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Dispersive SPE, an alternative to traditional SPE for extraction of 43 doping peptides from equine urine prior to LC–MS screening

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Abstract

Purpose

Dermorphin, growth hormone releasing peptide (GHRP), TB-500 and their analogues have been used illegally in the horse racing industry to improve the performance of the horses. This study aims to present dispersive solid phase extraction (dSPE) as an alternative to solid phase extraction (SPE) for the clean-up of equine urine samples prior to liquid chromatography combined with tandem mass spectrometry (LC–MS/MS) screening of 43 illegal performance enhancing peptides.

Methods

Sorbent types and mass, washing and eluting solvents were tested to obtain the optimal clean-up conditions for these peptides in horse urine matrices.

Results

The resulting dSPE clean-up method gave optimal recovery and reproducibility of 43 target peptides; for the first time dSPE is proven as a viable alternative to SPE and achieves limits of detection (LOD) that are sufficient for the screening of these peptides. The LODs for all dermorphin, TB-500 and GHRP peptides were 1 ng/mL. Recoveries of the 43 target analytes extracted from 3 spiked urine samples ranged from 8.9 to 58.8%. The intra-day and inter-day precision for all target analytes ranged from 0.6 to 24.1% and 1.4 to 27.8% respectively.

Conclusions

Using dSPE as a clean-up method, 43 peptide analytes of interest were successfully screened by LC–MS/MS.

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Acknowledgements

This work has been supported by funding from the ChemCentre and Edith Cowan University. We thank Dr. Rohan Steel from Racing Analytical Services Limited (RASL) (Victoria, Australia) for his inputs and providing peptide standards for our study.

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Authors and Affiliations

  1. School of Science, Edith Cowan University, Joondalup, WA, 6027, Australia

    Joanne Pugliese, Mary C. Boyce, Nathan G. Lawler & Thao T. Le

  2. Centre for Integrative Metabolomics and Computational Biology, Edith Cowan University, Joondalup, WA, 6027, Australia

    Mary C. Boyce & Nathan G. Lawler

  3. ChemCentre, Bentley, WA, 6102, Australia

    John Coumbaros & Thao T. Le

Authors
  1. Joanne Pugliese
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  2. Mary C. Boyce
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Corresponding author

Correspondence to Thao T. Le.

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The authors declare no conflict of interest.

Ethical approval

Throughout this study horse urine samples were used. All urine samples were supplied by the ChemCentre which were sourced from Murdoch Veterinary School as a waste product. All applicable national, and/or institutional guidelines for the care and use of animals were followed.

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Pugliese, J., Boyce, M.C., Lawler, N.G. et al. Dispersive SPE, an alternative to traditional SPE for extraction of 43 doping peptides from equine urine prior to LC–MS screening. Forensic Toxicol 38, 365–377 (2020). https://doi.org/10.1007/s11419-020-00524-z

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