Profiling the Steroid Sulfate Metabolome

Abstract

The study of androgenic anabolic steroids (AAS) and their metabolites form an important cornerstone of anti-doping analysis in equine sports. With the advancement of UHPLC-HRMS instruments focus has shifted toward the detection of free steroid metabolites and their intact phase II conjugates. The development of new tools to assist in this analysis of these compounds is of high priority. The work presented in this thesis aims to develop new methodologies for the detection and characterisation of AAS and their metabolites, with an emphasis on sulfate conjugates. Methodologies presented include a combination of UHPLC-MS, metabolomic based bioinformatics and synthetic chemistry techniques applied in a multidisciplinary way. Chapter One; a summary of the literature detailing the metabolism of AAS, past and current methods of detection in anti-doping laboratories. Adjacent techniques from different fields that are relevant or have potential application in anti-doping analysis are also highlighted. This includes the topics of metabolomics, different HRMS, non-targeted acquisition methods, associated data processing software, and intelligence based anti-doping approaches. Chapter Two; describes a new data analytical tool for the annotation of sulfated metabolites that utilises UHPLC-HRMS/MS instrumentation with a non-targeted data dependent acquisition method. This new tool was applied in two separate scenarios, firstly, to identify potential steroid sulfate biomarkers of testosterone propionate doping, and secondly, to qualitatively assess the selectivity of various sulfatase enzymes. Key to this method is the use of an automated k-means clustering algorithm to identify putative sulfated metabolites. This technique is unique as it also retains all other information in each metabolome, allowing for a full metabolomic analysis. In the second part of the chapter, the new analytical tool is applied to identify potential steroid sulfate metabolites of testosterone propionate doping in the urinary metabolome of three horses. From this a new semi-targeted steroidomic workflow is established, that highlights several new biomarkers that are likely related to doping. Chapter Three; describes a study detailing the development of a new type of stable isotope label for sulfate conjugate metabolites. The method is unique as it places the stable isotopes on the sulfate conjugate group. It is also readily accessible following a single one-pot synthetic step and simple SPE purification on small scales (5-10 mg). Its utility is demonstrated by the synthesis of a range of selectively labelled mono- and bis-conjugated compounds. The sulfate compounds synthesised are further analysed using pseudo-MS/MS/MS and energy-resolved collision induced dissociation (CID) mass spectrometry experiments, that use the labelled conjugates as probes to elucidate complex fragmentation relationships. Chapter Four; describes research that investigates the identity of a new steroid biomarker with energy resolved CID analysis. This chapter brings together work from chapter 2 and 3. Specifically, the new biomarker was discovered in chapter two and the methodology for its elucidation was developed in chapter three. It also further develops the energy-resolved CID analysis as a comprehensive analytical technique that is applicable to both, in vitro and in vivo samples, at biologically relevant concentrations. Chapter Five;describes research that extends the metabolomic techniques developed in this thesis to the analysis of a designer steroid, hemapolin. Firstly, a targeted analysis of a new class of sulfonate metabolites is established using UHPLC-HRMS/MS analysis. These metabolites can now be incorporated into anti-doping screening and confirmation procedures for future detection of hemapolin misuse. Following this a semi-targeted metabolomic analysis of the urinary metabolome was done to putative identify other long-term metabolites of hemapolin administration. Show more