Characterization of equine urinary metabolites of selective androgen receptor modulators (SARMs) S1, S4 and S22 for doping control purposes.

Selective androgen receptor modulators, SARMs, constitute a class of compounds with anabolic properties but with few androgenic side-effects. This makes them possible substances of abuse and the World Anti-Doping Agency (WADA) has banned the entire class of substances. There have been several cases of illicit use of aryl propionamide SARMs in human sports and in 2013, 13 cases were reported. These substances have been found to be extensively metabolized in humans, making detection of metabolites necessary for doping control. SARMs are also of great interest to equine doping control, but the in vivo metabolite pattern and thus possible analytical targets have not been previously studied in this species. In this study, the urinary metabolites of the SARMs S1, S4, and S22 in horses were studied after intravenous injection, using ultra high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QToF-MS). Eight different metabolites were found for SARM S1, nine for SARM S4, and seven for SARM S22. The equine urinary metabolite profiles differed significantly from those of humans. The parent compounds were only detected for SARMs S4 and S22 and only at the first sampling time point at 3 h post administration, making them unsuitable as target compounds. For all three SARMs tested, the metabolite yielding the highest response had undergone amide hydrolysis, hydroxylation and sulfonation. The resulting phase II metabolites (4-nitro-3-trifluoro-methyl-phenylamine sulfate for SARMs S1 and S4 and 4-cyano-3-trifluoro-methyl-phenylamine sulfate for SARM S22) are proposed as analytical targets for use in equine doping control.

Quantification of malachite green in fish feed utilising liquid chromatography-tandem mass spectrometry with a monolithic column.

The purpose of this study was to develop a rapid and sensitive method for the quantification of malachite green (MG) in fish feed using LC-ESI-MS/MS with a monolithic column as stationary phase. Fish feed was cleaned using ultrasonic assisted liquid-liquid extraction. The separation was achieved on a Chromolith® Performance RP-18e column (100 × 4.6 mm) using gradient mobile phase composition of methanol and 0.1% formic acid at the flow rate of 1.0 ml min⁻¹. The analyte was ionised using electrospray ionisation in positive mode. Mass spectral transitions were recorded in selected reaction monitoring (SRM) mode at m/z 329.78 → m/z 314.75 with a collision energy (CE) of 52% for MG. The system suitability responses were calculated for reproducibility tests of the retention time, number of theoretical plates and capacity factor. System validation was evaluated for precision, specificity and linearity of MG. The linearity and calibration graph was plotted in the range of 15.0-250 ng ml⁻¹ with the regression coefficient of >0.997. The lower limits of detection and quantification for MG were 0.55 and 1.44 ng ml⁻¹, respectively, allowing easy determination in fish feed with accuracy evaluated as a percentage recovery of 92.1% and precision determined as % CV of < 5. The method was also extended to the determination of MG in an actual fish feed. The sensitivity and selectivity of LC-ESI-MS/MS using monolithic column offers a valuable alternative to the methodologies currently employed for the quantification of MG in fish feeds.

Cortisol production in sheep is influenced by the functional expression of two cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17) isoforms.

In commercial production systems, the full expression of the genetic potential of an animal is limited by its intrinsic and extrinsic environment. It is therefore necessary to include robustness as a breeding goal because robustness is defined as the ability of an animal to express a high production potential in a wide variety of environmental conditions. The ability of mammals to produce sufficient cortisol on stimulation of the hypothalamic-pituitary-adrenal (HPA) axis is vital in its adaptation to stress. The biosynthesis of cortisol is dependent on the enzymatic activity of the microsomal enzyme, cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17). Two isoforms for sheep (Ovis aries) CYP17, previously identified in 2 independent studies, differ by 2 nucleotides, resulting in 2 AA differences (Ser210Gly and Tyr464Asn). The present study investigates the effect of these differences on cortisol production as a function of the HPA axis activity by comparing the catalytic activities of these isoforms. The activities of the CYP17 isoforms were compared by expressing the enzymes in vitro. The kinetic constants, Vmax and Km, which were determined for pregnenolone and progesterone (in the absence of cytochrome b(5)), showed no significant difference (P > 0.05) between the CYP17 isoforms. In contrast, a time course of the metabolism of pregnenolone, 17-hydroxypregnenolone, and progesterone, assayed in the presence and absence of ovine cytochrome b(5) overexpression, showed significant differences (P < 0.05) between the isoforms. Wild-type 1 CYP17 (WT1, GenBank accession number L40335) yielded more cortisol precursors than wild-type 2 (WT2, GenBank accession number AF251388). Site-directed mutagenesis indicated that a tyrosine residue at position 464 of WT1 increased the 17α-hydroxylation of progesterone compared with an asparagine residue at that position of WT2. In a subsequent insulin-induced hypoglycemic stress test, the presence of WT1 resulted in a greater cortisol output from the sheep adrenal than the presence of WT2, as homozygous WT1/WT1 sheep produced more cortisol than heterozygous WT1/WT2 sheep. The SNP located within the WT1 allele may therefore have a potential application in marker-assisted selection of sheep exhibiting a greater release of cortisol from the adrenal gland in response to stressors.