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Detection, quantification, and identification of dermorphin in equine plasma and urine by LC–MS/MS for doping control

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Abstract

Dermorphin is a unique opioid peptide that is 30–40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples. This paper describes the first liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for such a purpose. Equine plasma and urine samples were pre-treated with ethylenediamine tetra-acetic acid and urea prior to solid-phase extraction (SPE) on Oasis MCX cartridges. Resulting eluates were dried under vacuum and analyzed by LC–MS/MS for dermorphin. The matrix effect, SPE efficiency, intra-day and inter-day accuracy and precision, and stability of the analyte were assessed. The limit of detection was 10 pg/mL in plasma and 20 pg/mL in urine, and the limit of confirmation was 20 pg/mL in plasma and 50 pg/mL in urine. Dermorphin in plasma is stable at ambient temperature, but its diastereomer is unstable. With isotopically labeled dermorphin as an internal standard, the quantification range was 20–10,000 pg/mL in plasma and 50–20,000 pg/mL in urine. The intra-day and inter-day accuracy was from 91 % to 100 % for the low, intermediate, and high concentrations. The intra-day and inter-day coefficients of variation were less than 12 %. The method differentiates dermorphin from its diastereomer. This method is very specific for identification of dermorphin in equine plasma and urine, as assessed by BLAST search and targeted SEQUEST search, and by MS/MS spectrum library search. The method has been successfully applied to analysis of samples collected following dermorphin administration to research horses and of official post-race samples.

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Acknowledgments

Financial support was provided by The Pennsylvania Racing Commissions, and financial contributions were made by the Pennsylvania Harness Horsemen Association at Pocono Downs and Chester Downs, Meadows Standardbred Owners Association, Horsemen Benevolent and Protective Association at Penn National and Presque Isles Downs. The authors thank Hilary Goff, Deborah Tsang, and Jesse Vanderhoef for their excellent technical assistance.

The ProteinProspector software developed by Dr. Alma Burlingame and his group at the University of California, San Francisco, California was used in interpreting product ions of dermorphin. BLAST search was performed using NCBI BLAST server (National Center for Biotechnology Information, USA).

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Authors and Affiliations

  1. Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, 382 West Street Road, Kennett Square, PA, 19483, USA

    Fuyu Guan, Cornelius E. Uboh, Lawrence R. Soma, Mary Robinson & Xiaoqing Li

  2. Pennsylvania Equine Toxicology & Research Center, 220 East Rosedale Avenue, West Chester, PA, 19382, USA

    Cornelius E. Uboh

  3. New York Equine Drug Testing and Research Program, Morrisville State College, Ithaca, NY, 14850, USA

    George A. Maylin

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  1. Fuyu Guan
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  2. Cornelius E. Uboh
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Correspondence to Cornelius E. Uboh.

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Guan, F., Uboh, C.E., Soma, L.R. et al. Detection, quantification, and identification of dermorphin in equine plasma and urine by LC–MS/MS for doping control. Anal Bioanal Chem 405, 4707–4717 (2013). https://doi.org/10.1007/s00216-013-6907-0

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  • DOI: https://doi.org/10.1007/s00216-013-6907-0

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