Studies on neurosteroids XIX: Development and validation of liquid chromatography–tandem mass spectrometric method for determination of 5α-reduced pregnane-type neurosteroids in rat brain and serum

A sensitive liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method for the simultaneous determination of 5α-reduced pregnan-type neurosteroids, allopregnanolone (AP), epiallopregnanolone and 5α-dihydroprogesterone, in rat brain and serum has been developed and validated. The brain and serum steroids were extracted with methanol–acetic acid, purified using a Strata-X cartridge, derivatized with the permanently charged reagent, 2-hydrazino-1-methylpyridine (HMP), and subjected to LC-positive ESI-MS–MS. The limits of quantitation (LOQ) for brain (0.25 ng/g tissue) and serum (0.25 ng/ml) assays using the derivatization–ESI-MS–MS method are 60–150-fold lower than the LOQs for their atmospheric pressure chemical ionization-MS method without derivatization. [17α,21,21,21-2H4]-AP was used as an internal standard. This method allowed the reproducible and accurate quantification of the brain or serum neurosteroids using a 20 mg or 20 μl sample, respectively. That is, the intra- and inter-assay coefficients of variation were below 8.2 and 6.0%, respectively, and the % accuracy values were 98.5–103.0% for all the steroids in both the brain and serum. The application of the developed method to the analysis of changes in the brain and serum neurosteroid levels by immobilization stress and ethanol administration is also presented.