Determination of Organic Impurities and Subsidiary Colors in Ext. D&C Violet No. 2 (Acid Violet 43) by Ultra-High-Performance Liquid Chromatography.

Background: The U.S. Food and Drug Administration (FDA) batch certifies the anthraquinone color additive Ext. D&C Violet No. 2 (XV2, "Ext." stands for "External") to ensure that it meets requirements published in the Code of Federal Regulations (CFR). XV2 is manufactured by condensing 1,4-dihydroxy-9,10-anthracenedione (DHAQ) with p-toluidine (pT) followed by sulfonation of the condensation product at the ortho position of the pT group. Organic impurities include residual intermediates, DHAQ and pT, and reaction by-products, 1-hydroxy-9,10-anthracenedione (MHAQ) and p-toluidine-m-sulfonic acid (PTMS). Sulfonation of the condensation product at the meta position produces and isomeric subsidiary color (mXV2). Other subsidiary colors include a dye which is itself certifiable as D&C Green No. 5 (G5) and a sulfonated phthaloylcarbazole (AV43C).

Objective: This paper describes a simple and sensitive UHPLC method for the determination of CFR-specified organic impurities in XV2 samples submitted to the FDA for batch certification.

Methods: The UHPLC method uses a 1.7 mm particle size C-18 column with aqueous ammonium acetate and acetonitrile as eluants and photodiode array detection at two wavelengths. Analytes are identified by comparing their retention times and UV-visible spectra to those of reference standards.

Results: Method validation demonstrates linearity, limits of detection, limits of quantitation, accuracy, and precision. Excellent regression coefficients for the calibration curves were obtained, with values >0.999. Overall accuracy was 98.2-104.3% and precision was 0.0075--5.27% for all analytes.

Conclusion: The UHPLC method satisfies the accuracy and precision requisites for routine certification.

Highlights: The UHPLC method reported here can be used for the determination of CFR-specified organic impurities including intermediate starting materials, reaction by-products, and subsidiary colors in samples of XV2 submitted to the FDA for batch certification. Method's LOD is well below the CFR specification levels.