Advances in the detection of peptide hormones for anti-doping and other forensic purposes

Student thesis: Doctoral ThesisDoctor of Philosophy


The reliable detection of peptide hormones in biological matrices represents a major challenge for antidoping, clinical and forensic laboratories due to their small concentration in biological matrices, instability, and lack of knowledge about their metabolism or degradation. Both insulin and growth hormone releasing hormone (GHRH) and their synthetic analogues are prohibited in sport in and out of competition according to World Anti-Doping Agency (WADA) regulations, with a requirement on laboratories to detect very small concentrations in human blood and/or urine. However, the lack of knowledge about the metabolism of GHRH synthetic analogues and the loss/instability of insulins in biological matrices makes their analytical detection difficult. Hence, investigation of metabolism in the case of GHRH as well as insulin degradation products, especially in whole and in haemolysed blood, is needed. Indirect evidence of the prohibited administration of intact peptides via detection of their metabolites or degradation products in biological specimens may represent a valid alternative analytical methodology.

Previously identified in vitro metabolites of GHRH synthetic analogues (sermorelin, tesamorelin, CJC-1295, CJC-1295 with DAC) consisted of a library of nineteen selected peptides. These have been investigated starting from their chemical synthesis, purification, and characterisation. In addition, the commercially available metabolite (sermorelin-(3-29)-NH2) and all four parent peptide hormones have been included in this study. These 24 peptides have been used as reference materials to develop and validate a sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) method that enabled their detection in fortified urine samples at or below the WADA threshold (1 ng/mL). Solid phase extraction was optimised for sample preparation and target analytes were detected by LC-MS/MS. The method is currently undergoing validation in the Drug Control Centre, the WADA accredited anti-doping laboratory at King’s College London, prior to its implementation in the routine testing for GHRH analogues.

Insulin is unstable in both sample collection devices and biological matrices disappearing rapidly. Furthermore, commercial immunoassays fail to detect insulin analogues reliably in haemolysed blood. The aims of the second part of this PhD project were to investigate the causes of this disappearance, to identify any degradation products in haemolysed and whole blood and to use this information to develop improved methods for the detection of insulin analogues. This project focused on Humalog® (lispro), one of the insulin synthetic analogues. As a starting point, lispro was incubated in whole blood and haemolysed blood at room temperature. As a result, fifteen degradation products were identified by liquid chromatography-high resolution mass spectrometry and characterised by tandem MS. Chemical synthesis of the B-chain and some of its degradation products was straightforward while for one the synthesis was complicated by diketopiperazine by-product formation. This issue was resolved by using a new resin with more steric hindrance than the one used for the synthesis of the other B-chain degradation products. On the other hand, nine degradation products (A-chain) containing four cysteine residues proved to be a very challenging synthetic workflow. Three different chemical routes were designed and applied to achieve regioselective disulphide bonding. The nine degradation products were successfully synthesised, and then purified and characterised. The synthesised materials were employed to develop a sensitive LC-tandem MS analytical method for lispro detection in blood samples. Our findings will help to improve the analytical methods used to prove administration of lispro (and other insulin analogues) for anti-doping purposes or for the investigation of suspicious deaths associated with overdose.
Date of Award1 Oct 2023
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorVincenzo Abbate (Supervisor), Ivana Gavrilovic (Supervisor) & David Cowan (Supervisor)

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