Determination of stigmasterol primary oxidation products by high-performance liquid chromatography

A new specific chromatographic method for the determination of plant sterol primary oxidation products, hydroperoxides, was developed. Separation was performed by normal-phase high-performance liquid chromatography (NP-HPLC) using two types of detectors. A UV detector was used as a general detector and a fluorescence detector after post column diphenyl-1-pyrenylphosphine reagent addition (DPPP-FL detection) as a hydroperoxide-specific detector. Stigmasterol was used as a model compound. Hydroperoxides were obtained by photo-oxidation in dichloromethane in the presence of methylene blue as a sensitizer. Specificity of the DPPP-FL detection was confirmed by establishing the structures of the main hydroperoxides by gas chromatography mass spectrometry. 5α-Hydroperoxide, 7α-hydroperoxide and 6α- and 6β-hydroperoxides were identified by comparing mass spectral data and retention times with those of the corresponding hydroxy derivatives of cholesterol. Moreover, the suitability of the method for monitoring the formation of hydroperoxides during photo-oxidation was demonstrated. The results showed that the new NP-HPLC method with DPPP-FL detection provides information about the structures and amounts of different plant sterol hydroperoxides in a sample without prior derivatization, complex sample pre-treatment or heating.