Energy-Resolved Fragmentation Aiding the Structure Elucidation of Steroid Biomarkers.

The identification and confirmation of steroid sulfate metabolites in biological samples are essential to various fields, including anti-doping analysis and clinical sciences. Ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) is the leading method for the detection of intact steroid conjugates in biofluids, but because of the inherent complexity of biological samples and the low concentration of many targets of interest, metabolite identification based solely on mass spectrometry remains a major challenge. The confirmation of new metabolites typically depends on a comparison with synthetically derived reference materials that encompass a range of possible conjugation sites and stereochemistries. Herein, energy-resolved collision-induced dissociation (CID) is used as part of UHPLC-HRMS/MS analysis to distinguish between regio- and stereo-isomeric steroid sulfate compounds. This wholly MS-based approach was employed to guide the synthesis of reference materials to unambiguously confirm the identity of an equine steroid sulfate biomarker of testosterone propionate administration.

Synthesis of stable isotope labelled steroid bis(sulfate) conjugates and their behaviour in collision induced dissociation experiments.

Steroid bis(sulfate) metabolites derived from the two-fold sulfation of unconjugated precursors represent an important yet understudied portion of the steroid profile. The investigation of these compounds in fields such as medicine or anti-doping science relies on mass spectrometry (MS) as the principal tool to identify and quantify biomarkers of interest and depends in turn on access to steroid reference materials and their stable isotope labelled (SIL) derivatives. A new [18O] stable isotope label for sulfate metabolites is reported, which allows for the selective, late-stage and 'one-pot' synthesis of a variety of SIL-steroid conjugates suitable as MS probes and internal standards. The method is applied to more comprehensively study the MS behaviour of steroid bis(sulfate) compounds through collision-induced dissociation (CID) experiments.

Profiling Urinary Sulfate Metabolites With Mass Spectrometry.

The study of urinary phase II sulfate metabolites is central to understanding the role and fate of endogenous and exogenous compounds in biological systems. This study describes a new workflow for the untargeted metabolic profiling of sulfated metabolites in a urine matrix. Analysis was performed using ultra-high-performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with data dependent acquisition (DDA) coupled to an automated script-based data processing pipeline and differential metabolite level analysis. Sulfates were identified through k-means clustering analysis of sulfate ester derived MS/MS fragmentation intensities. The utility of the method was highlighted in two applications. Firstly, the urinary metabolome of a thoroughbred horse was examined before and after administration of the anabolic androgenic steroid (AAS) testosterone propionate. The analysis detected elevated levels of ten sulfated steroid metabolites, three of which were identified and confirmed by comparison with synthesised reference materials. This included 5α-androstane-3ß,17α-diol 3-sulfate, a previously unreported equine metabolite of testosterone propionate. Secondly, the hydrolytic activity of four sulfatase enzymes on pooled human urine was examined. This revealed that Pseudomonas aeruginosa arylsulfatases (PaS) enzymes possessed higher selectivity for the hydrolysis of sulfated metabolites than the commercially available Helix pomatia arylsulfatase (HpS). This novel method provides a rapid tool for the systematic, untargeted metabolic profiling of sulfated metabolites in a urinary matrix.

Synthesis of steroid bisglucuronide and sulfate glucuronide reference materials: Unearthing neglected treasures of steroid metabolism.

Doubly or bisconjugated steroid metabolites have long been known as minor components of the steroid profile that have traditionally been studied by laborious and indirect fractionation, hydrolysis and gas chromatography-mass spectrometry (GC-MS) analysis. Recently, the synthesis and characterisation of steroid bis(sulfate) (aka disulfate or bis-sulfate) reference materials enabled the liquid chromatography-tandem mass spectrometry (LC-MS/MS) study of this metabolite class and the development of a constant ion loss (CIL) scan method for the direct and untargeted detection of steroid bis(sulfate) metabolites. Methods for the direct LC-MS/MS detection of other bisconjugated steroids, such as steroid bisglucuronide and mixed steroid sulfate glucuronide metabolites, have great potential to reveal a more complete picture of the steroid profile. However, access to steroid bisglucuronide or sulfate glucuronide reference materials necessary for LC-MS/MS method development, metabolite identification or quantification is severely limited. In this work, ten steroid bisglucuronide and ten steroid sulfate glucuronide reference materials were synthesised through an ordered combination of chemical sulfation and/or enzymatic glucuronylation reactions. All compounds were purified and characterised using NMR and MS methods. Chemistry for the preparation of stable isotope labelled steroid {13C6}-glucuronide internal standards has also been developed and applied to the preparation of two selectively mono-labelled steroid bisglucuronide reference materials used to characterise more completely MS fragmentation pathways. The electrospray ionisation and fragmentation of the bisconjugated steroid reference materials has been studied. Preliminary targeted ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis of the reference materials prepared revealed the presence of three steroid sulfate glucuronides as endogenous human urinary metabolites.

SULFATION PATHWAYS: Alternate steroid sulfation pathways targeted by LC-MS/MS analysis of disulfates: application to prenatal diagnosis of steroid synthesis disorders.

The steroid disulfates (aka bis-sulfates) are a significant but minor fraction of the urinary steroid metabolome that have not been widely studied because major components are not hydrolyzed by the commercial sulfatases commonly used in steroid metabolomics. In early studies, conjugate fractionation followed by hydrolysis using acidified solvent (solvolysis) was used for the indirect detection of this fraction by GC-MS. This paper describes the application of a specific LC-MS/MS method for the direct identification of disulfates in urine, and their use as markers for the prenatal diagnosis of disorders causing reduced estriol production: STSD (steroid sulfatase deficiency), SLOS (Smith-Lemli-Opitz syndrome) and PORD (P450 oxidoreductase deficiency). Disulfates were detected by monitoring a constant ion loss (CIL) from the molecular di-anion. While focused on disulfates, our methodology included an analysis of intact steroid glucuronides and monosulfates because steroidogenic disorder diagnosis usually requires an examination of the complete steroid profile. In the disorders studied, a few individual steroids (as disulfates) were found particularly informative: pregn-5-ene-3ß,20S-diol, pregn-5-ene-3ß,21-diol (STSD, neonatal PORD) and 5α-pregnane-3ß,20S-diol (pregnancy PORD). Authentic steroid disulfates were synthesized for use in this study as aid to characterization. Tentative identification of 5ξ-pregn-7-ene-3ξ,20S-diol and 5ξ-pregn-7-ene-3ξ,17,20S-triol disulfates was also obtained in samples from SLOS affected pregnancies. Seven ratios between the detected metabolites were applied to distinguish the three selected disorders from control samples. Our results show the potential of the direct detection of steroid conjugates in the diagnosis of pathologies related with steroid biosynthesis.