title: Qualitative identification of growth hormone-releasing hormones in human plasma by means of immunoaffinity purification and LC-HRMS/MS.
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.
authors.affiliation: Institute for Biochemistry-Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Andre
authors.initials: A
authors.lastname: Knoop
authors.affiliation: Institute for Biochemistry-Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Thomas
authors.affiliation: Laboratoire d'Hormonologie, C.E.R. Groupe-Département Santé, Rue du Point du jour 8, 6900, Marloie, Belgium.
authors.forename: Eric
authors.initials: E
authors.lastname: Fichant
authors.affiliation: Laboratoire d'Hormonologie, C.E.R. Groupe-Département Santé, Rue du Point du jour 8, 6900, Marloie, Belgium.
authors.forename: Philippe
authors.initials: P
authors.lastname: Delahaut
authors.affiliation: Institute for Biochemistry-Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
authors.affiliation: Institute for Biochemistry-Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany. thevis@dshs-koeln.de.|European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany. thevis@dshs-koeln.de.
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D013007:Growth Hormone-Releasing Hormone / Q000097:blood*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D013007:Growth Hormone-Releasing Hormone
keywords: Doping; GHRH; Immunoaffinity; LC-HRMS/MS; MSIA™-Tips