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UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine

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Abstract

Recombinant human erythropoietin (rhEPO) analogues are known to have been used in horse sports for their assumed performance enhancing properties. Recently, several authors have published liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods for confirming the presence of rhEPO analogues in horse plasma. In the current study, an improved LC-MS/MS confirmatory procedure for rhEPO, darbepoetin (DPO) and continuous erythropoietin receptor activator (CERA) in horse plasma was developed and validated. The method was also adapted for and applied to urine samples for the first time. Similar to previously published plasma assays, the methods utilise size exclusion and immunoaffinity extraction prior to tryptic cleavage, enzymatic deglycosylation and LC-MS/MS analysis of the resulting signature tryptic peptides (rhEPO/CERA T5, rhEPO/CERA/DPO T6 and DPO T9). However, the novel application of UPLC chromatography significantly improves the run time of the method compared to nano- or micro-LC and its robustness compared to nano-LC. Furthermore, recombinant canine EPO was found to serve as an effective internal standard, thus allowing confidence in interpretation of the success/failure of every step in the procedure. Limits of detection for confirming the presence of rhEPO, CERA and DPO in plasma were 0.1, 0.25 and 0.05 ng mL−1, respectively, which were equal to or lower than limits achieved using previously published LC-MS/MS based methods. Limits of detection for confirming the presence of rhEPO, CERA and DPO in urine were 0.05, 0.15 and 0.025 ng mL−1 and the analysis of urine samples collected from horses administered rhEPO (Eprex™) or DPO (Aranesp™) demonstrated the use of this matrix as a suitable alternative in situations where plasma is not available.

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Acknowledgments

This work was funded by the British Horseracing Authority. The authors are grateful to Richard Kay from Quotient Bioresearch, UK and Polly Taylor from HFL Sport Science, UK as well as Terry Wan from the Racing Laboratory at the Hong Kong Jockey Club for technical advice throughout the study. Also, the authors would like to thank Ludovic Bailly-Chouriberry from LCH, France for useful discussions regarding validation guidelines for the use of protein mass spectrometry in animal sports.

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Authors and Affiliations

  1. HFL Sport Science (an LGC company), Newmarket Road, Fordham, Cambridgeshire, CB7 5WW, UK

    James P. Scarth, Cathrin Seibert, Pamela R. Brown, Phil Teale, Gavin J. Beamon & Clive M. Pearce

  2. HFL Sport Science (an LGC company), Alysheba Way, Lexington, KY, 40509, USA

    Richard A. Sams

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  1. James P. Scarth
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  2. Cathrin Seibert
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Correspondence to James P. Scarth.

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Scarth, J.P., Seibert, C., Brown, P.R. et al. UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine. Chromatographia 74, 593–608 (2011). https://doi.org/10.1007/s10337-011-2119-y

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  • DOI: https://doi.org/10.1007/s10337-011-2119-y

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