Quantification of 19-nortestosterone sulphate and boldenone sulphate in urine from male horses using liquid chromatography/tandem mass spectrometry.

The research article details an investigation into the misuse of the anabolic steroid 19-nortestosterone in male horses. The researchers created and tested a method for detecting 19-nortestosterone in horse urine, by analysing a substance created when the steroid is metabolized, and assessing the possibility of using this as a threshold substance to detect steroid misuse.

Research Goals

• The primary objective of this study was to develop and test a method of using liquid chromatography/tandem mass spectrometry (LC/MS/MS) to directly analyse 19-nortestosterone-17beta-sulphate, a metabolite of the anabolic steroid 19-nortestosterone.
• The researchers also aimed to investigate whether this method could be useful in detecting the misuse of 19-nortestosterone in male horses.

Methods and Approaches

• The researchers addressed the high concentration of the sulphate conjugates of female sex hormones in male equine urine, which could interfere with the analysis, by using a selective hydrolysis technique. They used glucuronidase extracted from Helix pomatia, a species of snail, which showed little to no activity for alkyl sulphates, including 19-nortestosterone-17beta-sulphate.
• The phenolic steroids were then removed, and the remaining substances were analysed using the LC/MS/MS method.
• This method also allowed for the quantification of the sulphate conjugate of boldenone, another anabolic steroid found naturally in male horses, which also has potential for abuse in sports.

Results

• The study found that 19-nortestosterone-17beta-sulphate is present in male horse urine as an endogenous substance – one that originates from within the organism.
• However, the researchers found that the levels of this substance in the urine of “normal” (i.e., non-steroid-using) horses were low.
• This indicates that, despite this substance’s presence as a naturally occurring compound, it might be possible to use its concentration levels as a threshold substance for detecting steroid misuse in male horses.
• The paper presents the results of this study, along with a detailed account of the development and validation of the LC/MS/MS method for detecting 19-nortestosterone-17beta-sulphate and boldenone sulphate in horse urine.