Determination of Aniline, 4-Aminoazobenzene, and 2-Naphthol in the Color Additive D&C Red No. 17 Using Ultra-High-Performance Liquid Chromatography.

Specifications in the U.S. Code of Federal Regulations for the color additive D&C Red No. 17 (R17, Colour Index No. 26100) limit the levels of the dye's intermediates, aniline (AN), 2-naphthol (ß-naphthol, BN), and 4-aminoazobenzene (4AAB), to 0.2, 0.2, and 0.1%, respectively. The present work reports the development and application of an ultra-HPLC method for the quantitative determination of these impurities in R17. A 1.7 µm particle size C-18 column was used with 0.2 M ammonium acetate and acetonitrile as the eluents. AN, BN, and 4AAB were quantified by using six-point calibration curves with data points (w/w) ranging from 0.01 to 0.25% for AN, 0.01 to 0.24% for BN, and 0.01 to 0.19% for 4AAB. The correlation coefficients ranged from 0.9992 to 0.9999. Limits of detection for the analytes ranged from 0.002 to 0.01%. Recoveries of the analytes ranged from 99.5 to 102%. Relative standard deviations ranged from 0.482 to 1.262%. The new method was applied to analyze portions from 22 batches of R17 submitted to the U.S. Food and Drug Administration for certification. It was found to be simpler to implement, faster, and more sensitive than the older gravity-elution column chromatography method, which it has replaced.

Determination of Organic Impurities in Anthraquinone Color Additives D&C Violet No. 2 and D&C Green No. 6 by Ultra-High Performance Liquid Chromatography.

A new practical and time-saving ultra-high performance liquid chromatography (UHPLC) method has been developed for determining the organic impurities in the anthraquinone color additives D&C Violet No. 2 and D&C Green No. 6. The impurities determined are p-toluidine, 1-hydroxyanthraquinone, 1,4-dihydroxyanthraquinone, and two subsidiary colors. The newly developed UHPLC method uses a 1.7-µ particle size C-18 column, 0.1 M ammonium acetate and acetonitrile as eluents, and photodiode array detection. For the quantification of the impurities, six-point calibration curves were used with correlation coefficients that ranged from 0.9974 to 0.9998. Recoveries of impurities ranged from 99 to 104%. Relative standard deviations ranged from 0.81 to 4.29%. The limits of detection for the impurities ranged from 0.0067% to 0.216%. Samples from sixteen batches of each color additive were analyzed, and the results favorably compared with the results obtained by gravity-elution column chromatography, thin-layer chromatography, and isooctane extraction. Unlike with those other methods, use of the UHPLC method permits all of the impurities to be determined in a single analysis, while also reducing the amount of organic waste and saving time and labor. The method is expected to be implemented by the U.S. Food and Drug Administration for analysis of color additive samples submitted for batch certification.

Conventional high-performance liquid chromatography versus derivative spectrophotometry for the determination of 1,3,6-pyrenetrisulfonic acid trisodium salt and 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt in the color additive D&C Green No. 8 (Pyranine).

Specifications in the U.S. Code of Federal Regulations for the color additive D&C Green No. 8 (Colour Index No. 59040) limit the levels of the subsidiary colors 1,3,6-pyrenetrisulfonic acid trisodium salt (P3S) and 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (P4S). The present paper describes a comparative study of two possible methods to replace the currently used multi-step TLC/spectrophotometry method of separating and quantifying the minor components P3S and P4S in G8. One of the new approaches uses conventional high-performance liquid chromatography (HPLC) and the other, derivative spectrophotometry. While the derivative spectrophotometric method was shown to be inadequate for the analysis of minor components overwhelmed by components of much higher concentration, the HPLC method was proven highly effective. The closely related, very polar compounds P3S and P4S were separated by the new HPLC method in less than 4 min using a conventional HPLC instrument. P3S and P4S were quantified by using five-point calibration curves with data points that ranged from 0.45 to 7.63% and from 0.13 to 1.82%, by weight, for P3S and P4S, respectively. The HPLC method was applied to the analysis of test portions from 20 batches of D&C Green No. 8 submitted to the U.S. Food and Drug Administration for certification.