Identification of erythropoietin mimetic peptide 1 linear form in a sealed vial and its administration study in horses for doping control purpose.

The erythropoietin mimetic peptide 1 linear form (EMP1-linear), GGTYSCHFGPLTWVCKPQGG-NH2 , was identified in an unknown preparation consisting of white crystalline powder contained in sealed glass vials using ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). The white crystalline powder, allegedly used for doping racehorses, was found to contain around 2% (w/w) of EMP1-linear. EMP1-linear can be cyclised in equine plasma at physiological temperature of 37°C by forming an intramolecular disulfide bond to give EMP1, which is a well-known erythropoiesis stimulating agent that can bind to and activate the receptor for cytokine erythropoietin (EPO). Thus, EMP1-linear is a prodrug of EMP1, which is a performance-enhancing doping agent that can be misused in equine sports. In order to identify potential target(s) for detecting the misuse of EMP1-linear in horses, an in vitro metabolic study using horse liver S9 fraction was performed. After incubation, EMP1-linear mainly existed in its cyclic form as EMP1, and four N-terminus truncated in vitro metabolites TYSCHFGPLTWVCKPQGG-NH2 (M1), SCHFGPLTWVCKPQGG-NH2 (M2), WVCKPQGG-NH2 (M3) and VCKPQGG-NH2 (M4) were identified. An intravenous administration study with the preparation of white crystalline powder containing EMP1-linear was also conducted using three retired thoroughbred geldings. EMP1 was detectable only in the postadministration plasma samples, whereas the four identified in vitro metabolites were detected in both postadministration plasma and urine samples. For controlling the misuse of EMP1-linear in horse, its metabolite M3 gave the longest detection time in both plasma and urine and could be detected for up to 4 and 27 h postadministration, respectively.

Long-term monitoring of IOX4 in horse hair and its longitudinal distribution with segmental analysis using liquid chromatography/electrospray ionization Q Exactive high-resolution mass spectrometry for the purpose of doping control.

IOX4, a hypoxia-inducible factor stabilizer, is classified as a banned substance for horses in both horse racing and equestrian sports. We recently reported the pharmacokinetic profiles of IOX4 in horse plasma and urine and also identified potential monitoring targets for the doping control purpose. In this study, a long-term longitudinal analysis of IOX4 in horse hair after a nasoesophageal administration of IOX4 (500 mg/day for 3 days) to three thoroughbred mares is presented for the first time for controlling the abuse/misuse of IOX4. Six bunches of mane hair were collected at 0 (pre), 1, 2, 3, and 6 month(s) postadministration. Our results showed that the presence of IOX4 was identified in all postadministration horse hair samples, but no metabolite could be detected. The detection window for IOX4 could achieve up to 6-month postadministration (last sampling point) by monitoring IOX4 in hair. In order to evaluate the longitudinal distribution of IOX4 over 6 months, a validated quantification method of IOX4 in hair was developed for the analysis of the postadministration samples. Segmental analysis of 2-cm cut hair across the entire length of postadministration hair showed that IOX4 could be quantified up to the level of 1.84 pg/mg. In addition, it was found that the movement of the incorporated IOX4 band in the hair shaft over 6 months varied among the three horses due to individual variation and a significant diffusion of IOX4 band up to 10 cm width was also observed in the 6-month postadministration hair samples.

Comprehensive metabolic study of IOX4 in equine urine and plasma using liquid chromatography/electrospray ionization Q Exactive high-resolution mass spectrometer for the purpose of doping control.

IOX4 is a hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitor, which was developed for the treatment of anemia by exerting hematopoietic effects. The administration of HIF-PHD inhibitors such as IOX4 to horses is strictly prohibited by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale. To the best of our knowledge, this is the first comprehensive metabolic study of IOX4 in horse plasma and urine after a nasoesophageal administration of IOX4 (500 mg/day, 3 days). A total of four metabolites (three mono-hydroxylated IOX4 and one IOX4 glucuronide) were detected from the in vitro study using homogenized horse liver. As for the in vivo study, post-administration plasma and urine samples were comprehensively analyzed with liquid chromatography/electrospray ionization high-resolution mass spectrometry to identify potential metabolites and determine their corresponding detection times. A total of 10 metabolites (including IOX4 glucuronide, IOX4 glucoside, O-desbutyl IOX4, O-desbutyl IOX4 glucuronide, four mono-hydroxylated IOX4, N-oxidized IOX4, and N-oxidized IOX4 glucoside) were found in urine and three metabolites (glucuronide, glucoside, and O-desbutyl) in plasma. Thus, the respective quantification methods for the detection of free and conjugated IOX4 metabolites in urine and plasma with a biphase enzymatic hydrolysis were developed and applied to post-administration samples for the establishment of elimination profiles of IOX4. The detection times of total IOX4 in urine and plasma could be successfully prolonged to at least 312 h.

Metabolic studies of selective androgen receptor modulators RAD140 and S-23 in horses.

This paper describes the studies of the in vitro biotransformation of two selective androgen receptor modulators (SARMs), namely, RAD140 and S-23, and the in vivo metabolism of RAD140 in horses using ultra-high performance liquid chromatography-high resolution mass spectrometry. in vitro metabolic studies of RAD140 and S-23 were performed using homogenised horse liver. The more prominent in vitro biotransformation pathways for RAD140 included hydrolysis, hydroxylation, glucuronidation and sulfation. Metabolic pathways for S-23 were similar to those for other arylpropionamide-based SARMs. The administration study of RAD140 was carried out using three retired thoroughbred geldings. RAD140 and the majority of the identified in vitro metabolites were detected in post-administration urine samples. For controlling the misuse of RAD140 in horses, RAD140 and its metabolite in sulfate form gave the longest detection time in hydrolysed urine and could be detected for up to 6 days post-administration. In plasma, RAD140 itself gave the longest detection time of up to 13 days. Apart from RAD140 glucuronide, the metabolites of RAD140 described herein have never been reported before.

Tiludronic acid can be detected in blood and urine samples from Thoroughbred racehorses over 3 years after last administration.

BACKGROUND: Administration of bisphosphonates, including tiludronic acid, to Thoroughbred racehorses below 3 and a half years of age is prohibited in most racing jurisdictions. OBJECTIVES: To determine if evidence of administration of tiludronic acid could be obtained from analysis of blood and urine samples beyond 40 days after administration. STUDY DESIGN: Retrospective cohort. METHODS: Horses maintained in a highly controlled environment and treated with Tildren®a were selected from clinical records. Twenty-four horses were identified, 21 of which were still in race training. Blood and urine samples were collected and analysed for the presence of tiludronic acid using ultra-high-performance liquid chromatography-high-resolution mass spectrometry. RESULTS: Tiludronic acid was detected in samples from every horse, including two that had been given a therapeutic dose of the drug 3 years prior to sample collection. The estimated concentrations of tiludronic acid in the blood collected at least 2 years post-administration were consistently very low (less than 0.3 ng/mL). The estimated concentrations in urine were less consistent and were generally lower than those in blood, although higher levels were inconsistently detected in individual horses (up to about 16 ng/mL almost 1 year post-administration in 1 horse and about 3.7 ng/mL at almost 3 years post-administration in another). MAIN LIMITATIONS: The study was performed in horses that are older than the primary target group. A single sample was obtained from most horses and so we cannot comment on elimination profiles. CONCLUSIONS: Evidence that a therapeutic dose of tiludronic acid has been administered to a horse can be obtained from detection of the drug in blood and urine samples over 3 years after it was administered.

Detection of bioactive peptides including gonadotrophin-releasing factors (GnRHs) in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS).

The use of bioactive peptides as a doping agent in both human and animal sports has become increasingly popular in recent years. As such, methods to control the misuse of bioactive peptides in equine sports have received attention. This paper describes a sensitive accurate mass method for the detection of 40 bioactive peptides and two non-peptide growth hormone secretagogues (< 2 kDa) at low pg/mL levels in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS). A simple mixed-mode cation exchange solid-phase extraction (SPE) cartridge was employed for the extraction of 42 targets and/or their in vitro metabolites from horse urine. The final extract was analyzed using UHPLC/HRMS in positive electrospray ionization (ESI) mode under both full scan and data independent acquisition (DIA, for MS2 ). The estimated limits of detection (LoD) for most of the targets could reach down to 10 pg/mL in horse urine. This method was validated for qualitative detection purposes. The validation data, including method specificity, method sensitivity, extraction recovery, method precision, and matrix effect were reported. A thorough in vitro study was also performed on four gonadotrophin-releasing factors (GnRHs), namely leuprorelin, buserelin, goserelin, and nafarelin, using the S9 fraction isolated from horse liver. The identified in vitro metabolites have been incorporated into the method for controlling the misuse of GnRHs. The applicability of this method was demonstrated by the identification of leuprorelin and one of its metabolites, Leu M4, in urine obtained after intramuscular administration of leuprorelin to a thoroughbred gelding (castrated horse).

Doping control analysis of antipsychotics and other prohibited substances in equine plasma by liquid chromatography/tandem mass spectrometry.

Antipsychotics are banned substances and considered by the Fédération Equestrian Internationale (FEI) to have no legitimate use in equine medicine and/or have a high potential for abuse. These substances are also prohibited in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering (published by the International Federation of Horseracing Authorities). Over the years, antipsychotics have been abused or misused in equestrian sports and horseracing. A recent review of literature shows that there is yet a comprehensive screening method for antipsychotics in equine samples. This paper describes an efficient liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous detection of over 80 antipsychotics and other prohibited substances at sub-parts-per-billion (ppb) to low-ppb levels in equine plasma after solid-phase extraction (SPE).

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.

Detection of seventy-two anabolic and androgenic steroids and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry in multiplexed targeted MS2 mode and gas chromatography-tandem mass spectrometry.

Anabolic and androgenic steroids (AAS) are banned substances in both human and equine sports. They are often administered intramuscularly to horses in esterified forms for the purpose of extending their time of action. The authors' laboratory has previously reported an UHPLC/HRMS method using quadrupole-Orbitrap mass spectrometer in full scan and parallel reaction monitoring (PRM) mode for the detection of 48 AAS and/or their esters in horse hair. However, two injections were required due to the long duty cycle time. In this paper, an UHPLC/HRMS method using multiplexed targeted MS2 mode was developed and validated to improve the coverage to 65 AAS and/or their esters in a single injection. In addition, a GC/MS/MS method in selected reaction monitoring (SRM) mode was developed to screen for another seven AAS and/or their esters not adequately covered by the UHPLC/HRMS method using the same sample extract after derivatisation with pentafluoropropionic anhydride. The UHPLC/HRMS and GC/MS/MS methods in combination allowed the detection of 72 AAS and/or their esters with estimated limits of detection down to sub to low ppb levels with good interday precision. Method applicability was demonstrated by the detection of boldione and 4-androstenedione in two out-of-competition hair samples and testosterone propionate in a referee hair sample.

Metabolic study of methylstenbolone in horses using liquid chromatography-high resolution mass spectrometry and gas chromatography-mass spectrometry.

Methylstenbolone (2,17α-dimethyl-5α-androst-1-en-17ß-ol-3-one) is a synthetic anabolic and androgenic steroid (AAS) sold as an oral 'nutritional supplement' under the brand names 'Ultradrol', 'M-Sten' and 'Methyl-Sten'. Like other AASs, methylstenbolone is a prohibited substance in both human and equine sports. This paper describes the studies of the in vitro and in vivo metabolism of methylstenbolone in horses using LC/HRMS, GC/MS and GC/MS/MS. Phase I in vitro metabolic study of methylstenbolone was performed using homogenised horse liver. Hydroxylation was the only biotransformation observed. Six in vitro metabolites were detected including four mono-hydroxylated metabolites, namely 16α/ß-hydroxymethylstenbolone (M1a, M1b), 20-hydroxymethylstenbolone (M1c), 6-hydroxymethylstenbolone (M1d), and two dihydroxylated methylstenbolone metabolites (M2c-M2d). An in vivo experiment was carried out using two retired thoroughbred geldings. Each horse was administered with 100 mg methylstenbolone supplement by stomach tubing daily for three consecutive days. Methylstenbolone and 14 metabolites were detected in the post-administration urine samples. The proposed in vivo metabolites included 16α/ß-hydroxymethylstenbolone (M1a, M1b), 20-hydroxymethylstenbolone (M1c), two dihydroxylated methylstenbolone (M2a, M2b), 17-epi-methylstenbolone (M3), methasterone (M4), 2,17-dimethylandrostane-16,17-diol-3-one (M5), dihydroxylated and reduced methylstenbolone (M6), 2α,17α-dimethylandrostane-3α,17ß-diol (M7), 2,17-dimethylandrostane-3,16,17-triol (M8a-M8c) and 2,17-dimethylandrostane-2,3,16,17-tetraol (M9), formed from hydroxylation, reduction and epimerisation. Methylstenbolone and ten of its metabolites could be detected in post-administration plasma samples. The highest concentration of methylstenbolone detected in urine was about 10-36 ng/mL at 3-4 h after the last administration, while the maximum concentration in plasma was about 0.4-0.7 ng/mL at 1 h after the last administration. For controlling the misuse of methylstenbolone, M8c and M9 gave the longest detection time in urine, while M4, M5 and M6 were the longest detecting analytes in plasma. They could be detected for up to 5 and 4.5 days respectively in urine and plasma. Apart from 16α/ß-hydroxymethylstenbolone (M1a, M1b), the methylstenbolone metabolites reported herein have never been reported before.

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.

Detection of anabolic and androgenic steroids and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry.

Anabolic and androgenic steroids (AASs) are a class of prohibited substances banned in horseracing at all times. The common approach for controlling the misuse of AASs in equine sports is by detecting the presence of AASs and/or their metabolites in urine and blood samples using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). This approach, however, often falls short as the duration of effect for many AASs are longer than their detection time in both urine and blood. As a result, there is a high risk that such AASs could escape detection in their official race-day samples although they may have been used during the long period of training. Hair analysis, on the other hand, can afford significantly longer detection windows. In addition, the identification of synthetic ester derivatives of AASs in hair, particularly for the endogenous ones, can provide unequivocal proof of their exogenous origin. This paper describes the development of a sensitive method (at sub to low parts-per-billion or ppb levels) for detecting 48 AASs and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Decontaminated horse hair was pulverised and subjected to in-situ liquid-liquid extraction in a mixture of hexane - ethyl acetate (7:3, v/v) and phosphate buffer (0.1M, pH 9.5), followed by additional clean-up using mixed-mode solid-phase extraction. The final extract was analysed using UHPLC-HRMS in the positive electrospray ionisation (ESI) mode with both full scan and parallel reaction monitoring (PRM). This method was validated for qualitative identification purposes. Validation data, including method specificity, method sensitivity, extraction recovery, method precision and matrix effect are presented. Method applicability was demonstrated by the successful detection and confirmation of testosterone propionate in a referee hair sample. To our knowledge, this was the first report of a comprehensive screening method for detecting as many as 48 AASs and/or their esters in horse hair. Moreover, retrospective analysis of non-targeted AASs and/or their esters was made feasible by re-examining the full scan UHPLC-HRMS data acquired.

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.

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.

Doping control analysis of lithium in horse urine and plasma by inductively coupled plasma mass spectrometry.

Lithium salts are commonly prescribed to treat bipolar disorder in humans. They are effective for the treatment of acute mania and the prophylaxis of manic relapses through long-term use. Although there is no reported legitimate therapeutic use of lithium in horses, its potential mood-stabilizing effect, low cost, and ready availability make lithium salt a potential agent of abuse in equine sports, especially for equestrian competition horses. Lithium can be found in soil, plants, and water, as such it is naturally present in the equine body, thus a threshold is necessary to control its misuse in horses. This paper describes the validation of quantification methods for lithium in equine urine and plasma using inductively coupled plasma mass spectrometry (ICP-MS). Based on a population study of lithium in horse urine and an administration study using a single oral dose of lithium chloride (100 mg) to mimic the daily lithium intake from a diet rich in lithium, a urinary threshold of 5 µg/mL was proposed. Applying this urinary threshold to two other administration studies (a single oral dose of 65 g of lithium chloride, and a single intravenous dose of 2.54 g of lithium chloride), excessive lithium in urine could be detected for 8 days and 2.5 days respectively. The concentrations of lithium in plasma following these three lithium chloride administration trials were also studied. Copyright © 2017 John Wiley & Sons, Ltd.

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.

In vitro metabolism studies of desoxy-methyltestosterone (DMT) and its five analogues, and in vivo metabolism of desoxy-vinyltestosterone (DVT) in horses.

The positive findings of norbolethone in 2002 and tetrahydrogestrinone in 2003 in human athlete samples confirmed that designer steroids were indeed being abused in human sports. In 2005, an addition to the family of designer steroids called 'Madol' [also known as desoxy-methyltestosterone (DMT)] was seized by government officials at the US-Canadian border. Two years later, a positive finding of DMT was reported in a mixed martial arts athlete's sample. It is not uncommon that doping agents used in human sports would likewise be abused in equine sports. Designer steroids would, therefore, pose a similar threat to the horseracing and equestrian communities. This paper describes the in vitro metabolism studies of DMT and five of its structural analogues with different substituents at the 17α position (RH, ethyl, vinyl, ethynyl and 2 H3 -methyl). In addition, the in vivo metabolism of desoxy-vinyltestosterone (DVT) in horses will be presented. The in vitro studies revealed that the metabolic pathways of DMT and its analogues occurred predominantly in the A-ring by way of a combination of enone formation, hydroxylation and reduction. Additional biotransformation involving hydroxylation of the 17α-alkyl group was also observed for DMT and some of its analogues. The oral administration experiment revealed that DVT was extensively metabolised and the parent drug was not detected in urine. Two in vivo metabolites, derived respectively from (1) hydroxylation of the A-ring and (2) di-hydroxylation together with A-ring double-bond reduction, could be detected in urine up to a maximum of 46 h after administration. Another in vivo metabolite, derived from hydroxylation of the A-ring with additional double-bond reduction and di-hydroxylation of the 17α-vinyl group, could be detected in urine up to a maximum of 70 h post-administration. All in vivo metabolites were excreted mainly as glucuronides and were also detected in the in vitro studies. Copyright © 2015 John Wiley & Sons, Ltd.

Doping control analyses in horseracing: a clinician's guide.

Doping(1) in sports is highly detrimental, not only to the athletes involved but to the sport itself as well as to the confidence of the spectators and other participants. To protect the integrity of any sport, there must be in place an effective doping control program. In human sports, a 'top-down' and generally unified approach is taken where the rules and regulations against doping for the majority of elite sport events held in any country are governed by the World Anti-Doping Agency (WADA). However, in horseracing, there is no single organisation regulating this form of equestrian sport; instead, the rules and regulations are provided by individual racing authorities and so huge variations exist in the doping control programs currently in force around the world. This review summarises the current status of doping control analyses in horseracing, from sample collection, to the analyses of the samples, and to the need for harmonisation as well as exploring some of the difficulties currently faced by racing authorities, racing chemists and regulatory veterinarians worldwide.