Three different experiments were performed in order to obtain the major carotenoid composition of the natural colorant annatto (E160b) through ToF-SIMS (time-of-flight secondary ion mass spectrometry) and XPS (X-ray photoelectron spectroscopy) analyses. In the first experiment, Bixa orellana seeds aril as well as its interior part were analyzed. The analysis of the seeds aril by ToF-SIMS gives the colorant fingerprint without any sample treatment, showing the presence of bixin and its characteristic fragments. The analysis performed in the interior part of the seeds indicates the presence of Fe. The second set of measurements was conducted on the seeds organic extract right after extraction revealing the same components observed by in situ measurement. A third set of measurements was performed aiming to determine the reason for the organic extract color shift observed after 3 months of exposure to ambient light at room temperature. In this case, it was possible to evidence the degradation of bixin by the loss of xylene molecules through ToF-SIMS and the probable carotenoid oxidation based on the C1s XPS spectrum of the degraded extract.