A RAPID METHOD FOR PROFILING THE PRODUCTS OF ANTIOXIDANT REACTIONS BY NEGATIVE-ION CHEMICAL-IONIZATION MASS-SPECTROMETRY

The antioxidant nutrients beta-carotene and alpha-tocopherol are thought to prevent oxidation of important biomolecules in vivo by trapping reactive free radicals. Conventional analytical methods for analyzing antioxidants and their products rely on time consuming chromatographic isolation followed by spectroscopic characterization. This approach is complicated by the instability of many of these products and the large amount of time required for isolation and characterization of multiple products. We have developed a negative ion chemical ionization mass spectrometry method to detect beta-carotene, alpha-tocopherol, and their reaction products. The method involves minimal sample handling and does not require compound isolation. Direct probe negative ion chemical ionization produces molecular anions with little or no fragmentation. Each ion signal in the resulting mass spectrum represents a compound or group of isomeric compounds in the original reaction mixture. Thus, a rapid ''snapshot'' of the reaction product profile is obtained within seconds. Application of this methodology to the analysis of beta-carotene oxidation in model chemical reaction systems and in microsomal membranes in vitro identified intact chain beta-carotene oxygenation products, carbonyl-containing chain cleavage products, and their oxygenation products. Similar analyses of alpha-tocopherol oxidations identified 8a-substituted tocopherones, alpha-tocopherolquinone and alpha-tocopherolquinone epoxides. Full-scan detection limits for the antioxidants and their products are in the picogram range. This method offers the first means for comprehensively profiling the fate of antioxidants in tissue samples.