Acetylpromazine, 1-{10-[3-(dimethylamino)propyl]-10H-phenothiazin-2-yl}ethenone, C19H22N2OS, 326.46 g·mol-1 is a phenothiazine derivative at one time used in human medicine as an antipsychotic medication but now predominantly used in veterinary medicine as a sedative/tranquilizer and referred to as acepromazine. In performance horses its use is regulated by using a 10 ng/mL threshold for the major urinary metabolite 2-(1-hydroxyethyl) promazine-sulfoxide (HEPS) in equine urine. To enable accurate quantitation of HEPS in equine urine we have synthesized and purified hydroxyethylpromazine sulfoxide-d4 (HEPS-d4) to be used as a stable isotopically labeled internal standard. Although labeled HEPS is commercially available (CAS 1346605-30-8), to the best of our knowledge there is no published synthetic procedure in the scientific literature. Here we demonstrate a viable synthetic procedure consisting of four major steps: (i) freebasing the Acepromazine maleate salt, (ii) H-D exchange of Acepromazine at room temperature, (iii) reduction of the ketone with NaBD4, and (iv) oxidation of the thioether via hydrogen peroxide and acetic acid. This deuterated internal standard will allow for precise LC/MS quantitation of HEPS at regulatory threshold concentrations, enabling accurate detection and quantitation of picogram/mL concentrations in equine urine samples, thereby supporting regulatory compliance for equine medication control programs.

Detomidine, 5-[(2,3-dimethylphenyl)methyl]-1H-imidazole, is a tranquilizer/sedative/analgesic widely used in equine medicine and regulated by several different analyte concentrations of detomidine and its hydroxydetomidine and carboxydetomidine metabolites in plasma and urine. Accurate regulatory quantitation of detomidine at low picogram/mL concentrations requires the availability of a stable isotope internal standard of detomidine; however, to the best of our knowledge, no certified deuterated internal standard of detomidine is commercially available and its synthesis has not been reported. Here, we report the first synthesis and characterization of detomidine-d, prepared in six sequential steps consisting of a deuterium-hydrogen exchange, diazotization, Grignard coupling, hydrogenolysis, and palladium-catalyzed deuterium-hydrogen exchange. This overall route gave detomidine-d in moderate chemical yield with each intermediate purified to > 90% purity as determined by RP-HPLC. Regiochemistry and isotopic enrichment were confirmed by 1H NMR and LC/MS, demonstrating a viable method for the incorporation of three deuterium atoms into the detomidine scaffold. This internal standard will allow for precise LC/MS quantitation of detomidine at regulatory threshold concentrations, enabling accurate detection and quantitation of picogram/mL concentrations in equine blood and urine samples, thereby supporting regulatory compliance for equine medication control programs.

Bupivacaine is a local anesthetic widely used in equine and human medicine. Use of bupivacaine in performance horses is regulated because its ability to block pain means that it can be misused for advantage in performance horses. In racing regulation, bupivacaine is classified by the Association of Racing Commissioners International (ARCI) as a Class 2 Penalty Class A Foreign substance, the detection of which can lead to significant penalties. In horses, bupivacaine is metabolized by Phase-I hydroxylation to yield 3-hydroxybupivacaine, which is then glucuronidated to yield the Phase-II metabolite bupivacaine-3-hydroxyglucuronide, which is excreted at relatively high concentrations in equine urine. Standard regulatory procedure during urinalysis is to perform an enzymatic hydrolysis, thereby enabling subsequent detection of 3-hydroxybupivacaine, the primary analyte used for bupivacaine regulation in urine samples from competition horses. We now report on the synthesis of 3-hydroxybupivacaine and deuterated 3-hydroxybupivacaine from piperidine-2-carboxylic acid in six successive steps with moderate yield. The compounds were characterized by 1H and 13C NMR and their purity ascertained by HPLC-MS. The deuterated bupivacaine and 3-hydroxybupivacaine were further confirmed by HRMS. The synthesis of these compounds provides certified reference standards and stable isotope-labeled internal standards for drug testing in competitive equine sports including horse racing.

Metformin is a widely prescribed oral antihyperglycemic agent and currently a first-line medication in the treatment of human type 2 diabetes, with a total of 92 million US prescriptions in 2022. The daily dose per human can be as much as 2.5 grams/day which is excreted largely unchanged into the environment. Metformin is chemically stable and a widely distributed environmental substance. Metformin therefore has the potential to be identified at trace levels in equine blood and urine samples as a result of random exposure to environmental metformin. Given these circumstances we have reviewed the scientific literature and calculated an irrelevant blood/plasma/serum concentration of metformin of 5 nanograms/ml. We now therefore propose this plasma concentration of metformin as an interim Screening Limit of Detection (SLOD) for metformin, below which concentration a blood/plasma/serum identification of metformin should not be considered appropriate for regulatory action.

This case report presents the first reported identifications of 20-hydroxyecdysone in post-event blood samples from an Endurance horse and a Harness horse racing in New South Wales, Australia.20-Hydroxyecdysone is a plant secondary metabolite structurally related to testosterone and used by plants to discourage insect predation.20-Hydroxyecdysone is found in many plants including spinach and is not infrequently identified at low concentrations in mammalian systems including in humans.Given its steroid-related structure, 20-hydroxyecdysone has been reported to enhance athletic performance in humans, although at this time 20-hydroxyecdysone is simply being monitored by the World Anti-Doping Agency (WADA) [31] .The Harness horse identification led the Harness Racing New South Wales (HRNSW) authorities to evaluate the home pastures of the horse in question, where they recovered at least three plants containing significant concentrations of 20-hydroxyecdysone.The HRNSW stewards were satisfied that the 20-hydroxyecdysone identification was due to "environmental contamination emanated from plants" in the trainer's establishment and did not impose a penalty on the trainer.These findings show that the pasture plant steroid 20-hydroxyecdysone is found in pasture plants from which it is bioavailable to equines and can present as low part per billion concentrations in equine blood samples, as has also been noted in humans.Based on these Australian identifications of 20-hydroxyecdysone in pasture plants and also in equine blood samples and the HRNSW decision not to penalize the trainer involved and the likelihood of no pharmacological response to pasture plant exposure to this substance we now present 2 parts per billion in equine blood/plasma as an appropriate regulatory cut-off or Screening Limit of Detection (SLOD) for 20-hydroxecdysone in equine blood/plasma.

Consistent with recently increased street availability and recreational use of the potent synthetic opioid fentanyl, there has been a parallel increase in trace level plasma identifications of fentanyl in racing horses, at times in association with trace level amounts of other human recreational substances such as the synthetic cathinone eutylone, currently classified as a designer drug.Fentanyl has three basic effects in the horse, i.e., an analgesic effect, a locomotor stimulation response and a potential endurance effect.Fentanyl is considered relatively straightforward to synthesize following a four-step procedure.Eutylone is the most frequently identified cathinone-related substance identified in the US and is considered a synthetic stimulant.Eutylone is inexpensive to produce and mimics the effects of cocaine, methamphetamine and 3,4-methylenedioxymethamphetamine, commonly known as ecstasy, and is, like fentanyl, a street marketed human recreational substance.Both substances are listed in horseracing as Class 1 with Penalty Class A substances, therefore having the highest penalties for identifications in horses given their stimulant properties, according to the ARCI (Association of Racing Commissioners International).Given that racing authorities recognize the potential for substances of "human use and addiction" to inadvertently transfer to racing horses, we were asked to develop an Irrelevant Plasma Concentration (IPC) for fentanyl in horses.Additionally, the finding of a trace level of eutylone along with a trace level of fentanyl increases the likelihood that these paired trace level identifications were caused by inadvertent transfer from a human using a combination of recreational substances.With regard to fentanyl, review of the published pharmacology of fentanyl suggested that locomotor responses disappear below plasma concentrations of 5 ng/mL, with the locomotor response peaking at 50 ng/mL.Similarly, the antinociceptive effects of fentanyl require concentrations above 6.5 ng/mL.An effective plasma concentration (EPC) of 25 ng/mL was therefore decided on.Dividing this EPC by 500, the conservative Toutain & Lassourd safety factor (SF) gives a 50 pg/ml IPC for fentanyl in the horse.This value was not exceeded by any of the low fentanyl concentrations identified in 125,000 post-race samples in the 2018-2022 time range.Plasma concentrations of fentanyl in the sub-40 pg/mL range are therefore pharmacologically irrelevant with a significant likelihood of transfer from recreational users.This IPC value is consistent with a number of recent trace level plasma fentanyl identifications in equine samples and judgements in these matters by regulatory authorities that the likely source of these trace level plasma fentanyl identifications was inadvertent transfer from human recreational users to the horses in question.The first detections of fentanyl detections in racehorses occurred in the period 1978-1983 corresponding to the introduction of sensitive radioimmunoassay screening methods.This contrasts with the more recent uptick in 2018-2022, apparently due to exposure of horses to inadvertent trace level transfers from recreational users of fentanyl.Eutylone continues to be a concern, but more information on its equine pharmacokinetics is necessary before similar development of EPC and IPC can be determined.

Gabapentin, 1-(Aminomethyl)cyclohexaneacetic acid, MW 171.240, is a frequently prescribed high dose human medication that is also used recreationally. Gabapentin is orally absorbed; the dose can be 3,000 mg/day and it is excreted essentially unchanged in urine. Gabapentin is stable in the environment and routinely detected in urban wastewater. Gabapentin randomly transfers from humans to racing horses and is at times detected at pharmacologically ineffective / trace level concentrations in equine plasma and urine. In Ohio racing between January 2019 and July 2020,18 Gabapentin identifications, all less than 2 ng/ml in plasma, were reported. These identifications were ongoing because the horsemen involved were unable to pin down and therefore avoid the source of these identifications. Given that 44 ng/ml or less is an Irrelevant Plasma Concentration (IPC) of Gabapentin in horses, we proposed a 5 ng/ml plasma interim Screening Limit of Detection for Gabapentin identifications in Ohio racing, and an essentially similar 8 ng/ml plasma Screening Limit of Detection was suggested by a scientific advisor to the Ohio Horse Racing Commission. As such, an analytical Screening Limit of 8 ng /ml in plasma is an appropriate and pharmacologically conservative analytical "cut-off" or Screening Limit of Detection (SLOD) for Gabapentin in equine competitive events to avoid the calling of "positive" identifications on random unavoidable trace level identifications of this widely prescribed human therapeutic medication in equine forensic samples.

At last 5Km in the endurance competition a gelding (Australian Stock Horse) with 10-years-old showed poor performance and exhausted. On clinical examination the horse presented exhausted, depression, lethargy and anorexia. After start the treatment the horse death. On necropsy showed multiples hemorrhages in the adrenal cortex and renal cortical with papillary necrosis, acute tubular necrosis. Histopathology showed: hemorrhages in adrenal cortex, capsule thickening and atrophy cortical with coagulation necrosis of zona glomerulosa, coagulation necrosis focal zona fasciculata, only congestion of the zona reticularis. In conclusion, we report a case of adrenocortical insufficiency syndrome in a resistance horse with rapid development of clinical signs, without response to emergency treatment, confirmed with laboratory, necropsy and histopathology results.

Based on structural similarities and equine administration experiments, Barbarin, 5-phenyl-2-oxazolidinethione from Brassicaceae plants, is a possible source of equine urinary identifications of aminorex, (R,S)-5-phenyl-4,5-dihydro-1,3-oxazol-2-amine, an amphetamine-related US Drug Enforcement Administration (DEA) controlled substance considered illegal in sport horses. We now report the synthesis and certification of d5 -barbarin to facilitate research on the relationship between plant barbarin and such aminorex identifications. D5 -barbarin synthesis commenced with production of d5 -2-oxo-2-phenylacetaldehyde oxime (d5 -oxime) from d5 -acetophenone via butylnitrite in an ethoxide/ethanol solution. This d5 -oxime was then reduced with lithium aluminum hydride (LiAlH4 ) to produce the corresponding d5 -2-amino-1-phenylethan-1-ol (d5 -phenylethanolamine). Final ring closure of the d5 -phenylethanolamine was performed by the addition of carbon disulfide (CS2 ) with pyridine. The reaction product was purified by recrystallization and presented as a stable white crystalline powder. Proton NMR spectroscopy revealed a triplet at 5.88 ppm for one proton, a double doublet at 3.71 ppm for one proton, and double doublet at 4.11 ppm for one proton, confirming d5 -barbarin as the product. Further characterization by high resolution mass spectrometry supports the successful synthesis of d5 -barbarin. Purity of the recrystallized product was ascertained by High Performance Liquid Chromatography (HPLC) to be greater than 98%. Together, we have developed the synthesis and full characterization of d5 -barbarin for use as an internal standard in barbarin-related and equine forensic research.

Zilpaterol is a β2-adrenergic agonist medication approved in certain countries as a cattle feed additive to improve carcass quality. Trace amounts of Zilpaterol can transfer to horse feed, yielding equine urinary “identifications” of Zilpaterol. These “identifications” occur because Zilpaterol is highly bioavailable in horses, resistant to biotransformation and excreted as unchanged Zilpaterol in urine, where it has a 5 day or so terminal half-life. In horses, urinary steady-state concentrations are reached 25 days (5 half-lives) after exposure to contaminated feed. Zilpaterol readily presents in horse urine, yielding clusters of feed related Zilpaterol identifications in racehorses. The first cluster, April 2013, involved 48 racehorses in California; the second cluster, July 2013, involved 15 to 80 racehorses in Hong Kong. The third cluster, March 2019, involved 24 racehorses in Mauritius; this cluster traced to South African feedstuffs, triggering an alert concerning possible Zilpaterol feed contamination in South African racing. The fourth cluster, September/October 2020 involved 18 or so identifications in French racing, reported by the French Laboratories des Courses Hippiques, (LCH), and in July 2021, a fifth cluster of 10 Zilpaterol identifications in South Africa. The regulatory approach to these identifications has been to alert horsemen and feed companies and penalties against horsemen are generally not implemented. Additionally, given their minimal exposure to Zilpaterol, there is little likelihood of Zilpaterol effects on racing performance or adverse health effects for exposed horses. The driving factor in these events is that Zilpaterol is dissolved in molasses for incorporation into cattle feed. Inadvertent incorporation of Zilpaterol containing molasses into horse feed was the source of the California and Hong Kong Zilpaterol identifications. A second factor in the 2019 Mauritius and 2020 French identifications was the sensitivity of testing for Zilpaterol in Mauritius and France, with the French laboratory reportedly testing at a “more sensitive level for Zilpaterol”. As of January 1st, 2021, the new FEI Atypical Finding (ATF) policy specifies Zilpaterol as a substance to be treated as an Atypical Finding (ATF), allowing consideration of inadvertent feed contamination in the regulatory evaluation of Zilpaterol identifications.

Diclazuril is a triazine-based antiprotozoal agent widely used in veterinary practice that may have clinical application in the treatment of bovine protozoal diseases. The present study reports on the bioavailability, pharmacokinetics, and metabolism of diclazuril and diclazuril sodium salt in cattle following administration of diclazuril suspended in water and by direct application of diclazuril sodium salt to the oral mucosa. Compared with diclazuril itself, the sodium salt formulation of diclazuril applied to the oral mucosa was rapidly and reliably absorbed. Plasma concentrations of diclazuril peaked at around 8 h after oral-mucosal administration of diclazuril sodium salt. On the contrary, application of diclazuril itself orally resulted in delayed and variable absorption. The mean bioavailability of diclazuril as pure powder was 42.5% relative to diclazuril sodium salt indicating approximately 2.5-fold increase in bioavailability of diclazuril as a sodium salt relative to diclazuril as a pure compound in cattle. The present study also reports finding of a previously unreported diclazuril metabolite at high concentrations in plasma especially after oral administration of diclazuril. Further studies, including synthesis and characterization of the novel described metabolite, are required to accurately determine aspects of the metabolism of diclazuril in cattle.

In veterinary medicine, a number of alpha-2 receptor agonists are marketed as sedatives/hypnotics and analgesics, with their principal use being the chemical restraint of large and small animals. Guanabenz (Wytensin((R))) is an alpha-2 adrenergic receptor agonist marketed for use in humans as an anti-hypertensive agent. Recent reports indicate that guanabenz has been administered to horses in small doses (0.04 mg/kg) for its anti-hypertensive effects. While this offers both benefits of sedation of the horse as well as amelioration of pulmonary hypertension during running exercise and consequent Exercise-Induced Pulmonary Hemorrhage (EIPH), guanabenz is currently proscribed in most racing jurisdictions and its administration to a racing horse can lead to penalties. The Association of Racing Commissioners International (ARCI) lists guanabenz as an ARCI Class 3 agent; Class 3 agents include bronchodilators, anabolic steroids and other drugs with primary effects on the autonomic nervous system, procaine, antihistamines with sedative properties and diuretics and includes amitraz, clonidine, xylazine, detomidine, medetomidine, and romifidine. Guanabenz is unique among alpha-2 agonists in that it differentiates into E- and Z-forms (Fig. 1), with the Z-form lacking hypotensive properties, yet with both E- and Z-forms able to afford relief to cellular stresses related to inflammation or degenerative diseases. The objective of the study was a preliminary description of the pharmacological properties of guanabenz in comparison with clonidine and a number of other alpha-2 agonists. The goal was clinical evaluation of their sedative, analgesic and related activities with the goal of increasing our understanding of the clinical use of such medications and also as a possible prophylaxis for Exercise-Induced Pulmonary Hemorrhage. The clinical study of guanabenz and clonidine was performed in a complete crossover strategy using quantitative markers of sedation, antinociception, heart rate, blood and urine glucose following administration of each compound in five horses. Amitraz, detomidine, medetomidine, romifidine, and xylazine were studied in one horse each. The sedation was quantified by measuring head droop and locomotor activity, while antinociception was measured by Hoof Withdrawal Reflex Latency. Heart rates, urine glucose, urine production and urine specific gravities were also determined by standard clinical chemistry techniques. Guanabenz serum levels and related urinary guanabenz glucuronide levels were determined by established Liquid Chromatography-tandem Mass Spectrometric (LC-MS) methods. In result the clinically effective doses (0.2 mg/kg) of guanabenz produced a rapid and intense sedative effect, with sagging of the lower lip, sunken eyelids, and marked head droop corresponding to plasma guanabenz concentrations that peaked at 120 ng/mL at 2.5 min post-injection (Fig. 2). The initial head height above the ground is considered 100 %, and head heights fell to values ranging 18-40 % with guanabenz, all of which are greater than a 50 % reduction in head height, considered a full clinically useful sedative effect. Despite the intensity of the sedation, all horses remained standing and were able to walk, and the sedation and head droop responses were rapidly reversed by intravenous administration of the alpha-2 receptor antagonist yohimbine, reversals occurring within 10 min of administration. As a pilot investigation this study was extended to six other members of the alpha-2 agonist group, clonidine administered to five horses, and amitraz, detomidine, medetomidine, romifidine, and xylazine to one horse each. Hoof Withdrawal Reflex Latency evaluation demonstrated the considerable analgesic properties of guanabenz, greater than the corresponding potencies among clonidine, detomidine, romifidine, medetomidine and xylazine. Heart rate monitoring showed guanabenz as possessing capacity for prolonged bradycardia, with effects of a single dose lasting for up to 3.5 hr, in contrast with clonidine (1 hr), amitraz (2 hr), detomidine (<1 hr), medetomidine (1 hr), romifidine (2 hr), and xylazine (<1 hr). Peak urine production following guanabenz administration occurred between 1.5 and 3.0 hr after administration (Fig. 6), as indicated by the steeper decline of the urine volume curve during that period. Urine specific gravity dropped to a low of about 1.006 at 2.0 hr after administration and remained at this level for similar to 1.0 hr. Urine pH remained at 8, and urine protein was negative throughout testing. The other alpha-2 agonists evaluated also caused an increased urine production with a concomitant decrease in specific gravity. The effect of guanabenz had the longest duration on increased urine volume, lasting about 3.0 hr. Xylazine had the shortest diuretic effect, persisting for only about 1.0 hr. Guanabenz along with romifidine and detomidine induced glucosuria whereas other alpha-2 agonists did not. Hyperglycemia and the corresponding glucosuria resulted in a significant diuresis, as shown by the cumulative urine volume. Guanabenz along with amitraz, detomidine and xylazine also produced measurable sedation presenting as reduced locomotor activity (Table 1). While all alpha-2 agonists showed qualitatively similar pharmacological responses, only guanabenz produced an intense and relatively prolonged antinociceptive response. The study is limited by the number of horses examined (five each for guanabenz and clonidine, five for repeat studies that included yohimbine antagonism, and one each for the other agonists). Study design was focused on clinical evaluation of agonist similarities and differences and thus did not specifically generate data for detailed statistical evaluation. In conclusion these studies show that a 100 mg IV dose of guanabenz rapidly induces clinically useful sedation, analgesia and antinociception effects that are more intense and considerably longer-lasting than those produced by other alpha-2 receptor agonists evaluated. Guanabenz also remains detectable in serum up to 8-hours following administration at doses as low as 0.04 mg/kg. In the work reported here, guanabenz administered at 0.2 mg/kg IV showed peak concentrations in serum of 120 ng/ mL at 2.5 min and was detectable for up to 4 hr with its glucuronide metabolite peaking at 120 min post-administration. Although we did not investigate the combination of guanabenz with opioid drugs such as butorphanol for pain management, guanabenz may well be a drug of choice among the other alpha-2 agonists evaluated in this study for administration with opioids for pain management based on maintaining maximum levels of analgesia for longer periods of time. These experiments suggest considerable clinical potential for guanabenz as a sedative and a relatively long-lasting analgesic in equine medicine. Based on these pharmacological properties, guanabenz and related alpha-2 agonists also have considerable potential for clinical use in equine medicine.

Dexamethasone (DXM) sodium phosphate is a widely used corticosteroid for inflammatory conditions in horses, regulated in racing jurisdictions in the USA by a 0.005 ng/ml serum/plasma threshold. This study seeks to describe serum concentrations of DXM at 48 and 72 h after intravenous administration of 20 mg DXM sodium phosphate over 1 to 5 days, and to identify a possible source of DXM overages. 74 horses (39 Thoroughbreds, 13 Standardbreds, 22 Quarter Horses) in active race training received 20 mg DXM sodium phosphate. Serum was collected before injection, at 48 and 72 h post last injection, and analysed by LC/MS-MS (limit of quantification (LOQ) = 2.5 pg/ml). No differences were identified by ANOVA (P≤0.05) for racing breeds, age, gender or the number of days of DXM sodium phosphate administration, so data were pooled for each time point. The DXM serum concentration at 48 h (mean ± standard deviation, range) was 2.18±1.56 pg/ml (<2.5 to 40 pg/ml). Summary statistics could not be derived for 72 h DXM serum concentration data owing to censored data, but ranged from <2.5 to 95.8 pg/ml. There was one extreme outlier (Tukey) at 48 h, and two extreme outliers at 72 h. A separate study was conducted using sedentary experimental horses to determine the likelihood that positive DXM samples could result from environmental transfer. Urine was collected from a mare 2 to 3 h post administration of 20 mg DXM. Hay with 100 ml of the DXM (17 ng/ml) containing urine was offered to each of six experimental horses and blood was collected at 0, 4, 8, 12, 16, 20 and 24 h. All six horses had plasma DXM concentration above the limit of detection and five of six had plasma DXM concentrations above the LOQ for at least one sample time.

The aim of this study is to report a case of iatrogenic poisoning by a turpentine-based commercial product in a 3-day-old foal and the successful treatment of this condition. A 3-day-old male foal presented with acute cardio-respiratory collapse, anaphylactic shock, severe respiratory distress, dyspnea and pulmonary edema. The animal’s history included previous administration of a commercial veterinary product (UNITIPAN). Clinical examination revealed: temperature: (39.9 °C), pulse: 135 bpm, respiration: 51 bpm, mucous membranes: moist and congested, capillary refill time: 3 sec or less. The foal was treated with emergency hydration, duphalyte, steroidal anti-inflammatory, atropine sulphate and antibiotic. After 6 h of treatment, the foal was stabilized and vital signs were normalized 8 h after treatment start.

Aminorex is a US DEA Schedule 1 controlled substance occasionally detected in racing horses. A number of aminorex identifications in sport horses were thought to have been caused by exposure to plant sources of aminorex. Glucobarbarin, found in plants of the Brassicaceae family, has been suggested as a potential proximate chemical source by being metabolized in the plant or the horse to aminorex. In Brassicaceae, glucobarbarin is hydrolyzed by myrosinase to yield barbarin, which serves as an insect repellant and/or attractant and is structurally related to aminorex. The synthesis, purification, and characterization of barbarin is now reported for use as a reference standard in aminorex related research concerning equine urinary identifications of aminorex and also for possible use in equine administration experiments. Synthesis of barbarin was performed via ring closure between phenylethanolamine and carbon disulfide in tetrahydrofuran with the catalyst pyridine under reflux. The reaction yielded a white crystalline substance that was purified and chemically characterized as barbarin for use as a Certified Reference Standard or for studies related to equine aminorex identification.

Starting in August 2015 Thoroughbred racing in Charles Town, West Virginia experienced a sequence of intermittent low concentration Naproxen identifications from 6.3 to 161 ng/ml of plasma (27.3 to 699 nM). These identifications were ongoing, indicating the horsemen were unaware of their origins. Naproxen is administered orally to horses at substantial doses and is chemically stable in the environment. These identifications are therefore most likely associated with exposure of these horses to environmental traces of Naproxen. Given the low concentrations of these identifications, we were asked to identify a Screening Limit of Detection (SLOD) below which these trace level Naproxen identifications would not be reported. Review of the data set suggested an SLOD of 200 ng/ml, while outlier analysis suggested an 'extreme' outlier level at 247 ng/ml, which figure was rounded up to 250 ng/ml Naproxen or 1.09 uM. This proposed SLOD is in good agreement with other US regulatory thresholds for therapeutic medications and this Interim Screening Limit of Detection was presented for review.

BACKGROUND:Aminorex, (RS)-5- Phenyl-4,5-dihydro-1,3-oxazol-2-amine, is an amphetamine-like anorectic and in the United States a Drug Enforcement Administration [DEA] Schedule 1 controlled substance. Aminorex in horse urine is usually present as a metabolite of Levamisole, an equine anthelmintic and immune stimulant. Recently, Aminorex identifications have been reported in horse urine with no history or evidence of Levamisole administration. Analysis of the urine samples suggested a botanical source, directing attention to the Brassicaceae plant family, with their contained GlucoBarbarin and Barbarin as possible sources of Aminorex. Since horsepersons face up to a 1 year suspension and a $10,000.00 fine for an Aminorex identification, the existence of natural sources of Aminorex precursors in equine feedstuffs is of importance to both individual horsepersons and the industry worldwide.RESULTS:Testing the hypothesis that Brassicaceae plants could give rise to Aminorex identifications in equine urine we botanically identified and harvested flowering Kentucky Barbarea vulgaris, ("Yellow Rocket") in May 2018 in Kentucky and administered the plant orally to two horses. Analysis of post-administration urine samples yielded Aminorex, showing that consumption of Kentucky Barbarea vulgaris can give rise to Aminorex identifications in equine urine.CONCLUSIONS:Aminorex has been identified in post administration urine samples from horses fed freshly harvested flowering Kentucky Barbarea vulgaris, colloquially "Yellow Rocket". These identifications are consistent with occasional low concentration identifications of Aminorex in equine samples submitted for drug testing. The source of these Aminorex identifications is believed to be the chemically related Barbarin, found as its precursor GlucoBarbarin in Kentucky Barbarea vulgaris and related Brassicaceae plants worldwide.

BACKGROUND:Methylprednisolone (MP) acetate is a commonly used corticosteroid for suppression of inflammation in synovial structures in horses. Its use is often regulated in equine sports by plasma MP concentrations.OBJECTIVES:To describe variability in MP plasma concentrations after MP acetate injection in different synovial structures and with co-administration with hyaluronic acid (HA).STUDY DESIGN:Field study in actively racing horses in three disciplines (Thoroughbred, Standardbred and Quarter Horse).METHODS:Seventy-six horses (15 Thoroughbreds, 20 Standardbreds and 41 Quarter Horses) were included in the study. Injection of any synovial structure with a total body dose of 100 mg MP acetate was permitted, data were grouped according to the synovial structure injected and co-administration with HA. Plasma was collected before injection and at 6 days post-injection. Per cent censored data (below the limit of quantification) for each synovial structure were determined, and summary statistics generated by Robust Regression on Order. Differences between synovial structures and co-administration with HA were identified by ANOVA with Tukey's post hoc testing.RESULTS:The MP plasma concentration at 6 days for injection for the entire group (mean ± standard deviation [s.d.], pg/mL) was 96 ± 104. Metacarpophalangeal (MCP) plasma concentrations contained 86% censored data and could not be included in the statistical analysis. The carpal joints (CJO) group had a lower plasma MP concentration (P<0.05) than the distal tarsal joints (DTJ) or medial femorotibial (MFT), the no HA (NHA) group had a lower plasma MP concentration (P<0.05) than HA.MAIN LIMITATIONS:The synovial structures injected varied by racing discipline, so this study was unable to identify any differences between disciplines.CONCLUSIONS:Practitioners should be aware that injection of DTJ, CS and MFT joints, and combining MP acetate with HA may prolong its clearance, and withdrawal times for competition in regulated equine sports.