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Neurosteroids: trophic and bevavioral effects

Deliverables

We have developed a very sensitive microassay, which allows to accurately measure low amounts of steroids in small tissue samples. It is based on the extraction of steroids from tissue, their separation by gas chromatography (GC) and their identification by mass spectrometry (MS). This method is not limited by the availability of selective antibodies, as this is the case for conventional radioimmunological procedures, and it allows simultaneously identifying and quantifying several steroids within a same tissue sample. The robustness of analytical criteria like reproducibility, linearity and precision has been validated. This new microassay may become an important diagnostic tool, because it allows for the first time to accurately measure femtomol amounts of steroids in small samples as for example biopsies, cerebrospinal fluid and cultured cells. The microassay of steroids by gas chromatography/mass spectrometry (GC/MS) is robust, accurate and extremely sensitive. It allows measuring concentrations of steroids in the femtomol range, which is about 100-times more sensitive than classical radioimmunoassays. Steroids are extracted from tissue samples by organic solvents and internal standards are added to the extracts for recovery calculations (preferentially deuterated steroids). Free steroids are separated from their sulphate and fatty acid esters by solid phase extraction by using mixtures of eluting solvents with different polarities. Free steroids: After filtration, steroids in this fraction are prepurified by HPLC. They are then converted to halogenous or sialylated derivatives, which improve resolution and the profile of their mass spectrum, characterised by abundant high mass ions. Sulphated steroids: The method we have set up allows hydrolysis and derivatisation of the conjugated steroids (sulphated forms of pregnenolone and dehydroepiandrosterone) in a single step by heptafluorobutyrate anhydride. The pre-purified and derivative steroids are then injected into the GC column. We have improved the injection step on the GC column (split less technique) and the detection procedure (selected ion monitoring).