MSACL 2026 Abstract

Enzymatic Hydrolysis Strategies for Extended Steroidomic Analysis in Urine: Comparative Evaluation from Controlled Optimization to Real Sample Application

Federico Ponzetto (1), Elias Villalobos (2), Alessia Melis (1), Laura Leoni (1), Veronica Pirana (1), Antonello Nonnato (3), Manuela Lucchiari (3), Fabio Settanni (3), Giulio Mengozzi (1,3)
(1) Clinical Biochemistry Laboratory, Department of Medical Sciences, University of Turin, Turin, Italy (2) Kura Biotech, Puerto Varas, Chile (3) Clinical Biochemistry Laboratory, City of Health and Science University Hospital, Turin, Italy

Federico Ponzetto, PhD (Presenter) University of Turin

Presenter Bio: Federico Ponzetto, PhD is a Tenure Track Researcher in Clinical Biochemistry and Clinical Molecular Biology at the Department of Medical Sciences, University of Turin, Italy. His research activity is mainly focused on steroid analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS), with particular expertise in targeted steroidomics, endogenous steroid profiling, and the development of high-throughput analytical workflows for both clinical endocrinology and anti-doping applications. He obtained his PhD at the Swiss Laboratory for Doping Analyses, University of Lausanne (Switzerland), where he developed blood steroid profiling strategies for the detection of testosterone misuse within the Athlete Biological Passport framework.
Dr. Ponzetto has more than 10 years of experience in mass spectrometry-based steroid analysis across multiple biological matrices, including serum, plasma, urine, saliva, and microsampling devices. His work has contributed to the implementation of innovative UHPLC-MS/MS methods for the quantification of endogenous steroids, phase II metabolites, glucocorticoids, and biomarkers relevant to endocrine disorders and doping control. He has authored over 30 peer-reviewed publications in the fields of clinical mass spectrometry, steroidomics, and endocrine laboratory medicine, and has served as principal investigator of international research projects funded by the World Anti-Doping Agency and Partnership for Clean Competition. His current research interests include advanced LC-MS/MS steroidomic platforms, adrenal and metabolic disorders, and automation of mass spectrometry workflows in routine laboratory medicine.

Relevant Financial Disclosures (within past 24 months, reported on May 26, 2026) Grant/Research Support Kura Biotech Discussing Products? YES, presenter will present educational content related to products/services of one or more ineligible company(ies) listed above. Using Generic Terms? NO Conflict mitigation IN PROCESS.

INTRODUCTION:
Urinary steroid profiling by LC-MS/MS enables a comprehensive and non-invasive assessment of steroid metabolism [1]. Accurate quantification requires efficient enzymatic hydrolysis of phase II conjugates, as steroid metabolites are extensively present as glucuronide and sulfate derivatives [2]. Recent advances in analytical workflows have highlighted the importance of integrating efficient enzymatic hydrolysis with LC-MS/MS methods to improve analytical performance and metabolite coverage [3].

OBJECTIVES:
This study aims to systematically evaluate the impact of enzymatic hydrolysis strategies on extended steroidomic analysis in urine by integrating controlled optimization experiments with real-sample performance. Specifically, the study (i) determines optimal hydrolysis conditions for conjugated steroids, (ii) compares the performance of different enzymatic systems, and (iii) assesses their effects on signal intensity, metabolite recovery, and analytical coverage across a broad panel of urinary steroids.

METHODS:
A two-phase experimental design was implemented. In the first phase, hydrolysis conditions were optimized in synthetic urine spiked with a comprehensive panel of glucuronide- and sulfate-conjugated steroids. A recombinant β-glucuronidase/arylsulfatase enzyme mixture (BGS, Kura Bioteh) was evaluated across different enzyme volumes (10, 20, and 30 µL), temperatures (35, 55, and 75 °C), and incubation times (30 min, 1 h, and 2 h). In the second phase, enzymatic performance was assessed in real urine samples from five individuals using three hydrolysis systems: recombinant β-glucuronidase/sulfatase (BGS), a β-glucuronidase/arylsulfatase mixture from Helix pomatia (H. pomatia, Roche), and β-glucuronidase from E. coli K-12 (E. coli, Roche).

An in-house developed comprehensive LC-MS/MS workflow was applied, enabling the detection of 143 free steroids and 50 conjugated steroids through two complementary analytical runs. The run targeting phase II metabolites was used to assess hydrolysis performance, while the run targeting free steroids was employed to evaluate the presence of free steroids following the three different hydrolysis procedures in real 24-hour urine samples.

For this approach, detected analyte peak areas were normalized to the signal of deuterated internal standards. Data analysis included global intensity comparison, fold-change analysis, dominant analyte counting, principal component analysis (PCA), and class-based evaluation.

RESULTS:
In synthetic urine, glucuronide-conjugated steroids showed near-complete hydrolysis under all tested conditions with BGS, indicating that enzymatic activity is not a limiting factor. In contrast, sulfate conjugates exhibited lower and more variable hydrolysis efficiencies, influenced by analyte structure, temperature, and incubation time. Increasing enzyme volume did not significantly improve hydrolysis, and optimal conditions for hydrolysis with BGS were identified as 10 µL of enzyme at 55 °C for 1 hour.
In real urine samples, all tested enzymes enabled the detection of 94 free steroid hormones. However, clear differences between enzymatic systems were observed: BGS provided the highest overall normalized signal intensity (mean ≈ 1.99), followed by H. pomatia (≈ 1.63). As expected, E. coli (≈ 1.42) resulted in lower signal intensity due to its activity being limited to glucuronide cleavage. At the analyte level, BGS was dominant for the majority of compounds (58 analytes), compared to H. pomatia (33) and E. coli (11). Fold-change analysis identified multiple metabolites significantly enriched under BGS, particularly among androgen and corticosteroid derivatives.

PCA revealed that 99.2% of the total variance was explained by the first principal component, indicating that enzymatic treatment primarily affects signal magnitude rather than qualitative metabolite profiles. Class-level analysis showed that androgens exhibited the largest differences between enzymes, while corticosteroids displayed moderate variability.

CONCLUSIONS:
This study demonstrates that glucuronide hydrolysis is rapid and robust across all tested conditions, whereas sulfate hydrolysis remains structurally dependent and represents the main limitation in the enzymatic deconjugation of urinary steroids. The obtained results indicate that dual-activity enzymatic systems are essential for comprehensive urinary steroid profiling. The recombinant BGS enzyme consistently outperformed both the H. pomatia enzyme mixture and E. coli β-glucuronidase in real urine samples, likely due to improved catalytic efficiency and balanced enzymatic activity. Notably, the recombinant β-glucuronidase/arylsulfatase formulation enables efficient one-step hydrolysis, allowing the simultaneous deconjugation of both glucuronide and sulfate phase II metabolites. Importantly, differences between enzymatic systems were predominantly quantitative, highlighting the impact of hydrolysis efficiency on analytical sensitivity rather than metabolite detectability. These findings support the use of optimized recombinant dual-activity enzymes as a robust strategy for extended steroidomic analysis in urine.

REFERENCES
[1] Araujo-Castro M, Valderrábano P, Escobar-Morreale HF, Hanzu FA, Casals G. Urine steroid profile as a new promising tool for the evaluation of adrenal tumors: literature review. Endocrine. 2021;72(1):40–48.
[2] Wang R, Hartmann MF, Wudy SA. Targeted LC-MS/MS analysis of steroid glucuronides in human urine. J Steroid Biochem Mol Biol. 2021 Jan;205:105774
[3] Zhong J, Ma X, Wang D, Luo W, Yin Y, Zou Y, Zhu Y, Li M, Xie S, Yu S, Qiu L. Liquid chromatography-tandem mass spectrometry-based urinary steroid profiling applied to primary aldosteronism diagnosis. J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Apr 1;1273:124930.