A new method was developed for the simultaneous determination of cholesterol and its oxidation products in eggs, using HPLC with UV and refractive index (RI) detectors, and HPLC interfaced with atmospheric pressure chemical ionization coupled to MS (HPLC-APCI-MS). The best conditions for direct saponification of the sample and extraction of the non-saponifiable material were defined using complete factorial designs with central points. The method showed accuracy and precision with a detection limit between 0.002 and 0.079 mu g/g. The oxides cholest-5-ene-3 beta,20 alpha-diol and cholest-5-ene-3 beta,25-diol identified by HPLC-UV-RI were not confirmed by HPLC-APCI-MS.416615622