[Detection of the annatto dye norbixin/bixin in cheese using derivative spectroscopy and high performance liquid...

A derivative spectroscopic method and a HPLC-method are described for the determination of the annatto dye-stuffs, norbixin and bixin, in cheese. Both methods enable a simple and quick sample preparation since the separation of beta-carotene and fat is not required. The sample preparation step consists of extraction with acetone, filtration, evaporation of the extract and separation of water residues by the addition of a few milliliters of absolute ethanol. This is followed by evaporation and extraction of the residual solution with chloroform/acetic acid (99.5 +/- 0.5) for the derivative spectroscopic method or with acetone for the HPLC method. The qualitative detection (detection limit greater than 0.67 mg/kg, depending on the genuine beta-carotene content) as well as the quantitative determination is possible by means of the derivative spectroscopic method. Therefore, this technique may be used within the rigorous Austrian regulations or for controlling the quantities and limits of annatto dye-stuffs in cheese, if its application is allowed. The method also has the advantage of quick detection (only 75 s) and saving of material used. The HPLC method allows for the separation and quantification of norbixin and bixin as well as the other carotenoids such as beta-carotene, beta-apo-8'-carotenal and beta-apo-8'-carotenoic acid--ethylester, which may also be found in varieties of cheese (detection limit of norbixin and bixin: 0.2 mg/kg). The time required for the separation of the above mentioned substances is 20 min and the HPLC method is proposed for the confirmation of low concentrations of these substances.