Studies on neurosteroids. V: Separation and characterization of pregnenolone 3-stearate in rat brains using high-performance liquid chromatography.

The separation and characterization of pregnenolone 3-stearate in rat brains are carried out using high-performance liquid chromatography (HPLC). The pregnenolone 3-stearate is obtained from a whole rat brain by extraction with ethyl acetate followed by silica gel column chromatography. The obtained fraction is derivatized with 1-dimethylaminonaphthalene-5-sulfonylhydrazine (dansylhydrazine) or 4-(N,N-dimethylaminosulfonyl)-7-hydrazino- 2,1,3-benzoxadiazole, and the derivative is separated by successive preparative HPLC with fluorescence detection. The chromatographic behaviors of both derivatives are identical to those of authentic samples. The former derivative obtained from the rat brain shows satisfactory mass spectral data, and after hydrolysis, dansylpregnenolone is confirmed by HPLC.