Investigations into the feasibility of routine ultra high performance liquid chromatography-tandem mass spectrometry analysis of equine hair samples for detecting the misuse of anabolic steroids, anabolic steroid esters and related compounds.

This research primarily focuses on assembling an effective method to detect the illegal administration of anabolic steroids and their esters in equine sports, using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to analyze horse mane hair samples.

Background and Research Objective

• The research addresses the challenge of detecting illicit use of anabolic steroids in horse racing; this is complicated by some steroids like testosterone and nandrolone, being naturally occurring or endogenous.
• Anabolic steroids are typically injected in an esterified form which enhances their effects and bioavailability compared to oral intake.
• The detection of an intact anabolic steroid ester in the system implies an illegal administration since these esterified forms do not occur naturally.
• While steroid esters can be detected in plasma samples, conventional methods using urine samples have not been successful.
• The key objective of this research was to analyze horse mane hair to detect the presence of anabolic steroid esters, potentially extending the window of detection for steroid abuse.

Methodology

• Equine mane hair samples were soaked in a particular buffer and the steroids, their esters, and related compounds were extracted through liquid-liquid extraction with a mix of hexane and ethyl acetate.
• Sample purification was conducted through solid phase extraction, followed by derivatisation with methoxylamine HCL for UHPLC-MS/MS analysis.
• This process was initially undertaken for four derivatives of testosterone and was later extended for an additional eighteen compounds.

Application and Findings

• The method’s effectiveness was tested by analyzing mane hair samples from a Thoroughbred mare given a 500mg dose of a mixed testosterone ester.
• Through this method, researchers successfully detected boldenone undecylenate and stanozolol in samples collected from suspicious screening results of post-race urine samples.
• The research also considered the use of segmental analysis for additional information about the timing of the steroid’s administration.

The study has successfully demonstrated the potential of using ultra high performance liquid chromatography-tandem mass spectrometry for analysing hair samples, as a feasible approach for detecting misuse of anabolic steroids, anabolic steroid esters and related compounds in equine sports.