Growth Hormone Isoform-Differential Mass Spectrometry for Doping Control Purposes

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 in enzymatic protein cleavage, followed by two-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 to independent confirmation, if combined with the antibody-based isoform differential test presently used in practice. To probe for 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 22 kDa fraction was within a range of 80-85%, either, or at 100%, the latter simulating an administration of 22 kDa-GH. Mean deviation of 22 kDa-fractions found from expectation was within less than 3%. Beyond this, results by antibody-free isoform-differential MS, as described, were in line with those of the WADA-approved antibody-based test for 18 native sera and 3positive 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. 20 kDa-GH as additional measurand, next to 22 kDa- and total-GH, however, provides useful extra-information, as it directly indicates presence or absence of a non-22 kDa-GH form.