Nano-electrospray mass spectrometry for the analysis of neurosteroids and related molecules

Abstract

Neurosteroids are steroids synthesised in the central and peripheral nervous systems. Known neurosteroids include pregnenolone, dehydroepiandrosterone (DHEA), progesterone and its reduced metabolites. It has been demonstrated that neurosteroids modulate neurotransmission by binding to neurotransmitter receptors, and exert physiological functions that are clearly different from those of endocrine steroids. The effects of neurosteroids on improving the memory of cognitively impaired aged rats, on the inhibition of aggressiveness in castrated male mice, and trophic effects on neuronal regeneration and remyelination have been documented. The local synthesis, selective interaction with neurotransmitter receptors and behavioural effects of neurosteroids strongly suggests that they may have important physiological or pathophysiological roles. There is an increasing need to develop methods to analyse these hormones with high sensitivity and high specificity.

In this thesis I focused on the development of methods combining nano-electrospray (ES) mass spectrometry with capillary column liquid chromatography (CLC) for the analysis of profiles of neurosteroids in rat brain. It was also an aim to make the methods applicable to a broad range of lipophilic biomolecules.

Initially, synthetic steroid sulphates and unconjugated oxosteroids (ketosteroids) were studied by nano-ES and tandem mass spectrometry. Steroid sulphates could be detected as deprotonated molecules in full range scanned spectra at a level of 1 pg/µL. Information about steroid structure was obtained from collision-induced dissociation (CID) spectra of 1 ng of steroid sulphate, while characterisation of the sulphate ester group required only 3 pg of material. Unconjugated oxosteroids were converted into their oximes which were detected as protonated molecules with 20 times higher sensitivity than the underivatised steroids. The detection limits for the oximes of 3-oxo-delta4, 20-oxo and 17-oxo steroids were 2.5, 5, and 25 pg/µL, respectively in full range scans. CID spectra of the protonated oximes provided valuable information regarding the position of oxo and hydroxyl group(s). These studies established a basis for determination and structure characterisation of neurosteroids from brain samples.

A procedure for CLC-ES mass spectrometry was then developed. A double splitter method was introduced which made it possible to use a pre-column for analyte focusing from large sample volumes. It also made it possible to operate the solvent pumps at flow rates compatible with gradient elution while the flow rates through the analytical column were compatible with micro-electrospray. The method was designed to be generally applicable to the analysis of biomolecules and its utilities were demonstrated by the analysis of steroid sulphates, in human plasma.

In the course of these studies, certain CLC-ES conditions were found to cause on-column chemical transformations of 3beta- hydroxy-delta5 steroid sulphates. Radical species generated from electrolysis of water and methanol in the solvent are proposed to be responsible for the formation of oxidised and methoxylated products of these steroids. Other analytes with double bonds were also transformed under these conditions. Thus, on-column electrochemistry can be an important source of artefacts in analyses by CLC-ES mass spectrometry. The reactions could be prevented by appropriate grounding.

The analysis of neurosteroids in rat brain required the development of an extraction, purification and subfractionation procedure. Brain steroids were extracted, and unconjugated neutral steroids and sulphated steroids were separated. The steroid sulphate fraction was then analysed by CLC-ES mass spectrometry. Endogenous sulphates of pregnenolone and DHEA were not detected at levels above the detection limit, 0.3 ng/g wet brain, while pregnenolone sulphate, added to brain extract at a level of 6.6 ng/g, was easily detected. The unconjugated oxosteroids were converted to their oximes, selectively isolated on a cation exchanger, and analysed by CLC-ES tandem mass spectrometry. The chromatograms showed the presence of progesterone, pregnenolone, pregnanolone isomers, DHEA and testosterone in rat brain. These steroids were characterised by tandem mass spectrometry, Based on the results of CLC-ES tandem mass spectrometry, the levels Of C21 and C19 steroids were estimated in the range of 0.04 - 20 ng/g wet brain. The levels of progesterone and testosterone showed a sex difference.

During the development of the above analytical methods, nano-ES mass spectrometry was applied to the characterisation of a lipophilic modulatory factor isolated from mouse brain. The factor, which activated the retinoid X receptor (RXR), was extracted from mouse brain incubates, purified by HPLC and analysed by nano-ES and tandem mass spectrometry. Accurate mass measurement and CID spectra of the purified active compound revealed that it was cis-4,7,10,13,16,19-docosahexaenoic acid.

In conclusion, the methods developed and described in this thesis are suitable for the analysis of sulphated steroids and oxosteroids, as well as other related compounds. With their high sensitivity the methods enable highly specific analysis of these important compounds from small amounts of sample.