Livestock Poisoning Research Articles

Distinguishing between toxic and non-toxic pyrrolizidine alkaloids and quantification by liquid chromatography–mass spectrometry

Pyrrolizidine alkaloids (PAs) are important plant toxins causing poisoning in livestock, leading to significant financial and production losses each year. It may also enter the human food chain as contaminants of grains, via animal products such as milk, eggs and honey or herbal remedies. Not all PAs are toxic and it is important to be able to distinguish between toxic and non-toxic PAs. We developed a sensitive and selective analytical method to determine toxic 1,2-unsaturated PAs concentrations in plant extracts by liquid chromatography–mass spectrometry (LC–MS) with electrospray ionization and precursor ion experiments. Multi-reaction-mode experiments were used to quantify the concentrations of the different unsaturated PAs and results were expressed as μg/g retrorsine equivalents. The results obtained on Crotalaria sphaerocarpa contaminating maize crops have important implications.

Isolation and identification of bacteria capable of degrading euptox A from Eupatorium adenophorum Spreng

Eupatorium adenophorum Spreng (E. adenophorum), an invasive plant which has caused widespread poisoning of livestock and crop failures in China. 9-oxo-10, 11-dehydroageraphorone (known as euptox A) is the main toxin causing a series of respiratory and digestive system diseases in animals. The purpose of the research is to isolate and identify the bacteria with the capacity of degrading euptox A, in which way could we lay the foundation in the utilization and prevention for E. adenophorum. Three strains of bacteria with the degradative characteristic are screened from E. adenophorum leaves, soil and goat's rumen respectively by nutrition enrichment. The 16S rDNA sequence of the three isolated strains (XC-07, XC-08, XC-09) were shown to be 97%, 100%, 100% similar to Stenotrophmonas sp., Klebsiella sp. and Pseudomonas sp., respectively, named as Stenotrophomonas sp. XC-07, Klebsiella sp. XC-08 and Pseudomonas sp. XC-09. The degradation rates of 3 strains to euptox A (45 mg/L) in 24 h are 91.2%, 94.3% and 93.2%, respectively, which are determined by HPLC.

The study of the Oxytropis kansuensis-induced apoptotic pathway in the cerebrum of SD rats.

BackgroundLocoweeds cause significant livestock poisoning and economic loss all over the world. Animals can develop locoism, a chronic neurological disease, after grazing on locoweeds. Oxytropis kansuensis is a variety of locoweed that contains swainsonine as its main toxic ingredient. The purpose of this study was to investigate the apoptotic pathway induced in the cerebrum by swainsonine.ResultsTwenty-four Sprague-Dawley rats were randomly divided into four groups (experimental groups I, II, III and a control group) and 6 SD rats of each group were feed in 3 cages separately. Rats were penned as groups and fed with feeds containing 15% (SW content 0.03‰), 30% (SW content 0.06‰), or 45% (SW content 0.09‰) O. kansuensis for experimental groups I, II, and III, respectively, or complete feed in the case of the control group. One hundred and nineteen days after poisoning, and all rats showed neurological disorders at different degrees, which were considered to be successful established a chronic poisoning model of O. kansuensis. rats were sacrificed and the expression of Fas, FasL, Bcl-2, Bax as well as cleaved caspase-3, -8 and -9 proteins in brain tissues were detected by Western blot. The results showed that SW treatment up-regulated Fas and Fas ligand (FasL) (P < 0.05), and that there was an increase in Bax and a decrease in Bcl-2 protein (P < 0.01). Moreover, SW treatment significantly increases the activation of caspase-3, 8 and -9, the key effectors in apoptosis pathway (P < 0.01).ConclusionOur data suggest that SW induces apoptosis in cells of the brain through death receptor and mitochondria-mediated, caspase-dependent apoptotic pathways in the brain tissue of SD rats.

Isolation of grayanotoxin I from Rhododendron species and quantification by densitometry

Species of the family Ericaceae are a potential source of toxic diterpenes, named grayanotoxins (GT). Grayanotoxin I (GT-1, Fig.) is one of the main compounds also known as acetylandromedol or andromedotoxin. GT-1 causes a number of livestock poisoning and food intoxication by toxic honey (“mad honey”) made by bees in the Black Sea region of Turkey. The symptoms of intoxication are related to the concentration and could cause a simple weakness up to cardiac problems.

Grazing on Microcystis aeruginosa and degradation of microcystins by the heterotrophic flagellate Diphylleia rotans

Cyanobacterial toxins can cause damage in aquatic ecosystems worldwide, as well as the poisoning of livestock, plants and humans when ingested in large amounts. Although many studies investigated grazing of harmful cyanobacteria by metazoan plankton, grazing of cyanobacteria by hetertophic flagellates is largely unexplored. This laboratory study investigated grazing of toxic Microcystis aeruginosa by the heterotrophic flagellate Diphylleia rotans isolated from a Saudi hypertrophic lake. D. rotans was able to feed on M. aeruginosa with estimated ingestion (10.2 to 16.5 prey flagellate −1d−1) and specific growth rates (0.71 to 0.99d−1) differed with the increase in the initial density of the flagellate. Grazing increased microcystin production within Microcystis cells during first two days of incubation, and afterwards induced the release of these toxins into the medium. The concentrations of releasing microcystin were strongly reduced in grazing-treated cultures compared to controls, indicating the degradation of microcystins by D. rotans growing under axenic conditions. Taken these results, D. rotans can play an important role in the reduction of Microcystis biomass and microcystin toxins, and thus could be used as a safe bioagent for the biocontrol of harmful algal blooms in aquatic environments.

Experimental rayless goldenrod (Isocoma pluriflora) toxicosis in horses

Rayless goldenrod (Isocoma pluriflora) sporadically poisons horses and other livestock in the southwestern United States. Similar to livestock poisoning by white snakeroot (Ageratina altissima) in the midwestern United States, previous research suggests that benzofuran ketones (BFK: tremetone, dehydrotremetone, 6-hydroxytremetone, and 3-oxyangeloyl-tremetone) are responsible for the toxicity of rayless goldenrod. However, experimental reproduction of rayless goldenrod-induced disease and detailed descriptions of poisoning in horses with known concentrations of tremetone and other BFK has not been documented. In this study four horses were fed increasing amounts of rayless goldenrod to obtain doses of approximately 0, 10, 30, and 60 mg BFK/kg BW for 14 days. After seven days of dosing the horse dosed with 60 mg BFK/kg BW horse developed depression, reluctance to eat, dehydration, trembling, and muscle fatigue. Biochemical alterations including increases in the serum enzyme activities of CK, AST, ALT, and LDH, and increased cardiac troponin I concentration, were also identified. Physiologically the clinically poisoned horse had decreased endurance seen as reluctance to perform on the treadmill with increased resting heart rate and a prolonged recovery of heart rate following treadmill exercise. The condition of the horse continued to decline and it was euthanized and necropsied on day 10. At necropsy the myocardium was pale and soft and many of the appendicular and large apical muscles were pale and moist. Histologically, the myocardium had extensive myocardial degeneration and necrosis with extensive fibrosis and multifocal mineralization. Several of the large appendicular muscles in this horse also had small foci of skeletal muscle degeneration and necrosis. Less severe myocardial changes were also identified in the horse dosed with 30 mg BFK/kg BW after 14 days of dosing. No clinical, biochemical or histologic changes were identified in the control horse and the horse dosed with 10 mg BFK/kg BW. These results suggest that doses of 60 mg BFK/kg BW for seven days produce extensive myocardial lesions in horses. The horse dosed with 30 mg BFK/kg BW developed less severe, but similar myocardial lesions over a longer duration, this suggests that poisoning may be cumulative and lower doses of longer duration are also toxic. Horses seem to be uniquely sensitive to rayless goldenrod-induced myocardial disease, therefore cardiac troponin I may be a useful marker of rayless goldenrod poisoning in horses. More work is needed to determine which BFK produce myocardial toxicity and better determine the effects of dose and duration on poisoning in horses.

Spatial variation of epoxyscillirosidine concentrations in &lt;i&gt;Moraea pallida&lt;/i&gt; (yellow tulp) in South Africa

Moraea pallida (yellow tulp) poisoning is economically the most important intoxication of livestock in South Africa. Poisoning varies according to locality, climatic conditions and growth stage of the plant. The primary objective of this study was to determine the concentration of the toxic principle, epoxyscillirosidine, in yellow tulp leaves and to ascertain the variability of epoxyscillirosidine concentrations within and between different locations. A secondary objective was to utilise Geographic Information Systems in an attempt to explain the variability in toxicity. Flowering yellow tulp plants were collected at 26 sampling points across 20 districts of South Africa. The leaves of five plants per sampling point were extracted and submitted for liquid chromatography/mass spectrometry analysis. A large variation in mean epoxyscillirosidine concentrations, ranging from 3.32 μg/g - 238.27 μg/g, occurred between different geographical regions. The epoxyscillirosidine concentrations also varied tremendously between individual plants (n = 5) collected at the same sampling point, with up to a 24 times difference between the lowest and highest concentration detected. No generalised correlation between epoxyscillirosidine concentrations and soil elemental concentrations could be established. However, samples obtained from the north-eastern part of the sampling region tended to have higher epoxyscillirosidine concentrations compared to samples obtained from the south-western part of the sampling region. Higher toxin concentrations in the north-east were associated with statistically significant higher soil concentrations of iron, bismuth, bromide, cadmium, chromium, rubidium, tellurium, thallium, titanium and zinc, whilst soil concentrations of strontium and soil pH, were significantly lower. This study corroborated the contention that epoxyscillirosidine concentration in yellow tulp fluctuates and may explain the variability in toxicity.

Phytochemical Studies of Malaysian Crotalaria Mucronata

Crotalaria sp. is a perennial subshrub-like herb with yellow flowers and leguminous pod. It has been shown to produce bioactive secondary metabolites and is often associated with livestock poisoning. The seeds of Crotolaria sp. contain pyrrolizidine alkaloids such as monocrotaline. Despite the toxicity nature of the plant, it has been in traditional medicine for the anti-inflammatory treatment. Previous study on Crotolaria sp. from other countries has reported the isolation of flavonoids, chalcones, terpenes, lectins, and polysaccharides. However, the phytochemical of the Malaysia Crotolaria has not been studied before. In the present study, Crotolaria mucronata has been collected from the hilly area in the Pahang state in Malaysia. The whole plant was dried and grounded and followed by ethanol extraction. The ethanolic crude extract was further partitioned into hexane, chloroform, and ethanol. The hexane extract was subjected to extensive chromatographic separation and the compounds isolated are characterized by various spectroscopic methods such as Nuclear Magnetic Resonance and mass spectrometry. The compounds identified thus far are unsaturated fatty acids, fatty acid esters, a straight chain alkane and 5hydroxy-3,4-dimethyl5-pentylfuran-2(5H)-one.

A liquid chromatography-electrospray tandem mass spectrometry method for the determination of multiple pesticide residues involved in suspected poisoning of non-target vertebrate wildlife, livestock and pets

A liquid chromatography tandem mass spectrometry (LC/MS/MS – triple quadrupole) method that facilitates the qualitative and quantitative determination of multiple pesticide residues that could be present in wild and domestic vertebrate animals has been developed. The method involves the direct analysis of crude extracts from a variety of test specimens taken from (non-target) birds and mammals suspected as being victims of accidental or deliberate exposure to pesticides. Performance of the method was validated following replicate analysis of pseudo-matrices (chicken muscle and chicken liver tissue) that had been fortified with 102 different pesticides and their degradation products at two different concentration levels (0.1 mg kg−1 and 1.0 mg kg−1). Residues detected were quantified following interpolation against external calibration curves obtained using matrix-matched standards that covered a residue concentration range between 0.025 μg ml−1 and 0.5 μg ml−1. Application of the method is demonstrated through inclusion of a few examples that show screening and confirmation results of pesticide residues detected in animals involved in suspected poisoning incidents.

Spontaneous and experimental poisoning by Marsdenia megalantha Goyder & Morillo in ruminants and a pig

Marsdenia megalantha is a rupicolous shrub with succulent roots from the semiarid region of Brazil that is known to cause fatal poisoning in livestock. We reported spontaneous cases of poisoning by M. megalantha roots in bovine, caprine, ovine, and equine species. The clinical and pathological findings of experimental administration of M. megalantha to sheep, goats, a calf and a pig are reported. Three goats, two sheep and a calf were dosed once orally with freshly chopped roots at dose of 25 g wet plant/kg bw; another sheep and a pig were dosed with 10 g wet plant/kg bw. Poisoning occurred in all of the animals except the three goats. Clinical signs of poisoning included tachycardia, opisthotonus, ruminal bloat, dyspnea, nystagmus, mydriasis, ataxia, and recumbence with paddling movements. Pathological evaluation showed segmental laminar neuronal necrosis and spongiosis in the telencephalic cortex and degeneration of Purkinje cells. The picrate paper procedure detected no cyanide in the plant roots, but the reaction used for nitrate detection gave a strongly positive response. In conclusion, M. megalantha is a poisonous plant that produces acute poisoning characterized mainly by nervous disturbances. Livestock producers should offer alternative food during the dry and early rainy seasons to avoid the poisoning by this plant.

Detection and localization of the endophyteUndifilum oxytropisin locoweed tissues

Poisoning of livestock owing to grazing on locoweeds results in significant economic losses in the western United States. Some Oxytropis spp. locoweeds contain a seed-transmitted endophytic fungus, Undifilum oxytropis, which produces the toxic alkaloid swainsonine. We sought to localize and characterize growth patterns of the fungus within leaves and petioles of Oxytropis lambertii Pursh and Oxytropis sericea Nutt. to help define the types of interactions between the fungus and its hosts. Vegetative hyphae were observed within locoweed tissues using integrated imaging. Topographical images from scanning electron microscopy revealed the presence of the endophyte in the pith tissue of petioles. The fungus was identified between plant cells but did not appear to penetrate host cells. Transmission electron microscopy images of thin sections revealed that hyphae were closely associated with host cell walls. Oxytropis sericea was innoculated with green fluorescent protein-transformed U. oxytropis and observed by confocal microscopy, confirming the presence of the endophyte hyphae in leaves and petioles. The fungus was identified only in the pith of petioles using fluorescence and in the vascular bundle throughout extracellular spaces in leaves. These results revealed no signs of a pathogenic interaction between plant and fungus and support the hypothesis of a mutualistic or commensal relationship.

Production of toxic pavettamine and pavettamine conjugates in the gousiekte-causing Fadogia homblei plant and its relation to the bacterial endosymbiont

Plant poisoning of livestock is responsible for considerable economic losses in southern Africa. Six plant species of the Rubiaceae family are known to cause gousiekte, a cardiac syndrome of ruminants induced by ingestion of the toxic compound pavettamine. Progress in understanding the etiology of this disease is largely hampered by the variable toxicity of the plants and the absence of a quantification method for pavettamine. The pavettamine concentration in leaf samples of Fadogia homblei, a known gousiekte causing plant, was analyzed by mass-spectrometry. In the most apical leaf pair, the highest concentration of pavettamine was detected. Distal leaves contained progressively less pavettamine. Besides a significant amount of free pavettamine, most pavettamine was found to occur in a conjugated form. To which molecules the pavettamine is conjugated remains unknown as is the function of conjugated pavettamine in the development of gousiekte. All know gousiekte-causing plants contain symbiotic bacteria in their leaves; it was hypothesized that these bacteria might be involved in the production of pavettamine. However, analysis of in vitro cultures of the F. homblei endosymbiont revealed no production of pavettamine. Pavettamine is therefore not produced by the bacteria alone. It is either the product of the interaction with the plant or solely produced by the host.

Detection of swainsonine and isolation of the endophyte Undifilum from the major locoweeds in Inner Mongolia

Locoweeds are Astragalus and Oxytropis species that contain the toxic alkaloid swainsonine, causing widespread poisoning of livestock in Inner Mongolia. Taxa (Astragalus, Oxytropis, Sphaerophysa, and Sophora species) suspected of causing locoism and/or poisoning in Inner Mongolia were surveyed for swainsonine and Undifilum, the fungal endophyte responsible for the production of swainsonine. Swainsonine was detected at concentrations greater than 0.01% in Astragalus variabilis and Oxytropis glabra. The endophyte Undifilum was detected by culturing and PCR in samples containing swainsonine concentrations greater than 0.01%. In some specimens of A. variabilis and O. glabra swainsonine was not detected or concentrations were less than 0.01%. In these samples the endophyte could not be cultured, but was detected by PCR. Additionally, contrary to previous reports the quinolizidine alkaloids, thermopsine, anagyrine, and lupanine, were not detected in O. glabra and Oxytropis ochrocephala, however the quinolizidine alkaloids, sophoridine, sophocarpine, and sophoramine were detected in Sophora alopecuroides as previously reported.

Spatial and Temporal Distribution of Nitrate Pollution in Groundwater of Abuja, Nigeria

Groundwater has been recognized as playing a very important role in the development of Abuja, Nigeria’s Capital as many households, private and government establishments depends solely on hand-dug wells and boreholes for their daily water needs. Exploitation of groundwater is rather delicate because of its potency to contamination and difficulty to remediate when compared to surface water. The purpose of this paper is to present the occurrence of nitrate in groundwater of Abuja and discuss the implication and sources of the nitrate. High nitrate level in drinking water leads to infant methaemoglobinaemia (blue-baby syndrome), gastric cancer, metabolic disorder and livestock poisoning. A simplified map of nitrate occurrences in Abuja indicates that some areas have nitrate concentration above the WHO and NSDWQ guide limit of 50 mg/l and it is dominant in the rainy season than dry season. The number of people drinking water with nitrate concentration above the permissible level cannot be quantified presently. The sources of nitrate in the groundwater were attributed to bedrock dissolution in the course of groundwater migration and more importantly anthropogenic activities such as on-site sanitation, waste dumpsites and agricultural chemicals. Water treatment by bio-denitrification and nitrate pollution control programs should be introduced at local, state and federal levels in order to educate people on the need to protect groundwater from nitrate pollution caused by agricultural activity and indiscriminate disposal of wastes.

Isolation and characterization of sodium 2-fluoroacetate from Mascagnia rigida using chromatography and infrared spectroscopy

Sodium monofluoroacetate was first identified in Dichapetalum cymosum, a South African plant that can cause livestock poisoning and death. After, several other plants also showed to contain this toxin, which leads to the “sudden death”. Mascagnia rigida, a well identified poisonous plant, commonly found in northeast of Brazil also cause sudden death in cattle, which shows clinical signs similar to those produced by the ingestion of plants that contain monofluoroacetate. Our aim was to identify the toxic compound present in the aqueous extract of M. rigida. For this purpose, the dried and milled plant was extracted; the extract was lyophilized and submitted to successive chromatographic process, until the desired purity of the active compound was achieved. The study of this material by planar chromatography and by infrared spectrometry indicated that the toxin can be a mixture of mono, di and trifluoroacetate.

Protein extraction methods for the two-dimensional gel electrophoresis analysis of the slow growing fungus Undifilum oxytropis

The fungus Undifilum oxytropis produces the toxin swainsonine and is symbiotic with locoweeds, which are toxic Oxytropis and Astragalus species. The genus Oxytropisincludes perennial legumes that are widespread in many rangeland regions around the world. Consumption of locoweeds causes significant livestock poisoning and severe economic losses. Information about swainsonine synthesis by the U. oxytropis endophyte in locoweeds is limited and the interactions between the fungus and locoweed plant are poorly understood. Since U. oxytropis is a slow growing fungus that does not readily sporulate, its genetic characterization has been limited. An understanding of its proteome can be an important component in its biological characterization. The goal of this study was to develop an efficient protein extraction method for U. oxytropis. To develop an optimized protein extraction protocol for U. oxytropis, five protein extraction methods were evaluated. Of the five procedures assessed, trichloroacetic acid (TCA) in acetone was shown to be the best method for the fungus. The U. oxytropis proteins extracted using the TCA-acetone method were further characterized using two dimensional gel electrophoresis (2-DE) followed by mass spectrometry. The high resolution of the 2-DE reference map provided a useful approach for proteomic analysis of slow growing fungi. Key words: Endophytes, mass spectrometry, protein extraction method, swainsonine, two-dimensional polyacrylamide gel electrophoresis, Undifilum oxytropis.

Environmental Factors Associated with Nitrate Poisoning in Livestock in Botswana

Some cases of sudden deaths in cattle, sheep and goats reported to the National Veterinary Laboratory, Gaborone were investigated. The clinical signs included: stupor, weakness, staggering, trembling, dyspnoea (difficulty in breathing), salivation, diarrhoea, and convulsions for those in extremis. The most consistent sign was the muddy coloured mucous membranes. Necropsy findings included: marked gastroenteritis and generalized congestion and petichiation in all internal body organs and chocolate brown coloured blood that clotted poorly. The presence of nitrate/nitrite in the blood of live animals, amniotic fluid, urine, ocular fluid, urine, drinking water and consumed forage were demonstrated by the diphenylamine test and subsequently confirmed by Gries test. Further confirmation of nitrate/ nitrite poisoning was by response to therapeutic intervention with methylene blue using the intravenous route of inoculation. Environmental factors that may have contributed to the poisoning episodes included, drought, high ambient temperatures, low cloud cover, soil leaching after flooding, soil moisture content and forage nitrogen content and natural geoformations associated with high nitrate content in the water. This is the first account of confirmed cases of nitrate/ nitrite poisoning in livestock in Botswana.

Locoweed dose responses to nitrogen: Positive for biomass and primary physiology, but inconsistent for an alkaloid

Plant communities may be influenced by toxic secondary metabolites or enhanced plant growth from plant-symbiont interactions. The C:N hypothesis predicts that carbon or nitrogen constrains plant secondary metabolite production, but it does not consider compounds produced by plant symbionts. Locoweeds are legumes that can have fungal endophyte alkaloid (swainsonine [SWA]) production, which causes livestock poisoning. We studied four locoweed taxa to test whether average SWA concentrations influenced SWA positive dose responses to N fertilizer. We measured locoweed leaf SWA, pigment concentrations and photosynthetic activity, and plant biomass dose responses to N supplementation for 3 mo in two greenhouse experiments. Leaf photosynthesis, leaf pigment concentrations, and plant biomass had positive, unsaturated dose responses across tested N doses. Although N enhanced primary growth, two moderate-SWA taxa (Astragalus mollissimus var. bigelovii and Oxytropis sericea) had negative SWA dose responses to increasing N, the high-SWA taxon (A. moll. var. mollissimus) had no SWA change, and the very low-SWA taxon (A. moll. var. matthewsii) had a transient positive dose response. Supplemented N led to positive dose responses for plant biomass and leaf photosynthesis and pigments, but SWA dose responses differed across locoweed taxa and time. At N levels that enhanced plant growth and reduced antioxidant protective systems, fungal endophyte alkaloid production was not strongly influenced. Production of SWA may be more strongly influenced by factors other than C:N supply (e.g., seasonality, plant age) in the locoweed-endophyte-Rhizobium complex.

Selenium fractions in organic matter from Se-rich soils and weathered stone coal in selenosis areas of China

A high degree of association between Selenium (Se) and organic matter has been demonstrated in natural environments, but Se fractions and speciation in organic matter is unclear. In this study, a method for quantifying organic matter associated with Se (OM-Se) was developed to investigate Se fractions in organic matter in Se-rich soils and weathered stone coal from Enshi, China, where Se poisoning of humans and livestock has been documented. Initially, Se was extracted using water and a phosphate buffer. Subsequently, OM-Se was extracted using NaOH, and then speciated into Se associated with fulvic acids (FA-Se) and humic acids (HA-Se). Both FA-Se and HA-Se were further speciated into the weakly bound and strongly bound fractions using a customized hydride generation reactor. The results show that FA-Se (1.91–479 mg kg −1) is the predominant form of Se in all Se-rich soils and the weathered stone coal samples, accounting for more than 62% of OM-Se (3.07–484 mg kg −1). Weakly bound FA-Se (1.33–450 mg kg −1) was prevalent in the total FA-Se, while weakly bound HA-Se (0.62–26.2 mg kg −1) was variable in the total HA-Se (1.15–32.5 mg kg −1). These data indicate that OM-Se could play a significant source and sink role in the biogeochemical cycling of Se in the supergene environment. Weakly bound FA-Se seems to act as a potential source for bioavailable Se, whereas strongly bound HA-Se is a possible OM-Se sink which is not readily transformed into bioavailable Se.

Rapid quantification of ionophores in feeds by liquid chromatography– tandem mass spectrometry

Ionophores are widely used in veterinary medicine as coccidiostats and for improving nutrient utilization in livestock production. Because of widespread use, ionophores sometimes cause poisoning in livestock. Quantifying concentration of these compounds in feeds for diagnostic purposes is needed. A method with a single step of solvent extraction was developed for rapid simultaneous quantification of monensin, lasalocid, salinomycin, and narasin in feeds by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The ionophores were extracted using methanol:water (90:10). With the high specificity and high sensitivity of tandem mass spectrometry, the extract was introduced for measurement without further processing. The effect of particle size of feeds on extraction efficiency was also investigated. It was found that feeds passing through a 1-mm filter or sieve show better quantitative extraction. Nigericin was used as internal standard for the measurement. The method was validated by fortification of the selected ionophore compounds in horse feed at different concentrations. The typical recovery rate was 69-122%. Meanwhile, various interlaboratory proficiency test samples of different matrices were also quantified as part of the procedure for method validation. A good agreement was found between results and the suggested values. The method is very sensitive, with detection limits between 0.018 µg/g and 0.056 µg/g for the compounds tested. Results showed that the lower limit of quantification was 0.2 µg/g for the ionophore compounds, which is much lower than the contents of the ionophores in medicated feeds, which is generally approximately 10-100 µg/g feed.