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Topic:Mass Spectrometry
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.
Airway Lipid Mediators in Horses: Baseline Bronchoalveolar Lavage Fluid Profiles and Effects of EPA and DHA-rich Microalgae Oil Supplementation. We investigated how dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affect airway inflammation, bronchoalveolar lavage fluid (BALF) lipid mediators and red blood cell fatty acids in horses. Increased BALF neutrophils are a hallmark of airway inflammation, commonly associated with equine asthma. The long-chain n-3 PUFAs EPA and DHA are essential nutrients and precursors of specialized pro-resolving mediators (SPMs) that regulate inflammatory responses. In this prospective field trial, we assigned horses to three diets, including...
Detection of Cyclosporine in Horse Hair After Ocular Implant Administration. This study describes the detection and temporal distribution of cyclosporine in equine hair following ocular implant administration in a Thoroughbred gelding. Mane hair samples were collected at 172- and 217-day post-implantation and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after alkaline hydrolysis and liquid-liquid extraction. The method achieved an estimated limit of detection of 0.1 pg/mg. Cyclosporine was detected in hair at both sampling time points, with segmental analysis revealing distribution patterns consistent with systemic exposure and incorporation ...
Detection and confirmation of hypoxia-inducible factor activating agents in equine plasma and urine by liquid chromatography coupled to mass spectrometry. Hypoxia-inducible factor (HIF) activating agents can promote the transcription of the erythropoietin gene, enhancing an athlete's performance, and are therefore prohibited in sports. A liquid chromatography coupled to high-resolution mass spectrometry (LCHRMS) method was developed for the detection and confirmation of 11 HIF-activating substances in equine plasma and urine. Ammonium formate added to the mobile phase system suppressed the responses of all the analytes in either positive or negative ion mode and thus was not used. The starting organic percentage in the mobile phase gradient and ...
High-Throughput Equine Plasma Profiling Using Laser-Assisted Rapid Evaporative Ionisation Mass Spectrometry. Remnants of erythrocyte removal from blood constitute therapeutic plasma-a water-based solution containing mainly platelets, extracellular vesicles (EVs), protein and lipid complexes and ions. Plasma-based therapies, including their use to accelerate wound healing in human and animal patients, are increasingly gaining prominence in regenerative medicine; however, methods for the optimal preparation of individual plasma are still under development. Rapid and accurate analysis of plasma composition is essential for the development of therapeutic applications and for ensuring the quality of plasm...
Simple and efficient HPLC-MS/MS method for simultaneous extraction and quantification of anthelmintics in equine manure. Anthelmintic treatment is a common practice in horses. The presence of pharmaceutical residues in fields and pastures is well-documented affecting various organisms, like nematodes. This study describes the development and validation of a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method using multiple reaction monitoring (MRM) for extracting and determining the concentrations of four commonly used anthelmintics in horse manure. Manure was initially dried, then spiked with various concentrations (10-10,000 ng) of anthelmintics and extracted using an acetonit...
Detection of ionisable lipids in equine plasma by supported liquid extraction and liquid chromatography-high-resolution tandem mass spectrometry for doping control of mRNA agents. Messenger ribonucleic acid (mRNA) has emerged as a new class of therapeutic agent with unprecedented potential. In particular, the lipid nanoparticle (LNP)-mRNA formulations enabled safe and effective delivery of mRNA in vivo, leading to the success of mRNA vaccines during the COVID-19 pandemic. To control the potential misuse of LNP-mRNA agents in equine sports, a simple and sensitive method has been developed for the detection of ionisable lipids in equine plasma using supported liquid extraction (SLE) followed by liquid chromatography-high-resolution tandem mass spectrometry. To overcome th...
Administration Study of Bedinvetmab, an Antinerve Growth Factor Monoclonal Antibody, in Horses for Doping Control Purposes. Monoclonal antibodies (mAbs) targeting nerve growth factor (NGF) represent a novel therapeutic approach for managing acute and chronic pain in humans and animals. However, their potential misuse as pain-masking agents in competitive equine sports raises significant welfare and integrity concerns. To enhance understanding of the elimination profile of anti-NGF mAb if misused in horses, this study describes the first administration of bedinvetmab (a canine anti-NGF mAb) in three geldings, establishing its plasma elimination profile following subcutaneous administration. Bedinvetmab plasma concen...
Metabolomic Analysis of the Effects of Cysteamine Zinc on the Composition and Amino Acid Profile of Mare’s Milk. This study aims to investigate the effects of cysteamine zinc supplementation on milk production, composition, amino acid profile, and metabolites in mares. Building on prior experimental findings, a dose of 7 mg/kg body weight of CS-Zn was selected for the experimental group, which was compared with a control group. Milk samples were collected at various time points, and milk yield was recorded each time. Routine analysis of milk components, as well as the determination of milk metabolites and amino acids, were performed. The results indicated that, compared to the control group, the experime...
OPTIMIZING EXTRACTION AND CONCENTRATION METHODS FOR LC-MS/MS ANALYSIS OF VETERINARY PHARMACEUTICALS IN EQUINE-ASSOCIATED ENVIRONMENTAL MATRICES. This study developed a method for the simultaneous quantification of six potentially environmentally toxic veterinary drugs: benzylpenicillin, acepromazine, flunixin, dexamethasone, ketamine and romifidine, in soils, sediments, equine faeces and natural waters. Three methods were compared for their extraction from solid matrices: Ultrasonic extraction (USE), QuEChERS and Accelerated Solvent Extraction (ASE). ASE method was optimized according to temperature and extraction cycles. Quantification was performed by LC-MS/MS. LC and MS parameters were optimized. The method was validated according t...
Derivatisation-free FIA-MS/MS assay for rapid simultaneous screening of atypical myopathy biomarkers and acylcarnitine profiles from dried blood spots. Atypical myopathy (AM) is a frequently fatal, toxin-induced disease in horses, characterised by acute progression and mortality rates exceeding 70% within 72 h. The disease is caused by ingestion of seedlings or seeds of certain maple species that contain the toxins hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG). Early diagnosis depends on reliable detection of these compounds and their metabolites. However, most available LC-MS/MS assays are limited by derivatisation requirements and dependence on serum and urine matrices. Here, a rapid derivatisation-free flow injection analysis...
Proteomic signatures of equine dental tooth tissues in ageing and disease. Ageing and dental disease in horses lead to structural and functional deterioration of dental tissues, yet their molecular signatures remain poorly characterised. Understanding how these processes alter the protein composition of enamel, dentin, cementum and pulp is essential for improving equine oral health and identifying biomarkers of degeneration. Objective: To characterise the proteomic profiles of equine dental tissues and determine age- and disease-associated changes, with the aim of identifying proteins consistently altered across conditions. Methods: Ex vivo proteomic study. Methods: ...
LC-HRMS detection of three ionizable lipids in equine plasma: surrogate markers for long-term LNP-mediated gene-doping surveillance. Lipid nanoparticles (LNPs) are essential delivery vehicles for messenger ribonucleic acid (mRNA)-based therapies. However, their potential misuse as gene-doping agents in horse racing and equestrian sports necessitates robust analytical surveillance. This study presents a highly sensitive, Orbitrap technology-driven liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for the simultaneous detection of three clinically relevant ionizable lipids: ALC-0315, SM-102, and DLin-MC3-DMA (MC3), which are critical components of approved RNA therapeutics and vaccines. Following a modi...
Sensitive and selective quantification of elcatonin in equine plasma and urine using LC-FAIMS-MS/MS. The use of drugs for enhancing bone strength is prohibited under international regulations governing horse racing and equestrian sports. Elcatonin, a synthetic peptide with analgesic and bone-strengthening properties, is routinely monitored for compliance. However, its cyclic structure and high molecular weight hinder reliable detection. In this study, we developed and validated a sensitive liquid chromatography-high-field asymmetric waveform ion mobility spectrometry-tandem mass spectrometry method for quantifying elcatonin in equine plasma and urine. Sample preparation was optimised via weak...
Integrative Phosphoproteomic Profiling Reveals Stage-Specific Signalling and Metabolism in Equine Melanocytic Neoplasm. Equine melanocytic neoplasms (EMN) are aggressive tumours characterised by high metastatic potential and limited therapeutic options available. However, the molecular mechanisms underlying their progression remain poorly understood. This study therefore presents the integrative phosphoproteomic analysis of EMN tissue, with the aim of elucidating stage-specific alterations in signalling pathways and metabolism. Nineteen tissue samples from grey horses were categorised as normal-stage (n = 6), early-stage EMN (n = 7), and severe-stage EMN (n = 6) and subjected to in-depth analysis us...
Evaluating Protein Enrichment Methods to Improve Biomarker Discovery in Equine Cerebrospinal Fluid. Cerebrospinal fluid (CSF) is a valuable source of biomarkers for neurological diseases, but detection of low-abundance proteins is often masked by highly abundant proteins. Enrichment strategies can enhance proteomic coverage and improve biomarker discovery, yet comparative evaluations of such methods in equine CSF are limited. Objective: This study compared the ProteoMiner Small-Capacity Enrichment Kit and the PreOmics Enrich-iST Kit for their ability to deplete high-abundance proteins and enhance detection of low-abundance proteins relevant to neuropathology. Methods: Equine CSF samples were...
Pharmacokinetics of Intramuscularly Administered Ketamine at Subanaesthetic Dosage in Horses. Subanaesthetic doses of ketamine (0.5 mg kg) provide analgesia in several species, but there is limited information on the pharmacokinetics and clinical effects of intramuscularly administered ketamine in horses. This study investigated the pharmacokinetics of ketamine and norketamine, and evaluated clinical effects, ataxia, and vital parameters, following intramuscular and intravenous administration of 0.5 mg kg ketamine in nine healthy horses, using a randomized two-period crossover design with a 1-week washout period. Plasma concentrations were analyzed using Ultra-High Performance ...
Synthesis and Characterization of Deuterated Detomidine for Use in Equine Medication Regulation. 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. ...
Supramolecular Solvent Extraction for Doping Control Analysis of Prohibited Substances in Horse Urine. Despite the recent success in introducing supramolecular solvents (SUPRAS)-based extraction to drug analysis, its application and robustness in day-to-day regular urine testing have yet to be demonstrated. Moreover, the applicability of SUPRAS in equine doping control testing remains unexplored. In this work, we have successfully developed for the first time a simple, rapid, inexpensive, and environmentally friendly SUPRAS extraction method for analyzing 76 prohibited substances of different classes (selective androgen receptor modulators, hypoxia-inducible factor prolyl hydroxylase inhibitors...
Analysis of lipid mediator profiles in the blood of horses with equine asthma using UPLC-MS/MS. Equine asthma (EA) is a chronic inflammatory disease of the lower respiratory tract in horses, which is mainly triggered by dusty hay. EA is becoming increasingly acknowledged as a valuable animal model for studying human asthma. Research on the pathogenesis of EA has primarily focused on the associated cells and cytokines involved. However, the role of inflammation-related lipid mediators (LM) in the development and progression of equine asthma remains elusive. LMs are categorized into pro-inflammatory arachidonic acid (AA)-derived leukotrienes (LTs) and prostaglandins (PGs), as well as infla...
Untargeted LC-HRMS metabolomic analysis reveals exercise-induced biochemical alterations in endurance Arabian horses. BACKGROUND: Endurance exercise induces extensive metabolic remodeling in equine athletes, yet the global metabolic responses in purebred Arabian horses remain insufficiently characterized. This study aimed to investigate plasma metabolomic alterations associated with prolonged aerobic exercise using untargeted liquid chromatography–high-resolution mass spectrometry (LC-HRMS). RESULTS: Plasma samples from twelve trained Arabian horses were collected before and after 80-km endurance rides and analyzed using both hydrophilic interaction (HILIC) and reversed-phase (C18) chromatographic platforms...
Monitoring trace minerals and heavy metals in liver of free-living large herbivores in the Netherlands. Trace minerals are essential for animal health but can also, together with heavy metals, have a negative impact, making their monitoring crucial to assess animal health. These elements were examined through a long-term post-mortem monitoring system based on routine liver sampling for Heck cattle, Konik horses and red deer in place at the Oostvaardersplassen nature reserve in the Netherlands, using data from this system to determine reference intervals and investigate trends in liver trace element concentrations. Throughout the monitoring programme, inductively coupled plasma mass spectrometry ...
Metabolomics reveals early predictors of blastocyst formation in equine ICSI-derived embryos. Equine in vitro embryo production (IVP) via ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) has gained prominence in sport-horse breeding due to high success rates, efficient semen utilization, and year-round embryo production, including from mares with fertility challenges or postmortem. However, assessing viability of early-stage ICSI-produced equine embryos remains challenging due to subjective morphological evaluations and limited visibility of developmental features. Non-invasive metabolomic analysis of culture media offers a promising alternative to enhance embryo selectio...
Proteomic profiling of equine airway mucus reveals compositional changes in asthmatic phenotypes. Mucus hypersecretion and accumulation are hallmark features of equine asthma (EA), a meaningful respiratory disorder in horses occurring in mild to moderate (MEA) and severe (SEA) forms. Changes of the proteomic composition of airway mucus in EA are poorly understood. Using label-free quantitative liquid chromatography-mass spectrometry, we analyzed airway mucus from SEA (n = 10), MEA (n = 6), and healthy (n = 8) horses. We identified and quantified 2,275 proteins including gel-forming mucins MUC5AC and MUC5B and membrane-bound mucins MUC1 and MUC4. Compared with healthy controls, ...
Pharmacokinetics of Intragastric Ursodeoxycholic Acid and Its Impact on Bile Acid Profiles in Horses. Ursodeoxycholic acid (UDCA), a secondary bile acid (BA) with therapeutic applications, is standard therapy for cholestatic hepatopathies in humans. In recent years, its use has been increasingly explored in equine medicine for similar indications. Pharmacokinetic data for UDCA in horses are currently lacking. This study aimed to describe the pharmacokinetic parameters following a single intragastric administration of 15 mg/kg in nine healthy, fasted horses and to characterize the changes in their BA profiles. Plasma concentrations of UDCA were measured at specific time points using liquid ch...
Comparative Analysis of Extracellular Vesicle Isolation From Equine Serum and Plasma Using Two Isolation Methods With Structural and Proteomic Validation. Extracellular vesicles (EVs) are promising biomarkers and mediators of intercellular communication, but their isolation from equine biofluids remains challenging. This study compared two isolation workflows-size-exclusion chromatography (SEC) and differential ultracentrifugation followed by SEC (UC + SEC)-to evaluate their efficiency, reproducibility, and the proteomic composition of EVs derived from equine serum and plasma. Blood from six healthy horses was processed to obtain platelet-free plasma and serum. EVs were isolated using SEC or UC + SEC and characterized by transmission ele...
Detection and Longitudinal Monitoring of Cyclosporine in Horse Plasma and Urine After Ocular Implant Administration. This paper describes the detection and longitudinal monitoring of cyclosporine in plasma and urine after subconjunctival implant administration in a horse. Sensitive liquid chromatography tandem mass spectroscopy (LC-MS/MS) methods for detecting cyclosporine in horse plasma and urine have been developed and validated, with estimated limits of detection down to 1 pg/mL in both matrices. The developed methods enabled longitudinal monitoring of cyclosporine levels in blood and urine samples collected over 6 months from a horse that had received an ocular cyclosporine implant.
Cell Surface Vimentin Is an Attachment Factor That Facilitates Equine Arteritis Virus Infection In Vitro. Our laboratory identified the susceptible allelic variant of equine CXCL16 protein (EqCXCL16S) as an entry receptor for equine arteritis virus (EAV). However, EAV has a broad host cell tropism and infects cells that lack EqCXCL16S. Thus, we hypothesized that EAV interacts with other host cell protein(s) that facilitate EAV infection. A virus overlay protein-binding assay in combination with a Far-Western blot from EAV-susceptible equine pulmonary artery endothelial cells (EECs) and equine dermal fibroblasts (E. Derm) identified a 57 kDa protein, present in the membrane fraction of the protein ...
Equine fetal-sex determination from mid-gestation to term using serum conjugated steroid profiling by liquid chromatography-tandem mass spectrometry. Refined profiling of conjugated estrogens and androgens during equine pregnancy using liquid chromatography-tandem mass spectrometry (LC-MS/MS) could provide accurate fetal-sex determination. Current methods for fetal-sex prediction remain limited by timing, accuracy, and operator expertise. This study investigated sex-specific differences in maternal conjugated steroid profiles to develop a reliable, non-invasive predictive method. Samples were collected from 141 mares of various breeds starting at sixteen weeks of pregnancy. The samples were pooled according to gestational stage, divided int...
Pharmacokinetics and Safety of a Single Subcutaneous or Intramuscular Dose of Ketamine in Healthy Horses. Pharmacokinetics (PK) of intramuscular (IM) and subcutaneous (SC) ketamine in horses has not been described. This study aimed to evaluate the PK and safety of ketamine and its metabolites after a single SC or IM administration. In Phase 1, two horses received 0.5 or 1 mg/kg of ketamine via SC and IM routes. In Phase 2, eight horses received 0.5 mg/kg IM. Plasma or serum concentrations of ketamine and major metabolites were determined by a validated liquid chromatography-mass spectrometry method at baseline and selected intervals post-administration. Subcutaneous administration resulted in ...
Dynamic DIA proteomics of mare’s milk reveals distinct colostrum signatures and stage-specific lactation profiles. This study used data-independent acquisition (DIA) proteomics to analyze the changing protein content of mare's milk (n = 3) throughout the entire lactation period (days 1-120), with samples taken at seven different times. Principal component analysis (PCA) revealed that the protein composition of colostrum on day 1 differed markedly from that of later lactation stages. Colostrum-specific proteins such as IgG, IgM, IgE, AGPAT1, NPVF and CAMK2G were associated with rapid energy supply, neurodevelopment and immune defense. In contrast, mature milk was enriched in proteins involved in sustained...
