Simultaneous separation and determination of 16 testosterone and nandrolone esters in equine plasma using ultra high performance liquid chromatography-tandem mass spectrometry for doping control.

This research explores a method for detecting and identifying 16 different testosterone and nandrolone esters used for doping in racehorses. By utilising ultra high performance liquid chromatography and tandem mass spectrometry, the method can ascertain the presence of these substances, thus aiding with the creation of doping control regulations.

Explanation of the Research

• The article focuses on discovering a process to identify 16 types of testosterone and nandrolone esters found in equine plasma. These substances have proven useful in heightening the biological concentration of steroids in racehorses, enhancing their athletic performances, and thus is of interest in establishing anti-doping regulations.
• The newly presented method uses ultra high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). This technology combination provides a sensitive, robust, and reproducible means for separating, screening, quantifying, and confirming the presence of these substances in equine plasma.

Methodology

• Extraction of analytes from the equine plasma was done through liquid-liquid extraction, using a mix of methyl tert-butyl ether and ethyl acetate (50:50, v/v).
• Separation occurred on a sub-2 micron C(18) column, with detection achieved by a triple-quadrupole mass spectrometer under positive electrospray ionization mode with selected reaction monitoring (SRM).
• The mobile phase used 2 mM ammonium formate and methanol. For quantification, deuterium-labeled testosterone enanthate and testosterone undecanoate were utilized as dual-internal standards.
• The method’s limits of detection (LOD) and quantification (LOQ) ranged from 25-100 pg/mL and 100-200 pg/mL respectively, and the linear dynamic range of quantification stood at 100-10,000 pg/mL.
• The validity of the analytes’ presence was confirmed by matching the retention time with mass spectrometric ion ratios that resulted from MS/MS product ions, with the limit of confirmation (LOC) within 100-500 pg/mL.

Notability of Research

• This research provides a solid foundation for further understanding and controlling doping in horse racing. This approach is notable because it provides a highly sensitive and selective method for the detection of doping agents.
• The method developed and presented can play a significant role in supporting regulatory authorities in their efforts to maintain clean and fair competitions in racehorse events.