Developing innovative markers to enhance the detection windows for small peptide hormones

Abstract

Peptide hormones, listed among the prohibited substances by the World Anti-Doping Agency (WADA), present challenges in detection. Peptide molecules are known to have low detecting stability due to their short half-life and low concentration in the body. Additionally, transportation and storage of urine samples may lead to alterations in these substances. The World Anti-Doping Agency provides guidelines for urine sample transportation, emphasizing concerns about temperature and time. However, fluctuating temperatures during transport, especially in hot weather and unforeseen circumstances, can pose challenges. In this study, Ibutamoren, Ipamorelin, GHRP-2, GHRP-6, and Hexarelin were selected from prohibited peptide molecules and incubated in urine for detection after 0 and 48 hours at 60 degrees Celsius. GHRP-2 was selected for further study with its notable rate of decrease and presence in the Adverse Analytical Findings (AAFs) of the 2021 Anti-Doping Testing Figures. The study utilized Q-Exactive mass spectrometry to observe potential altered products in GHRP-2 incubated samples. The ion mass spectrum of the altered product was analyzed through Q-Exactive LC-MS/MS and included fragment ions of GHRP-2, suggesting the that the altered product is likely originated from GHRP-2. The findings of this study can suggest a strategy to enhance the detection window after secretion, even in the presence of potential temperature fluctuations in the sample.