Dissertation: Method Development for Mass Spectrometric Detection of Neurosteroids in Cerebrospinal Fluid as Potential Biomarkers of Cognitive Disorders

Neurosteroids (NS) are a special of group of endogenous steroids, which are synthesized in the central (CNS) or peripheral nervous system (PNS), which is not only a source but also a target for NS. As they are discussed as potential biomarkers for various cognitive disorders, research interest in this topic remarkably increases. In this context CSF becomes an interesting specimen for NS analysis, as it is the only accessible matrix which most likely reflects brain metabolism in living human beings. Analysis of NS is truly challenging for different reasons, such as low amounts of available matrix, ultra-trace-levels of expected steroids, and high structural similarity of endogenous steroids. Therefore, the aim of this work was the development of a comprehensive and sensitive method for profiling of an as large as possible set of NS in one aliquot of human CSF. For method development and evaluation, a set of fifty-one steroids, including (pro-)gestagens, androgens, corticoids, estrogens and steroid sulfates was selected, based on comprehensive literature research of neurosteroids already determined in human CSF and expanded with steroids covering major peripheral pathways of biosynthesis and metabolism. Profiling of endogenous steroids is well-established with gas chromatography (tandem) mass spectrometry (GC-MS(/MS)), (ultra) high performance liquid chromatography (tandem) mass spectrometry ((U)HPLC-MS(/MS)), or immunoassays (IA) – each method offering different advantages or disadvantages. As IA, HPLC and GC based methods were reported for ascending number of analytes that were detected, GC-MS was initially selected within this project regarding the selected large set of analytes. Analysis with GC-MS requires derivatization of analytes, therefore several well-established derivatization procedures were examined. Overall, none of the derivatization procedures provided acceptable results for further method development and analyte quantification, due to formation of multiple derivatives of some steroids and occurrence of coelution of side and main peaks of different endogenous steroids with same m/z. As alternative approach supercritical fluid chromatography (tandem) mass spectrometry (SFC-MS(/MS)) presents new and promising application opportunities and currently experiences a comeback to analytical science was therefore investigated for steroid profiling. Full method development included investigation of chromatographic and mass spectrometric parameters, followed by robustness testing. One of the unique features of SFC instrumentation is the opportunity to use a different make-up solution for ionization than mobile phase. Considering the rather poor ionization capacity of steroids, the different ion sources ESI, APCI, and APPI were tested for their suitability in combination with different make-ups. Finally, with its unique selectivity SFC-MS/MS was able and to overcome the derivatization issues of GC-MS and to distinguish between all selected steroids. Method development resulted in three optimized methods on different columns with ESI ionization that offer excellent separation in reasonable time and can be easily operated in one sequence as high-throughput approach. SFC-MS/MS further scores with facilitated sample preparation and reduced time expenses compared to GC-MS, as no time-consuming derivatization step and/or sulfate cleavage is required. In addition, the development of SFC-based methods also contributes to environmentally sustainable chemical research for the future. Altogether, considering the requirements of this project, the high potential of SFC-MS/MS in endogenous steroid profiling and its superiority towards GC-MS was demonstrated. In a subsequent project, the method will be validated with the CSF matrix. Prospectively, expansion of this method to other body fluids like blood seems also very promising. In the future, the SFC-ESI-MS/MS method will offer the opportunity to quantify a large set of NS within the rare specimen human CSF and may contribute to clarification of knowledge on metabolism and functions of NS.