Determination of combined benzidine in FD & C yellow no. 5 (tartrazine), using a highly sensitive analytical method

53 samples of FD & C Yellow No. 5 (tartrazine; Colour Index No. 19140) were examined for combined benzidine. These samples, which represent separate lots from 12 dye distributors, were submitted to the US FDA for certification between 28 February 1990 and 27 June 1991. A method was developed to reduce the dye matrix with dithionite so that combined benzidine present in the form of azo or disazo dyes would be converted to free benzidine. Reduction was followed by extraction, diazotization and coupling with pyrazolone T, and the total benzidine present was quantitated as benzidine-pyrazolone T disazo dye (BZPT) by HPLC with detection at 500 nm. The limit of quantitation for benzidine in FD & C Yellow No. 5 by this method is 5 ng/g. 25 samples of FD & C Yellow No. 5 were found to contain 7-83 ng/g of combined benzidine that was released by dithionite reduction. 23 of these samples were from the same manufacturer. The identify of the BZPT from two FD & C Yellow No. 5 samples was confirmed by spectral analysis using HPLC with a photodiode array detector.