Glycine‐modified growth hormone secretagogues identified in seized doping material

Abstract

A number of unknown pharmaceutical preparations seized by Danish customs authorities were submitted for liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis. Comparison with reference standards unequivocally identified the content of the powders as analogs of the growth hormone secretagogues GHRP-2 (Pralmorelin), GHRP-6, Ipamorelin, and modified growth hormone releasing factor (modified GRF 1-29), which can be used as performance-enhancing substances in sports. In all cases, the detected modification involved the addition of an extra glycine amino acid at the N-terminus, and analytical methods targeting growth hormone secretagogues should hence be updated accordingly.

Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide and non-peptide doping agents by means of LC-HRMS

“…From 2016 to 2017, the number of GHRF testing was increased by 17% [14]. Recently, glycine-modified analogues of GHRP-2, GHRP-6, and ipamorelin were identified in seized material [15][16][17]. All of these compounds are classified as non-threshold substances and are prohibited by WADA at all times [18] (in-and out-ofcompetition).…”

Analysis of seized peptide and protein‐based doping agents using four complimentary methods: Liquid chromatography coupled with time of flight mass spectrometry, liquid chromatography–ultraviolet, Bradford, and immunoassays

“…The use of LC-TOF-MS for detection of peptides and proteins offers the possibility of structural elucidation in samples with unknown peaks, either by using the exact mass to identify mass changes of known peptide modifications or by fragmenting the peptide and identifying the individual amino acid residues by de novo sequencing. 2 However, it can be difficult to have a general sample preparation that provides optimal ionization of all peptides and proteins, and thus, it is possible to have false negatives if the substance is not ionized properly. It therefore compliments the LC-UV method, where the analytes are identified in solution.…”

Annual banned‐substance review – Analytical approaches in human sports drug testing

“…The best possible analyte recovery is one major aspect of initial testing procedures, and improving the enrichment of peptidic substances from biological matrices by using ion‐pair SPE was demonstrated by Judak et al Acidifying urine with formic acid or trifluoroacetic acid was shown to enhance the recovery of nine model peptides, allowing for lowering the test methods’ LODs in doping controls as is frequently required to cope with new additions to the illicit drug market such as the recently detected analogs of growth hormone releasing peptides (GHRPs). On various occasions, the detection of GHRPs and a synthetic version of the GH releasing hormone (GHRH) modified at the N‐terminus by attaching an additional glycine residue was reported . Whether this modification is eliminated by in vivo metabolic reactions yielding established target analytes of GHRPs and GHRH remains to be shown; if the modification is sufficiently resistant to proteolytic activities in vivo, test methods will potentially necessitate adaptation.…”