A sensitive new LC-MS/MS method detected ghrelin in blood at very low levels but found no endogenous ghrelin in urine, contradicting earlier studies and raising questions about urine-based doping tests for this peptide.
0 pg/mL urinary ghrelinDespite a detection limit of 30-50 pg/mL, no endogenous ghrelin was found in urine — contradicting earlier immunoassay-based studies
What the researchers found
LC-MS/MS methods were successfully validated for ghrelin (G) and desacyl ghrelin (DAG) in plasma and urine. Limits of detection were 30-50 pg/mL with recoveries of 45-50% and imprecisions of 3-24%. Plasma quantification showed accuracies of ~100% for G and ~106% for DAG.
Healthy volunteer plasma levels ranged from 30-100 pg/mL for ghrelin and 100-1200 pg/mL for desacyl ghrelin. Critically, no endogenous ghrelin or desacyl ghrelin was detected in urine by this mass spectrometry approach, despite adequate sensitivity — contradicting earlier studies that used ligand binding assays. The method performed well at just 5% of WADA's minimum required performance level of 2 ng/mL in urine.
Why it matters
Ghrelin is a WADA-banned substance, but detecting its misuse requires reliable analytical methods. This study reveals that urine-based ghrelin testing — the standard approach for most doping controls — may be fundamentally flawed because ghrelin doesn't appear to be present in urine at detectable levels. This has significant implications for how anti-doping labs approach peptide hormone testing.
The numbers in context
LOD 30-50 pg/mL; recovery 45-50%; imprecision 3-24%; blood ghrelin 30-100 pg/mL; DAG 100-1200 pg/mL; WADA MRPL 2 ng/mL
How the study worked
Two LC-MS/MS strategies were developed: a bottom-up approach (peptide digestion) for plasma and a top-down approach (intact peptide analysis) for urine. Both used solid-phase extraction for sample enrichment and cleanup. Methods were validated for limits of detection, recovery, precision, and accuracy. Plasma from healthy volunteers was analyzed as proof of concept.
Who was studied
Healthy volunteers (number not specified)
What this study cannot tell us
Only a small number of healthy volunteers were tested — no samples from individuals who actually used exogenous ghrelin were available. The absence of ghrelin in urine contradicts earlier immunoassay-based findings but needs independent confirmation. The study did not assess whether exogenously administered ghrelin might appear in urine at higher concentrations than endogenous levels.
How to read the evidence
This is a method development and validation study with proof-of-concept testing in healthy volunteers. The analytical validation is rigorous, but the clinical and anti-doping implications need confirmation in larger studies including ghrelin-dosed subjects.
When this study was published
Published in 2021, this study reflects the current shift toward mass spectrometry-based peptide detection in anti-doping science. Its finding about urinary ghrelin absence remains an active topic in the field.
The bigger picture
As peptide hormones become increasingly common as both therapeutic agents and potential doping substances, anti-doping laboratories need to evolve from traditional immunoassay methods to more specific mass spectrometry approaches. This study exemplifies how improved analytical specificity can overturn previous findings and force a reassessment of testing strategies — a pattern likely to repeat as more peptide hormones come under anti-doping scrutiny.
Questions still open
- Were earlier reports of urinary ghrelin based on immunoassay cross-reactivity rather than actual ghrelin presence?
- Would exogenously administered ghrelin at doping-relevant doses produce detectable urinary metabolites?
- Should anti-doping testing for ghrelin shift entirely from urine to blood-based methods?
Common questions
Why is ghrelin banned in sports?
Why does it matter that no ghrelin was found in urine?
Read the original research
Determination of ghrelin and desacyl ghrelin in human plasma and urine by means of LC-MS/MS for doping controls.
Drug testing and analysis, 13(11-12), 1862-1870
Citation
Thomas, Andreas; Krombholz, Sophia; Wolf, Carina; Thevis, Mario. (2021). Determination of ghrelin and desacyl ghrelin in human plasma and urine by means of LC-MS/MS for doping controls.. Drug testing and analysis, 13(11-12), 1862-1870. https://doi.org/10.1002/dta.3176