Profiling Urinary Sulfate Metabolites With Mass Spectrometry

Christopher C J Fitzgerald; Rikard Hedman; Dimanthi R Uduwela; Bettina Paszerbovics; Adam J Carroll; Teresa Neeman; Adam Cawley; Lance Brooker; Malcolm D McLeod

doi:10.3389/fmolb.2022.829511

Profiling Urinary Sulfate Metabolites With Mass Spectrometry

Front Mol Biosci. 2022 Feb 23:9:829511. doi: 10.3389/fmolb.2022.829511. eCollection 2022.

Authors

Christopher C J Fitzgerald¹, Rikard Hedman¹, Dimanthi R Uduwela¹, Bettina Paszerbovics¹, Adam J Carroll¹, Teresa Neeman¹, Adam Cawley², Lance Brooker³, Malcolm D McLeod¹

Affiliations

¹ Research School of Chemistry, Australian National University, Acton, ACT, Australia.
² Australian Racing Forensic Laboratory, Racing NSW, Sydney, NSW, Australia.
³ Australian Sports Drug Testing Laboratory, National Measurement Institute, Sydney, NSW, Australia.

Abstract

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-3β,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.

Keywords: anti-doping; mass spectrometry; metabolomics; steroid; sulfatase; sulfate ester; sulfation.