title: Immunoaffinity purification of peptide hormones prior to liquid chromatography-mass spectrometry in doping controls.
abstract: For most peptide hormones prohibited in elite sports the concentrations in plasma or urine are very low (pg/mL). Accordingly, hyphenated purification and enrichment steps prior to mass spectrometric detection are required to obtain sufficient doping control assays. Immunoaffinity purification in combination with nano-scale liquid chromatography coupled to high resolution/high accuracy mass spectrometry was found to have the potential of providing the necessary sensitivity and unambiguous specificity to produce reliable results. With the presented methodology 12 prohibited peptides (porcine insulin, Novolog, Apidra, Lantus DesB30-32 metabolite, Humalog and human insulin, Synacthen (synthetic ACTH analogue), luteinizing hormone-releasing hormone (LH-RH), growth hormone releasing hormone (GH-RH(1-29)) and CJC-1295 (GH-RH analogue), LongR(3)-IGF-1 and IFG-1) were simultaneously purified from plasma/serum or urine. With limits of detection for each target compound ranging in the low pg/mL level (urine), the method enables the determination of urinary peptides at physiologically relevant concentrations. For each class of peptides an appropriate antibody and a respective internal standard was implemented ensuring robust analysis conditions. Due to the fast and simple sample preparation procedure (∼25 samples per day) and the fact that all materials are commercial available, the implementation of the methodology to laboratories from other analytical fields (forensics, pharmacokinetic sciences, etc.) is enabled.
authors.affiliation: Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Germany. a.thomas@biochem.dshs-koeln.de
authors.forename: Andreas
authors.initials: A
authors.lastname: Thomas
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
authors.forename: Philippe
authors.initials: P
authors.lastname: Delahaut
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods* / Q000592:standards; D004300:Doping in Sports; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives / Q000097:blood / Q000302:isolation & purification / Q000652:urine; D006801:Humans; D007118:Immunoassay / Q000379:methods* / Q000592:standards; D061267:Insulin Aspart / Q000097:blood / Q000652:urine; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000097:blood / Q000302:isolation & purification / Q000652:urine; D036361:Peptide Hormones / Q000097:blood / Q000302:isolation & purification* / Q000652:urine; D058573:Performance-Enhancing Substances / Q000097:blood / Q000302:isolation & purification / Q000652:urine; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015813:Substance Abuse Detection / Q000379:methods*; D013997:Time Factors
chemical_list: C502076:CJC 1295; D010446:Peptide Fragments; D036361:Peptide Hormones; D058573:Performance-Enhancing Substances; D013007:Growth Hormone-Releasing Hormone; D061267:Insulin Aspart