Determination of ghrelin and desacyl ghrelin in human plasma and urine by means of LC-MS/MS for doping controls / Andreas Thomas, Sophia Krombholz, Carina Wolf, Mario Thevis. - (Drug Testing and Analysis (2021) 11 October)
- PMID: 34633773
- DOI: 10.1002/dta.3176
The hunger hormone ghrelin (G) is classified as prohibited substance in professional sport by the World Anti-Doping Agency (WADA), due to its known growth hormone releasing properties. The endogenous bioactive peptide consists of 28 amino acids with a caprylic acid attached to serine at position 3. Within this study it was aimed to develop methods to determine G and desacyl ghrelin (DAG) in plasma and urine by means of LC-MS/MS. Two strategies were applied with a bottom-up approach for plasma and top-down analyses for urine. Both sample preparation procedures were based on solid-phase extraction for enrichment and sample clean-up. Method validation showed good results for plasma and urine with limits of detection (LODs) for G and DAG between 30 and 50 pg/mL, recoveries between 45-50 %, and imprecisions (intra- and inter-day) between 3 - 24 %. Plasma analysis was also valid for quantification with accuracies determined with ~100 % for G and ~106 % for DAG. The minimum required performance level for doping control laboratories is set to 2 ng/mL in urine, and the herein established method yielded acceptable results even at 5 % of this level. As proof-of-concept, plasma levels (G and DAG) of healthy volunteers were determined and ranged between 30 and 100 pg/mL for G and 100 - 1200 pg/mL for DAG. In contrast to earlier reported studies using ligand binding assays for urinary G and DAG, in this mass spectrometry-based study no endogenous urinary G and DAG were found, although the LODs should enable this.