Quantification of neurosteroids in rat plasma and brain following swim stress and allopregnanolone administration using negative chemical ionization gas chromatography/mass spectrometry

A simplified method for the quantitative analysis of neurosteroids in rat p lasma and brain is described. The method uses negative chemical ionization gas chromatography/mass spectrometry and involves the synthesis of pentaflu orobenzyloxime/trimethylsilyl ether derivatives with excellent chromatograp hic and electron-capturing properties. Deuterium-labeled analogs of the ste roids of interest were synthesized and used as internal standards. The ster oids (allopregnanolone, epiallopregnanolone, pregnenolone, testosterone, an d dehydroepiandrosterone) were isolated from the plasma or brain matrix by a rapid and straightforward solid-phase extraction procedure. The mass spec trometer was operated in a selective ion monitoring mode, allowing for pico grams of neurosteroids to be quantified from biological extracts. The metho d was linear (typical R-2 = 0.999) over the concentration range (100 to 800 0 pg from 0.3 mi plasma and 250 to 8000 pg from 100 mg brain tissue) with g ood precision and accuracy. In experimental protocols, the procedure was su itable for measuring concentrations of endogenous neurosteroids in rat plas ma and brain. Significant elevations (P < 0.001) were observed in the front al cortex for allopregnanolone and pregnenolone following a swim stress and for allopregnanolone and epiallopregnanolone following allopregnanolone in jection (8 mg/kg, sc). The present method allows accurate determination of neurosteroids and will be helpful in elucidating the role of neurosteroids in health and disease. (C) 2000 Academic Press.