Studies on neurosteroids .4. Quantitative determination of pregnenolone in rat brains using high-performance liquid chromatography

The quantitative determination of pregnenolone in rat brain was done using reverse phase high performance liquid chromatography with fluorescence detection and the internal standard method. The desired fraction was obtained from a rat brain with the combined use of a Bond Elut C8 cartridge for the solid-phase extraction and a lipophilic gel (piperidino-hydroxypropyl Sephadex LH-20) for the ion-exchange chromatography. The fraction was derivatized with 1-anthroyl cyanide followed by purification with 2 successive silica gel columns to remove the excess or decomposed reagent. Separation of the compounds was performed on a J'sphere ODS-L80 column and the calibration graph was linear from 10 to 60 ng/tube (ca. 1 g tissue of brain). The method was applied to the determination of pregnenolone in rat brains of Wistar and Sprague-Dawley strains, most of which showed much lower amounts than that previously reported.