Development and validation of general urine screening method for performance enhancing drugs in racehorses utilizing liquid chromatography-high resolution mass spectrometry (LC-HRMS).

Overview

• This research article reports the development and validation of a rapid and automated urine screening method to detect multiple performance-enhancing drugs in racehorses using liquid chromatography-high resolution mass spectrometry (LC-HRMS).
• The method effectively identifies over 250 prohibited drugs and their metabolites with high sensitivity and specificity in a short analysis time.

Background and Objective

• Drug regulatory authorities face challenges due to the complexity and continual updates in the market of performance-enhancing drugs used in horse racing.
• There is a need for a comprehensive screening assay that can quickly and reliably detect a wide range of prohibited substances in racehorses.
• The objective of this study was to develop and validate a high-throughput, automated LC-HRMS-based screening method for urine samples that covers multiple drug classes.

Methodology

• Sample Preparation:
  • Uses a small urine volume (250 µL) which improves efficiency and reduces sample handling.
  • Urine is subjected to enzymatic hydrolysis to release drug metabolites that may be conjugated.
  • Supported liquid extraction (SLE) performed using Biotage Isolute plates integrated with the Biotage Extrahera automated system ensures reproducible and high-throughput extraction.
  • Extracts are evaporated and reconstituted in 96-well plates, enabling compatibility with automated UHPLC injection systems.
• Analytical Instrumentation:
  • Chromatography performed on Vanquish UHPLC system coupled with an ACE Excel column provides high-resolution separation of analytes.
  • Detection carried out using Thermo Fisher Q-Exactive high-resolution mass spectrometer equipped with Heated Electrospray Ionization (HESI) source.
  • Full-scan mass spectrometry mode enables simultaneous detection and identification of a large number of drug targets without pre-selection.
• Identification Criteria:
  • Drugs and metabolites are identified based on matching retention time and accurate mass measurement.
  • Mass tolerance window is set at +/- 5 ppm to ensure confident identification and reduce false positives.

Validation

• The method was validated for over 250 drug targets and/or their metabolites.
• Key performance parameters assessed include:
  • Sensitivity: Ability to detect low concentrations of drugs to avoid false negatives.
  • Specificity: Ability to correctly identify true drug signals and exclude interferences.
  • Extraction Recovery: Efficiency of the sample preparation process in recovering analytes from urine.
  • Precision: Repeatability and reproducibility of the measurements across runs and samples.
• The total run time for the analytical method was optimized to 7 minutes, facilitating high-throughput screening.

Significance and Advantages

• The described method allows for rapid and automated analysis, supporting large numbers of samples typically encountered in racing regulation environments.
• The minimal sample volume requirement and use of 96-well plate formats streamline laboratory workflows.
• LC-HRMS full-scan capability theoretically allows incorporating an unlimited number of drug targets, providing flexibility to adapt to emerging doping compounds.
• High sensitivity and specificity ensure reliability and robustness when used for initial screening before confirmatory testing.
• This method addresses ongoing challenges related to the evolving landscape of performance-enhancing drugs in equine sports by providing a comprehensive, adaptable approach.