Analysis of methyloxime derivatives of intact esters of testosterone and boldenone in equine plasma using ultra high performance liquid chromatography tandem mass spectrometry.

This research presents a method for analyzing steroid esters in horse blood samples using advanced techniques in chromatography and mass spectrometry. The method aids in detecting the illegal use of anabolic steroids in horses, even when the steroids are naturally occurring in the body.

Overview of the Research

• The primary focus of this research was on the analysis of certain esters of testosterone and boldenone in horse blood. Ester compounds of these anabolic steroids are often administered through intramuscular injections, and their detection can provide evidence of illegal steroid use.
• The researchers adopted an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MSMS) method to analyze these steroid esters. This sophisticated technique separates and identifies chemical compounds based on their mass and charge.
• During the process, the esters were extracted from horse blood, after which they were treated with methoxyamine hydrochloride to create methyloxime derivatives. A substance called metenolone enanthate served as an internal standard for the analysis.

Findings

• The researchers managed to establish a detection limit for all the analyzed steroid esters at 1-3 picograms per milliliter (pg/mL). This is based on a signal to noise ratio of 3:1. The lower limit for their quantification was 5 pg/mL when extracting from 2 milliliters of blood.
• Recovery rates were high, ranging from 85-97%, except for testosterone decanoate, which had a recovery rate of 62%. This indicates the effectiveness of the extraction and quantification process during the analysis.
• Additionally, the intra-day coefficient of variation for the analysis was less than 9.2% at 40 pg/mL and less than 6.0% at 400 pg/mL. This implies a high precision in the method developed by the researchers.
• Ultimately, this method successfully confirmed the presence of testosterone esters in injected horse blood samples. This serves as proof of its effectiveness in detecting illegal use of steroids in racehorses.

Implications

• This research provides a reliable method to detect illegal steroid use in animals, particularly racehorses. Given that these steroids may occur naturally in the horse body, the method’s ability to discern between naturally occurring steroids and injected ones is crucial.
• The adoption of such methods could lead to more fair play in competitive racing and other horse-related sports.