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

Abstract

A highly specific method is described for measuring the testosterone:epitestosterone ratio in equine urine by gas chromatography-mass spectrometry (GC-MS) with stable isotope internal standards. The procedure was based on Serdolit Pad-1 resin extraction, enzymatic hydrolysis, and chemical derivatisation prior to instrumental analysis. The mixed derivatives, 3-trimethylsilyl-17-pentafluorophenyldimethylsilyl ether (3-TMS-17-flophemesyl) testosterone and epitestosterone, were found to have excellent analytical properties. The specificity of the derivatisation method exploits a unique feature of steroids: the selective exchange of the alcoholic flophemesyl ether for the trimethylsilyl ether. The sensitivity and specificity of the mixed 3-TMS-17-flophemesyl derivatives allow adequate determinations of testosterone and epitestosterone, even in urine from mares, in 5 ml samples. The repeatability of testosterone and epitestosterone was 6.2 and 5.7%, respectively, and their reproducibility was in the range of 6.4–8.7%.