title: Determination of IGF-1 and IGF-2, their degradation products and synthetic analogues in urine by LC-MS/MS.
abstract: Peptide analysis in doping controls by means of nano-UPLC coupled high resolution/high mass accuracy mass spectrometry provides the state-of-the-art technique in modern sports drug testing. The present study describes a recent application of this technique for the qualitative determination of different urinary insulin-like growth factor (IGF) related peptides. After simultaneous isolation by solid phase extraction and magnetic particle-based immunoaffinity purification, target analytes (IGF-1, IGF-2, Des1-3-IGF-1, R(3)-IGF-1 and longR(3)-IGF-1) were separated by nano-liquid chromatography prior to mass spectrometric detection. Endogenously produced IGF-1 and IGF-2, as well as the degradation product Des1-3-IGF-1, were frequently detected in urine samples from healthy volunteers in a concentration range of 20-400 pg mL(-1). The impact of IGF binding proteins (IGFBPs), being also present in urine, was potentially estimated by an additional ultrafiltration step in the sample preparation procedure. The synthetic analogue longR(3)-IGF-1, which is assumed to be subject to misuse by cheating athletes, was also analysed and detected in fortified urine samples. Besides the intact molecule, an N-terminally truncated degradation product Des1-10-longR(3)-IGF-1 was identified as the more stable target for doping controls using urine samples. The method was validated for qualitative purposes considering the parameters specificity, limit of detection (20-50 pg mL(-1)), recovery (10-35%), precision (<20%), linearity, robustness and stability.
authors.affiliation: Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany. a.thomas@biochem.dshs-koeln.de
authors.forename: Andreas
authors.initials: A
authors.lastname: Thomas
authors.forename: Maxie
authors.initials: M
authors.lastname: Kohler
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: D002853:Chromatography, Liquid / Q000379:methods*; D004300:Doping in Sports; D005260:Female; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000652:urine*; D007335:Insulin-Like Growth Factor II / Q000652:urine*; D057230:Limit of Detection; D008297:Male; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors; D014462:Ultrafiltration / Q000379:methods
chemical_list: D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II