In this application, we propose the development of selective and sensitive methodology for the quantitative analysis of urine for anabolic steroids. The methodology will be based on electron capture negative ionization (ECNI) or electron impact mass spectrometry with sample introduction by gas chromatographic inlet. A major thrust of the proposal is based on the novel strategy of transforming the analyte to an electrophilic species via an oxidation reaction to a transformed structure. A parallel approach involves adding electrophilic moieties to the parent structure via more conventional derivatization reactions. Preliminary experiments have shown that the oxidative approach permits remarkable discrimination against biochemical background in the detection of dexamethasone in a biological sample matrix; these experiments also have demonstrated an unusually high ECNI response from selected oxidatively modified steroids. The proposal consists of two major parts. The first will exploit the analytical advantage of ECNI mass spectrometry by assessing both the conventional derivatization approach as well as the oxidative modification approach to prepare the anabolic steroids for analysis. In parallel with this practical study of analyte modification for purposes of developing a quantitative assay, we also will pursue a fundamental investigation of structure vs ECNI response. This investigation involving model compounds will be directed toward understanding the basis for the dramatic increase in the electron-capture cross section of certain steroid structures. Other efforts in these two parts of the proposed work will involve optimization of the chemical oxidation conditions, simplification of sample processing, preparation and evaluation of suitable internal standards, and statistical evaluation of preliminary results from the developing methodologies. Successful completion of the work proposed in the fundamental study of structure vs ECNI response will lead to a greater understanding of the electron-capture process and provide a basis for rationally extending ECNI analytical methodology to a larger scope of compounds of biomedical importance.
