Determination of Seven Manufacturing Impurities in FD&C Red No. 40 by Ultra-High-Performance Liquid Chromatography

Background: The U.S. Food and Drug Administration batch-certifies color additives to ensure that each lot meets published specifications for coloring food, drugs, and cosmetics. Objective: An ultra-high-performance LC (UHPLC) method was developed to determine seven manufacturing impurities in the monoazo color additive FD&C Red No. 40 (R40). The analytes consist of two intermediates, an impurity originating from one intermediate, a reaction by-product, and three subsidiary colors. The intermediates are 4-amino-5-methoxy-2-methylbenzenesulfonic acid [cresidine-p-sulfonic acid (CSA)] and 6-hydroxy-2-naphthalene sulfonic acid sodium salt (SS). The impurity originating from the intermediate SS is 6,6'-oxybis[2-naphthalenesulfonic acid] disodium salt. The reaction by-product is 4,4'-(diazoamino)bis[5-methoxy-2-methylbenzenesulfonic acid disodium salt. The subsidiary colors are sodium salts of CSA coupled with 2-naphthol-3,6-disulfonic acid, 2-naphthol-6,8-disulfonic acid, or 2-naphthol. Methods: Samples of R40 were dissolved in an ammonium acetate buffer modified to pH 9.2, filtered, and analyzed by UHPLC. Quantitation of the analytes was performed by calibration in the presence of the color additive matrix. Results: UHPLC validation studies showed linear calibration curves (R2 = 0.9999), good recovery (95-121%) and precision (RSDs = 1.0-6.3%), and LOQs ranging from 0.002 to 0.030%. Survey analyses of 31 samples from 11 manufacturers yielded results by the new UHPLC method and a previously used HPLC method that are consistent within experimental error. Conclusions: The new UHPLC method provides faster analysis time, improved separation, and similar sensitivity compared to the HPLC method. Highlights: An UHPLC method was developed and validated to determine seven manufacturing impurities in R40 submitted to the FDA for batch certification.