Unravelling androgens in sport: Altrenogest shows strong activation of the androgen receptor in a mammalian cell bioassay.

Altrenogest is a commonly used progestogen for the suppression of oestrus and associated distracting behaviours that interfere with training and performance of female racehorses. The steroid is derived from 19-nor testosterone and is structurally similar to the anabolic androgenic steroid, trenbolone. In this study, the relative androgen potency of altrenogest was determined by a kidney (HEK293) cell androgen bioassay. The HEK293 bioassay shows that in its pure form, altrenogest has a high relative potency compared with testosterone but is not as strong as ß-trenbolone. Our results also show that altrenogest is able to activate the androgen receptor at the concentrations relevant to the administration regime of racehorses and retains its activity ex vivo. Thus, we show unequivocally that altrenogest, a progestogen used widely in female racehorses, acts as a strong androgen in a mammalian cell bioassay.

Doping control analysis of total arsenic in equine plasma.

Arsenic can be easily found in our surrounding environment. Because of its ubiquitous nature, horse urine and blood invariably contain low levels of arsenic. Nevertheless, inorganic arsenic, despite its general use as a tonic for horses, is an effective doping agent having a deleterious effect because of its ability to induce gastroenteritis. The misuse of arsenic in horseracing has been controlled by an international urinary threshold of total arsenic at 0.3 µg/mL. However, an equivalent threshold for total arsenic in plasma is yet to be established. In this study, an inductively coupled plasma-mass spectrometry method has been developed for quantifying total arsenic in equine plasma. Statistical analysis determined that the data from a population study of 1,552 post-race and out-of-competition plasma samples fits a Gaussian mixture model with two Gaussian components. A rounded-up provisional threshold for plasma total arsenic at 2.5 ng/mL was subsequently established. Results from administration trials with a sodium arsanilate-containing supplement showed that both urinary and plasma arsenic was significantly elevated after administration. The maximum urinary detection time was around 22 h based on the international threshold. However, the maximum plasma detection time would be longer than 73 h if the provisional threshold of 2.5 ng/mL was adopted. In view of the high discrepancy between the urine and plasma detection times, a revised plasma threshold of 15 ng/mL is proposed to afford a comparable detection time in both matrices. The risk of a normal sample exceeding the proposed plasma total arsenic threshold is practically zero.

A high-throughput and broad-spectrum screening method for analysing over 120 drugs in horse urine using liquid chromatography-high-resolution mass spectrometry.

A high-throughput method has been developed for the doping control analysis of 124 drug targets, processing up to 154 horse urine samples in as short as 4.5 h, from the time the samples arrive at the laboratory to the reporting deadline of 30 min before the first race, including sample receipt and registration, preparation and instrument analysis and data vetting time. Sample preparation involves a brief enzyme hydrolysis step (30 min) to detect both free and glucuronide-conjugated drug targets. This is followed by extraction using solid-supported liquid extraction (SLE) and analysis using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). The entire set-up comprised of four sets of Biotage Extrahera automation systems for conducting SLE and five to six sets of Orbitrap for instrumental screening using LC-HRMS. Suspicious samples flagged were subject to confirmatory analyses using liquid chromatography-triple quadrupole mass spectrometry. The method comprises 124 drug targets from a spectrum of 41 drug classes covering acidic, basic and neutral drugs. More than 85% of the targets had limits of detection at or below 5 ng/mL in horse urine, with the lowest at 0.02 ng/mL. The method was validated for qualitative identification, including specificity, sensitivity, extraction recovery and precision. Method applicability was demonstrated by the successful detection of different drugs, namely (a) butorphanol, (b) dexamethasone, (c) diclofenac, (d) flunixin and (e) phenylbutazone, in post-race or out-of-competition urine samples collected from racehorses. This method was developed for pre-race urine testing in Hong Kong; however, it is also suitable for testing post-race or out-of-competition urine samples, especially when a quick total analysis time is desired.

Doping control study of AICAR in post-race urine and plasma samples from horses.

Acadesine, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, commonly known as AICAR, is a naturally occurring adenosine monophosphate-activated protein kinase (AMPK) activator in many mammals, including humans and horses. AICAR has attracted considerable attention recently in the field of doping control because of a study showing the enhancement of endurance performance in unexercised or untrained mice, resulting in the term 'exercise pill'. Its use has been classified as gene doping by the World Anti-Doping Agency (WADA), and since it is endogenous, it may only be possible to control deliberate administration of AICAR to racehorses after establishment of an appropriate threshold. Herein we report our studies of AICAR in post-race equine urine and plasma samples including statistical studies of AICAR concentrations determined from 1,470 urine samples collected from thoroughbreds and standardbreds and analyzed in Australia, France, and Hong Kong. Quantification methods in equine urine and plasma using liquid chromatography-mass spectrometry were developed by the laboratories in each country. An exchange of spiked urine and plasma samples between the three countries was conducted, confirming no significant differences in the methods. However, the concentration of AICAR in plasma was found to increase upon haemolysis of whole blood samples, impeding the establishment of a suitable threshold in equine plasma. A possible urine screening cut-off at 600 ng/mL for the control of AICAR in racehorses could be considered for adoption. Application of the proposed screening cut-off to urine samples collected after intravenous administration of a small dose (2 g) of AICAR to a mare yielded a short detection time of approximately 4.5 h. Copyright © 2017 John Wiley & Sons, Ltd.

Interlaboratory trial for the measurement of total cobalt in equine urine and plasma by ICP-MS.

Cobalt is an essential mineral micronutrient and is regularly present in equine nutritional and feed supplements. Therefore, cobalt is naturally present at low concentrations in biological samples. The administration of cobalt chloride is considered to be blood doping and is thus prohibited. To control the misuse of cobalt, it was mandatory to establish an international threshold for cobalt in plasma and/or in urine. To achieve this goal, an international collaboration, consisting of an interlaboratory comparison between 5 laboratories for the urine study and 8 laboratories for the plasma study, has been undertaken. Quantification of cobalt in the biological samples was performed by inductively coupled plasma-mass spectrometry (ICP-MS). Ring tests were based on the analysis of 5 urine samples supplemented at concentrations ranging from 5 up to 500 ng/mL and 5 plasma samples spiked at concentrations ranging from 0.5 up to 25 ng/mL. The results obtained from the different laboratories were collected, compiled, and compared to assess the reproducibility and robustness of cobalt quantification measurements. The statistical approach for the ring test for total cobalt in urine was based on the determination of percentage deviations from the calculated means, while robust statistics based on the calculated median were applied to the ring test for total cobalt in plasma. The inter-laboratory comparisons in urine and in plasma were successful so that 97.6% of the urine samples and 97.5% of the plasma samples gave satisfactory results. Threshold values for cobalt in plasma and urine were established from data only obtained by laboratories involved in the ring test. Copyright © 2017 John Wiley & Sons, Ltd.

In vitro phase I metabolism of selective estrogen receptor modulators in horse using ultra-high performance liquid chromatography-high resolution mass spectrometry.

Selective estrogen receptor modulators (SERMs) are chemicals that possess the anti-oestrogenic activities that are banned 'in' and 'out' of competition by the World Anti-Doping Agency (WADA) in human sports, and by the International Federation of Horseracing Authorities (IFHA) in horseracing. SERMs can be used as performance-enhancing drugs to boost the level of androgens or to compensate for the adverse effects as a result of extensive use of androgenic anabolic steroids (AASs). SERMs have indeed been abused in human sports; hence, a similar threat can be envisaged in horseracing. Numerous analytical findings attributed to the use of SERMs have been reported by WADA-accredited laboratories, including 42 cases of tamoxifen and 2 cases of toremifene in 2014. This paper describes the identification of the in vitro phase I metabolites of tamoxifen and toremifene using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS), with an aim to identify potential screening targets for doping control in equine sports. A total of 13 and 11 in vitro metabolites have been identified for tamoxifen and toremifene, respectively, after incubation with homogenized horse liver. The more prominent in vitro biotransformation pathways include N-desmethylation, hydroxylation, and carboxylation. In addition, this is the first report of some novel metabolites for both tamoxifen and toremifene with hydroxylation occurring at the N-methyl moiety. To our knowledge, this is the first study of the phase I metabolism of tamoxifen and toremifene in horses using homogenized horse liver. Copyright © 2017 John Wiley & Sons, Ltd.

Simultaneous detection of recombinant growth hormones in equine plasma by liquid chromatography/high-resolution tandem mass spectrometry for doping control.

Growth hormone (GH), or somatotropin, is a protein that may enhance physical performance and facilitate growth and wound healing. For this reason, growth hormones and their recombinant analogues are prohibited in human sports by the World Anti-Doping Agency (WADA) and in horseracing under Article 6 of the International Agreement on Breeding, Racing and Wagering published by the International Federation of Horse Racing Authorities (IFHA). Identifying the illicit use of GHs in both human athletes and racehorses is challenging, especially when differentiating between endogenous and exogenous GHs, and between analogues of GH from different species. This paper describes a multiplexed mass spectrometric method for the simultaneous detection of three recombinant GHs, namely recombinant equine GH (reGH), recombinant human GH (rhGH) and recombinant porcine GH (rpGH), in equine plasma. Recombinant chicken GH (rcGH) was used as an internal standard. The recombinant GHs were extracted from equine plasma by automated C4 solid-phase extraction after ammonium sulfate precipitation, and then cleaned up by chloroform/methanol precipitation before trypsination. Proteotypic peptides were analyzed by liquid chromatography/high-resolution tandem mass spectrometry (LC-MS/HRMS). The limits of detection were estimated to be approximately 0.5ng/mL for reGH, 2.5ng/mL for rhGH and 1.25ng/mL for rpGH. Confirmation at 1ng/mL for reGH and 5ng/mL each for rhGH and rpGH was successfully achieved by comparing the retention times and relative abundances of three major product-ions of the respective standards in accordance with industry criteria published by the Association of Official Racing Chemists. The developed method requires less plasma (2mL) and has a shorter turnaround time as compared with other published mass spectrometric methods, and demonstrates good precision and reproducibility. To our knowledge, this is the first reported method for the simultaneous detection of different recombinant GHs (reGH, rhGH and rpGH) at low ng/mL level in horse plasma samples.

Doping control analysis of 46 polar drugs in horse plasma and urine using a 'dilute-and-shoot' ultra high performance liquid chromatography-high resolution mass spectrometry approach.

The high sensitivity of ultra high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) allows the identification of many prohibited substances without pre-concentration, leading to the development of simple and fast 'dilute-and-shoot' methods for doping control for human and equine sports. While the detection of polar drugs in plasma and urine is difficult using liquid-liquid or solid-phase extraction as these substances are poorly extracted, the 'dilute-and-shoot' approach is plausible. This paper describes a 'dilute-and-shoot' UHPLC-HRMS screening method to detect 46 polar drugs in equine urine and plasma, including some angiotensin-converting enzyme (ACE) inhibitors, sympathomimetics, anti-epileptics, hemostatics, the new doping agent 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), as well as two threshold substances, namely dimethyl sulfoxide and theobromine. For plasma, the sample (200µL) was protein precipitated using trichloroacetic acid, and the resulting supernatant was diluted using Buffer A with an overall dilution factor of 3. For urine, the sample (20µL) was simply diluted 50-fold with Buffer A. The diluted plasma or urine sample was then analysed using a UHPLC-HRMS system in full-scan ESI mode. The assay was validated for qualitative identification purpose. This straightforward and reliable approach carried out in combination with other screening procedures has increased the efficiency of doping control analysis in the laboratory. Moreover, since the UHPLC-HRMS data were acquired in full-scan mode, the method could theoretically accommodate an unlimited number of existing and new doping agents, and would allow a retrospectively search for drugs that have not been targeted at the time of analysis.

Generation of phase II in vitro metabolites using homogenized horse liver.

The successful use of homogenized horse liver for the generation of phase I in vitro metabolites has been previously reported by the authors' laboratory. Prior to the use of homogenized liver, the authors' laboratory had been using mainly horse liver microsomes for carrying out equine in vitro metabolism studies. Homogenized horse liver has shown significant advantages over liver microsomes for in vitro metabolism studies as the procedures are much quicker and have higher capability for generating more in vitro metabolites. In this study, the use of homogenized liver has been extended to the generation of phase II in vitro metabolites (glucuronide and/or sulfate conjugates) using 17ß-estradiol, morphine, and boldenone undecylenate as model substrates. It was observed that phase II metabolites could also be generated even without the addition of cofactors. To the authors' knowledge, this is the first report of the successful use of homogenized horse liver for the generation of phase II metabolites. It also demonstrates the ease with which both phase I and phase II metabolites can now be generated in vitro simply by using homogenized liver without the need for ultracentrifuges or tedious preparation steps.

Controlling the misuse of cobalt in horses.

Cobalt is a well-established inducer of hypoxia-like responses, which can cause gene modulation at the hypoxia inducible factor pathway to induce erythropoietin transcription. Cobalt salts are orally active, inexpensive, and easily accessible. It is an attractive blood doping agent for enhancing aerobic performance. Indeed, recent intelligence and investigations have confirmed cobalt was being abused in equine sports. In this paper, population surveys of total cobalt in raceday samples were conducted using inductively coupled plasma mass spectrometry (ICP-MS). Urinary threshold of 75 ng/mL and plasma threshold of 2 ng/mL could be proposed for the control of cobalt misuse in raceday or in-competition samples. Results from administration trials with cobalt-containing supplements showed that common supplements could elevate urinary and plasma cobalt levels above the proposed thresholds within 24 h of administration. It would therefore be necessary to ban the use of cobalt-containing supplements on raceday as well as on the day before racing in order to implement and enforce the proposed thresholds. Since the abuse with huge quantities of cobalt salts can be done during training while the use of legitimate cobalt-containing supplements are also allowed, different urinary and plasma cobalt thresholds would be required to control cobalt abuse in non-raceday or out-of-competition samples. This could be achieved by setting the thresholds above the maximum urinary and plasma cobalt concentrations observed or anticipated from the normal use of legitimate cobalt-containing supplements. Urinary threshold of 2000 ng/mL and plasma threshold of 10 ng/mL were thus proposed for the control of cobalt abuse in non-raceday or out-of-competition samples.

Syntheses, structures, physical properties, and electronic properties of some AMUQ3 compounds (A = alkali metal, M = Cu or Ag, Q = S or Se).

The seven new isostructural quaternary uranium chalcogenides KCuUS 3, RbCuUS 3, RbAgUS 3, CsCuUS 3, CsAgUS 3, RbAgUSe 3, and CsAgUSe 3 were prepared from solid-state reactions. These isostructural materials crystallize in the layered KZrCuS 3 structure type in the orthorhombic space group Cmcm. The structure is composed of UQ 6 octahedra and MQ 4 tetrahedra that share edges to form (2) infinity[UMQ 3 (-)] layers. These layers stack perpendicular to [010] and are separated by layers of face- and edge-sharing AQ 8 bicapped trigonal prisms. There are no Q-Q bonds in the structure, so the formal oxidation states of A/U/M/Q may be assigned as 1+/4+/1+/2-, respectively. CsCuUS 3 shows semiconducting behavior with thermal activation energy E a = 0.14 eV and sigma 298 = 0.3 S/cm. From single-crystal absorption measurements in the near IR range, the optical band gaps of these compounds are smaller than 0.73 eV. The more diffuse 5f electrons play a much more dominant role in the optical properties of the AMUQ 3 compounds than do the 4f electrons in the AMLnQ 3 compounds (Ln = rare earth). Periodic DFT spin band-structure calculations on CsCuUS 3 and CsAgUS 3 establish two energetically similar antiferromagnetic spin structures and show magnetic interactions within and between the layers of the structure. Density-of-states analysis shows M-Q orbital overlap in the valence band and U-Q orbital overlap in the conduction band.

Syntheses, crystal structures, and physical properties of La5Cu6O4S7 and La5Cu6.33O4S7.

Single crystal and bulk powder samples of the quaternary lanthanum copper oxysulfides La5Cu6.33O4S7 and La5Cu6O4S7 have been prepared by means of high-temperature sealed-tube reactions and spark plasma sintering, respectively. In the structure of La 5Cu6.33O4S7, Cu atoms tie together the fluorite-like (2)infinity[La5O4S(5+)] and antifluorite-like (2) infinity[Cu6S6(5-)] layers of La5Cu6O4S7. The optical band gap, E g, of 2.0 eV was deduced from both diffuse reflectance spectra on a bulk sample of La5Cu6O4S7 and for the (010) crystal face of a La 5Cu6.33O4S7 single crystal. Transport measurements at 298 K on a bulk sample of La 5Cu 6O 4S 7 indicated p-type metallic electrical conduction with sigma electrical =2.18 S cm(-1), whereas measurements on a La 5Cu6.33O4S7 single crystal led to sigma electrical =4.5 10(-3) S cm(-1) along [100] and to semiconducting behavior. In going from La 5Cu6O4S7 to La5Cu6.33O4S7, the disruption of the (2)infinity[Cu6S6(5-)] layer and the decrease in the overall Cu(2+)(3d(9)) concentration lead to a significant decrease in the electrical conductivity.

On the anisotropy of the magnetic properties of CsYbZnSe3.

DC magnetic susceptibility measurements on CsYbZnSe 3 show a broad magnetic transition at approximately 10 K and pronounced differences between zero-field-cooled and field-cooled data that lead to experimental effective magnetic moments of 4.26(5) BM and 4.39(4) BM, respectively. Specific heat measurements confirm that there is neither long-range ordering nor a phase transition between 1.8 and 380 K. First-principles electronic structure calculations with and without inclusion of spin-orbit coupling effects show that the spins of CsYbZnSe 3 prefer to orient along [010] rather than along either [100] or [001] of this orthorhombic material and that the spin exchange between adjacent Yb3+ ions along [100] is substantially antiferromagnetic. The magnetic properties of CsYbZnSe 3 are best described by an Ising uniform antiferromagnetic chain model.

Syntheses, Crystal and Band Structures, and Magnetic and Optical Properties of New CsLnCdTe(3) (Ln = La, Pr, Nd, Sm, Gd-Tm, and Lu).

A series of new quaternary semiconductor materials CsLnCdTe(3) (Ln = La, Pr, Nd, Sm, Gd-Tm, and Lu) was obtained from high-temperature solid-state reactions by the reactive halide flux method. These compounds belong to the layered KZrCuS(3) structure type and crystallize in the orthorhombic space group Cmcm (No. 63). Their structure features two-dimensional infinity(2)[LnCdTe(3)-] layers of edge- and vertex-sharing LnTe(6) octahedra with Cd atoms filling the tetrahedral interstices, which stack along b-axis. The Cs atoms are located between the infinity(2)[LnCdTe(3)-] layers and are surrounded by eight Te atoms to form a CsTe(8) bicapped trigonal prism. Such Te layers are more flexible than the Se analogues in the isostructural CsLnMSe(3) to accommodate nearly the entire Ln series. Theoretical studies performed on CsTmCdTe(3) show that the material is a direct band gap semiconductor and agrees with the result from a single-crystal optical absorption measurement. Magnetic susceptibility measurements show that the CsLnCdTe(3) (Ln = Pr, Nd, Gd, Dy, Tm) materials exhibit temperature-dependent paramagnetism and obey the Curie-Weiss law, whereas CsSmCdTe(3) does not.

Zinc oxide nanorod growth on gold islands prepared by microsphere lithography on silicon and quartz.

Gold islands, vapor deposited on silicon and quartz by microsphere lithography patterning, are used to nucleate arrays of ZnO nanorods. ZnO is grown on approximately 0.32 microm2 Au islands by carbothermal reduction in a tube furnace. Scanning electron microscopy (SEM) and energy dispersive atomic X-ray spectroscopy (EDS) confirm that the gold effectively controls the sites of nucleation of ZnO. Atomic force microscopy (AFM) shows that approximately 30 nm diameter nanorods grow horizontally, along the surface. Alloy droplets that are characteristic of the vapor-liquid-solid (VLS) mechanism are observed at the tips of the nanorods. The spatial growth direction of VLS catalyzed ZnO nanorods is along the substrate when they nucleate from gold islands on silicon and quartz. The energy of adhesion of the VLS droplet to the surface can account for the horizontal growth.

Syntheses, structures, physical properties, and theoretical studies of CeMxOS (M = Cu, Ag; x approximately 0.8) and CeAgOS.

Black single crystals of the two nonstoichiometric cerium coinage-metal oxysulfide compounds CeCu(x)OS and CeAg(x)OS (x approximately 0.8) have been prepared by the reactions of Ce2S3 and CuO or Ag2O at 1223 or 1173 K, respectively. A black powder sample of CeAgOS has been prepared by the stoichiometric reaction of Ce2S3, CeO2, Ag2S, and Ag at 1073 K. These isostructural materials crystallize in the ZrSiCuAs structure type with two formula units in the tetragonal space group P4/nmm. Refined crystal structure results and chemical analyses provide evidence that the previously known anomalously small unit-cell volume of LnCuOS for Ln = Ce (Ln = rare-earth metal) is the result of Cu vacancies and the concomitant presence of both Ce3+ and Ce4+. Both CeCu(0.8)OS and CeAgOS are paramagnetic with mu(eff) values of 2.13(6) and 2.10(1) mu(B), respectively. CeCu(0.8)OS is a p-type semiconductor with a thermal activation energy Ea = 0.22 eV, sigma(electrical) = 9.8(1) 10(-3) S/cm at 298 K, and an optical band gap Eg < 0.73 eV. CeAgOS has conductivity sigma(conductivity) = 0.16(4) S/cm and an optical band gap Eg = 0.71 eV at 298 K. Theoretical calculations with an on-site Coulomb repulsion parameter indicate that the Ce 4f states are fully spin-polarized and are not localized in CeCuOS, CeCu(0.75)OS, or CeAgOS. Calculated band gaps for CeCu(0.75)OS and CeAgOS are 0.6 and 0.8 eV, respectively.