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 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.