Determination of benzidine in the food colours tartrazine and sunset yellow FCF, by reduction and derivatization followed by high-performance liquid chromatography

Free and bound benzidine, a non-sulphonated aromatic amine (NSAA), were determined in the food colours tartrazine and sunset yellow FCF. Bound amines were released by reducing with sodium dithionite, then total NSAAs were extracted into chloroform, transferred to aqueous acid solution and diazotized with sodium nitrite before coupling with 2-naphthol-3,6-disulphonic acid, disodium salt (R-salt). Coloured benzidine and aniline derivatives (BZDRS and ANRS) were analysed using reversed-phase ion pair high-performance liquid chromatography (HPLC) and an absorbance detector set at 548nm. Levels of total benzidine were similar to those reported in studies conducted in the USA, and ranged from < 5 to 270ng/g. Total aniline was also determined (0.2-188 mug/g). Recoveries of benzidine with this method were found to be lower than expected (average ca 46% ), but were reproducible. Detection limits were 15-20ng BZDRS/g (3-4ng benzidine/g). Mass spectrometry (LC-MS) was evaluated for identifying and determining purity of the standards, but difficulties were encountered when the methodology was applied to commercial samples.