Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry

A selective and extremely sensitive procedure has been developed and optimi zed, using high-performance liquid chromatography (HPLC), specific derivati zation and gas chromatography-mass spectrometry (GC-MS), to simultaneously quantify very small amounts of different neurosteroids from rat brain. Unco njugated and sulfated steroids in brain extracts were separated by solid-ph ase extraction. The unconjugated fraction was further purified by HPLC, the steroids being collected in a single fraction, and the sulfated fraction w as solvolyzed. All steroids were derivatized with heptafluorobutyric acid a nhydride and analyzed by GC-MS (electron impact ionization) using selected- ion monitoring. High sensitivity and accuracy were obtained for all steroid s. The detection limits were 1 pg for pregnenolone (PREG), dehydroepiandros terone (DHEA) and their sulfate esters PREG-S and DHEA-S, 2 pg for progeste rone (PROG) and 5 pg for 3 alpha,5 alpha-tetrahydroprogesterone (3 alpha,5 alpha-THP). In a pilot study on a rat brain, the concentrations of PREG-S a nd DHEA-S were 8.26+/-0.80 and 2.47+/-0.27 ng/g, respectively. Those of PRE G, DHEA and PROG were 4.17+/-0.22, 0.45+/-0.02 and 1.95+/-0.10 ng/g, respec tively. Good linearity and accuracy were observed for each steroid. The met hodology validated here, allows femtomoles of neurosteroids, including the sulfates, found in small brain samples (at least equal to 10 mg) to be quan tified simultaneously. (C) 2000 Elsevier Science B.V. All rights reserved.