Novel Biomarkers in Recombinant Human Growth Hormone Detection. LC-MRM-MS Method for Fibronectin Quantification in AntiDoping Routin

Code: 17A07GR 

The World Anti-Doping Agency (WADA) establishes two procedures to detect recombinant human growth hormone abuse (rhGH) in sports. The first, the “hGH Isoforms Test”, is based on the separate measurement of growth hormone (GH) isoforms using immunoassays. The second, the “hGH Biomarkers Test”, involves the analysis of two GH-responsive proteins, IGF-1 and P-III-NP, using either immunoassays and/or mass spectrometry (MS)-based techniques. Although being currently applied in routine analysis in anti-doping laboratories, both present some limitations. Main drawback of the “hGH isoforms Test” is the short window of opportunity whereas for the “hGH biomarkers Test” is their dependence of age and sex parameters and the more rapid elimination of IGF-1 compared to P-III-NP reducing retrospectivity.

Recent studies have described fibronectin (FN) as a long-term biomarker of rhGH abuse. These results have been obtained applying enzyme-linked immunoassays (ELISA) to serum and dried blood spots (DBS). Thus, the inclusion of FN in the evaluation of potential rhGH misuse would improve detection of cheating athletes. However, immunological tests suffer from some limitations that MS-based methodologies could overpass. This project aims to develop and validate a liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) method to measure FN concentrations in serum and DBS. The potential advantages of the MS-based
methodology will be assessed and special focus will be done on pre-analytical effects of storage conditions and the introduction of freeze-thaw cycles. Preliminary results from our laboratory show that FN determination is affected by these pre-analytical effects. The new method will be used to quantify FN in samples from an administration study with subjects treated with rhGH and in a control group of healthy subjects to obtain preliminary reference values and preliminary potential decision limits.

Main Findings:

Detection of recombinant human growth hormone (rhGH) abuse in sports is one of the major concerns of anti-doping agencies. The discovery of new biomarkers targeting rhGH misuse would help to improve the current methods carried out routinely in anti-doping laboratories. Recently, immunoreactive fibronectin (FN) protein has been described as a potential biomarker of rhGH abuse that could increase the window of opportunity of the already approved biomarkers of rhGH misuse, IGF-1 and P-III-NP.  Traditionally, immunoassays have been the method of choice to measure protein levels in biological samples. However, these antibody-based approaches could present analytical issues, as cross-reactivity, dependence on tertiary/quaternary protein structures or interferences with endogenous antibodies, which could impact final determination. To overcome immunoassay drawbacks, this project aimed to develop a mass spectrometry-based method to quantify FN in blood samples. A panel of four tryptic peptides (out of six initially proposed) located in different domains of FN sequence was selected to be used as surrogates of FN protein for quantification. The digestion process with trypsin was improved in view of the matrix characteristics and protein expected levels (in the range of µg/mL). Once optimized, an LC-MRM-MS method was developed and validated in accordance to the World Anti-Doping Agency in the Code International Standard for Laboratories (ISL) in terms of specificity, carryover, LoD (1.2 - 2.2 fmol/µL), LoQ (range 3 - 6 fmol/μL), linearity (mean r2 > 0.99), precision (within-run 3.0 - 3.9 %; between-run 9.5 - 44.3 %), accuracy (-16.7 - 5.7 %), recovery (mean 87.7 %), matrix effect (mean 14.6 %) and stability (-15.4 - 18.3 %). This procedure was applied to a set of samples from two different studies. The first was an administration study in which 10 subjects were treated for three consecutive days with a moderate dose of rhGH (0.027 mg/kg) and compared with two untreated subjects. The analysis of FN concentration using the mass spectrometry validated method indicated that no differences were present due to the drug treatment, in disagreement with previous results by immunoassay. For the second study, and because FN has been described as a potential biomarker of Dystrophic Muscular Duchenne (DMD), a population of DMD (mostly teenagers) patients was compared with a group of healthy control children. Similarly, no differences in FN levels were detected between both groups. However, when these samples were tested by immunoassay, significant differences were observed between both groups.  

These apparent divergences between results obtained with mass spectrometry and those using immunoassay could be potentially explained by the high structural and functional complexity of FN, which could impact differently depending on the antibodies’ selectivity used in immunoanalytical methods. In summary, our results spotlight the importance of method selection in peptide/protein quantification when intended to be used for bioanalytical discriminating purposes.