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Anal Bioanal Chem. 2016 May;408(12):3145-53. doi: 10.1007/s00216-016-9377-3. Epub 2016 Feb 15.

Qualitative identification of growth hormone-releasing hormones in human plasma by means of immunoaffinity purification and LC-HRMS/MS.

Author information

1
Institute for Biochemistry-Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
2
Laboratoire d'Hormonologie, C.E.R. Groupe-Département Santé, Rue du Point du jour 8, 6900, Marloie, Belgium.
3
Institute for Biochemistry-Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany. thevis@dshs-koeln.de.
4
European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany. thevis@dshs-koeln.de.

Abstract

The use of growth hormone-releasing hormones (GHRHs) is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). The aim of the present study was to develop a method for the simultaneous detection of four different GHRHs and respective metabolites from human plasma by means of immunoaffinity purification and subsequent nano-ultrahigh performance liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry. The target analytes included Geref (Sermorelin), CJC-1293, CJC-1295, and Egrifta (Tesamorelin) as well as two metabolites of Geref and CJC-1293, which were captured from plasma samples using a polyclonal GHRH antibody in concert with protein A/G monolithic MSIA™ D.A.R.T.'S® (Disposable Automation Research Tips) prior to separation and detection. The method was fully validated and found to be fit for purpose considering the parameters specificity, linearity, recovery (19-37%), lower limit of detection (<50 pg/mL), imprecision (<20%), and ion suppression/enhancement effects. The analytes' stability and metabolism were elucidated using in vitro and in vivo approaches. EDTA blood samples were collected from rats 2, 4, and 8 h after intravenous administration of GHRH (one compound per test animal). All intact substances were detected for at least 4 h but no anticipated metabolite was confirmed in laboratory rodents' samples; conversely, a Geref metabolite (GHRH3-29) was found in a human plasma sample collected after subcutaneous injection of the drug to a healthy male volunteer. The obtained results demonstrate that GHRHs are successfully detected in plasma using an immunoaffinity-mass spectrometry-based method, which can be applied to sports drug testing samples. Further studies are however required and warranted to account for potential species-related differences in metabolism and elimination of the target analytes.

KEYWORDS:

Doping; GHRH; Immunoaffinity; LC-HRMS/MS; MSIA™-Tips

PMID:
26879649
PMCID:
PMC4830873
DOI:
10.1007/s00216-016-9377-3
[Indexed for MEDLINE]
Free PMC Article

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