Identification and Determination of the Isomer and Subsidiary Color in Food Blue No. 2 (Indigo Carmine) by LC/MS and HPLC

Abstract

CFR and JECFA specify that the total color in FD&C Blue No. 2 (B2; Indigo Carmine, Indigotine, Food Blue No. 2) is not less than 85%, its isomer (B2iso) in B2 is not more than 18%, and its subsidiary color (B2sub) in B2 is not more than 2% (CFR) or 1% (JECFA). Japan's Specifications and Standards for Food Additives, 7^th Edition, specifies that the total color in B2 is not less than 85.0% and other color materials in B2 are not detected by paper chromatography. LC/MS and HPLC were employed to identify and determine the main component (B2m) of B2, B2iso, and B2sub. The pseudo molecular ions (B2m and B2iso: [M-2Na+H]^-, m/z=421; B2sub: [M-Na]^-, m/z=341) of each color material were obtained and identified by LC/MS based on their absorptions and mass spectra. The contents of B2iso and B2sub in B2 samples (certified samples from fiscal year 1998 to fiscal year 2002) were determined by HPLC using calibration curves for the standards of B2m and B2iso. The contents of B2iso in most samples were less than 10%, and the contents of B2sub in all samples were not more than 1%. All of them were within the regulatory limits set by the CFR and JECFA.