Abstract: AimTo simultaneously determine three unconjugated neurosteroids, dehydroepiandrosterone (DHEA), pregnenolone (PREG), allopregnenolone (AP), from several brain regions of the rat. MethodsNeurosteroids were isolated separately in a two steps procedure by using ethyl acetate-n-hexane (90∶10) as the first step to extract the unconjugated steroids, then the steroid fractions were further purified by SPE. All steroids were derivatized with 2-nitro-4-trifluoromethylphenylhydrazine (2-NFPH) and analyzed by HPLC-MS (APCI) using selected-ion monitoring. Methyltestosterone was chosen as the internal standard. ResultsThe linear calibration curve of DHEA was obtained in the concentration range of 0.030-2.00 μg·L^-1. The linear calibration curves of PREG and AP were obtained in the concentration range of 0.025-2.00 μg·L^-1. The concentrations of DHEA, PREG and AP in male rat brain regions were (0.70±0.23), (4.8±1.9), (1.1±0.6) ng·g^-1 for frontal cortex, (0.57±0.28), (6±3), (0.5±0.3) ng·g^-1 for hippocampus, (1.5±1.0), (9±5), (1.4±0.9) ng·g^-1 for amygdale, (0.52±0.14), (7.7±2.8), (0.5±0.6) ng·g^-1 for striatum, (2.9±1.6), (18±9), (1.6±1.3) ng·g^-1 for nucleus accumbens, (4.0±2.0), (27±12), (0.8±0.5) ng·g^-1 for pituitary gland, (1.7±1.2), (16±10), and (0.8±0.7) ng·g^-1 for hypothalamus, respectively. ConclusionGood linearity and accuracy were observed for each steroid. The procedure was suitable for measuring concentrations of the unconjugated steroids in rat brain simultaneously.