Identification and differentiation of somapacitan, a long-acting growth hormone derivative, and recombinant human growth hormone in equine plasma by LC-HRMS for doping control purpose.

Overview

• This research developed a sensitive and specific test method to identify and distinguish between somapacitan, a long-acting growth hormone derivative, and recombinant human growth hormone (rhGH) in horse plasma.
• The method uses liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) to support doping control in equine sports.

Background

• Somapacitan and recombinant human growth hormone (rhGH) are protein-based therapeutics primarily used to treat growth disorders and growth hormone deficiency in humans.
• Both substances have the potential to enhance athletic performance in horses and are therefore banned substances in horseracing and equestrian sports.
• International bodies regulating horse sports, such as the International Federation of Horseracing Authorities and the Fédération Equestre Internationale, prohibit these substances at all times.

Objective of the Study

• To develop and validate a robust analytical method capable of detecting and differentiating somapacitan and rhGH in equine plasma.
• This method would aid doping control laboratories in identifying illegal use of these growth hormones in horses.

Methodology

• Sample Preparation:
  • Equine plasma samples undergo ammonium sulfate precipitation to precipitate proteins.
  • C4 solid-phase extraction is used to purify the samples further.
  • Chloroform/methanol precipitation is applied for additional cleanup.
  • Protein samples are subjected to trypsin digestion, breaking the proteins into peptide fragments.
• Analytical Technique:
  • LC-HRMS (Liquid Chromatography – High Resolution Mass Spectrometry) is used to analyze the resulting peptide fragments.
  • The approach focuses on unique peptide fragments generated from somapacitan and rhGH to discriminate between the two hormones.

Key Findings

• Unique Peptide Identification:
  • The T10 peptide fragment is unique to somapacitan and can be detected with its side chain structure intact, allowing specific identification.
  • For rhGH, its own unique T10 peptide fragment was also detected, enabling differentiation from somapacitan.
• Sensitivity and Limits:
  • Limit of Identification (LOI, confirmation level):
    • Somapacitan: 5 ng/mL
    • rhGH: 2 ng/mL
  • Limit of Detection (LOD, minimal detectable amount):
    • Somapacitan: 5 ng/mL
    • rhGH: 1 ng/mL
• Enhanced Confirmation:
  • Peptides T1, T8, and T9 are shared between somapacitan and rhGH.
  • Using these peptides, the identification of somapacitan can be achieved with increased sensitivity (LOI down to 2 ng/mL and LOD down to 1 ng/mL).

Validation and Robustness

• The method was extensively validated demonstrating:
  • Specificity – accurately distinguishing somapacitan from rhGH and other proteins.
  • Identification capability – reliable confirmation of the presence of these hormones at low concentrations.
  • Robustness – method stability under different experimental conditions.
  • Precision and reproducibility – consistent results across multiple tests.

Applications and Implications

• The validated LC-HRMS method enables doping control laboratories to detect illegal administration of somapacitan and rhGH in horses with high sensitivity and specificity.
• This tool supports enforcement against the misuse of performance-enhancing growth hormones in equestrian sports.
• The methodology can be applied to:
  • Analyze post-administration samples collected during planned somapacitan and rhGH administration trials.
  • Test samples from future suspected doping cases in horse competitions.