High Performance Liquid Chromatographic Determination of Subsidiary Colors in FD&C Red No. 3

Abstract

Abstract Two rapid, sensitive, reproducible methods that use a Zorbax C-8 reverse phase column and high performance liquid chromatography are described for the determination of the subsidiary colors in FD&C Red No. 3. With the first method, 8 subsidiary colors (fluorescein, 2'-iodofluorescein, 4'-iodofluorescein, 2',5'-diiodofluorescein, 2',7'-diiodofluorescein, 4',- 5'-diiodofluorescein, 2',4',7'-triiodofluorescein, and 2',4',5'-triiodofluorescein) are eluted in a reproducible pattern by increasing the organic nature of a buffered mobile phase. Method 1 is capable of quantitating all the subsidiary colors except 4'-iodofluorescein and 4',5'-diiodofluorescein. If these 2 subsidiary colors are seen, the sample must be run again by method 2, which uses a different program and solvent system to quantitate them. The average recoveries for the 6 subsidiary colors quantitatively determined in FD&C Red No. 3 by method 1 ranged from 96 to 98%. The average recoveries for 4'-iodofluorescein and 4',5'-diiodofluorescein, quantitatively determined in FD&C Red No. 3 by method 2, were 101 and 103%, respectively. The amounts of the 6 subsidiary colors recovered by method 1 were 0.04-7.4% by weight of the total sample. The amounts of the 2 subsidiary colors recovered by method 2 were 0.13-2.6% by weight of the total sample.