Mass spectrometry is an analytical technique used to measure the mass-to-charge ratio of ions, providing detailed information about the molecular composition of samples. In equine research, mass spectrometry is employed to analyze a wide range of biological materials, including blood, tissues, and other bodily fluids. This technique is utilized for various applications, such as identifying biomarkers for disease, studying metabolic pathways, and assessing nutritional and environmental influences on horse health. By enabling precise molecular characterization, mass spectrometry aids in the understanding of equine physiology and pathology. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and findings of mass spectrometry in equine science.
Bundgaard L, Årman F, Åhrman E, Walters M, Auf dem Keller U, Malmström J, Jacobsen S.In human proteomics, substantial efforts are ongoing to leverage large collections of mass spectrometry (MS) fragment ion spectra into extensive spectral libraries (SL) as a resource for data independent acquisition (DIA) analysis. Currently, such initiatives in equine research are still missing. Here we present a large-scale equine SL, comprising 6394 canonical proteins and 89,329 unique peptides, based on data dependent acquisition analysis of 75 tissue and body fluid samples from horses. The SL enabled large-scale DIA-MS based quantification of the same samples to generate a quantitative eq...
Manikantan V, Ripley NE, Nielsen MK, Dangoudoubiyam S.Parascaris spp. represent a significant threat to equine health worldwide, particularly in foals. The long-term survival of parasites in the host necessitates persistent modulation of the host immune response. Intercellular communication achieved through the exchange of molecules via extracellular vesicles (EVs) released from the parasite could be a crucial factor in this regard. This study aimed to isolate and characterize EVs released by adult male and female Parascaris worms and conduct a proteomic analysis to identify sex-specific proteins and potential immunomodulatory factors. Methods: L...
Kwak YB, Stambler I, Yoo HH, Yoon J.Red ginseng (RG), a traditional herbal remedy, has garnered attention owing to its diverse health benefits resulting from its complex composition. However, extensive research is needed to substantiate the efficacy of RG and understand the underlying mechanisms supporting these benefits. This study aimed to identify potential biomarkers and investigate the impact of RG on related metabolic pathways in horse plasma using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. Unassigned: Ten horses were divided into control and RG groups, with the latter administered RG at a dose of ...
Philip M, Kal AKK, Subhahar MB, Karatt TK, Graiban FM, Ajeebsanu MM, Joseph M, Jose SV.Phosphodiesterase 4 (PDE4) inhibitors are a newer class of drugs that induce bronchodilation and have anti-inflammatory effects, making them susceptible to misuse as performance enhancers in competitive sports. Methods: This study explores the metabolic conversion of PDE4 inhibitor ibudilast in thoroughbred horses after oral administration and in vitro using equine liver microsomes and Cunninghamella elegans. A liquid chromatography-high resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. Results: A total of 20 in vivo metabolites wer...
Cheung HW, Wong KS, Tam JCL, Farrington AF, Bond AJ, Wan TSM, Ho ENM.Erythropoiesis-stimulating agents (ESAs) continue to be a significant threat to the integrity of human and equine sports. Besides conventional direct testing, monitoring the biomarkers associated with the effects of ESAs may provide a complementary approach via indirect detection to enhance doping control. In this study, we applied label-free proteomics to discover plasma protein biomarkers in Thoroughbred geldings after administration with a long-acting form of recombinant human erythropoietin (rhEPO), methoxy polyethylene glycol epoetin beta, Mircera. Increased haematocrit, haemoglobin and r...
Delcourt V, Pinetre J, Chabot B, Barnabé A, Cacault M, Loup B, Becher F, Fenaille F, Popot MA, Garcia P, Bailly-Chouriberry L.Data-independent acquisition (DIA) methods employing a scanning quadrupole were recently described across multiple platforms. These strategies display remarkable performances in untargeted proteomics studies thanks to rapid duty cycles, leading to ultrashort liquid chromatography gradients while maintaining enough data points per peaks when coupled to fast-scanning mass analyzer. In this article, we perform the evaluation of three data acquisition strategies named diaPASEF,slicePASEF, and prmPASEF on a trapped ion mobility spectrometry quadrupole-time-of-flight (TIMS-Q-TOF) mass spectrometer f...
Kong FKW, Wong ASY, Cheung RKK, Wan TSM, Ho ENM.In order to control the potential misuse of perfluorocarbons as an oxygen carrier in equine sports, a simple and sensitive method for detecting perfluorocarbons in equine plasma by gas chromatography/tandem mass spectrometry using negative chemical ionization with methane as reagent gas has been developed and fully validated. The method covers seven perfluorocarbons, which are the active components in blood substitute products, and shows good sensitivity and robustness. Limits of detection as low as 0.01 ng/mL could be achieved by the method. To the best of our knowledge, this is the first r...
Klingberg J, Richards S, Hochwallner T, Kennan L, Keledjian J.Bisphosphonate drugs and myo-inositol trispyrophosphate are of concern to the racing industry and have been listed as prohibited substances in equine sports. The current bisphosphonate plasma screening analysis employed at the Australian Racing Forensic Laboratory involves the use of sequential solid-phase extraction procedures, passing the samples through a mixed mode cartridge, followed by a weak anion exchange cartridge. The eluates collected following the second extraction are then methylated and analysed by liquid chromatography-mass spectrometry. Under these extraction conditions, some b...
Ishii H, Shigematsu R, Takemoto S, Ishikawa Y, Mizobe F, Nomura M, Arima D, Kunii H, Yuasa R, Yamanaka T, Tanabe S, Nagata SI, Yamada M, Leung GN. The use of osilodrostat, developed as a medication for Cushing's disease but categorized as an anabolic agent, is banned in horses by both the International Federation of Horseracing Authorities and the Fédération Equestre Internationale. For doping control purposes, elimination profiles of hydrolyzed osilodrostat in horse urine were established and the detectability of free forms of osilodrostat and its major metabolite, mono-hydroxylated osilodrostat (M1c), was investigated. Post-administration urine samples obtained from a gelding and three mares were analyzed to establish the eliminatio...
Ishii H, Shigematsu R, Takemoto S, Ishikawa Y, Mizobe F, Nomura M, Arima D, Kunii H, Yuasa R, Yamanaka T, Tanabe S, Nagata SI, Yamada M, Leung GN.Osilodrostat, used to treat Cushing's disease, exhibits an anabolic effect, leading to its classification as a prohibited substance in horseracing and equestrian sports. This study reports the characterization of osilodrostat metabolites in horse urine and elucidates its metabolic pathways for the first time for doping control purposes. Methods: Osilodrostat was administered nasoesophageally to four thoroughbreds (one gelding and three mares) at a dose of 50 mg each. Potential metabolites were extensively screened via our developed generic approach employing differential analysis to identify m...
Lawson EF, Ghosh A, Grupen C, Netherton J, Aitken RJ, Smith ND, Lim R, Drury HR, Pickford R, Gibb Z, Baker M, Tanwar PS, Swegen A.In many mammals, the lipid platelet-activating factor (PAF) has important functions in female reproduction and fertility. This study shows that PAF is present in the reproductive tissues of mares and is involved in processes related to ovulation and early pregnancy. Platelet-activating factor (PAF) has been implicated in a number of reproductive processes ranging from ovulation to embryo motility but has not been widely explored in the mare. To identify the presence and examine the role of PAF in the equine periconception processes, targeted mass spectrometry coupled with chromatographic separ...
Li X, Du M, Liu Y, Wang M, Shen Y, Xing J, Zhang L, Zhao Y, Bou G, Bai D, Dugarjaviin M, Xia W.During follicular development, changes in the composition of the follicular fluid are synchronized with the development of oocytes. Our aim was to screen the key factors affecting oocyte maturation and optimize the in vitro culture protocol by understanding the changes of proteins and metabolites in follicular fluid. Follicles are divided into three groups according to their diameter (small follicle fluid (SFF): 10 mm < d < 20 mm; medium follicle fluid (MFF): 20 mm < d < 30 mm; large follicle fluid (LFF): 30 mm < d). Proteins and metabolites from the follicular fluid w...
Michanek P, Bröjer J, Lilliehöök I, Fjordbakk CT, Löwgren M, Hedeland M, Bergquist J, Ekstrand C.Canagliflozin (CFZ) is a sodium-glucose cotransporter-2 inhibitor that has shown promising results as a drug for the treatment of insulin dysregulation in horses. Even though CFZ is used clinically, no pharmacokinetic data has previously been published. In this study, the pharmacokinetics of CFZ after administration of a single oral dose of 1.8 mg/kg in eight healthy Icelandic horses was examined. Additionally, the effect of treatment on glucose and insulin levels in response to a graded glucose infusion was investigated. Plasma samples for CFZ quantification were taken at 0, 0.33, 0.66, 1, ...
Richards S, Palmer D, Cawley A, Wainscott M, Keledjian J.An improved screening workflow and a robust capillary flow LC-MS confirmatory method for the detection of recombinant human erythropoietin (rHuEPO) has been implemented to increase the sensitivity of rHuEPO detection and to reduce the number of suspect samples committed to confirmatory testing. The influence of repeated dosing of epoetin-β on the detection window of rHuEPO in equine plasma was assessed using the optimised method. Samples were initially assessed using an economical R&D Human EPO Duo-Set ELISA Development System. Samples indicating a result greater than the batch baseline were ...
Ren H, Wen X, He Q, Yi M, Dugarjaviin M, Bou G.The reproductive performance of horse sperm and donkey sperm has been reported to differ. Sperm proteins play a crucial role in sperm viability and fertility. Although differences between species are known, no prior study has investigated disparities in the sperm proteome between horses and donkeys. Therefore, this study characterized and compared the sperm proteomes of horses and donkeys using 4D-DIA mass spectrometry technology. We identified 3436 proteins in horse sperm and 3404 proteins in donkey sperm. Of these, 3363 proteins were expressed in both horse and donkey sperm, with 73 proteins...
Lomnicka I, Dubey S, Waller P, Vora D, Dirikolu L.The screening of drugs in plasma and urine often requires initial extraction (such as liquid-liquid extraction and solid-phase extraction) before the samples are submitted to instrumental analyses. These extraction procedures are often laborious and time-consuming. In this manuscript, a high-throughput automated assay based on liquid chromatography-high-resolution mass spectrometry (LC-HRMS) suitable for use as an initial testing procedure covering multiple classes of compounds prohibited in horse racing is described. The assay requires a 600-μL plasma aliquot, which is subjected to solid pha...
Portaels J, Van Crombrugge E, Van Den Broeck W, Lagrou K, Laval K, Nauwynck H.Numerous Aspergillus fumigatus (Af) airborne spores are inhaled daily by humans and animals due to their ubiquitous presence. The interaction between the spores and the respiratory epithelium, as well as its impact on the epithelial barrier function, remains largely unknown. The epithelial barrier protects the respiratory epithelium against viral infections. However, it can be compromised by environmental contaminants such as pollen, thereby increasing susceptibility to respiratory viral infections, including alphaherpesvirus equine herpesvirus type 1 (EHV-1). To determine whether Af spores di...
Gray B, Lubbock K, Love C, Ryder E, Hudson S, Scarth J.The analytical approaches taken by laboratories to implement robust and efficient regulation of horseracing medication and doping control are complex and constantly evolving. Each laboratory's approach will be dictated by differences in regulatory, economic and scientific drivers specific to their local environment. However, in general, laboratories will all be undertaking developments and improvements to their screening strategies in order to meet new and emerging threats as well as provide improved service to their customers. In this paper, the published analytical advances in horseracing me...
Teles Filho ACA, Sanchez DJD, Viana AGA, Sheheryar S, Guerreiro DD, Bustamante-Filho IC, Martins AMA, Sousa MV, Ricart CAO, Fontes W, Moura AA.The present study was conducted to investigate the global proteome of 8-day-old equine blastocysts. Follicular dynamics of eight adult mares were monitored by ultrasonography and inseminated 24 h after the detection of a preovulatory follicle. Four expanded blastocysts were recovered, pooled, and subjected to protein extraction and mass spectrometry. Protein identification was conducted based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, and PepExplorer). Enrichment analysis was performed using g:Profiler, Panther, and String platforms. After the elimina...
Cutler C, Viljanto M, Hincks P, Habershon-Butcher J, Scarth J, van Eenoo P.S-23 is an arylpropionamide selective androgen receptor modulator that has been investigated in animal models for use as a male hormonal contraceptive but is not yet available therapeutically. S-23 is available alongside other selective androgen receptor modulators (SARMs) to purchase online via uncontrolled sites, sold as supplement products. It has been detected in several human doping cases, highlighting the importance of identifying the best analytical targets for equine doping control. The purpose of this study was to investigate the detection of S-23 and its phase I metabolites in equine...
Kabi L E, Zor TA, Göktaş EF, Güneş E, Yatanaslan L, Tektaş MH, İnceman B, Tufan M.The use of prohibited substances in horse racing is a major concern that jeopardizes both the fairness of competitions and the health of horses. This problem can stem from the use of licensed drugs for animal health, as well as unlicensed substances. Horse doping laboratories monitor the potential use of these substances in racehorses within the framework of regulations set by the International Federation of Horse Racing Authority. In this context, sildenafil and its major metabolite n-desmethyl sildenafil were detected in a post-race horse urine sample sent to the Pendik Veterinary Control In...
Uchida T, Kisugi T, Ishii H, Yamada M, Kinoshita K, Leung GN.To uphold the integrity of horseracing and equestrian sports, it is critical for an equine doping control laboratory to develop a comprehensive screening method to cover a wide range of target substances at the required detection levels in equine urine. Methods: The procedure involved the enzymatic hydrolysis of 3 mL urine samples followed by solid-phase extraction using HF Bond Elut C18 cartridge. The resulting extracts were then separated on a C18 reversed-phase column and analyzed using liquid chromatography/high-resolution mass spectrometry (LC/HRMS) in both electrospray ionization positiv...
Wong ASY, Yuen BP, Wong COL, Kong FK, So YM, Kwok WH, Brooks L, Wan TSM, Ho EN.Bisphosphonates and myo-inositol trispyrophosphate (ITPP) are two classes of difficult-to-detect polar drugs that are prohibited under the rules of racing. ITPP is a drug capable of increasing the amount of oxygen in hypoxic tissues, and studies have shown that administration of ITPP increases the maximal exercise capacity in mice. The properties of ITPP make it an ideal candidate as a doping agent to enhance performance in racehorses. In recent years, ITPP had indeed been detected in racehorses and confiscated items. As for bisphosphonates, it is especially critical to control their use as si...
Da Silva E, Martín-Cano FE, Gómez-Arrones V, Gaitskell-Phillips G, Alonso JM, Rey J, Becerro L, Gil MC, Peña FJ, Ortega-Ferrusola C.Equine endometritis is one of the main causes of subfertility in the mare. Unraveling the molecular mechanisms involved in this condition and pinpointing proteins with biomarker potential could be crucial in both diagnosing and treating this condition. This study aimed to identify the endometritis-induced changes in the endometrial proteome in mares and to elucidate potential biological processes in which these proteins may be involved. Secondly, biomarkers related to bacterial endometritis (BE) in mares were identified. Uterine lavage fluid samples were collected from 28 mares (14 healthy: ne...
Ferran AA, Roques BB, Chapuis L, Kuroda T, Lacroix MZ, Toutain PL, Bousquet-Melou A, Lallemand EA.Benzylpenicillin (BP) is a first-line antibiotic in horses but there are discrepancies between manufacturers and literature recommendations regarding dosing regimen. Objectives of this study were to evaluate pharmacokinetics and local tolerance of four different formulations of BP in adult horses, and to suggest optimized dosing regimen according to the formulation. Unassigned: A cross-over design was used in 3 phases for the intramuscular injection of three different products: procaine BP alone, procaine BP/ benzathine BP combination or penethamate hydriodide were administered IM in the glute...
Helmes E, Montgomery J, Alarcio G, Mendoza HG, Blea JA, Beal PA, Moeller BC.There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which m...
Knych HK, Weiner D, McKemie DS, Traynham M, Blea J.Butorphanol is commonly administered, both by the intravenous and intramuscular routes, to racehorses to facilitate handling for diagnostic procedures. As the administration of butorphanol for therapeutic purposes is considered appropriate, in order to avoid inadvertent positive drug tests, a thorough understanding of the pharmacokinetics of this drug is necessary. In the current study, 12, exercised Thoroughbred horses were administered a single intramuscular dose of 0.1 mg/kg butorphanol, and serum and urine samples were collected at various times post drug administration for determination...
Kwak YB, Yoon J, Yoo HH.SR9009, a peroxisome proliferator-activated receptor δ (PPARδ) agonist, is known for its potential benefits in energy homeostasis. It failed to receive the United States Food and Drug Administration (USFDA) approval and its illegal distribution has raised concerns. As a result, it has been classified as a prohibited substance by the World Anti-Doping Agency and the International Federation of Horseracing Authorities (IFHA). This study emphasizes the application of the in-silico molecular networking technology to analyze phase I drug metabolites in horses, distinguishing it from conventional ...
O'Kennedy MM, Roth R, Ebersohn K, du Plessis LH, Mamputha S, Rutkowska DA, du Preez I, Verschoor JA, Lemmer Y.A safe, highly immunogenic multivalent vaccine to protect against all nine serotypes of African horse sickness virus (AHSV), will revolutionise the AHS vaccine industry in endemic countries and beyond. Plant-produced AHS virus-like particles (VLPs) and soluble viral protein 2 (VP2) vaccine candidates were developed that have the potential to protect against all nine serotypes but can equally well be formulated as mono- and bi-valent formulations for localised outbreaks of specific serotypes. In the first interferon α/β receptor knock-out (IFNAR-/-) mice trial conducted, a nine-serotype (nona...
Demir C, Brereton RG, Dumasia MC.After oral administration of quinine sulfate to a thoroughbred mare, seven urine samples were obtained over a 45.5 h period. Using gas chromatography -electron impact ionization and positive-ion chemical ionization mass spectrometry, quinine and five putative metabolites were detected and tentatively identified in enzyme-hydrolysed post-administration urine; all metabolites involved some form of oxidation. The parent drug could be detected for about 16 h and some phase I biotransformation products for up to 40 h post-administration.
Marland A, Sarkar P, Leavitt R, Lee-Ruff E, Ramnauth J.A method for the extraction of oxaprozin from equine urine and serum and its quantitation by high-performance liquid chromatography-ultraviolet detection is presented. Confirmation of oxaprozin in postadministration extracts was accomplished by gas chromatographic- mass spectrometric analysis of methylated extracts or liquid chromatography with tandem mass spectrometry daughter ion mass spectra of underivatized extracts. Daypro, a formulation of oxaprozin, was administered orally at a dose of 4.8 g to four standardbred mares. Urine and serum samples were collected to 120 h postadministration. ...
Viljanto M, Cutler C, Taylor P, Habershon-Butcher J, Gray B.MK-0677 (ibutamoren) is an orally active non-peptide growth hormone secretagogue that binds to the ghrelin receptor stimulating the secretion of endogenous growth hormone. It is one of the most prevalent performance-enhancing compounds currently available online and is potentially subject to abuse both in human and equine sports. The aim of the current study was to investigate whether it could be detected in equine hair following oral administration of MK-0677 mesylate to a Thoroughbred racehorse. MK-0677 and its O-dealkylated metabolite were extracted using an existing method for prohibited s...
Rumpler MJ, Sams RA, Colahan P.We describe a validated, rapid, sensitive, and specific UHPLC-MS/MS method to detect and quantify glycopyrrolate in 0.5 mL of horse urine. Further, we investigated the elimination of glycopyrrolate in urine after both intravenous and oral administration of clinically relevant doses to Thoroughbred horses. Quantification was performed by weighted, linear regression analysis using a deuterated analogue of glycopyrrolate as internal standard (IS). The method was characterized by a linear range of 5-2500 pg/mL, a lower limit of quantification of 5 pg/mL and a limit of detection of 1 pg/mL. The int...
Valverde A, Cribb N, Arroyo L.Twelve adult horses were randomly assigned to 2 groups in a prospective experimental trial. A pneumatic tourniquet (425 mmHg) was placed, under sedation, proximal to the carpus on one randomly chosen thoracic limb. A cephalic vein catheter was placed distal to the tourniquet to establish an intravenous regional limb perfusion technique (IVRLP) with morphine (0.1 mg/kg) diluted with saline 0.9% to 0.1 mL/kg, and the tourniquet left in place for 30 minutes. Horses were euthanized at 1 h (Group I) or 6 h (Group II) following the IVRLP and synovial fluid from the radiocarpal, intercarpal, metacarp...
Knych HK, Katzman S, McKemie DS, Arthur RM, Blea J.Lidocaine is a local anesthetic that is sometimes administered in combination with epinephrine. The addition of epinephrine increases the time lidocaine remains at the site of administration, thus prolonging the duration of effect. Due to their potential to prevent the visual detection of lameness, the administration of local anesthetics is strictly regulated in performance and racehorses. Recent reports of positive regulatory findings for lidocaine in racehorses suggests a better understanding of the behavior of this drug is warranted. The objective of the current study was to describe serum ...
Kuroda T, Minamijima Y, Niwa H, Mita H, Tamura N, Fukuda K, Toutain PL, Ohta M.Fosfomycin (FOM) is an approved veterinary medicinal product for large animals in Japan, but Clinical breakpoint (CBP) for antimicrobial susceptibility test (AST) is not defined for animals. This study aimed at conducting a pharmacokinetics/pharmacodynamics (PK/PD) analysis to determine the PK/PD cutoff for the CBP in horses. Drug concentrations following single intravenous administration (IV) of 20 mg/kg body weight (BW) FOM in nine horses were measured using liquid chromatography/mass spectrometry. The data were modelled using a nonlinear mixed-effects model, followed by Monte Carlo simulati...
Knych HK, Weiner D, McKemie DS, Traynham M, Blea J.Butorphanol is commonly administered, both by the intravenous and intramuscular routes, to racehorses to facilitate handling for diagnostic procedures. As the administration of butorphanol for therapeutic purposes is considered appropriate, in order to avoid inadvertent positive drug tests, a thorough understanding of the pharmacokinetics of this drug is necessary. In the current study, 12, exercised Thoroughbred horses were administered a single intramuscular dose of 0.1 mg/kg butorphanol, and serum and urine samples were collected at various times post drug administration for determination...
Kwak YB, Yoon J, Yoo HH.SR9009, a peroxisome proliferator-activated receptor δ (PPARδ) agonist, is known for its potential benefits in energy homeostasis. It failed to receive the United States Food and Drug Administration (USFDA) approval and its illegal distribution has raised concerns. As a result, it has been classified as a prohibited substance by the World Anti-Doping Agency and the International Federation of Horseracing Authorities (IFHA). This study emphasizes the application of the in-silico molecular networking technology to analyze phase I drug metabolites in horses, distinguishing it from conventional ...
Ferran AA, Roques BB, Chapuis L, Kuroda T, Lacroix MZ, Toutain PL, Bousquet-Melou A, Lallemand EA.Benzylpenicillin (BP) is a first-line antibiotic in horses but there are discrepancies between manufacturers and literature recommendations regarding dosing regimen. Objectives of this study were to evaluate pharmacokinetics and local tolerance of four different formulations of BP in adult horses, and to suggest optimized dosing regimen according to the formulation. Unassigned: A cross-over design was used in 3 phases for the intramuscular injection of three different products: procaine BP alone, procaine BP/ benzathine BP combination or penethamate hydriodide were administered IM in the glute...
Helmes E, Montgomery J, Alarcio G, Mendoza HG, Blea JA, Beal PA, Moeller BC.There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which m...
Uchida T, Kisugi T, Ishii H, Yamada M, Kinoshita K, Leung GN.To uphold the integrity of horseracing and equestrian sports, it is critical for an equine doping control laboratory to develop a comprehensive screening method to cover a wide range of target substances at the required detection levels in equine urine. Methods: The procedure involved the enzymatic hydrolysis of 3 mL urine samples followed by solid-phase extraction using HF Bond Elut C18 cartridge. The resulting extracts were then separated on a C18 reversed-phase column and analyzed using liquid chromatography/high-resolution mass spectrometry (LC/HRMS) in both electrospray ionization positiv...
Teles Filho ACA, Sanchez DJD, Viana AGA, Sheheryar S, Guerreiro DD, Bustamante-Filho IC, Martins AMA, Sousa MV, Ricart CAO, Fontes W, Moura AA.The present study was conducted to investigate the global proteome of 8-day-old equine blastocysts. Follicular dynamics of eight adult mares were monitored by ultrasonography and inseminated 24 h after the detection of a preovulatory follicle. Four expanded blastocysts were recovered, pooled, and subjected to protein extraction and mass spectrometry. Protein identification was conducted based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, and PepExplorer). Enrichment analysis was performed using g:Profiler, Panther, and String platforms. After the elimina...
Kabi L E, Zor TA, Göktaş EF, Güneş E, Yatanaslan L, Tektaş MH, İnceman B, Tufan M.The use of prohibited substances in horse racing is a major concern that jeopardizes both the fairness of competitions and the health of horses. This problem can stem from the use of licensed drugs for animal health, as well as unlicensed substances. Horse doping laboratories monitor the potential use of these substances in racehorses within the framework of regulations set by the International Federation of Horse Racing Authority. In this context, sildenafil and its major metabolite n-desmethyl sildenafil were detected in a post-race horse urine sample sent to the Pendik Veterinary Control In...
Lomnicka I, Dubey S, Waller P, Vora D, Dirikolu L.The screening of drugs in plasma and urine often requires initial extraction (such as liquid-liquid extraction and solid-phase extraction) before the samples are submitted to instrumental analyses. These extraction procedures are often laborious and time-consuming. In this manuscript, a high-throughput automated assay based on liquid chromatography-high-resolution mass spectrometry (LC-HRMS) suitable for use as an initial testing procedure covering multiple classes of compounds prohibited in horse racing is described. The assay requires a 600-μL plasma aliquot, which is subjected to solid pha...
Ishii H, Shigematsu R, Takemoto S, Ishikawa Y, Mizobe F, Nomura M, Arima D, Kunii H, Yuasa R, Yamanaka T, Tanabe S, Nagata SI, Yamada M, Leung GN. The use of osilodrostat, developed as a medication for Cushing's disease but categorized as an anabolic agent, is banned in horses by both the International Federation of Horseracing Authorities and the Fédération Equestre Internationale. For doping control purposes, elimination profiles of hydrolyzed osilodrostat in horse urine were established and the detectability of free forms of osilodrostat and its major metabolite, mono-hydroxylated osilodrostat (M1c), was investigated. Post-administration urine samples obtained from a gelding and three mares were analyzed to establish the eliminatio...
Philip M, Kal AKK, Subhahar MB, Karatt TK, Graiban FM, Ajeebsanu MM, Joseph M, Jose SV.Phosphodiesterase 4 (PDE4) inhibitors are a newer class of drugs that induce bronchodilation and have anti-inflammatory effects, making them susceptible to misuse as performance enhancers in competitive sports. Methods: This study explores the metabolic conversion of PDE4 inhibitor ibudilast in thoroughbred horses after oral administration and in vitro using equine liver microsomes and Cunninghamella elegans. A liquid chromatography-high resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. Results: A total of 20 in vivo metabolites wer...
Cheung HW, Wong KS, Tam JCL, Farrington AF, Bond AJ, Wan TSM, Ho ENM.Erythropoiesis-stimulating agents (ESAs) continue to be a significant threat to the integrity of human and equine sports. Besides conventional direct testing, monitoring the biomarkers associated with the effects of ESAs may provide a complementary approach via indirect detection to enhance doping control. In this study, we applied label-free proteomics to discover plasma protein biomarkers in Thoroughbred geldings after administration with a long-acting form of recombinant human erythropoietin (rhEPO), methoxy polyethylene glycol epoetin beta, Mircera. Increased haematocrit, haemoglobin and r...
Kwak YB, Stambler I, Yoo HH, Yoon J.Red ginseng (RG), a traditional herbal remedy, has garnered attention owing to its diverse health benefits resulting from its complex composition. However, extensive research is needed to substantiate the efficacy of RG and understand the underlying mechanisms supporting these benefits. This study aimed to identify potential biomarkers and investigate the impact of RG on related metabolic pathways in horse plasma using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. Unassigned: Ten horses were divided into control and RG groups, with the latter administered RG at a dose of ...
Bundgaard L, Årman F, Åhrman E, Walters M, Auf dem Keller U, Malmström J, Jacobsen S.In human proteomics, substantial efforts are ongoing to leverage large collections of mass spectrometry (MS) fragment ion spectra into extensive spectral libraries (SL) as a resource for data independent acquisition (DIA) analysis. Currently, such initiatives in equine research are still missing. Here we present a large-scale equine SL, comprising 6394 canonical proteins and 89,329 unique peptides, based on data dependent acquisition analysis of 75 tissue and body fluid samples from horses. The SL enabled large-scale DIA-MS based quantification of the same samples to generate a quantitative eq...
Manikantan V, Ripley NE, Nielsen MK, Dangoudoubiyam S.Parascaris spp. represent a significant threat to equine health worldwide, particularly in foals. The long-term survival of parasites in the host necessitates persistent modulation of the host immune response. Intercellular communication achieved through the exchange of molecules via extracellular vesicles (EVs) released from the parasite could be a crucial factor in this regard. This study aimed to isolate and characterize EVs released by adult male and female Parascaris worms and conduct a proteomic analysis to identify sex-specific proteins and potential immunomodulatory factors. Methods: L...
Philip M, Karakka Kal AK, Subhahar MB, Karatt TK, Graiban FM, Ajeebsanu MM, Joseph M, Jose SV.The phosphodiesterase 4 (PDE4) inhibitors constitute a relatively modern class of medications that are known for inducing bronchodilation and exhibiting anti-inflammatory properties within the body. Due to these properties, there is concern regarding their potential misuse as performance-enhancing substances in competitive sports. This study delves into the metabolic conversion of roflumilast in thoroughbred horses following oral administration and in vitro experimentation using equine liver microsomes and Cunninghamella elegans. High-performance liquid chromatography coupled with a Q Exactiv...
Nomura M, Kuroda T, Ohta M, Kusano K, Minamijima Y, Nagata S.Salbutamol is a short-acting and selective beta-2 adrenergic agonist. Inhaled (IH) administration of salbutamol is widely used to control lower respiratory tract disease in horses. Here, we estimated the pharmacokinetic parameters of salbutamol after a single intravenous (IV) or IH administration in six horses, and we statistically analysed the detection times with various dosing regimens. Plasma and urine concentrations of salbutamol were measured by liquid chromatography-tandem mass spectrometry, and data were modelled by using a nonlinear mixed effect model followed by Monte Carlo simulatio...
Elbourne M, Keledjian J, Cawley A, Fu S.Altrenogest, also known as allyltrenbolone, is a synthetic form of progesterone used therapeutically to suppress unwanted symptoms of estrus in female horses. Altrenogest affects the system by decreasing levels of endogenous gonadotrophin and luteinizing and follicle-stimulating hormones, which in turn decreases estrogen and mimics the increase of progesterone production. This results in more manageable mares for training and competition alongside male horses while improving the workplace safety of riders and handlers. However, when altrenogest is administered, prohibited steroid impurities su...
Kwok KY, Kwok WH, Wan TSM, Brooks L, Popot MA, Jaubert M, Bailly-Chouriberry L, Heffron BT, Choo CK, Tso J, Tso R, McKenzie B, Selvadurai N, Batty D....The use of alkalinising agents prior to racing for manipulating performance in the horse has been identified since the 1990s. To mitigate the risk, an international threshold for available carbon dioxide in equine plasma based on analyses using the Beckman Synchron EL-ISE analyser was adopted in 1994 by the International Federation of Horseracing Authorities (IFHA) and revised from 37 to 36 mM in 2004. In 2009, the technical support for the above instrument was discontinued by its manufacturer. Based on the same measurement principle (i.e., ion selective electrode), the Beckman DxC600 analys...
So YM, Kwok WH, Ching CCW, Wong COL, Wan TSM, Ho ENM.Anabolic-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 duration of action. As such, the detection of intact esters of endogenous steroids in hair is particularly advantageous, as it can provide unequivocal proof of their external origin. Compared with urine and blood matrices, hair in general allows a longer detection window as long as the administered drug can be incorporated into hair and has not degraded. This can be useful in doping control of equi...
Jensen A, Clarke EJ, Nugent Z, Paice E, Gringel I, Yamamoto K, Rocchigiani G, Peffers AJ, Cooper L, Peffers MJ.Equine dental diseases significantly impact a horse's overall health, performance and quality of life. They can result in secondary infections and digestive disturbances, potentially leading to colic. A recently described disease affecting the incisors of horses is equine odontoclastic tooth resorption and hypercementosis (EOTRH). Understanding EOTRH is crucial for early diagnosis, effective management and prevention of its severe consequences. Objective: To determine proteomic differences in incisor cementum in horses with and without clinical EOTRH. Methods: Comparative and observational cli...
Perera TRW, de Ruijter-Villani M, Gibb Z, Nixon B, Sheridan A, Stout TAE, Swegen A, Skerrett-Byrne DA.Embryo-maternal signaling during the establishment of pregnancy in horses remains one of the biggest mysteries in large animal physiology. Early pregnancy loss represents a major source of economic loss to the breeding industry. This study aimed to investigate the systemic changes associated with early pregnancy by mapping the proteome of blood plasma at 14 days in pregnant and non-pregnant mares. Methods: Plasma proteomes were analysed in commercially bred pregnant (n = 17) and non-pregnant (n = 17) Thoroughbred mares at 14 days after ovulation, using high-resolution mass spectrometry. D...
The AnalystJanuary 23, 2025
doi: 10.1039/d4an01501k
Göktaş EF, Kabil E, Söylemez Yeşilçimen E, Dirikolu L.Various technical methodologies are required to accurately detect substances of different chemical and pharmacological properties in biological samples, which are increasing in number and variety daily. Therefore, laboratories where many samples and different factors are analyzed simultaneously need methods with easy sample preparation, short analysis times and low analysis costs. In this study, the objective was to scan substances susceptible to chemical degradation, amenable to analysis without hydrolysis, and exhibiting short-term stability by employing a straightforward, expeditious, and c...
