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Rationale: Selective androgen receptor modulators (SARMs) are prohibited in sports due to their performance enhancing ability. It is important to investigate the metabolism to determine appropriate targets for doping control. This is the first study where the equine metabolites of SARMs S1, S4 (Andarine) and S22 (Ostarine) have been studied in plasma., Methods: Each SARM was administered to three horses as an intravenous bolus dose and plasma samples were collected. The samples were pretreated with protein precipitation using cold acetonitrile before separation by liquid chromatography. The mass spectrometric analysis was performed using negative electrospray, quadrupole time-of-flight mass spectrometry operated in MS(E) mode and triple-quadrupole mass spectrometry operated in selected reaction monitoring mode. For the quantification of SARM S1, a deuterated analogue was used as internal standard., Results: The numbers of observed metabolites were eight, nine and four for the SARMs S1, S4 and S22, respectively. The major metabolite was formed by the same metabolic reactions for all three SARMs, namely amide hydrolysis, hydroxylation and sulfonation. The values of the determined maximum plasma concentrations were in the range of 97-170 ng/mL for SARM S1, 95-115 ng/mL for SARM S4 and 92-147 ng/mL for SARM S22 and the compounds could be detected for 96 h, 12 h and 18 h, respectively., Conclusions: The maximum plasma concentration of SARMs S1, S4 and S22 was measured in the first sample (5 min) after administration and they were eliminated fast from plasma. The proposed targets to be used in equine doping control are the parent compounds for all three SARMs, but with the metabolite yielding the highest response as a complementary target. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Rationale: The issue of illicit performance enhancement spans human and animal sport in presumably equal measure, with prohibited substances and methods of doping conveying both ways. Due to the proven capability of unbound ionic cobalt (Co(2) (+) ) to stimulate erythropoiesis in humans, both human and equine anti-doping regulations have listed cobalt as a banned substance, and in particular in horse drug testing, thresholds for cobalt concentrations applying to plasma and urine have been suggested or established. Recent reports about the finding of substantial amounts of undeclared nickel in arguably licit performance- and recovery-supporting products raised the question whether the ionic species of this transition metal (Ni(2) (+) ), which exhibits similar prolyl hydroxylase inhibiting properties to Co(2) (+) , has been considered as a substitute for cobalt in doping regimens., Methods: Therefore, a pilot study with 200 routine post-competition doping control horse urine samples collected from animals participating in equestrian, gallop, and trotting in Europe was conducted to provide a first dataset on equine urinary Ni(2) (+) concentrations. All specimens were analyzed by conventional inductively coupled plasma mass spectrometry (ICP-MS) to yield quantitative data for soluble nickel., Results: Concentrations ranging from below the assay's limit of quantification (LOQ, 0.5 ng/mL) up to 33.4 ng/mL with a mean value (± standard deviation) of 6.1 (±5.1) ng/mL were determined for the total nickel content., Conclusions: In horses, nickel is considered a micronutrient and feed supplements containing nickel are available; hence, follow-up studies are deemed warranted to consolidate potential future threshold levels concerning urine and blood nickel concentrations in horses using larger sets of samples for both matrices and to provide in-depth insights by conducting elimination studies with soluble Ni(2) (+) -salt species. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Rationale: Potentially performance-enhancing agents, particularly anabolic agents, are advertised and distributed by Internet-based suppliers to a substantial extent. Among these anabolic agents, a substance referred to as LGD-4033 has been made available, comprising the core structure of a class of selective androgen receptor modulators (SARMs)., Methods: In order to provide comprehensive analytical data for doping controls, the substance was obtained and characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography/electrospray ionization high resolution/high accuracy tandem mass spectrometry (LC/ESI-HRMS). Following the identification of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile, the substance was subjected to in vitro metabolism studies employing human liver microsomes and Cunninghamella elegans (C. elegans) preparations as well as electrochemical metabolism simulations., Results: By means of LC/ESI-HRMS, five main phase-I metabolites were identified as products of liver microsomal preparations including three monohydroxylated and two bishydroxylated species. The two most abundant metabolites (one mono- and one bishydroxylated product) were structurally confirmed by LC/ESI-HRMS and NMR. Comparing the metabolic conversion of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile observed in human liver microsomes with C. elegans and electrochemically derived metabolites, one monohydroxylated product was found to be predominantly formed in all three methodologies., Conclusions: The implementation of the intact SARM-like compound and its presumed urinary phase-I metabolites into routine doping controls is suggested to expand and complement existing sports drug testing methods., (Copyright © 2015 John Wiley & Sons, Ltd.)

Rationale: Bupivacaine is a local anaesthetic prohibited in equine sports. It is highly metabolized in the horse but a thorough description of its metabolite profile is lacking. An administration study should find appropriate analytical targets for doping control. Furthermore, knowledge of an in vitro system for production of metabolites would be beneficial., Methods: Marcain® (bupivacaine hydrochloride) was administered subcutaneously to a horse and urine samples were collected. In vitro metabolic systems consisting of the fungi Cunninghamella elegans and Cunninghamella blakesleeana were incubated with bupivacaine and bupivacaine-d(9). Samples were analyzed directly after dilution or cleaned up using liquid-liquid extraction. Separation was achieved with liquid chromatography. Mass spectrometric analysis was performed using positive electrospray ionization with both a tandem quadrupole and an ion trap instrument using MS(n) and hydrogen/deuterium exchange., Results: In horse urine, seven phase I metabolites were found: 3'- and 4'-hydroxybupivacaine, N-desbutylbupivacaine, two aliphatically hydroxylated metabolites, one N-oxide, and dihydroxybupivacaine. Sulfated hydroxybupivacaine and glucuronides of 3'- and 4'-hydroxybupivacaine and of dihydroxybupivacaine were also detected. All these metabolites were previously undescribed in the horse, except for 3'-hydroxybupivacaine. 3'- and 4'-Hydroxybupivacaine were designated as appropriate targets for doping control. Interestingly, all the equine phase I metabolites were also detected in the samples from C. elegans and C. blakesleeana., Conclusions: The qualitative aspects of the metabolism of bupivacaine in the horse have been investigated with many novel metabolites described. The fungi C. elegans and C. blakesleeana have proven to be relevant models for mammalian metabolism of bupivacaine and they may in the future be used to produce analytical reference materials., (Copyright © 2012 John Wiley & Sons, Ltd.)

Cunninghamella elegans is a filamentous fungus that has been shown to biotransform drugs into the same metabolites as mammals. In this paper we describe the use of C. elegans to aid the identification of clemastine metabolites since high concentrations of the metabolites were produced and MS(n) experiments were facilitated. The combination of liquid chromatography and tandem mass spectrometry with two different ionization techniques and hydrogen/deuterium exchange were used for structural elucidation of the clemastine metabolites. Norclemastine, four isomers of hydroxylated clemastine, and two N-oxide metabolites were described for the first time in C. elegans incubations. The N-oxidations were confirmed by hydrogen/deuterium exchange and deoxygenation (-16 Da) upon atmospheric pressure chemical ionization mass spectrometry. By MS(n) fragmentation it was concluded that two of the hydroxylated metabolites were oxidized on the methylpyrridyl moiety, one on the aromatic ring with the chloro substituent, and one on the aromatic ring without the chlorine., (Copyright 2010 John Wiley & Sons, Ltd.)

For the first time chemical derivatization of isomeric drug glucuronides with 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC) has been successfully applied as a tool for determining the site of conjugation. This provides a way to differentiate between glucuronide isomers containing aliphatic and phenolic hydroxyl groups. The analyses were performed with liquid chromatography/electrospray ion trap mass spectrometry (LC/ESI-MSn). DMISC has previously been shown to react selectively with phenols in estrogens, thus improving sensitivity in ESI-MS. The model compounds selected for this study were commercially available standards of formoterol, morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). Formoterol glucuronides were produced with an enzymatic method in house. Both formoterol and morphine possess one phenolic and one aliphatic hydroxyl group where glucuronidation could take place. The product ion mass spectra of the native morphine glucuronides were indistinguishable due to the initial neutral loss of monodehydrated glucuronic acid (176 u). However, a significant difference between the isomers was observed with DMISC derivatization, as only the form with a free phenol, M6G, gave a detectable reaction product. Formoterol formed two detectable glucuronide isomers in the enzymatic reaction. Their respective sites of conjugation could not be directly determined from the product ion spectra. Reaction with DMISC, however, gave a detectable product with only one of the isomers. Based on previous experience of the preferred DMISC reactions with phenols, and interpretation of the fragmentation pattern of the derivative, it was concluded that the reactive isomer had a free phenol, and was thus conjugated on the aliphatic chain.

This paper describes a way of differentiating between the three isomers of estriol glucuronide by the use of chemical derivatization and liquid chromatography/electrospray tandem mass spectrometry (MS/MS). In their native form, these isomers gave rise to almost identical product ion spectra, involving the neutral loss of 176 Da (i.e. monodehydrated glucuronic acid), which made it impossible to determine the position of conjugation by MS/MS alone. In order to change the fragmentation pathways, positive charges were introduced into the analytes by chemical derivatization. The following reagents were tested: 2-chloro-1-methylpyridinium iodide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2-picolylamine. Interestingly, derivatization using a combination of all three reagents gave a selective fragmentation pattern that could differentiate between the isomers estriol-16-glucuronide and estriol-17-glucuronide. Estriol-3-glucuronide, which lacks a free phenolic group, could be differentiated through a different type of reaction product when exposed to 2-chloro-1-methylpyridinium iodide. Furthermore, in order to assist structural assignment of the fragments, their accurate masses were determined using a hybrid quadrupole time-of-flight mass spectrometer and fragmentation pathways were elucidated by the use of MS3 on an ion trap mass spectrometer., (Copyright 2006 John Wiley & Sons, Ltd.)

The metabolism of clemastine was studied in dogs, horses, and humans after a single dose of Tavegyl. The urine collected was extracted by solid-phase extraction or hydrolyzed with beta-glucuronidase and then extracted by liquid-liquid extraction, prior to analysis for unchanged drug and phase I and II metabolites by liquid chromatography/tandem mass spectrometry. The metabolites were identified by their molecular mass and interpretation of the product ion spectra, since no standard substances were available. Unchanged drug was recovered in urine samples from dogs and humans, but not from horses. In dogs and humans, the phase I metabolite, norclemastine, was identified, and clemastine metabolites with one and two additional oxygens were found in all three species. In horses and dogs monohydroxylation on one of the aromatic rings or the adjacent methyl group was favored while, in humans, the additional oxygen was positioned on either the aromatic or the aliphatic part of the structure, and the aliphatic reaction seemed to result in at least three isomers. In the metabolites with two additional oxygens, both the oxygens were found on the aliphatic fragment in humans and dogs, whereas they were situated on the aromatic part of the structure in horses. In human patients, glucuronidated monohydroxyclemastine was recovered, and in urine from horses both mono- and dihydroxyclemastine glucuronides were identified, while phase II metabolites could not be recovered from the dog urine. Clemastine metabolism in dogs and horses has, to our knowledge, not been studied before, and new metabolites from humans are presented in this article. Thus, the metabolites described in the present work have not been previously reported in the literature., (Copyright 2004 John Wiley & Sons, Ltd.)