DETERMINATION OF INTERMEDIATES AND SUBSIDIARY COLORS IN THE COLOR ADDITIVE FD&C RED NO. 4 (PONCEAU SX) USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Abstract

Specifications in the U.S. Code of Federal Regulations for the color additive FD&C Red No. 4 (Color Index 14700) limit the levels of the intermediates, 5-amino-2,4-dimethyl-1-benzenesulfonic acid (AMBSA) and 4-hydroxy-1-naphthalenesulfonic acid (HNSA), and of subsidiary colors. The present study reports the development of a high-performance liquid chromatography (HPLC) method for the quantitative determination of these intermediates and subsidiary colors in one analysis. The most commonly-found subsidiary color, 3-[(2,4-dimethyl-6-sulfophenyl)azo]-4-hydroxy-1-naphthalenesulfonic acid (6SSC), is determined and is used as a secondary reference material to determine the quantities of other subsidiary colors present. High-speed countercurrent chromatography was used to obtain purified HNSA and 6SSC for use as reference materials. AMBSA, HNSA, and 6SSC were quantified by using five-point calibration curves with data points that ranged from 0.028–0.234%, 0.031–0.252%, and 0.113–2.036% by weight, respectively. The HPLC method is rapid (50 min for the total analysis cycle) and simple to implement. It was applied to the analysis of test portions from 23 lots of FD&C Red No. 4 submitted to the U.S. Food and Drug Administration (FDA) for certification and was later implemented by the FDA for routine batch certification of FD&C Red No. 4.