Detection and confirmation of hypoxia-inducible factor activating agents in equine plasma and urine by liquid chromatography coupled to mass spectrometry.

Overview

• This study developed and validated a method using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) to detect and confirm substances that activate hypoxia-inducible factor (HIF) in horse plasma and urine.
• HIF-activating agents are performance-enhancing substances that stimulate erythropoietin gene transcription and are banned in sports.

Background

• Hypoxia-inducible factors (HIFs) regulate cellular responses to low oxygen and can enhance erythropoietin (EPO) production.
• Increased EPO stimulates red blood cell production, improving oxygen delivery and athletic performance.
• Because of their performance-enhancing effects, HIF-activating agents are prohibited in competitive sports, including horse racing.

Aims and Objectives

• Develop a sensitive and reliable analytical method to detect 11 specific HIF-activating substances in equine plasma and urine.
• Optimize chromatographic and mass spectrometric conditions to maximize detection while minimizing interference and carryover.
• Validate limits of detection (LOD) and limits of confirmation (LOC) for the substances.
• Apply the method to real samples from horses administered a HIF activator.

Methodology

• Analytical Technique:
  • Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) was employed.
  • Both positive and negative ion modes were tested; negative ion mode was predominantly used for detection without matrix interference.
• Sample Preparation:
  • Solid-phase extraction (SPE) was used to extract analytes from plasma and urine matrices.
  • Optimization of mobile phase:
    • Ammonium formate, initially considered for the mobile phase, suppressed analyte signals and was therefore excluded.
    • The initial organic content in the mobile phase gradient and sample reconstitution solutions were optimized to reduce analyte carryover between runs.
• Calibration and Validation:
  • Ten calibration levels spanning 10 to 10,000 pg/mL were prepared for all 11 analytes in both plasma and urine.
  • Limits of detection (LOD) were evaluated using HRMS:
    • 10 to 50 pg/mL for plasma samples.
    • 10 to 200 pg/mL for urine samples.
  • Limits of confirmation (LOC), assessed by parallel reaction monitoring:
    • 50 to 200 pg/mL for plasma (with exceptions for daprodustat and IOX 3 at 500 pg/mL).
    • 20 to 500 pg/mL for urine (exceptions for daprodustat and IOX 3 at 1000 pg/mL).

Results

• The method effectively detected and confirmed all 11 HIF-activating agents with minimal interference from plasma or urine matrices.
• No significant carryover was observed after optimization of chromatographic conditions.
• LC-HRMS sensitivity was sufficient for detection at low picogram-per-milliliter levels.
• The method was successfully applied to plasma and urine samples from a horse orally administered JNJ 42041935, a known HIF activator, demonstrating practical applicability.

Significance and Applications

• The developed method enables robust doping control testing for HIF-activating substances in equine sports.
• It enhances the ability of regulatory agencies to detect prohibited erythropoietin-stimulating drugs, thus preserving fair competition.
• The approach can be adapted for use in other biological matrices or species with similar doping control needs.

Summary

• This research successfully established a highly sensitive and specific LC-HRMS method for detecting 11 HIF activators in horse plasma and urine.
• Careful optimization of sample preparation and chromatographic parameters minimized matrix effects and analyte carryover.
• The validated method supports doping control efforts and was shown to work on samples from treated horses, paving the way for effective anti-doping monitoring in equine athletes.