Comprehensive metabolic study of nicotine in equine plasma and urine using liquid chromatography/high-resolution mass spectrometry for the identification of unique biomarkers for doping control.

Nicotine is classified as a stimulant, and its use is banned in horse racing and equestrian sports by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. Because nicotine is a major alkaloid of tobacco leaves, there is a potential risk that doping control samples may be contaminated by tobacco cigarettes or smoke during sample collection. In order to differentiate the genuine doping and sample contamination with tobacco leaves, it is necessary to monitor unique metabolites as biomarkers for nicotine administration and intake. However, little is known about the metabolic fate of nicotine in horses. This is the first report of comprehensive metabolism study of nicotine in horses. Using liquid chromatography/electrospray ionization high-resolution mass spectrometry, we identified a total of 17 metabolites, including one novel horse-specific metabolite (i.e., 4-hydroxy-4-(3-pyridyl)-N-methylbutanamide), in post-administration urine samples after nasoesophageal administration of nicotine to three thoroughbred mares; eight of these compounds were confirmed based on reference standards. Among these metabolites, N-hydroxymethylnorcotinine was the major urinary metabolite in equine, but it could only be tentatively identified by mass spectral interpretation due to the lack of reference material. In addition, we developed simultaneous quantification methods for the eight target analytes in plasma and urine, and applied them to post-administration samples to establish elimination profiles of nicotine and its metabolites. The quantification results revealed that trans-3'-hydroxycotinine could be quantified for the longest period in both plasma (72 h post-administration) and urine (96 h post-administration). Therefore, this metabolite is the most appropriate monitoring target for nicotine exposure for the purpose of doping control due to its long detection times and the availability of its reference material. Further, we identified trans-3'-hydroxycotinine as a unique biomarker allowing differentiation between nicotine administration and sample contamination with tobacco leaves.

A sensitive HPLC-MS/MS method for the detection, resolution and quantitation of cathinone enantiomers in horse blood plasma and urine.

Resolution of cathinone enantiomers in equine anti-doping analysis is becoming more important to distinguish the inadvertent ingestion of plant-based products from those of deliberate administration of designer synthetic analogs. With this in mind, a rapid and sensitive method was developed and validated for the detection, resolution and quantitative determination of cathinone enantiomers in horse blood plasma and urine. The analytes were recovered from the blood plasma and urine matrices by using a liquid-liquid extraction after adjusting the pH to 9. The recovered analytes were derivatized with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide, a chiral derivatizing agent analogous to Marfey's reagent. The resulting diastereoisomers were baseline resolved under a reversed-phase liquid chromatographic condition. Derivatization of the analytes not only allowed the separation of the enantiomers using cost-effective traditional liquid chromatography conditions and reversed-phase columns but also increased the sensitivity, at least to an order of magnitude, when tandem mass spectrometry is used for the detection. A limit of detection of 0.05 ng/mL was achieved for cathinone enantiomers for both matrices. Acceptable intraday and interday precision and accuracy along with satisfactory dilution accuracy and precision were observed during the method validation. The method suitability was tested using the post administration urine samples collected after single doses of cathinone and ephedrine as single-enantiomeric form and methcathinone as racemic form. Finally, a proof of concept of the isomeric ratio in urine samples to distinguish the presence of cathinone as a result of accidental ingestion of plant-based product from that of an illicit use of a designer product is demonstrated. To the best of our knowledge, this is the first such work where cathinone enantiomers were resolved and quantified in horse blood plasma and urine at sub nanogram levels.

Metabolic study of GW1516 in equine urine using liquid chromatography/electrospray ionization Q-Exactive high-resolution mass spectrometry for doping control.

RATIONALE: The use of GW1516, a peroxisome proliferator-activated receptor δ (PPAR δ) agonist, is strictly prohibited in both horseracing and equestrian competitions. However, little is known about its metabolic fate in horses. To the best of our knowledge, this is the first reported metabolic study of GW1516 in equine urine. METHODS: Urine samples obtained from a thoroughbred after nasoesophageal administration with GW1516 were protein-precipitated and the supernatants were subsequently analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) with a Q-Exactive mass spectrometer. Monoisotopic ions of GW1516 and its metabolites were monitored from the full-scan mass spectral data of pre- and post-administration samples. A quantification method was developed and validated to establish the excretion profiles of GW1516, its sulfoxide, and its sulfone in equine urine. RESULTS: GW1516 and its nine metabolites [including GW1516 sulfoxide, GW1516 sulfone, 5-(hydroxymethyl)-4-methyl-2-(4-trifluoromethylphenyl)thiazole (HMTT), methyl 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylate (MMTC), 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylic acid (MTTC), and M1 to M4] were detected in post-administration urine samples. GW1516 sulfoxide and GW1516 sulfone showed the longest detection times in post-administration urine samples and were therefore recommended as potential screening targets for doping control purposes. Quantitative analysis was also conducted to establish the excretion profiles of GW1516 sulfoxide and GW1516 sulfone in urine. CONCLUSIONS: For the purposes of doping control of GW1516, the GW1516 sulfoxide and GW1516 sulfone metabolites are recommended as the target analytes to be monitored in equine urine due to their high specificities, long detection times (1 and 4 weeks, respectively), and the ready availability of their reference materials.

Equine metabolism of the selective androgen receptor modulator AC-262536 in vitro and in urine, plasma and hair following oral administration.

AC-262536 is one of a number of selective androgen receptor modulators that are being developed by the pharmaceutical industry for treatment of a range of clinical conditions including androgen replacement therapy. Though not available therapeutically, selective androgen receptor modulators are widely available to purchase online as (illegal) supplement products. The growth- and bone-promoting effects, along with fewer associated negative side effects compared with anabolic-androgenic steroids, make these compounds a significant threat with regard to doping control in sport. The aim of this study was to investigate the metabolism of AC-262536 in the horse following in vitro incubation and oral administration to two Thoroughbred horses, in order to identify the most appropriate analytical targets for doping control laboratories. Urine, plasma and hair samples were collected and analysed for parent drug and metabolites. Liquid chromatography-high-resolution mass spectrometry was used for in vitro metabolite identification and in urine and plasma samples. Nine phase I metabolites were identified in vitro; four of these were subsequently detected in urine and three in plasma, alongside the parent compound in both matrices. In both urine and plasma samples, the longest detection window was observed for an epimer of the parent compound, which is suggested as the best target for detection of AC-262536 administration. AC-262536 and metabolites were found to be primarily glucuronide conjugates in both urine and plasma. Liquid chromatography-tandem mass spectrometry analysis of post-administration hair samples indicated incorporation of parent AC-262536 into the hair following oral administration. No metabolites were detected in the hair.

Application of a non-target variable data independent workflow (vDIA) for the screening of prohibited substances in doping control testing.

A non-target variable Data Independent Acquisition (vDIA) workflow based on accurate mass measurements using a Q Exactive OrbiTrap is presented for the first time for equine doping control testing. The vDIA workflow uses a combination of MS1 events (1 to 2) and multiple vDIA events to cover the analytes of interest. The workflow basically captures a digital image of a sample allowing all relevant MS1 and MS2 data to be recorded. In theory, the workflow can accommodate an unlimited number of analytes as long as they are amenable to the sample extraction protocol and fall within the mass limits of the workflow. Additional targets fulfilling the above requirements can be added without changing any settings. The performance of the vDIA workflow was illustrated by applying it to two screening methods in horse urine, with one workflow covering 331 basic drugs and the other covering 45 quaternary ammonium drugs (QADs). Both screening methods have good detection sensitivity with 84% of the basic drugs having Limits of Detection (LoDs) of ≤ 1 ng/mL and 84% of the QADs having LoDs of ≤ 0.4 ng/mL. Other method characteristics including retention reproducibility, method precision and false hit rate will also be presented.

Detection and identification of ACP-105 and its metabolites in equine urine using LC/MS/MS after oral administration.

ACP-105 is a novel nonsteroidal selective androgen receptor modulator (SARM) with a tissue-specific agonist effect and does not have side effects associated with the use of common androgens. This research reports a comprehensive study for the detection of ACP-105 and its metabolites in racehorses after oral administration (in vivo) and postulating its structures using mass spectrometric techniques. To obtain the metabolic profile of ACP-105, a selective and reliable LC-MS/MS method was developed. The chemical structures of the metabolites were determined based on their fragmentation pattern, accurate mass, and retention time. Under the current experimental condition, a total of 19 metabolites were detected in ACP-105 drug administered equine urine samples. The study results suggest the following: (1) ACP-105 is prone to oxidation, which gives corresponding monohydroxylated, dihydroxylated, and trihydroxylated metabolites; (2) along with oxidation, there is a possibility of elimination of water molecule (dehydration) from the third position of the tropine moiety, resulting in the dehydrated analogs of corresponding monohydroxylated, dihydroxylated, and trihydroxylated metabolites; (3) from the study on the metabolites using LC-MS/MS, it is clear that the fragmentation pattern is identical and a great number of fragment ions are common in all the metabolites and the parent drug. (4) The ACP-105 and its metabolites were detected for up to 72 h; thus, the result is a valuable tool for evaluating its use and/or misuse in sport.

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.

Evaluation of podocin in urine in horses using qualitative and quantitative methods.

No sensitive method for diagnosing early kidney dysfunction in horses has been identified so far. Many studies carried out in humans and small animals show that podocin can be useful to diagnose various kidney diseases, mainly affecting the glomeruli. The aim of this study was to perform a qualitative and quantitative analysis of podocin in urine samples obtained from healthy horses, horses with clinical kidney dysfunction and horses at risk of acute kidney injury. The study objectives aimed to assess: (1) whether the selected podocin tryptic peptide for LC-MS-MRM allows for podocin detection in horse; and (2) whether the species-specific ELISA test makes this detection possible as well;, (3) whether the chosen methods are sensitive enough to detect kidney dysfunction and glomerular injury, (4) whether the results of the tests applying both methods correspond with one another, (5) whether the results correlate with the hematological and biochemical data. The signals that may indicate the presence of trypsin fragments of podocin were found in three healthy horses, all the horses diagnosed with kidney dysfunction and half of the animals at risk for acute kidney injury. The concentration of podocin, diagnosed with the ELISA test was as follows: from 0.19 to 1.2 ng/ml in healthy animals, from 0.19 to 20.0 ng/ml in AKI horses, from 0.29 to 5.71 ng/ml in horses at risk for acute kidney injury. The results of both methods corresponded significantly. Podocin may be a potential biomarker of clinical kidney disease in horses and may be used in the detection of glomerular injury. However, its use is limited by the possibility of physiological podocyturia. LC-MS-MRM seems to be a more sensitive method to evaluate the presence of podocin than the ELISA test, whilst selected tryptic peptides of podocin appear to apply to horses. The ELISA test showed greater effectiveness in excluding the disease than in confirming it.

Determination of grapiprant plasma and urine concentrations in horses.

OBJECTIVE: Non-steroidal anti-inflammatory drugs are inhibitors of cyclooxygenase (COX) in tissues and used as therapeutic agents in different species. Grapiprant, a member of the piprant class of compounds, antagonizes prostaglandin receptors. It is a highly selective EP4 prostaglandin E2 receptor inhibitor, thereby limiting the potential for adverse effects caused by wider COX inhibition. The objectives of this study were to determine if the approved canine dose would result in measurable concentrations in horses, and to validate a chromatographic method of analysis for grapiprant in urine and plasma. STUDY DESIGN: Experimental study. ANIMALS: A total of six healthy, adult mixed-breed mares weighing 502 ± 66 (397-600) kg and aged 14.8 ± 5.3 (6-21) years. METHODS: Mares were administered one dose of 2 mg kg-1 grapiprant via nasogastric tube. Blood and urine samples were collected prior to and up to 48 hours after drug administration. Drug concentrations were measured using high-performance liquid chromatography. RESULTS: Grapiprant plasma concentrations ranged from 71 to 149 ng mL-1 with the mean peak concentration (106 ng mL-1) occurring at 30 minutes. Concentrations were below the lower limit of quantification (50 ng mL-1) in four of six horses at 1 hour and in all six horses by 2 hours after drug administration. Grapiprant urine concentrations ranged from 40 to 4077 ng mL-1 and were still detectable at 48 hours after administration. CONCLUSIONS AND CLINICAL RELEVANCE: Currently, there are no published studies looking at the pharmacodynamics of grapiprant in horses. The effective concentration needed to control pain in dogs ranges 114-164 ng mL-1. Oral administration of grapiprant (2 mg kg-1) in horses did not achieve those concentrations. The dose was well tolerated; therefore, studies with larger doses could be conducted.

The intravenous pharmacokinetics of butorphanol and detomidine dosed in combination compared with individual dose administrations to exercised horses.

In equine and racing practice, detomidine and butorphanol are commonly used in combination for their sedative properties. The aim of the study was to produce detection times to better inform European veterinary surgeons, so that both drugs can be used appropriately under regulatory rules. Three independent groups of 7, 8 and 6 horses, respectively, were given either a single intravenous administration of butorphanol (100 µg/kg), a single intravenous administration of detomidine (10 µg/kg) or a combination of both at 25 (butorphanol) and 10 (detomidine) µg/kg. Plasma and urine concentrations of butorphanol, detomidine and 3-hydroxydetomidine at predetermined time points were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The intravenous pharmacokinetics of butorphanol dosed individually compared with co-administration with detomidine had approximately a twofold larger clearance (646 ± 137 vs. 380 ± 86 ml hr-1  kg-1 ) but similar terminal half-life (5.21 ± 1.56 vs. 5.43 ± 0.44 hr). Pseudo-steady-state urine to plasma butorphanol concentration ratios were 730 and 560, respectively. The intravenous pharmacokinetics of detomidine dosed as a single administration compared with co-administration with butorphanol had similar clearance (3,278 ± 1,412 vs. 2,519 ± 630 ml hr-1  kg-1 ) but a slightly shorter terminal half-life (0.57 ± 0.06 vs. 0.70 ± 0.11 hr). Pseudo-steady-state urine to plasma detomidine concentration ratios are 4 and 8, respectively. The 3-hydroxy metabolite of detomidine was detected for at least 35 hr in urine from both the single and co-administrations. Detection times of 72 and 48 hr are recommended for the control of butorphanol and detomidine, respectively, in horseracing and equestrian competitions.

Effect of a commercial anionic dietary supplement on urinary pH and concentrations of electrolytes and pH in blood of horses.

Aims: To compare urine urinary pH, blood pH and concentration of electrolytes in blood of healthy horses fed an anionic salt supplement to achieve diets with a dietary cation-anion difference (DCAD) of -40 or 0 mEq/kg DM, with horses a fed a diet with a DCAD of 85 mEq/kg DM.Methods: Eight healthy horses received each of three diets in a randomised crossover design. Diets consisted of grass hay and concentrate feed, with a varying amount of an anionic supplement to achieve a DCAD of 85 (control), 0 or -40 mEq/kg DM. They were fed for 14 days each with a washout period of 7 days between. Urine pH was measured daily and blood samples were collected on Days 0, 7 and 14 of each study period for the measurement of pH and concentration of electrolytes.Results: Four horses voluntarily consumed the anionic supplement with their feed, but four horses required oral supplement administration via dose syringe. During the study period mean urine pH was lower in horses fed diets with a DCAD of 0 (6.91; SD 0.04) and -40 (6.83; SD 0.04) mEq/kg DM compared to the control diet (7.30; SD 0.04). Compared with horses fed the control diet, mean urine pH was lower in horses fed the 0 and -40 mEq/kg DM diets on Days 1-12 and 14 (p < 0.05) of the study period. On Day 13 it was only lower in horses fed the -40 mEq/kg DM diet (p < 0.01). Urine pH was similar for horses fed the 0 and -40 mEq/kg DM diets (p = 0.151). The DCAD of the diet had no effect on blood pH, ionised Ca or anion gap. Mean concentrations of bicarbonate in blood were affected by diet (p = 0.049); they were lower when horses were fed the 0 mEq/kg diet relative to the control diet on Day 14.Conclusions and clinical relevance: The anionic supplement reduced urine pH in horses fed diets with a DCAD of 0 or -40 mEq/kg DM compared with 85 mEq/kg DM. However as urinary pH did not fall below pH 6.5, the pH below which calcium carbonate uroliths do not form, this reduction in urine pH is unlikely to be clinically significant. The supplement was variably palatable and showed minimal promise as an effective urinary acidifier at the doses administered in this study.

Bioformation of boldenone and related precursors/metabolites in equine feces and urine, with relevance to doping control.

Boldenone (1-dehydrotestosterone) is an exogenous anabolic-androgenic steroid (AAS) but is also known to be endogenous in the entire male horse and potentially formed by microbes in voided urine, the gastrointestinal tract, or feed resulting in its detection in urine samples. In this study, equine fecal and urine samples were incubated in the presence of selected stable isotope labeled AAS precursors to investigate whether microbial activity could result in 1-dehydrogenation, in particular the formation of boldenone. Fecal matter was initially selected for investigation because of its high microbial activity, which could help to identify potential 1-dehydrogenated biomarkers that might also be present in low quantities in urine. Fecal incubations displayed Δ1-dehydrogenase activity, as evidenced by the use of isotope labeled precursors to show the formation of boldenone and boldione from testosterone and androstenedione, as well as the formation of Δ1-progesterone and boldione from progesterone. Unlabeled forms were also produced in unspiked fecal samples with Δ1-progesterone being identified for the first time. Subsequent incubation of urine samples with the labeled AAS precursors demonstrated that Δ1-dehydrogenase activity can also occur in this matrix. In all urine samples where labeled boldenone or boldione were detected, labeled Δ1-progesterone was also detected. Δ1-progesterone was not detected any non-incubated urine samples or following an administration of boldenone undecylenate to one mare/filly. Δ1-progesterone appears to be a candidate for further investigation as a suitable biomarker to help evaluate whether boldenone present in a urine sample may have arisen due to microbial activity rather than by its exogenous administration.

Investigation of the metabolism of the selective androgen receptor modulator LGD-4033 in equine urine, plasma and hair following oral administration.

LGD-4033 is one of a number of selective androgen receptor modulators (SARMs) that are being developed by the pharmaceutical industry to provide the therapeutic benefits of anabolic androgenic steroids, without the less desirable side effects. Though not available therapeutically, SARMs are available for purchase online as supplement products. The potential for performance enhancing effects associated with these products makes them a significant concern with regards to doping control in sports. The purpose of this study was to investigate the metabolism of LGD-4033 in the horse following oral administration, in order to identify the most appropriate analytical targets for doping control laboratories. LGD-4033 was orally administered to two Thoroughbred horses and urine, plasma and hair samples were collected and analysed for parent drug and metabolites. LC-HRMS was used for metabolite identification in urine and plasma. Eight metabolites were detected in urine, five of which were excreted only as phase II conjugates, with the longest detection time being observed for di- and tri-hydroxylated metabolites. The parent compound could only be detected in urine in the conjugated fraction. Seven metabolites were detected in plasma along with the parent compound where mono-hydroxylated metabolites provided the longest duration of detection. Preliminary investigations with hair samples using LC-MS/MS analysis indicated the presence of trace amounts of the parent compound and one of the mono-hydroxylated metabolites. In vitro incubation of LGD-4033 with equine liver microsomes was also performed for comparison, yielding 11 phase I metabolites. All of the metabolites observed in vivo were also observed in vitro.

Cobalt accumulation in horses following repeated administration of cobalt chloride.

OBJECTIVE: To monitor cobalt concentrations in urine, red blood cells and plasma after chronic parenteral administration of cobalt chloride evaluate these results against the current International Federation of Horseracing Authorities thresholds for detecting cobalt misuse. DESIGN: Eight mares were randomly assigned to four treatment groups, with two mares in each group: Group 1 - control group, Group 2 - 25 milligrams cobalt intravenously as CoCl2 weekly, Group 3 - 50 milligrams cobalt intravenously as CoCl2 weekly, and Group 4 - 25 milligrams cobalt intravenously mid-week and at the end of the week. Urine and blood samples were collected before each weekly administration so that trough levels were assessed. In the group receiving two doses per week, urine and blood were collected prior to the dose given at the end of each week. Samples were initially collected at time zero then weekly for 10 weeks. Three further collections of urine and blood were made at days 81, 106 and 127. METHODS: Urine creatinine measurements to assess horse hydration status were performed by the Jaffe reaction method. Cobalt determinations in plasma, blood and urine were by inductively coupled plasma-mass spectrometry. Haematocrit concentrations, used to calculate red cell cobalt levels, were performed using a microhematocrit centrifuge. Statistical analyses were conducted in Genstat (v17, VSNi). RESULTS: Marked cobalt accumulation was evident with increasing cobalt concentrations for all sample matrices in specimens collected immediately prior to cobalt administration. Correlation between the sample matrices improved when urine cobalt concentration was adjusted for creatinine level. Red cell cobalt levels remained elevated for at least 12 weeks after cessation of administration, consistent with the lifespan of the red cell. There was no significant change in haematocrit concentrations for the duration of the study. CONCLUSION: The current urine cobalt threshold was only effective at detecting acute cobalt exposure while the plasma cobalt threshold was able to consistently identify chronic high-level cobalt exposure and potential cobalt misuse. The threshold values legislated for urine cobalt do not correlate with those set for plasma. The acute nature of urinary cobalt excretion provides a relatively small window through which cobalt administration is detected. Plasma and red cell cobalt concentrations can provide a clearer picture of potential cobalt misuse.

The diagnostic performance of human urinary dipsticks to estimate urine pH, specific gravity (SpG), and protein in horses: are they reliable?

BACKGROUND: Urinalysis is a critical diagnostic test which is performed in routine veterinary medicine practice. In this diagnostic test, semiquantitative measurement of urine biochemical substances is carried out using urinary dipstick. In the current study, we evaluated the diagnostic performance of human urinary dipsticks to estimate pH, specific gravity (SpG), and protein in 80 urine specimens collected from horses. These parameters were measured using two commercial human dipsticks (KP and MN in abbreviation) and quantitative reference methods. The reference methods for pH, SpG, and protein were pH meter, handheld refractometer, and pyrogallol red method, respectively. The correlation between the semiquantitative dipstick analysis and quantitative reference methods was determined using Spearman's rank correlation coefficient. RESULTS: In general, our results revealed that the both human urinary dipsticks are unreliable tests for urinary pH, SpG, and protein content in horses. The analysis indicated that there was a poor correlation between the urine dipsticks and reference method (KP: rS = 0.534 and MN: rs = 0.485, Ps < 0.001) for protein. Additionally, there was a weak correlation between the results of pH measured using the urine dipsticks and reference method (KP: rS = 0.445 and MN: rs = 0.370, Ps < 0.001). Similar findings were obtained for SpG (KP: rS = 0.285, MN: rs = 0.338, Ps < 0.001). The estimation of proteinuria using the human dipsticks in horses lacked specificity, as many false positive protein results were obtained. CONCLUSION: We observed that the human commercial urinary dipsticks used in this study were not reliable to correctly estimate urine protein, SpG, and pH in horses.

A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry.

Bioactive peptides possess pharmacological effects and can be illicitly used in sports. To deter such misuse, an untargeted method using high resolution mass spectrometry (HRMS) has been developed for comprehensive detection of multitudinous exogenous peptides in equine plasma and urine. Forty-four peptides were extracted using mixed-mode solid-phase extraction (SPE) from plasma and urine, separated with a hydrophilic interaction liquid chromatography (HILIC) column, and detected on an HRMS instrument. Ammonium formate as a mobile phase additive had effects on HILIC retention and charge state distribution of the peptides. The acetonitrile percentage in the reconstitution solution affected the solubility of peptide neat standards and peptides in plasma and urine extracts differently. The stability of the peptides in plasma at ambient temperature was assessed. The limit of detection (LOD) was 10-50 pg/mL for most of the peptides in plasma, and ≤ 500 pg/mL for the remaining. LOD was 100-400 pg/mL for the majority of the analytes in urine, and ≤ 4000 pg/mL for the others. The method was used successfully to analyze incurred plasma and urine samples from research horses administered dermorphin. Even in the absence of reference standards, dermorphin metabolites (aFGYPS-NH2 , YaFG, and YaF) were identified. These results demonstrate that data generated with this method can be retrospectively reviewed for peptides that are unknown at the time of sample analysis without requiring re-analysis of the sample. This method provides a powerful novel tool for detection of numerous bioactive peptides and their metabolites in equine plasma and urine for doping control.

Detection, pharmacokinetics, and selected pharmacodynamic effects of methamphetamine following a single transmucosal and intravenous administration to exercised Thoroughbred horses.

Methamphetamine is a central and peripheral nervous system stimulant. There is only a single study that describes exposure to and disposition of this compound in horses. The potential for abuse and inadvertent exposure in equine athletes along with the limited data available necessitates further study. The objectives of the current study were to describe drug and metabolite concentrations, develop an analytical method that could be used to regulate its use, and describe selected pharmacodynamic effects. In phase 1, six horses were randomized into three transmucosal dose groups (n = 2/group; 0.5, 1.0 or 10 mg). In phase 2, horses received a single 10 mg intravenous dose. In phase 3, the effects of urinary pH on elimination were studied. Blood and urine samples were collected for up to 72 hours post drug administration. Concentrations of methamphetamine were measured using liquid chromatography-tandem mass spectrometry. Methamphetamine was below the limit of detection (LOD) in blood by 2, 4, and 18 hours following transmucosal administration of 0.5, 1, and 10 mg, respectively. Following intravenous administration, methamphetamine fell below the LOD between 12 and 18 hours. Following urinary acidification, methamphetamine fell below the limit of quantitation (LOQ) by 12 hours. In urine, methamphetamine was no longer detected at 48, 48, and 72 hours in the 0.5, 1, and 10 mg transmucosal groups and 18 hours in the intravenous group. Increased urinary pH resulted in urinary concentrations of methamphetamine falling below detectable levels by 48 hours post transmucosal administration. While the number of animals was small, behavioral, stimulatory, and cardiac effects were minimal.

Tracking main environmental factors masking a minor steroidal doping effect using metabolomic analysis of horse urine by liquid chromatography-high-resolution mass spectrometry.

There is an urgent need to implement holistic and untargeted doping control protocols with improved discriminatory power, compared to conventional methods that only target doping agents. Metabolomics, which aims to characterize all metabolites present in biological matrices, could fulfill this need. In this context, the aim of this study was to evaluate the impact of environmental factors on the ability to obtain a metabolic signature of stanozolol administration in horse doping situation. Urine samples from 16 horses breeded in two different places were collected over a one-year period, before, during and seven months after the administration of stanozolol, a horse doping agent. Metabolomic analysis was performed using ultra-high pressure reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (MS). Results showed a major impact of the nutritional regimen, drug administration (for de-worming purpose) and breeding place on the metabolite profiles of horse urines, which hampered the detection of metabolic perturbations induced by stanozolol administration. After having used MS/MS experiments to characterize some MS features related to these environmental factors, we showed that highlighting and then removing the features impacted by these confounding factors before performing supervised multivariate statistical analyses could address this issue. In conclusion, adequate consideration should be given to environmental and physiological factors; otherwise, they can emerge as confounding factors and conceal doping administration.

Pilot study to quantify the time to clear dexamethasone from plasma and urine of adult horses following a single nebulisation.

OBJECTIVE: To quantify the time to clear dexamethasone from plasma and urine of horses following a single nebulisation. DESIGN: Experimental using six Standardbred mares. METHODS: Dexamethasone sodium phosphate (0.04 mg/kg) diluted in 0.9% sodium chloride was administered as an aerosol using a Flexineb E2® nebuliser. Blood samples (0, 2, 4, 6, 8, 10, 12, 24, 32, 48, 72 and 96 h) and urine samples (0, 1, 4, 8, 24, 32, 48, 72 and 96 h) were collected for analysis using liquid chromatography mass spectrometry. RESULTS: Maximum plasma concentrations (tmax ) were reached by the earliest detection point (2 h) after nebulisation (0.6-1.8 ng/mL), but was no longer detectable at 48 h. However, in one horse 0.1 ng/mL was found at 96 h after three consecutive readings of 0 ng/mL. The tmax in urine was reached by the earliest collection point (1 h) after nebulisation (3.2-23.8 ng/mL), but was no longer present in urine at 72 h in five horses, while detectable levels (0.1 ng/mL) were still present at 96 h in one horse. CONCLUSIONS: A single dose of 0.04 mg/kg of DSP administered as an aerosol through a FlexinebE2® mask was no longer detectable in blood at 48 h in six horses tested, but one horse returned a reading of 0.1 ng/mL at 96 h after having no detectable levels. Dexamethasone was not detectable in urine at 72 h in five horses but was detectable at a low concentration (0.1 ng/mL) at 96 h in one horse.

Pharmacokinetics, metabolism and excretion of celecoxib, a selective cyclooxygenase-2 inhibitor, in horses.

Celecoxib, a nonsteroidal anti-inflammatory drug, is frequently used to treat arthritis in humans with minimal gastrointestinal side effect compared to traditional NSAIDs. The primary aim of this study was to determine the pharmacokinetic profile of celecoxib-a selective cyclooxygenase-2 (COX-2) inhibitor in horses. Six horses were administered a single oral dose of celecoxib at 2 mg/kg (body weight). After oral dosing, the drug reached a maximum concentration (mean ± SD) in blood of 1,088 ± 324 ng/ml in 4.58 hr. The elimination half-life was 13.60 ± 3.18 hr, and the area under the curve was 24,142 ± 1,096 ng hr ml-1 . The metabolism of celecoxib in horses was via a single oxidative pathway in which the methyl group of celecoxib is oxidized to a hydroxymethyl metabolite and is further oxidized to form a carboxylic acid metabolite. Celecoxib is eliminated mainly through faeces as unchanged drug and as metabolites in urine. Therefore, instructions for a detection time following therapeutic dosing of celecoxib can be set by the racing practitioner and veterinarians to control illegal use in horse racing based on the results of this study.