Detection and characterization of 20-oxosteroids in rat brains using LC-electron capture APCI-MS after derivatization with 2-nitro-4-trifluoromethylphenylhydrazine

The detection and characterization of 20-oxosteroids in rat brains by liquid chromatography (LC)-electron capture atmospheric pressure chemical ionization (ECAPCI)-mass spectrometry (MS) is described. In the present method, oxosteroids were derivatized with a highly electron-affinitive reagent, 2-nitro-4-trifluoromethylphenylhydrazine, to convert them to the corresponding hydrazones. The derivatized steroids showed over 20-fold higher sensitivity in ECAPCI-MS than intact steroids measured in positive-APCI-MS. A brain sample was homogenized, purified with two disposable cartridges, derivatized and subjected to LC-ECAPCI-MS. Pregnenolone, progesterone, 5alpha-dihydroprogesterone, allopregnanolone and epiallopregnanolone were clearly detected with a 50 mg sample of the brain tissue.