Development of a Samll Peptide, 2 kDa mixed standard

Code: 17A11CG 

An increasing number of peptidic analytes are being added to the WADA prohibited list, multi-residue testing procedures have become routine for the detection of many of these compounds and their metabolites by LCHRMS or LCMSMS in anti-doping laboratories. We propose to develop a multi-analyte mixed standard, covering the majority of lutenising hormone releasing hormones (LHRHs), growth hormone releasing peptides (GHRPs), growth hormone secretagogues (GHSs) and their metabolites. The multi-analyte standard would be available to all WADA accredited laboratories as a standardised mix across all laboratories world-wide.

Main Findings: 

The aim of this study was to prepare a small peptide (< 2 kDa) mixed standard and determine the best storage conditions to maintain the stability of all peptides within the standard. In this study, ASDTL commissioned Auspep Pty Ltd (Tullamarine, Australia) to synthesise and prepare two small peptide (<2 kDa) mixed standards. The first (SPMIX1) contains 41 small peptides and metabolites, while the second (SPMIX2) contains the same 41 small peptides but also utilises bovine insulin to act as a carrier peptide to minimise any binding of the peptides to the glass vials. The peptides were selected based on published metabolism studies to include the parent peptides and the main metabolites excreted. Minor metabolites and parent peptides that were found to be completely metabolised were not included so that the cost of the mixed standard could remain as low as possible to ensure all WADA accredited laboratories would have access.
Initial homogeneity testing was performed on both SP Mixes. The results obtained for the three peptides for which isotopically labelled internal standards were available - Leuprolide, GHRP-2 and GHRP-2 (1-3)-OH, were used to evaluate the homogeneity of SPMIX1 and SPMIX2 as they provided the most precise data set. The repeatability precision of the measurements was sufficiently small as to show a statistically significant inhomogeneity between vials. Analysis of variance (ANOVA) fo the homogeneity results obtained for each analyte was used to indicate, if at a level of confidence of 95%, whether the variability observed between-bottles was significantly different to the variability observed within bottles (duplicate analysis). The uncertainty in the mass of peptide in each vial due to inhomogeneity was conservatively estimated to be ≤2.52 % for SPMIX1 and ≤ 2.16 % for SPMIX2.
Stability assessment was carried out over a 12 month period under the following 4 different conditions for both SPMIX1 and SPMIX2: 1) in solution at -20°C; 2) in solution at -80°C; 3) dried prior to storage at -20°C; and 4) dried prior to storage at -80°C. The majority of peptides were best stored at  -80°C either in solution or dried, except for Hexarelin, its metabolites and AOD-9604 (7-16). At  -80°C, there was very little difference over the 12 month period between being stored in solution or dried for either SMPIX1 or SPMIX2, with dried peptides only marginally better. However for Hexarelin and its metabolites, drying the solutions prior to storage resulted in a decrease in analyte concentration, especially when stored at  -20°C with losses of 10-30% observed in both SP MIXees. Losses of around 15% were observed for AOD-9604 (7-16), when stored in solution at  -20°C but were not observed to the same extent under the other conditions tested.
The mixed peptide standards were distributed to all WADA accredited laboratories during the 38th Manfred Donike Workshop 2020. Each laboratory was asked to assess both SP MIXes against in-house mix standards with respect to the peptides included in the mix, the addition of a cattier peptide and whether it would be cost effective to purchase SP MIXes instead of buying individual standards. Eleven laboratories reported results and found the quality of the SP MIXes compared well to what they currently used and indicated they would be prepared to purchase SP MIXes in the future, provided additional peptides are added so that they are similar in composition to their current in-house peptide standard mixes. Only 2 laboratories noted a preference for bovine insulin to be added as a peptide carrier.