title: Development of an absolute quantification method targeting growth hormone biomarkers using liquid chromatography coupled to isotope dilution mass spectrometry.
abstract: A method to perform absolute quantification of two biomarkers (IGF-1 and IGFBP-3) of growth hormone abuse has been developed. Isotope dilution is used with synthetically labelled peptides as internal standards. Peptide selection and multiple reaction monitoring design are discussed. A simple sample preparation based on the reduction and alkylation of cysteine residues followed by tryptic digestion provides a sufficient digestion of proteins. Serum samples fortified with increasing amounts of target proteins are analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a triple quadrupole mass spectrometer. Specificity is ensured by the selection of sequences with no homology in BLAST, as well as retention time deviation check, and ion ratio monitoring. Linearity is studied in terms of calibration curves. These curves for IGFBP-3 and IGF-1 are generated with mean slopes of 0.055 and 0.065, intercepts of 0.107 and -0.011, and with coefficients of correlation of 0.95 and 0.98, respectively. These curves result from the addition of proteins to the serum. Risks of variations related to potential matrix effects are therefore reduced, as well as probable variations related to the digestion steps. The working concentration ranges are 4-10 ng/microl for IGFBP-3 and 2-8 ng/microl for IGF-1. Preliminary data regarding repeatability show that relative standard deviations (RSDs) range between 13 and 32% for IGFBP-3 and between 7 and 29% for IGF-1.
authors.affiliation: University of Liège, Chemistry Department, Mass Spectrometry Laboratory, Allée de la Chimie 3, B-6c Sart-Tilman, B-4000 Liège, Belgium. s.kirsch@ulg.ac.be
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Kirsch
authors.forename: Joëlle
authors.initials: J
authors.lastname: Widart
authors.forename: Joel
authors.initials: J
authors.lastname: Louette
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Focant
authors.forename: Edwin
authors.initials: E
authors.lastname: De Pauw
mesh_terms: D015415:Biomarkers / Q000032:analysis*; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D004300:Doping in Sports; D013006:Growth Hormone / Q000097:blood*; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000032:analysis*; D007334:Insulin-Like Growth Factor I / Q000032:analysis*; D007553:Isotope Labeling / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015415:Biomarkers; D018972:Insulin-Like Growth Factor Binding Protein 3; D007334:Insulin-Like Growth Factor I; D013006:Growth Hormone