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Analysis of metabolites and metabolic pathways of troponin activator in rats using UHPLC-MS

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Abstract

Troponin activators, including Reldesemtiv and Tirasemtiv, represent a class of drugs that enhance the contractility of cardiac muscle and delay the onset of muscle fatigue. Consequently, they were classified as prohibited substances by the World Anti-Doping Agency (WADA) in 2024. This study aimed to develop an ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS)–based method to quantify Reldesemtiv and Tirasemtiv levels in rat urine, blood, and feces over a 72-h period following transgastric administration. Additionally, Compound Discoverer 3.3 was utilized to investigate the metabolites and metabolic pathways of these two drugs in vivo. Twelve metabolites of Reldesemtiv and seven metabolites of Tirasemtiv were identified. Notably, ten of the twelve Reldesemtiv metabolites had not been previously reported in the literature. Using UHPLC-MS, the concentration of each metabolite in urine and blood was measured at various time points, enabling the generation of a metabolic profile. Six metabolites of Reldesemtiv are detectable 72 h after administration, while Tirasemtiv and its metabolite T-M1a were also detectable at the same time point. This analytical method can characterize the metabolic profiles of the drugs and their metabolites, as well as identify unknown and long-lived metabolites in drug testing. These capabilities are critical for the quantitative analysis of troponin activators within complex biological matrices, supporting their study in physiological and doping-control contexts.

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All data generated or analyzed during this study are included in the published article and its supplementary materials.

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Acknowledgements

The authors would like to thank the Hebei Institute for Drug and Medical Device Control for providing technical and application support.

Funding

This work was supported by the Centralized Guided Local Science and Technology Development Funds Project (Nos. 246Z7772G, 226Z7708G).

Author information

Author notes

  1. Mingxue Yang, Xu Cao, Zhaobo Wu, Xiaochen Huang, Aoxin Ma, and Yunda Li contributed equally to this work.

Authors and Affiliations

  1. School of Public Health, Hebei Medical University, Shijiazhuang, 050017, China

    Mingxue Yang, Xu Cao, Zhaobo Wu, Aoxin Ma & Kaoqi Lian

  2. Hebei Research Institute of Sports Science, Shijiazhuang, 050017, China

    Xiaochen Huang, Yunda Li & Guojun Li

  3. Hebei Key Laboratory of Environment and Human Health, Shijiazhuang, 050017, China

    Kaoqi Lian

  4. Hebei Key Laboratory of Early Life Health Promotion, Hebei Medical University, Shijiazhuang, 050031, China

    Kaoqi Lian

Authors
  1. Mingxue Yang
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  2. Xu Cao
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  3. Zhaobo Wu
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  4. Xiaochen Huang
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  6. Yunda Li
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  7. Guojun Li
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Contributions

Responsible for the methodology, data analysis, and the initial drafting of the manuscript: M.Y.; quantitative method establishment and methodological validation, writing—review and editing: X.C.; played a role in the formal analysis: Z.W.; contributed to the supervision of the project: X.H.; contributed to validation: A.M.; responsible for the methodology: Y.L.; conducted the supervision, funding acquisition, resource allocation, and manuscript review: G.L.; conducted the funding acquisition, manuscript review, and provided supervision: K.L. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Guojun Li or Kaoqi Lian.

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Ethical approval

All experimental procedures were conducted in strict compliance with the National Guidelines for Humane Treatment of Laboratory Animals (GB/T 35823–2018) and were approved by the Institutional Animal Care and Use Committee (IACUC) of Hebei Institute for Drug Control (Approval No LL2024- 03).

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The authors declare no competing interests.

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Yang, M., Cao, X., Wu, Z. et al. Analysis of metabolites and metabolic pathways of troponin activator in rats using UHPLC-MS. Anal Bioanal Chem (2025). https://doi.org/10.1007/s00216-025-06031-7

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