Advances in the detection of growth hormone releasing hormone synthetic analogs

The administration of growth hormone releasing hormone (GHRH) and its synthetic analogs is prohibited by the World Anti-Doping Agency (WADA). Although there is evidence of their use, based on admissions and intelligence, they do not appear to have been found in anti-doping samples by WADA accredited laboratories. This might be due to their small concentration in urine and limited knowledge about their metabolism, especially for unapproved synthetic analogs. This study investigates the in vitro metabolism and detection of four of the larger GHRH synthetic analogs (sermorelin, tesamorelin, CJC-1295, and CJC-1295 with drug affinity complex) in fortified urine. Nineteen major in vitro metabolites were identified, selected for synthesis, purified, and characterized in house. These were used as reference materials to spike into urine together with commercially available parent peptides and a metabolite of sermorelin (sermorelin(3-29)-NH₂ ) to develop a sensitive liquid chromatography-tandem mass spectrometry method for their detection to help prove GHRH administration. Limits of detection of the target peptides were generally 1 ng/ml (WADA required performance limit) or less.