Liquid chromatography – high resolution mass spectrometry-based metabolomic approach for the detection of Continuous Erythropoiesis Receptor Activator effects in horse doping control.

The research article investigates whether potential metabolomic changes could be used to detect the illegal use of Erythropoiesis Stimulating Agents (ESAs), developed to enhance athlete’s endurance, in horse doping control. The study utilised high-resolution mass spectrometry and liquid chromatography to identify any potential biomarkers and found the approach could detect use of these agents for significantly longer than traditional methods.

Research Methodology

• The study was carried out on three thoroughbred horses, each administered with 4.2μg/kg of Mircera, also known as a Continuous Erythropoiesis Receptor Activator (CERA), and are a type of ESA.
• Blood and urine samples from the subjects were collected for analysis.
• The haematological parameters were also monitored throughout the study, this included the concentration of CERA in the plasma which was measured using an Enzyme-Linked Immunosorbent Assay (ELISA).
• With the use of Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC-HRMS), urine and plasma metabolic fingerprints of the subject horses were recorded in both positive and negative mode.

Data Analysis and Results

• The data collected from the samples were normalized and analysed using multivariate statistics to create Orthogonal Partial Least Squares (OPLS) models.
• The study found that the concentration of Hemoglobin and hematocrit in the horses significantly increased after the administration of CERA. Interestingly, reticulocytes did not show a similar increase.
• The concentration of CERA in the plasma had a high peak followed by a slow decrease until it became undetectable after an unidentified number of days (represented by D in the abstract).
• The OPLS models constructed from the multivariate statistics were able to discriminate between pre and post-administration plasma and urine samples up to 74 days after administration. This is 43 days longer than the detection capability of the conventional ELISA method.
• Through the reduction and detailed examination of the variables (ions), the researchers identified some potential candidate biomarkers.

Conclusion

• The research successfully identified potential biomarkers that could indicate the use of ESAs like CERA in horses.
• The LC-HRMS-based metabolomic approach proposed by the study proved superior to the traditional ELISA method, with the potential to detect the misuse of ESAs in horse doping control for up to 74 days post administration.
• The findings of this study pave the way for further research that could lead to enhanced methods for detecting doping in sport animals.