A broad-spectrum peptide screening method using an optimized solid-phase extraction and liquid chromatography-high-field asymmetric ion mobility spectrometry-mass spectrometry for doping control in equine urine.
Abstract: The abuse of prohibited peptide-based drugs with a broad spectrum of chemical characteristics poses a significant concern for the horseracing industry. Recently, there has been a notable increase in positive cases of small-peptide drugs reported in equine and canine sports. In addition to small peptides, large peptides (over 2 kDa) with structural diversity have also entered the market in increasing numbers as drugs for humans and livestock. However, the simultaneous analysis of both small- and large-peptide-based drugs is still challenging. In this study, a screening method was developed to cover 74 analytes, including peptides, their catabolites, and/or their mimetics, with molecular weights ranging from 0.3 kDa to greater than 5 kDa. The simultaneous extraction of both small and large peptides was achieved using a weak cation-exchange solid-phase extraction cartridge with a mixture of different pore sizes (suitable for large peptides), followed by analysis using liquid chromatography high-field asymmetric ion mobility spectrometry tandem mass spectrometry (LC-FAIMS-MS/MS). For method validation, the limits of detection (LoDs), reproducibility, recovery, matrix effect, selectivity, and carryover were evaluated. Remarkably, the LoDs of ∼80% of the analytes were less than or equal to 50 pg ml, with the lowest LoD (1 pg ml) being observed for selected peptides in horse urine. These results indicate a substantial advancement in achieving comprehensive coverage for both small and large peptides with high sensitivity for the purpose of doping control in horseracing and equestrian sports.
Publication Date: 2024-11-06 PubMed ID: 39503331DOI: 10.1039/d4ay01477dGoogle Scholar: Lookup
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- Journal Article
Summary
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The study develops a method for detecting a wide range of peptide-based performance-enhancing drugs in horse urine, to ensure fairness in horse racing and equestrian sports. The new screening method covers 74 substances, including small and large peptides, and their metabolites or mimics.
Context
- The presence of prohibited peptide-based drugs in horse racing has been a significant issue. These drugs have a broad chemical range and are used as performance enhancers in horse and dog sports.
- There has been a surge in the number of cases where small-peptide drugs have been detected in equine and canine sports.
- Large peptides, which are over 2 kiloDaltons in molecular weight, are also increasingly being used in the market for humans and livestock.
- It has been challenging to simultaneously analyze both small and large peptide-based drugs.
Purpose of the Study
- The research aims to develop a method capable of screening both small and large peptides. The method covers a breadth of 74 analytes, including peptides, their metabolites, and/or their mimetics, with molecular weights ranging from 0.3 kiloDaltons to more than 5 kiloDaltons.
Methodology
- The researchers utilized a weak cation-exchange solid-phase extraction cartridge with a mix of different pore sizes, suitable for large peptides, to simultaneously extract all peptides.
- Analysis was done using sophisticated technology known as Liquid Chromatography High-field Asymmetric Ion Mobility Spectrometry Tandem Mass Spectrometry (LC-FAIMS-MS/MS).
Validation of the Method
- The method was validated by understanding the limits of detection (LoDs), reproducibility, recovery, the matrix effect, selectivity, and carryover.
- Notably, the LoDs of around 80% of the analytes were equal to or less than 50 picograms per milliliter, with the lowest LoD being 1 picogram per milliliter, as observed for specific peptides in horse urine.
Significance of the Study
- The results indicate a massive development in achieving comprehensive coverage for both small and large peptides with high sensitivity.
- This development is vital for the purpose of doping controls in horse racing and equestrian sports and encourages fairness in these sports.
Cite This Article
APA
Ohnuma K, Hirano-Kodaira M, Bannai M, Shimizu Y, Yamada M, Kinoshita K, Ngai-Wa Leung G, Ishii H.
(2024).
A broad-spectrum peptide screening method using an optimized solid-phase extraction and liquid chromatography-high-field asymmetric ion mobility spectrometry-mass spectrometry for doping control in equine urine.
Anal Methods.
https://doi.org/10.1039/d4ay01477d Publication
Researcher Affiliations
- Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Japan. k-ohnuma@lrc.or.jp.
- Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Japan. k-ohnuma@lrc.or.jp.
- Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Japan. k-ohnuma@lrc.or.jp.
- Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Japan. k-ohnuma@lrc.or.jp.
- Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Japan. k-ohnuma@lrc.or.jp.
- Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Japan. k-ohnuma@lrc.or.jp.
- Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Japan. k-ohnuma@lrc.or.jp.
- Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Japan. k-ohnuma@lrc.or.jp.
Citations
This article has been cited 1 times.- Králík F, Kvíčalová A, Salaďáková A, Kuchař M, Setnička V. Growth Hormone-Releasing Peptides: Investigation of Their Secondary Structure, Thermal Stability, and Model Membrane Interactions. Chirality 2026 Feb;38(2):e70083.
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