Abstracts

Rationale: Neurosteroids are important modulators of neuronal activity. Allopregnanolone, a metabolite of progesterone, and other endogenous neurosteroids are of particular interest in epilepsy. Allopregnanolone has a stereospecific affinity for the GABA-A receptor and has been found to be a potent anticonvulsant in animal models. The measurement of neurosteroids in biological samples requires a sensitive and specific assay. Although prior studies have demonstrated the feasibility of measuring endogenous neurosteroids in CSF and serum, background interferences from biological matrices limit sensitivity. Also, allopregnanolone is one of four possible pregnanolone stereoisomers produced from progesterone by stereospecific isoenzymes and the complete separation of these pregnanolone isomers has not been achieved. Our method for the separation and measurement of allopregnanolone and other pregnanolones has the advantage of extremely high chromatographic resolving power, sufficient to separate pregnanolone isomers, as well as very high sensitivity. Methods: Samples were heated with triethylamine sulfate to denature any protein in the biological matrix. Steroids were extracted using a C18 column followed by a Sephadex LH-20 column to further purify the pregnanolone fraction containing allopregnanolone and its three stereoisomers isopregnanolone, epipregnanolone, and pregnanolone as well as pregnenolone, a precursor of progesterone. After further purification by HPLC, samples were derivatized in two steps, first with methoxyamine hydrochloride, then tert-butyldimethylsilyl imidazole. The derivatized samples were passed through a Lipidex 5000 column, dried and reconstituted in hexane for GC-MS analysis. Results: Using our method, we were able to achieve excellent chromatographic separation and quantification of individual pregnanolone isomers and pregnenolone. By monitoring the ion m/z 404 for the pregnanolone isomers, and m/z 402 for pregnenolone, these species were characterized in a single run (Figure 1). The use of the methyloxime t-BDMS derivative (MO-tBDMS) afforded greater specificity, and improved sensitivity over other derivatives we tested, because it increases the molecular weight of the steroids and directs the fragmentation to the t-BDMS group. Our limit of detection, for a mixture of pregnanolones and pregnenolone standards in a serum matrix, was 300 femtograms on column. The recovery of each steroid was excellent and gave good linearity over a very low concentration range (Figure 2). Sample concentrations ranged 0.6 to 40 picograms on column. Conclusions: We have developed a new assay for the measurement of low concentrations of pregnanolones and pregnenolone that features a solid-phase extraction step, followed by isolation and purification of these steroids using a combination of a lipophilic gel chromatography and HPLC. This has been a distinct and novel feature of our assay development that renders the samples of sufficient purity to permit detection and quantification of these neurosteroids in small quantities of serum and CSF.