis of acid orange 7 and SII in blusher products. J Appl Pharm Sci, 2019; 9(07):100–105.

Copyright: The Author(s). This is an open access article distributed under the Creative Commons Attribution Non-Commercial License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Barot J, Bahadur A. Toxic impacts of C.I. acid orange 7 on behavioural, haematological and some biochemical parameters of labeo rohita fingerlings. Int J Sci Res Environm Sci, 2015; 3:284-90. https://doi.org/10.12983/ijsres-2015-p0284-0290

Bhawani S, Nageshwari HG, Mamatha G, Venu M, Krishna SM, Krishna MKS. Analytical method development and validation of Bendamustine in bulk using RP-HPLC. Pharm Res, 2018; 2:000158. https://doi.org/10.23880/OAJPR-16000158

Boeniger M. Carcinogenicity and metabolism of azo dyes, especially those derived from benzidine. NIOSH (NTIS), Springfield, VA, 1980.

Calbiani F, Careri M, Elviri L, Mangia A, Pistarà L, ZagnoniI. Development and in-house validation of a liquid chromatography-electrospray-tandem mass spectrometry method for the simultaneous determination of Sudan I, Sudan II, Sudan III and Sudan IV in hot chilli products. J Chromatogr A, 2004; 1042:123-30. https://doi.org/10.1016/j.chroma.2004.05.027

Ding Y, Sun C, Xu X. Simultaneous identification of nine carcinogenic dyes from textiles by liquid chromatography/electrospray ionization mass spectrometry via negative/positive ion switching mode. Eur J Mass Spectrom, 2009; 15:705-13. https://doi.org/10.1255/ejms.1032

Dolan JW. System suitability. 2004; 17(6):328-32. Available via http://alfresco.ubm-us.net/alfresco_images/pharma/2014/08/22/351c1a16- 183a-48dd-9241-4277b40c2168/article-98295.pdf (Accessed 16 July 2018).

Ertas E, Ozer H, Alasalvar CA. Rapid HPLC method for determination of sudan dyes and para red in red chilli pepper. Food Chem, 2007; 105:756-60. https://doi.org/10.1016/j.foodchem.2007.01.010

Fang G, Wu Y, Dong X, Liu C, He S, Wang S. Simultaneous determination of banned acid orange dyes and basic orange dyes in foodstuffs by liquid chromatography-tandem electrospray ionization mass spectrometry via negative/positive ion switching mode. J Agric Food Chem, 2013; 61:3834-41; doi:10.1021/jf400619y. https://doi.org/10.1021/jf400619y

Gonzalez AG, Herrador MA. A practical guide to analytical method validation, including measurement uncertainty and accuracy profiles. Trends Anal Chem, 2007; 26(3):227-38. https://doi.org/10.1016/j.trac.2007.01.009

Hueper WC. Occupational and environmental cancers of the urinary system. Br J Surg, 1970; 57:940-5.

International Conference on Harmonization. Guidance for industry: Q2B validation of analytical procedures: methodology. p. 10, 1996. Available via www.fda.gov/cedr/guidance/index.htm. (Accessed 16 July 2018)

Ma M, Luo X, Chen B, Su S, Yao S. Simultaneous determination of water-soluble and fat-soluble synthetic colorants in foodstuff by high-performance liquid chromatography-diode array detection-electrospray mass spectrometry. J Chromatogr A, 2006; 1103:170-6. https://doi.org/10.1016/j.chroma.2005.11.061

Magnusson B, Örnemark U. (eds.). Eurachem guide: The fitness for purpose of analytical methods-a laboratory guide to method validation and related topics. 2014. Available via http://www.eurachem.org. (Accessed 30 July 2018)

Miller JN, Miller JC. Statistics and chemometrics for analytical chemistry. Prentice Hall, UK, 2010.

National Standard 2760-2011. Using standard of food additives. National Standard of People's Republic of China, 2011.

Pielesz A, Baranowska I, Rybak A, Włochowicz A. Detection and determination of aromatic amines as products of reductive splitting from selected azo dyes. Ecotoxicol Environ Safety, 2002; 53:42-7. https://doi.org/10.1006/eesa.2002.2191

Purba NBR, Rohman A, Martono S. The optimization of HPLC for quantitative analysis of acid orange 7 and Sudan II in cosmetic products using box behnken design. Int J Appl Pharm, 2019; 11:130-7. https://doi.org/10.22159/ijap.2019v11i2.31285

Robens JF. Thirteen-week subchronic toxicity studies of Direct Blue 6, Direct Black 38, and Direct Brown 95 dyes. Toxicol Appl Pharmacol, 1980; 54:431-42. https://doi.org/10.1016/0041-008X(80)90170-2

SCCS, Scientific Committee on Consumer Safety Opinion On Acid Orange. Luxembourg, European Union. 2014. pp. 6-8. Available via https://ec.europa.eu/health/scientific_committees/consumer_safety/docs/ sccs_o_158.pdf. (Accessed 16 July 2018)

Snyder LR, Kirkland JJ, Dolan JW. Introduction to modern liquid chromatography. 3rd edition, John Wiley & Sons Inc. Publication, USA, 2010. https://doi.org/10.1002/9780470508183

Sun HW, Wang FC, Ai LF. Determination of banned 10 azo-dyes in hot chili products by gel permeation chromatography-liquid chromatography-electrospray ionization-tandem mass spectrometry. J Chromatogr A, 2007; 1164:120-8. https://doi.org/10.1016/j.chroma.2007.06.075

USP 27/NF 22. United States pharmacopoeial convention. Rockville, MD: United States Pharmacopeial Convention, p. 2281, 2003.

Yang Y, Zhang J, Yin J, Yang Y. Fast simultaneous determination of eight sudan dyes in chili oil by ultra-high-performance supercritical fluid chromatography. J Anal Methods Chem, 2019; 2019:3731028; doi:10.1155/2019/3731028. https://doi.org/10.1155/2019/3731028

Yoshioka N, Ichihashi K. Determination of 40 synthetic food colors in drinks and candies by high-performance liquid chromatography using a short column with photodiode array detection. Talanta, 2008; 74:1408-13. https://doi.org/10.1016/j.talanta.2007.09.015

Zatar NA. Simultaneous determination of seven synthetic water-soluble food colorants by ion-pair reversed-phase high-performance liquid chromatography. J Food Technol, 2007; 5:220-4.

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