Determination of testosterone:epitestosterone ratio after pentafluorophenyldimethylsilyl-trimethylsilyl derivatisation using gas chromatography-mass spectrometry in equine urine.

The research describes an effective method for determining the ratio of testosterone to epitestosterone in horse urine using gas chromatography-mass spectrometry.

Research Objective and Methodology

• The study aimed to develop a highly specific method for measuring the testosterone:epitestosterone ratio in equine, or horse, urine. This method employs gas chromatography-mass spectrometry (GC-MS) with stable isotope internal standards.
• This process involves extraction using Serdolit Pad-1 resin, followed by enzymatic hydrolysis. Then the samples undergo chemical derivatisation before instrumental analysis. The procedure aims to increase the efficiency of identifying and quantifying these two hormonal substances.
• Two mixed derivatives, 3-trimethylsilyl-17-pentafluorophenyldimethylsilyl ether testosterone and epitestosterone, were found to possess excellent analytical properties, making them ideal for this scientific approach.

Specificity of Derivatisation Method

• The research highlights the specificity of the derivatisation method, which makes use of a unique characteristic of steroids: the selective exchange of the alcoholic flophemesyl ether for the trimethylsilyl ether.
• This specificity guarantees the accuracy of the resulted figures as it minimizes the potential for other substances to interfere with the analysis.

Sensitivity and Sample Size

• The sensitivity and specificity of the mixed 3-TMS-17-flophemesyl derivatives allow for adequate determinations of testosterone and epitestosterone even in small urine samples (as small as 5 ml), including urine from mares (female horses).
• This indicates that the methodology presents viable applications in practical settings, where sample sizes may be limited.

Repeatability and Reproducibility

• The study reported promising repeatability results for testosterone and epitestosterone measurements, standing at 6.2% and 5.7% respectively.
• Similarly, the reproducibility of the technique ranged between 6.4 and 8.7%.
• These findings help in distinguishing this method as reliable, indicating that it can consistently produce accurate results across different running sessions.