Horse metabolism and the photocatalytic process as a tool to identify metabolic products formed from dopant substances: the case of sildenafil.

Two horses were treated with sildenafil, and its metabolic products were sought in both urine and plasma samples. Prior to this, a simulative laboratory study had been done using a photocatalytic process, to identify all possible main and secondary transformation products, in a clean matrix; these were then sought in the biological samples. The transformation of sildenafil and the formation of intermediate products were evaluated adopting titanium dioxide as photocatalyst. Several products were formed and characterized using the HPLC/HRMS(n) technique. The main intermediates identified in these experimental conditions were the same as the major sildenafil metabolites found in in vivo studies on rats and horses. Concerning horse metabolism, sildenafil and the demethylated product (UK 103,320) were quantified in blood samples. Sildenafil propyloxide, de-ethyl, and demethyl sildenafil, were the main metabolites quantified in urine. Some more oxidized species, already formed in the photocatalytic process, were also found in urine and plasma samples of treated animals. Their formation involved hydroxylation on the aromatic ring, combined oxidation and dihydroxylation, N-demethylation on the pyrazole ring, and hydroxylation. These new findings could be of interest in further metabolism studies.

Rapid test by liquid chromatography/tandem mass spectrometry to evaluate equine urine reactivity towards 17beta-OH steroids.

Bacteria frequently found in equine urine samples may cause degradation of 17beta-OH steroids. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to evaluate the microbiological contamination of equine urine as a marker of poor storage conditions. Norethandrolone was used as the internal standard, and the linearity, sensitivity, precision and accuracy of the method were evaluated. 17beta-OH oxidation was demonstrated for testosterone, nandrolone, trenbolone and boldenone, but did not occur in alpha-epimers such as alpha-boldenone and epitestosterone, demonstrating the stereoselectivity of the reaction. A rapid test was performed by spiking one of the four 17beta-OH steroids in samples of diluted equine urine. The steroids were transformed into their respective ketones in the presence of bacterial activity. The test allows direct injection of diluted samples into the LC/MS system, without the need for prior extraction. Results show that the best method of storage is freezing at -18 degrees C. Urine specimens should be analyzed as soon as possible after thawing. This allows bacterial degradation of equine urine to be arrested temporarily, so that the urine can be used for qualitative or quantitative analysis of 17beta-OH steroids.