Evidence of boldenone, nandrolone, 5(10)-estrene-3ß-17α-diol and 4-estrene-3,17-dione as minor metabolites of testosterone in equine.

The detection of boldenone, nandrolone, 5(10)-estrene-3ß,17α-diol, and 4-estrene-3,17-dione in a urine sample collected from a gelding having been treated with testosterone (500 mg 'Testosterone Suspension 100', single dose, injected intramuscularly) in 2009 led the authors' laboratory to suspect that these 'testicular' steroids could be minor metabolites of testosterone in geldings. Administration trials on six castrated horses with Testosterone Suspension 100 confirmed that low levels of boldenone, nandrolone, 5(10)-estrene-3ß,17α-diol, and 4-estrene-3,17-dione could indeed be detected and confirmed in the early post-administration urine samples from all six geldings. Although boldenone has been reported to be present in urine after testosterone administration, there has been no direct evidence reported that boldenone, nandrolone, 5(10)-estrene-3ß,17α-diol, and 4-estrene-3,17-dione are metabolites of testosterone in geldings. Subsequent in vitro experiments involving the incubation of testosterone with horse liver microsomes, liver, adipose and muscle tissues, and adrenal cortex homogenates failed to provide evidence that these four substances are minor metabolites of testosterone. An administration trial using 'Testosterone Suspension 100' supplemented with 13 C-labelled testosterone (500 mg, 1:1 ratio, injected intramuscularly) was performed. The similarities of the excretion curves of 12 C-testosterone and 13 C-testosterone in urine suggest that there was minimal kinetic isotope effect. 13 C-Labelled boldenone, nandrolone and 4-estrene-3,17-dione were detected but not 5(10)-estrene-3ß,17α-diol and its 13 C-counterpart. This is the first unequivocal evidence of boldenone, nandrolone and 4-estrene-3,17-dione being metabolites of testosterone in geldings. In view of these results, caution should be exercised when interpreting findings of boldenone, nandrolone and/or 4-estrene-3,17-dione together with a relatively high level of testosterone in gelding urine. Copyright © 2017 John Wiley & Sons, Ltd.

Metabolic study of androsta-1,4,6-triene-3,17-dione in horses using liquid chromatography/high resolution mass spectrometry.

Androsta-1,4,6-triene-3,17-dione (ATD) is an irreversible steroidal aromatase inhibitor and is marketed as a supplement. It has been reported to effectively reduce estrogen biosynthesis and significantly increase the levels of endogenous steroids such as dihydrotestosterone and testosterone in human. ATD abuses have been reported in human sports. Its metabolism in human has been studied, and the in vitro metabolic study of ATD in horses has been reported, however, little is known about its biotransformation and elimination in horses. This paper describes the in vitro and in vivo metabolism studies of ATD in horses, with an objective of identifying the target metabolites with the longest detection time for controlling ATD abuse. In vitro metabolism studies of ATD were performed using homogenized horse liver. ATD was found to be extensively metabolized, and its metabolites could not be easily characterized by gas chromatography/mass spectrometry (GC/MS) due to insufficient sensitivity. Liquid chromatography/high resolution mass spectrometry (LC/HRMS) was therefore employed for the identification of in vitro metabolites. The major biotransformations observed were combinations of reduction of the olefin groups and/or the keto group at either C3 or C17 position. In addition, mono-hydroxylation in the D-ring was observed along with reduction of the olefin groups and/or the keto group at C17 position. Fourteen in vitro metabolites, including two epimers of androsta-1,4,6-trien-17-ol-3-one (M1a, M1b), androsta-4,6-diene-3,17-dione (M2), boldione (M3), androsta-4,6-diene-17ß-ol-3-one (M4), androsta-4,6-diene-3-ol-17-one (M5), boldenone and epi-boldenone (M6a, M6b), four stereoisomers of hydroxylated androsta-1,4,6-trien-17-ol-3-one (M7a to M7d), and two epimers of androsta-1,4-diene-16α,17-diol (M8a, M8b), were identified. The identities of all metabolites, except M1a, M5, M7a to M7d, were confirmed by matching with authentic reference standards using LC/HRMS. For the in vivo metabolism studies, two thoroughbred geldings were each administered with 800 mg of ATD by stomach tubing. ATD, and twelve out of the fourteen in vitro metabolites, including M1a, M1b, M2, M4, M5, M6, M7a to M7d, M8a and M8b, were detected in post-administration urine. Two additional urinary metabolites, namely stereoisomers of hydroxylated androsta-4,6-dien-17-ol-3-one (M9a, M9b), were tentatively identified by mass spectral interpretation. Elevated level of testosterone was also observed. In post-administration blood samples, only the parent drug, M1b and M2 were identified. This study showed that the detection of ATD administration would be best achieved by either monitoring the metabolites M1b (androsta-1,4,6-trien-17ß-ol-3-one) or M4 (both excreted as sulfate conjugates) in urine, which could be detected for up to a maximum of 77 h post-administration. The analyte of choice for plasma is M1b, which could be detected for up to 28 h post administration.

Metabolic studies of formestane in horses.

Formestane (4-hydroxyandrost-4-ene-3,17-dione) is an irreversible steroidal aromatase inhibitor with reported abuse in human sports. In 2011, our laboratory identified the presence of formestane in a horse urine sample from an overseas jurisdiction. This was the first reported case of formestane in a racehorse. The metabolism of formestane in humans has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the in vitro and in vivo metabolic studies of formestane in horses, with the objective of identifying the target metabolite with the longest detection time for controlling formestane abuse. In vitro metabolic studies of formestane were performed using homogenized horse liver. Seven in vitro metabolites, namely 4-hydroxytestosterone (M1), 3ß,4α-dihydroxy-5ß-androstan-17-one (M2a), 3ß,4ß-dihydroxy-5ß-androstan-17-one (M2b), 3ß,4α-dihydroxy-5α-androstan-17-one (M2c), androst-4-ene-3α,4,17ß-triol (M3a), androst-4-ene-3ß,4,17ß-triol (M3b), and 5ß-androstane-3ß,4ß,17ß-triol (M4) were identified. For the in vivo studies, two thoroughbred geldings were each administered with 800 mg of formestane (32 capsules of Formadex) by stomach tubing. The results revealed that the parent drug and seven metabolites were detected in post-administration urine. The six in vitro metabolites (M1, M2a, M2b, M2c, M3a, and M3b) identified earlier were all detected in post-administration urine samples. In addition, 3α,4α-dihydroxy-5α-androstan-17-one (M2d), a stereoisomer of M2a/M2b/M2c, was also identified. This study has shown that the detection of formestane administration would be best achieved by monitoring 4-hydroxytestosterone (M1) in the glucuronide-conjugated fraction. M1 could be detected for up to 34 h post-administration. In blood samples, the parent drug could be detected for up to 34 h post administration.

Doping control analysis of TB-500, a synthetic version of an active region of thymosin ß4, in equine urine and plasma by liquid chromatography-mass spectrometry.

A veterinary preparation known as TB-500 and containing a synthetic version of the naturally occurring peptide LKKTETQ has emerged. The peptide segment (17)LKKTETQ(23) is the active site within the protein thymosin ß(4) responsible for actin binding, cell migration and wound healing. The key ingredient of TB-500 is the peptide LKKTETQ with artificial acetylation of the N-terminus. TB-500 is claimed to promote endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, collagen deposition and decrease inflammation. In order to control the misuse of TB-500 in equine sports, a method to definitely identify its prior use in horses is required. This study describes a method for the simultaneous detection of N-acetylated LKKTETQ and its metabolites in equine urine and plasma samples. The possible metabolites of N-acetylated LKKTETQ were first identified from in vitro studies. The parent peptide and its metabolites were isolated from equine urine or plasma by solid-phase extraction using ion-exchange cartridges, and analysed by liquid chromatography-mass spectrometry (LC/MS). These analytes were identified according to their LC retention times and relative abundances of the major product ions. The peptide N-acetylated LKKTETQ could be detected and confirmed at 0.02 ng/mL in equine plasma and 0.01 ng/mL in equine urine. This method was successful in confirming the presence of N-acetylated LKKTETQ and its metabolites in equine urine and plasma collected from horses administered with a single dose of TB-500 (containing 10mg of N-acetylated LKKTETQ). To our knowledge, this is the first identification of TB-500 and its metabolites in post-administration samples from horses.

Control of the misuse of bromide in horses.

Bromide is a sedative hypnotic. Due to its potential use as a sedative or calmative agent in competition horses, a method to control bromide is needed. Colorimetric method had been employed in the authors' laboratory from 2003 for the semi-quantification of bromide in equine plasma samples. However, the method was found to be highly susceptible to matrix interference, and was replaced in 2008 with a more reliable inductively coupled plasma-mass spectrometry (ICP/MS) method. Equine plasma was protein-precipitated using trichloroacetic acid, diluted with nitric acid, and then submitted directly to ICP/MS analysis. Since bromide is naturally occurring in equine plasma, a threshold is necessary to control its misuse in horses. Based on population studies (n = 325), a threshold of 90 µg/mL was proposed (with a risk factor of less than 1 in 10 000). Using the ICP/MS screening method, equine plasma samples with bromide greater than 85 µg/mL would be further quantified using the more accurate ICP/MS standard addition method. Confirmation of bromide was achieved by gas chromatography-mass spectrometry (GC-MS), with the bromide detected as its pentafluorobenzyl derivative. A sample is considered positive if its plasma bromide concentration exceeds the threshold (90 µg/mL) plus the measurement uncertainty of the quantification method (8 µg/mL at 99% 1-tailed confidence level) and its presence is confirmed using the GC-MS method. Following oral administration of potassium bromide (60 g each) to two geldings, plasma bromide levels peaked after approximately 2 hours at about 300 µg/mL, and then remained above the threshold for 8 and 13 days respectively.