Profiling Urinary Sulfate Metabolites With Mass Spectrometry

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.

Potential of measured relative shifts in collision cross section values for biotransformation studies

“…3 . Phase II biotransformations such as sulfation and glucuronidation were not considered for this assessment since the major fragment ion from such metabolites is often only the parent drug or corresponding precursor following the characteristic neutral loss of the sulfate (80 Da, SO 3 ) or the glycone (176 Da, C 6 H 8 O 6 ), respectively [ 38 – 41 ]. One prerequisite for the assessment of biotransformation-specific shifts in relative TW CCS N2, meas on fragment ion level was the presence of common fragment ions between the metabolite and parent drug or precursor (with and without the biotransformation).…”

Structural Investigation of Betulinic Acid Plasma Metabolites by Tandem Mass Spectrometry

“…The fragmentation spectrum and proposed fragmentation pathway are depicted in Figure 6 and Figure 7 . The fragment ion at m / z 455.35 is 80 Da less than the parent ion, a typical fragment for the sequencing of sulfoconjugates, which corresponds to the cleavage of SO 3 [ 51 ] and most importantly brings solid evidence regarding the proposed sulfoconjugated structure. Further, m / z 455.35 could generate: (i) the fragment at m / z 411.36 (−44 Da) which corresponds to the removal of CO 2 , as presented in the fragmentation scheme of BA; (ii) the fragment at m / z 351.31 (−104 Da) by the simultaneous loss of HCOOH, C 3 H 4 and H 2 O, which in turn could produce the fragment at m / z 309.26 (−42 Da) by the removal of three CH 2 groups, which generates the fragment at m / z 281.23 (−28 Da) by the elimination of other two CH 2 groups, lastly forming two consecutive ring cleavage ions at m / z 255.21 and 241.20 corresponding to the elimination of cycle E; (iii) m / z 373.27 (−82 Da), corresponding to the simultaneous cleavage of C 3 H 4 and three CH 2 groups, which in turn could produce m / z 357.28 (−16 Da), corresponding to the additional removal of O, as already presented in the fragmentation scheme of BA.…”

High-Resolution Mass Spectrometry for Human Exposomics: Expanding Chemical Space Coverage

“…For instance, metabolites of exogenously derived chemicals are pivotal in assessing exposures. Various enzyme digestion methods, including the analysis of phase II sulfated metabolites through sulfatase treatment, are utilized for exogenous metabolite analysis . The many technical aspects, from laboratory measurements to data analytics, are discussed at length in the following sections as they function as an integral companion to the burgeoning HRMS capacity.…”