Metabolite profiling of biological steroids in pathological tissue specimen

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We have developed a highly sensitive and specific method for the analysis of steroid profiles in biological samples including pathological tissue specimens. After the addition of deuterium-labeled internal standards, biological samples were saponified with 1N ethanolic KOH, extracted with n-hexane followed by n-hexane:ethylacetate (1:1), and derivatized into picolinyl esters before analysis by high performance liquid chromatography-positive electrospray ionization-tandem mass spectrometry (HPLC-PESI-MS/MS). Cholesterol related sterols, oxysterols, adrenal steroids and neurosteroids were quantified simultaneously. The effects of formaldehyde fixation and Mayer's hematoxylin staining on the steroid analysis of pathological tissue specimen were examined. These procedures did not affect significantly the results of the present analysis.