Detection of seventy-two anabolic and androgenic steroids and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry in multiplexed targeted MS2 mode and gas chromatography-tandem mass spectrometry.

The research paper presents the development of an improved method for the detection of anabolic and androgenic steroids (AAS) in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry and gas chromatography-tandem mass spectrometry. This developed technique is capable of detecting 72 different steroids in a single sample run.

Use of Anabolic and Androgenic Steroids in Equine Sports

• The paper begins with establishing the context, explaining how Anabolic and Androgenic Steroids (AAS) are forbidden substances in both human and equine sports. These substances, often administered intramuscularly in esterified forms, are designed to increase muscle strength and performance.
• Noting the need for effective detection methods to ensure the integrity of the sport and health of the animals, the authors highlight their previous work with an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS) method which successfully detected 48 different types of AAS and/or their esters in horse hair samples.

Improvement in Steroid Detection: Single-Injection Multiplexed Targeted MS Mode

• The research paper presents an advanced version of the previous method where an UHPLC/HRMS process employs multiplexed targeted MS mode.
• This advanced method allows the detection of 65 different AAS in a single injection, which offers an improvement in efficiency and detection scope compared to the older two-injection method.

Gas Chromatography-Tandem Mass Spectrometry (GC/MS/MS) Method

• In addition to the UHPLC/HRMS method, the authors also establish a GC/MS/MS method to detect seven additional AAS substances that were not within the detection range of the UHPLC/HRMS method.
• This secondary method involves the use of selected reaction monitoring (SRM) mode and requires chemical modification of the sample with pentafluoropropionic anhydride before analysis.

Results and Implications

• As a result, the combination of these two methods allows the detection of a total of 72 different AAS and/or their esters from a single sample.
• The newly developed method delivers highly accurate and precise results with limits of detection down to the sub to low ppb levels.
• The authors verified the effectiveness of the method by testing hair samples from out-of-competition horses and confirming the presence of specific AAS substances.