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.
Highlights
The study of phase II metabolism is essential to understand the biochemical role and fate of endogenous and exogenous compounds and is dominated by the two major classes of conjugates: sulfates and glucuronides (Schänzer, 1996)
This directed research towards liquid chromatographymass spectrometry (LC-MS) techniques, which permit the direct detection of the intact sulfate conjugates, allowing the sulfate fraction to be targeted during analysis
Intact phase II anabolic androgenic steroid (AAS) metabolites such as sulfate esters play an important role in anti-doping analysis as long-term biomarkers for both exogenous and endogenous steroids (Schänzer et al, 2013; Piper et al, 2016; Kiousi et al, 2021)
Summary
The study of phase II metabolism is essential to understand the biochemical role and fate of endogenous and exogenous compounds and is dominated by the two major classes of conjugates: sulfates and glucuronides (Schänzer, 1996). Approaches to examine the sulfate metabolome employed gas chromatography-mass spectrometry (GC-MS) techniques, these suffered from various limitations due to the polar character of the conjugates, challenges with reliable enzymatic or chemical cleavage of sulfate esters, and loss of information about conjugation sites and levels (Gomes et al, 2009). This directed research towards liquid chromatographymass spectrometry (LC-MS) techniques, which permit the direct detection of the intact sulfate conjugates, allowing the sulfate fraction to be targeted during analysis. Approaches based on tandem mass spectrometry include, multiple reaction monitoring (MRM), precursor ion scanning, or constant ion loss (CIL) scanning, which have all been used to selectively and directly detect sulfate conjugated metabolites (Bowers and Sanaullah, 1996; Bean and Henion, 1997; Hintikka et al, 2008; Gómez et al, 2013; Balcells et al, 2017a; McLeod et al, 2017; Schänzer and Thevis, 2017)
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