Profiling Urinary Sulfate Metabolites With Mass Spectrometry

Area of Science:

• Metabolomics
• Analytical Chemistry
• Biochemistry

Background:

• Urinary phase II sulfate metabolites are crucial for understanding biological compound metabolism.
• Existing methods for profiling sulfated metabolites can be limited.
• A need exists for rapid, untargeted analysis of these compounds.

Purpose of the Study:

• To develop and validate a novel workflow for untargeted metabolic profiling of urinary sulfated metabolites.
• To apply the workflow to identify equine steroid metabolites and assess sulfatase enzyme activity.

Main Methods:

• Ultra-high-performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with data-dependent acquisition (DDA).
• Automated script-based data processing pipeline with k-means clustering for sulfate identification.
• Application to equine urine post-testosterone propionate administration and human urine with different sulfatase enzymes.

Main Results:

• Detected ten elevated sulfated steroid metabolites in horse urine after testosterone propionate administration.
• Identified a novel equine metabolite: 5α-androstane-3β,17α-diol 3-sulfate.
• Demonstrated higher selectivity of Pseudomonas aeruginosa arylsulfatases (PaS) for human urinary sulfated metabolites compared to Helix pomatia arylsulfatase (HpS).

Conclusions:

• The novel UHPLC-HRMS/MS workflow enables rapid, systematic, and untargeted metabolic profiling of urinary sulfated metabolites.
• The method successfully identified new equine steroid metabolites and differentiated sulfatase enzyme activities.
• This approach offers a valuable tool for metabolomic research involving sulfated compounds in biological matrices.