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Development and validation of a liquid chromatography–mass spectrometry procedure after solid-phase extraction for detection of 19 doping peptides in human urine

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Abstract

A liquid chromatography–tandem mass spectrometry method was developed and used to simultaneously detect 19 small peptide hormones prohibited in sport and their main metabolites in urine after solid-phase extraction. Detection was achieved using a triple-quadrupole mass spectrometric detector coupled with an electrospray ionization interface after chromatographic separation with an octadecyl column based on fused-core particle technology. Sample pretreatment was performed by solid-phase extraction. The extraction procedure was optimized by comparison of different sorbents and washing/elution protocols. The best results were obtained using a mixed-mode weak cation exchange sorbent, two washing steps (ultrapurified water and methanol), and elution using 300 mM ammonium formate in 25 % ammonia/methanol (10/90) or 25 % ammonia/10 % formic acid/methanol (8/12/80). The procedure was validated in terms of sensitivity (lower limits of detection: 0.05–2.0 ng/ml depending on the target analyte), specificity, recovery [>60 %, coefficient of variation (CV) <15 % except for TB500 17–23 fragment, AOD9604, and ARA290 for which recovery was <50 %), ion suppression/enhancement (<35 %), robustness, carryover, stability of the target analytes [stable for at least for 2 days (25 °C, 2 weeks (4 °C), 2 months (−20 °C)], and repeatability of retention times (CV <0.1 %) and relative abundances of the selected ion transitions (CV <15 %). The suitability of the method was confirmed by analyzing spiked and excreted urines, the latter collected after intravenous injection of 0.1 mg of GHRP-2.

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Acknowledgments

This work has been supported by a Research Grant of the Italian Department of Health (“Ministero della Salute, Commissione per la vigilanza sul doping e sulla tutela sanitaria delle attività sportive”).

Conflict of interest

The authors declare no conflict of interest.

Compliance with ethical standards

In this study, human urine samples were used. The real samples obtained from excretion studies of GHRP-2 were authorized by WAADS and provided to all WADA-accredited laboratories; the handling of all samples was conducted in accordance with the relevant rules.

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Authors and Affiliations

  1. Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, 00197, Rome, Italy

    Monica Mazzarino, Valeria Calvaresi, Xavier de la Torre, Graziella Parrotta, Chiara Sebastianelli & Francesco Botrè

  2. Dipartimento di Medicina Sperimentale, “Sapienza” Università di Roma, Viale Regina Elena 324, 00161, Rome, Italy

    Francesco Botrè

Authors
  1. Monica Mazzarino
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  2. Valeria Calvaresi
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  3. Xavier de la Torre
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  6. Francesco Botrè
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Correspondence to Francesco Botrè.

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Mazzarino, M., Calvaresi, V., de la Torre, X. et al. Development and validation of a liquid chromatography–mass spectrometry procedure after solid-phase extraction for detection of 19 doping peptides in human urine. Forensic Toxicol 33, 321–337 (2015). https://doi.org/10.1007/s11419-015-0279-4

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  • DOI: https://doi.org/10.1007/s11419-015-0279-4

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