[Determination of neurosteroids in rat brain regions by liquid chromatography/negative atomspheric pressure...

A simplified method has been established using liquid chromatography-negative atomspheric pressure ionization mass spectrometry (LC-MS) to simultaneously determine two conjugated neurosteroids from rat brain regions. Neurosteroids were separately isolated in a two-step procedure using chloroform/2-butanol (50: 50, v/v) where the first step was to extract sulfated steroids, then steroid fractions were purified by solid phase extraction (SPE), and finally the sulfated steroid was solvolyzed. Estrogen sulfate was chosen as internal standard. All steroids were derivatized with 2-nitro-4-trifluoromethylphenylhydrazine (2NFPH) and analyzed by LC-MS using selected-ion monitoring. LC-MS was performed on an Agilent 1100 liquid chromatograph-mass spectrometer with atmospheric pressure chemical ionization (APCI) interface, and the hydrazones of oxosteroids was analyzed in a negative-ion mode. A Zorbax SB C18 column was used with a flow rate of 1 mL/min at 40 degrees C. The mobile phase consisted of acetonitrile and distilled water. The concentrations of PREGS and DHEAS in male rat brain regions were (4.14 +/- 1.33 ) ng/g and (2.26 +/- 0.76) ng/g (pituitary gland), (1.98 +/- 1.13) ng/g and (1.80 +/- 0.93) ng/g (hypothalamus), (1.08 +/- 0.48 ) ng/g and (0.81 +/- 0.23) ng/g (frontal cortex), (0.72 +/- 0.19) ng/g and (0.77 +/- 0.12) ng/g (hippocampus), (1.70 +/- 0.45) ng/g and (1.44 +/- 0.71 ) ng/g (amygdale), (0.92 +/- 0.27) ng/g and (0.85 +/- 0.44) ng/g (striatum), (3.62 +/- 1.77) ng/g and (3.17 +/- 2.11) ng/g (nucleus accumbens), respectively. Good linearity and accuracy were observed for each steroid. The procedure was suitable for measuring concentrations of the sulfated steroids in rat brain regions simultaneously.