Growth hormone isoform‐differential mass spectrometry for doping control purposes

Abstract

Mass spectrometry (MS) allows for monitoring growth hormone (GH) isoform compositions at high specificity. It is demonstrated that this capability can be used to reliably detect alterations as elicited by (putative) doping with 22 kDa-GH. Sample treatment consists of enzymatic protein cleavage, followed by 2-step liquid chromatography clean-up, prior to analysis by MS. The protocol does not depend on antibodies for analyte extraction at any stage. Therefore, MS opens an opportunity for independent confirmation, if combined with the antibody-based isoform differential test presently used in practice. To check the fitness-for-purpose of this concept, GH-free serum was spiked with pure 22 kDa- and 20 kDa-GH covering a representative range of concentrations (0.5-9.4 μg/L), while the 22kDa fraction was within a range of 80%-85%, or at 100%, the latter simulating an administration of 22 kDa-GH. Mean deviation of 22 kDa-fractions found was within less than 3% for samples with total GH>= 1 μg/L . Beyond this, results by antibody-free isoform-differential MS, as described, were in line with those of the World Anti-Doping Agency-approved antibody-based test for 18 native sera and 3 positive controls. In this context, relating 22 kDa-GH to total-GH rather than 22 kDa+20 kDa was considered as an alternative strategy to earlier approaches. However, 20 kDa-GH as an additional measurand, next to 22 kDa- and total-GH, provides useful extra information, as it directly indicates the presence or absence of a non-22 kDa-GH form.

Longitudinally monitoring of P‐III‐NP, IGF‐I, and GH‐2000 score increases the probability of detecting two weeks’ administration of low‐dose recombinant growth hormone compared to GH‐2000 decision limit and GH isoform test and micro RNA markers

“…21 In fact MS is regarded as the gold standard in sports medicine because of the specificity and possibility to simultaneously quantify different proteins. 21 In fact MS is regarded as the gold standard in sports medicine because of the specificity and possibility to simultaneously quantify different proteins.…”

“…Instead of using different immunoassay kit, mass spectrometric (MS) methods may be promising for isoform confirmation. 21 In fact MS is regarded as the gold standard in sports medicine because of the specificity and possibility to simultaneously quantify different proteins. 22 MS methods for plasma/serum determination of IGF-I has been developed.…”

Candidate High-Resolution Mass Spectrometry-Based Reference Method for the Quantification of Procalcitonin in Human Serum Using a Characterized Recombinant Protein as a Primary Calibrator

“… 22 − 24 The SIL protein added at the beginning of the sample preparation process is ideal for protein quantification with the bottom-up approach. 25 , 27 It compensates for the bias caused by incomplete digestion or material loss during sample preparation and LC-MS/MS analysis. 24 , 26 These limitations have been underlined in a previous study in which PCT was quantified through peptide-based calibration using SIL peptides as internal standards.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing

“…Advancing the testing and detection capabilities concerning the misuse of growth hormone (GH) and its releasing factors has continued to be of particular interest to doping control laboratories despite the fact that at least the extent to which GH itself possesses performance‐enhancing properties is still unclear . The currently employed isoform test targeting the relative abundance of different GH variants in human serum has been model and template for a recently presented mass spectrometry‐based approach, which complements routinely applied tests by providing quantitative information on serum concentrations of 22 kDa, 20 kDa, and “total” GH . Here, 500 μL of serum is enriched with 15 N‐labeled 22 kDa and 20 kDa GH as internal standards, and the entire specimen is subjected to enzymatic hydrolysis using activated trypsin.…”

“…87 The currently employed isoform test targeting the relative abundance of different GH variants in human serum has been model and template for a recently presented mass spectrometry-based approach, which complements routinely applied tests by providing quantitative information on serum concentrations of 22 kDa, 20 kDa, and "total" GH. 88 Modifications of illicit peptidic drugs, mostly by N-terminally added amino acid residues, have been reported also for the growth hormone releasing peptide (GHRP)-2, GHRP-6, and ipamorelin. 39 Until the metabolism of these compounds is clarified, the structural alteration needs to be taken into consideration in routine doping control test methods, such as the multi-analyte procedure presented by Cuervo et al 89 and acetonitrile (solvent B).…”