Simultaneous determination of selected ionophoric coccidiostats and amino acids in feed premixes using high-performance liquid chromatography-ultraviolet detection method.

The combination of ionophoric coccidiostats and amino acids (AAs) is important in poultry feeding to enhance immunity and improve the growth and feed efficiency of birds suffering from coccidiosis. A simple, rapid, and economical high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method for the simultaneous determination of three ionophoric coccidiostats, namely salinomycin (SAL), maduramicin (MAD), and monensin (MON) in addition to three AAs; L-tryptophan (L-TRP), alpha-ketoleucin (KLEU), and L-valine (L-VAL) in feed premixes was developed and validated. Chromatographic separation was achieved in less than 12 min using a phenyl hexyl column with a mobile phase consisting of acetonitrile/methanol/water (25:20:55, v/v/v) adjusted to pH 3 using phosphoric acid. Isocratic elution was performed at a flow rate of 1 mL/min with UV detection at 210 nm. The method showed good linearity in the ranges 0.50-5.0 mg/mL for MON, 0.20-2.0 mg/mL for MAD and SAL, 10.0-100.0 µg/mL for L-TRP and KLEU, and 50.0-500.0 µg/mL for VAL. The developed method was successfully applied to determine the studied analytes in feed premixes with good recoveries and precision. The good validation criteria of the proposed method allow its utilization in quality control laboratories.

Tetracyclines in pasteurized milk commercialized in São Paulo, Brazil: occurrence and dietary risk assessment based on single and combined exposure with polyether ionophores.

Tetracyclines and polyether ionophores are veterinary drugs frequently used in animals reared for food production. Despite the benefits, residues can be harmful for human health if levels are higher than those considered safe. The aim of this study was to evaluate, by LC-MS/MS, tetracyclines residues in pasteurised milk samples marketed in São Paulo state, Brazil and estimate the exposure of the adult/adolescent/children population. Within the eight antibiotics monitored two were detected, tetracycline and 4-epitetracycline, found in 22.5% and 12.5% of the samples, respectively. Since the same samples had been previously tested for polyether ionophore residues, risk assessment based on dietary exposure to tetracyclines when considered individually and in combination with the ionophores was performed. The daily intakes were below the acceptable daily intake (ADI) for tetracycline. Considering the levels of monensin reported in a previous study, the combined exposure with tetracycline and 4-epi-tetracycline also indicated low potential health concerns.

Inhibition of platelet-activating factor (PAF)-induced platelet aggregation by fatty acids from human saliva.

Experiments were undertaken to identify the nature of a previously identified inhibitor of PAF-induced platelet aggregation (PA) in human saliva. Human saliva fractionated by preparative thin layer chromatography (TLC) yielded a fraction that co-migrated with fatty acids (FAs) and inhibited PAF-induced aggregation of platelets. Synthetic FAs tested for their capacities to inhibit 0.1 nM PAF-induced PA showed that only the cis-unsaturated compounds were inhibitory with activities of some of the polyunsaturated FAs (PUFA) reaching almost 100% at 20 µM. Eicosapentanoic acid (EPA) and 8,11,14-eicosatrienoic acid also deaggregated the PAF-induced aggregates. With the exception of oleic acid (OLA), cis-monounsaturated FAs, and elaidic acid, the trans isomer of OLA, were poor inhibitors. In a direct comparison with other platelet agonists, ADP, thrombin, and ionophore A23187, the active saliva fraction and selected individual FAs inhibited, to greater or lesser extent, PA induced by each of the agonists. EPA, OLA, linoleic acid (LNA), and the active saliva fraction were potent inhibitors of ADP-induced PA, EPA completely inhibited thrombin-induced PA and the saliva fraction showed only weak - moderate inhibitory activity to both thrombin- and ionophore A23187-induced PA. Other reports of endogenous PAF inhibitors in mammalian tissues are compared to the present results. PAF can trigger and amplify inflammatory cascades suggesting a possible modulation role for cis-unsaturated FAs in some diseases.

Simultaneous immunodetection of ionophore antibiotics, salinomycin and narasin, in poultry products and milk.

Rabbit polyclonal antibodies were generated against the ionophore antibiotic salinomycin (SAL) as a determinant of the BSA-SAL conjugate. The homologous ELISA format was found to be preferred for similar recognition of SAL and narasin (NAR) with IC50 values of 0.55 and 0.57 ng mL-1, respectively. Both analytes could be determined in the range of 0.1-2.7 ng mL-1 (IC20-IC80) with a detection limit of 0.03 ng mL-1. To analyze matrices, individual pretreatment of samples was required. For chicken muscles, simple buffer extraction was sufficient to recover 87-110% of ionophores. Extraction with acetonitrile followed by evaporation of the solvent was best for recovering 67-108% SAL and NAR from egg homogenate. A feature of the extraction of ionophores from milk was the elimination of fat-mediated interference by organic solvation. It was found that the absence of Na+ and K+ ions during reconstitution of sample extracts was a key factor contributing to the increase in the average recovery of ionophores from 32% to 93%. Thanks to this special pretreatment and improved recovery, the developed immunoassay method was suitable for the analysis of ionophore antibiotics SAL and NAR in a milk matrix, which has not been previously reported.

Polyether ionophores residues in pasteurized milk marketed in the state of São Paulo, Brazil: Occurrence and exposure assessment.

This paper aimed to survey the occurrence of six polyether ionophores (lasalocid, maduramycin, monensin, narasin, salinomycin and semduramycin) in pasteurized milk using a validated method by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). For that, 40 samples of pasteurized milk, from seven brands, collected in six locations (supermarkets, grocery stores and greengrocers) in the metropolitan region of São Paulo and Campinas, São Paulo State, Brazil, were evaluated. Sampling included whole (18), skimmed (8) and semi-skimmed (10) milk as well as lactose-free (4) products. Only monensin antibiotic residues were found in the analyzed samples. This ionophore was confirmed in 45% of the samples and quantified in 32.5% at concentrations ranging from 0.1 to 0.27 µg kg-1. The positive samples were whole or semi-skimmed milk. Considering the levels of monensin reported in the present study and data on milk consumption in Brazil, an exposure assessment indicated that the intake of this antibiotic does not represent a risk to health.

Occurrence and Concentrations of Residues of Tetracyclines, Polyether Ionophores, and Anthelmintics in Livers of Chickens Sold in the Informal Market in Gauteng Province, South Africa.

ABSTRACT: The occurrence, concentrations, and variables associated with tetracycline, polyether ionophore, and anthelmintic residues in the livers of chickens sold in the informal market in South Africa were determined. Ultrahigh-performance liquid chromatography-tandem mass spectrometry was used to simultaneously analyze for four tetracyclines, five polyether ionophores, and six anthelmintic residues. The study determined the presence of residues in liver samples at both the limit of quantifications (LOQ) and concentrations over the maximum residue limit (MRL), i.e., noncompliant. Doxycycline (tetracycline group) was detected in 24 (24.5%) of 98 chicken livers, and 15 (15.3%) of the 98 were noncompliant. Mean ± standard deviation (SD) concentrations of 919.04 ± 1,081.30 ppb (LOQ) and 1,410.57 ± 108.89 ppb (MRL) were obtained. Maduramicin was detected in 27 (27.6%) of 98 chicken livers, and 19 (19.4%) of 98 were noncompliant. The mean ± SD for LOQ was 117.96 ± 84.56 ppb, and MRL was 153.21 ± 76.29 ppb. The concentrations of residues of doxycycline and maduramicin in chicken livers varied significantly across townships. Lasalocid was found in 31 (31.6%) of 98 samples, of which 5 (5.1%) had concentrations above the MRL. The mean ± SD concentration of lasalocid was 62.90 ± 170.84 ppb for samples in which lasalocid was quantified and 310.16 ± 356.68 ppb for noncompliant samples. Detectable concentrations of praziquantel, closantel, and rafoxanide (anthelmintics) residues were found in 3 (3.1%), 1 (1.0%), and 2 (2.0%) of 98 chicken livers, respectively. The presence of residues of three classes of veterinary drugs in chicken livers poses food safety implications to consumers and indicates a need for enhanced regulatory enforcement in controlling these drugs in South Africa.

A new sample treatment strategy based on simultaneous supramolecular solvent and dispersive solid-phase extraction for the determination of ionophore coccidiostats in all legislated foodstuffs.

A single-step sample treatment, for the simultaneous extraction and clean-up for the determination of ionophore coccidiostats in EU legislated foodstuffs, is here proposed. The treatment is based on the combination of: (i) a supramolecular solvent with restricted access properties (SUPRAS-RAM), spontaneously formed by the addition of hexanol, water and THF to the sample; and (ii) dispersive solid phase extraction (dSPE). The SUPRAS-RAM extract was directly compatible with LC-MS/MS and no further re-extraction, evaporation or cleanup procedures were necessary. SUPRAS-RAM efficiently extracted the ionophores (recoveries in milk, eggs, fat, liver, kidney, and chicken and beef muscle were in the range 71-112%) and removed proteins and carbohydrates, whereas dSPE removed fats and other lipophilic compounds. The method was validated following the European Commission Decision 2002/657/EC. Detection limits (0.004-0.07 µg kg-1) were far below the maximum residue limits (1-150 µg kg-1). Method analytical and operational characteristics were suitable for routine determination of ionophores.

Polyether ionophores residues in Minas Frescal cheese by UHPLC-MS/MS.

An analytical method was developed and validated for the determination of three polyether ionophores (monensin, lasalocid, and salinomycin) in 60 samples of Brazilian Minas Frescal cheese by UHPLC-MS/MS. Linearity ranged from 1 to 8 µg kg-1 for monensin and salinomycin, and from 0.50 to 4 µg kg-1 for lasalocid. Limits of detection and quantitation were 0.50 µg kg-1 and 1 µg kg-1, respectively, for both monensin and salinomycin, and 0.25 µg kg-1 and 0.50 µg kg-1, respectively, for lasalocid. Recoveries were between 69% and 84% with coefficients of variation up to 16.28% for repeatability and 13.79% for intermediate precision. A total of 60 samples of Minas Frescal cheese were analysed and only monensin residues were found. Monensin was detected in 55% of the samples and quantified in 5 of them at mean levels varying from 1.00 to 1.73 µg kg-1. The proposed method demonstrated the suitability for monitoring these substances in cheese.

A multi-class multi-residue method for the analysis of polyether ionophores, tetracyclines and sulfonamides in multi-matrices of animal and aquaculture fish tissues by ultra-high performance liquid chromatography tandem mass spectrometry.

An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) based multiclass multi-residue method for the simultaneous analysis of 5 polyether ionophores, 4 tetracycline and 10 sulfonamides in animal and aquaculture fish tissues was developed and validated. Sample extraction and clean-up were based on a modified QuEChERS method. The method was validated using an in-house validation based on performance characteristics modified from Commission Decision 2002/657/EC. Both matrix effect and uncertainties associated with sample preparation and instrumental analysis were minimised by the use of matrix-matched calibrations. Recoveries of analytes were generally satisfactory and typically fell between 80% and 113%. The repeatability and intermediate reproducibility measured as relative standard deviations were in most cases less than 15% (n = 63). The decision limit (CCα) and detection capability (CCß) ranged from 110.7 to 125.8 and 121.5 to 151.7 µg kg-1 for tetracyclines, 113.4 to 118.3 and 116.8 to 126.5 µg kg-1 for sulfonamides and 50.8 to 52.4 and 51.5 to 55.6 µg kg-1 for polyether ionophores, respectively. The method displayed its fitness for purpose through satisfactory results obtained in international proficiency testing schemes. The method was applied to animal and aquaculture fish tissues obtained from different sources in South Africa. Polyether ionophores were predominantly detected in samples in the range 4.26-290.10 µg/kg. Oxytetracycline was found in one porcine liver sample; however, none of the targeted analytes were present above the detection limit in the aquaculture samples.

Occurrence of ß-lactam and polyether ionophore antibiotics in lagoon water and animal manure.

The occurrence of micropollutants in agricultural wastes is an emerging area of interest due to the potential impact of these compounds on the environment. A sensitive and reliable analytical method using liquid chromatography-electrospray tandem mass spectrometry has been developed and validated for the determination of three ß-lactam and three polyether ionophore antibiotics in lagoon water and animal manure matrices. The method was applied to evaluate the occurrence of these compounds from participating farms in northern Colorado. Seven of the 19 lagoon water samples and two of the six animal manures showed detectable. The three targeted ß-lactams (cephapirin, penicillin G, cloxacillin) were found at 0.97-43.31 µg/L in the lagoon water samples. Of the three targeted polyether ionophores, only monensin (94 to 1077 µg/L) was detected in the beef runoff pond water samples. Only cloxacillin was measured in the dairy animal manure samples at levels from 8.09 to 45.20 µg/kg. No cephapirin, penicillin G, cloxacillin, salinomycin, or narasin A were detected in any solid animal manure sample. These results indicate that elevated concentrations of ß-lactam and ionophore compounds might be found in lagoon or runoff pond waters and solid animal manures compared to surface waters, which these compounds are used in veterinary applications.

Method development for the analysis of ionophore antimicrobials in dairy manure to assess removal within a membrane-based treatment system.

Ionophore antimicrobials are heavily used in the livestock industries, both for preventing animal infection by coccidia protozoa and for increasing feed efficiency. Ionophores are excreted mostly unmetabolized and are released into the environment when manure is land-applied to fertilize croplands. Here, an analytical method was optimized to study the occurrences of five ionophore residues (monensin, lasalocid, maduramycin, salinomycin, and narasin) in dairy manure after solid-liquid separation and further treatment of the liquid manure by a membrane-based treatment system. Ionophore residues from the separated solid manure (dewatered manure) and suspended solids of manure slurry samples were extracted using ultrasonication with methanol, followed by sample clean-up using solid phase extraction (SPE) and subsequent analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The use of an ethyl acetate and methanol (1:1 v:v) mixture as an SPE eluent resulted in higher recoveries and lower method quantitation limits (MQL), when compared to using methanol. Overall recoveries from separated solid manure ranged from 73 to 134%. Liquid manure fractions were diluted with Nanopure™ water and cleaned up using SPE, where recoveries ranged from 51 to 100%. The developed extraction and LC-MS/MS methods were applied to analyze dairy manure samples subjected to an advanced manure treatment process involving a membrane-based filtration step (reverse osmosis). Monensin and lasalocid were detected at higher concentrations in the suspended solid fractions (4.40-420 ng/g for lasalocid and 85-1950 ng/g for monensin) compared to the liquid fractions (

Liquid to liquid extraction and liquid chromatography-tandem mass spectrometry determination of hainanmycin in feed.

Hainanmycin is a new veterinary polyether antibiotic and has few sensitive analytical method in present days. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) relying on multiple reaction monitoring (MRM) detection was developed for analysis of hainanmycin in animal feed. Feed samples were extracted with ethyl acetate and purified by two steps of liquid-liquid extraction (LLE) to get rid of water solvable matrix and lipids one by one. The final simple was analyzed by LC-MS/MS. The LC mobile phase was composed of 0.1% aqueous formic acid and 0.1% formic acidified acetonitrile by gradient elution. Average recoveries ranged from 74.22% to 87.85%, as determined by spiking with 2.0 (LOQ) ∼2500µgkg-1 of hainanmycin. The inter-day and intra-day coefficient of variation was 9.21% to 11.77% and 7.67% to 13.49%, respectively. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.36µgkg-1 and 2.0µgkg-1, respectively.

Impact of ionophore monensin on performance and Cu uptake in earthworm Eisenia andrei exposed to copper-contaminated soil.

Exposure of beneficial soil organisms to chemical mixtures is of great concern and can result in unexpected deleterious consequences. We investigated the effects of concurrent soil contamination with monensin, a veterinary pharmaceutical and feed additive, and copper, on earthworm copper uptake and reproductive success. The animals were exposed for 14 or 28 days to both substances and the results showed that the Cu body burden of earthworms increases in the presence of monensin. The harmful effects of Cu on earthworm cocoon production were considerably higher when monensin was also present in the soil. To localise the copper in earthworm tissues, histological staining was performed using two different dyes (rubeanic acid and 5-4-(p-dimethylaminobenzylidene)-rhodanine). Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to quantify the Cu levels in the tissues. Cu was found predominantly in the gut wall. The Cu content in the body wall was at least ten times lower compared to the gut, but was proportional to the level of soil contamination. Concurrent soil contamination with monensin and copper resulted in higher earthworm Cu levels and in decreased reproductive success of these important soil decomposers.

The effect of composting on the persistence of four ionophores in dairy manure and poultry litter.

Manure composting is a well-described approach for stabilization of nutrients and reduction of pathogens and odors. Although composting studies have shown that thermophilic temperatures and aerobic conditions can increase removal rates of selected antibiotics, comparable information is lacking for many other compounds in untreated or composted manure. The objective of this study was to determine the relative effectiveness of composting conditions to reduce concentrations of four widely used ionophore feed supplements in dairy manure and poultry litter. Replicate aliquots of fresh poultry litter and dairy manure were amended with monensin, lasalocid, salinomycin, or amprolium to 10mgkg(-1)DW. Non-amended and amended dairy manure and poultry litter aliquots were incubated at 22, 45, 55, or 65°C under moist, aerobic conditions. Residue concentrations were determined from aliquots removed after 1, 2, 4, 6, 8, and 12weeks. Results suggest that the effectiveness of composting for contaminant reduction is compound and matrix specific. Composting temperatures were not any more effective than ambient temperature in increasing the rate or extent of monensin removal in either poultry litter or dairy manure. Composting was effective for lasalocid removal in poultry litter, but is likely to be too slow to be useful in practice (8-12weeks at 65°C for >90% residue removal). Composting was effective for amprolium removal from poultry litter and salinomycin in dairy manure but both required 4-6weeks for >90% removal. However, composting did not increase the removal rates or salinomycin in poultry litter or the removal rates of lasalocid or amprolium in dairy manure.

Transformation of ionophore antimicrobials in poultry litter during pilot-scale composting.

Ionophores are the second top selling class of antimicrobials used in food-producing animals in the United States. In chickens, ionophores are used as feed additives to control coccidiosis; up to 80% of administered ionophores are excreted in the litter. Because poultry litter is commonly used to fertilize agricultural fields, ionophore residues in litter have become contaminants of emerging concern. This study aims to develop a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to quantify ionophores, and identify their transformation products (TPs) in poultry litter after on-farm pilot-scale composting. The validation parameters of the optimized method showed good accuracy, ranging from 71 to 119% recovery and relative standard deviation (precision) of ≤19% at three different concentration levels (10, 50 and 100 µg/kg). Monensin, salinomycin and narasin, were detected in the poultry litter samples prior to composting at 290.0 ± 40, 426 ± 46, and 3113 ± 318 µg kg(-1), respectively. This study also aims to investigate the effect of different composting conditions on the removal of ionophores, such as the effect of turning or aeration. Results revealed a 13-68% reduction in ionophore concentrations after 150 d of composting, depending on whether the compost was aerated, turned, or subjected to a combination of both aeration and turning. Three transformation products and one metabolite of ionophores were identified in the composted litter using high-resolution liquid chromatography with quadrupole time-of-flight mass spectrometry (LC-QToF/MS).

The Chloride Anion Acts as a Second Messenger in Mammalian Cells - Modifying the Expression of Specific Genes.

BACKGROUND/AIMS: Cystic Fibrosis (CF) is caused by mutations in the CFTR gene, encoding a cAMP-activated chloride (Cl-) channel. We have previously demonstrated that the expression of several genes can be modulated by the CFTR activity; among them, SRC, MTND4, CISD1, and IL1B. However, the CFTR signalling mechanism involved in the expression of CFTR-dependent genes is unknown. The aim of this work was to determine if intracellular chloride (Cl-)i might function as a second messenger modulating the expression of specific genes. METHODS: Differential display (DD) was applied to IB3-1 cells (CF cells), cultured under conditions that produce different intracellular Cl- concentrations ([Cl-]i), to analyse their expression profile. RESULTS: Several differentially expressed gene products were observed by using DD, suggesting the presence of chloride-dependent gene expression. Two cDNA fragments, derived from differentially expressed mRNAs and showing opposed response to Cl-' were isolated, cloned, sequenced and its Cl- dependency validated by reverse transcription quantitative-PCR (RT-qPCR). We identified the gene RPS27, which encodes the multifunctional ribosomal protein RPS27, also known as metallopanstimulin-1 (MPS-1), and the gene GLRX5, encoding glutaredoxin-related protein 5, as chloride-dependent genes. RPS27 was negatively regulated with increased [Cl-]i, approximately from 25-75 mM Cl- (EC50 = 46 ± 7 mM), and positively regulated from 75-125 mM Cl- (EC50 = 110 ± 11 mM) (biphasic response). In contrast, GLRX5 was positively modulated by [Cl-]i, showing a typical sigmoidal dose-response curve from 0-50 mM Cl-, reaching a plateau after 50 mM Cl- (EC50 ∼ 34 mM). CONCLUSION: The results suggest the existence of chloride-dependent genes. The Cl- anion, therefore, might act as a second messenger for channels or receptors able to modulate the intracellular Cl- concentration, regulating in turn the expression of specific genes.

Degradation and dissipation of the veterinary ionophore lasalocid in manure and soil.

Lasalocid is a veterinary ionophore antibiotic used for prevention and treatment of coccidiosis in poultry. It is excreted from the treated animals mostly in its active form and enters the environment with the use of contaminated manure on agricultural land. To properly assess the risk that lasalocid poses to the environment, it is necessary to know its environmental concentrations as well as the rates of its degradation in manure and dissipation in soil. These values are still largely unknown. A research was undertaken to ascertain the rate of lasalocid degradation in manure under different storage conditions (aging in a pile or composting) and on agricultural soil after using lasalocid-contaminated manure. The results have shown that there is considerable difference in lasalocid degradation between aging manure with no treatment (t1/2=61.8±1.7 d) and composting (t1/2=17.5±0.8 d). Half-lives in soil are much shorter (on average 3.1±0.4 d). On the basis of the measured concentrations of lasalocid in soil after manure application, we can conclude that it can potentially be harmful to soil organisms (PEC/PNEC ratio of 1.18), but only in a worst-case scenario of using the maximum permissible amount of manure and immediately after application. To make certain that no harmful effects occur, composting is recommended.

Polymeric optical sensors for selective and sensitive nitrite detection using cobalt(III) corrole and rhodium(III) porphyrin as ionophores.

Cobalt(III) 5,10,15-tris(4-tert-butylphenyl) corrole with a triphenylphosphine axial ligand and rhodium(III) 5,10,15,20-tetra(p-tert-butylphenyl) porphyrin are incorporated into plasticized poly(vinyl chloride) films to fabricate nitrite-selective bulk optodes via absorbance measurements. The resulting films yield sensitive, fast and fully reversible response toward nitrite with significantly enhanced nitrite selectivity over other anions including lipophilic anions such as thiocyanate and perchlorate. The selectivity patterns differ greatly from the Hofmeister series based on anion lipophilicity and are consistent with selectivity obtained with potentiometric sensors based on the same ionophores. The optical nitrite sensors are shown to be useful for detecting rates of emission of nitric oxide (NO) from NO releasing polymers containing S-nitroso-N-acetyl-DL-penicillamine.

Comparison of triple quadrupole mass spectrometry and Orbitrap high-resolution mass spectrometry in ultrahigh performance liquid chromatography for the determination of veterinary drugs in sewage: benefits and drawbacks.

This paper presents a comparison of triple quadrupole tandem mass spectrometry (MS/MS) and Orbitrap high-resolution mass spectrometry (HRMS) combined to ultrahigh performance liquid chromatography for the determination of glucocorticoids and polyether ionophores in sewage, in order to show the major benefits and drawbacks for each mass spectrometry analyser. Overall, HRMS measurements have enhanced performance in terms of confirmatory capabilities than MS/MS measurements. Moreover, similar limits of quantification, limits of detection, linear range and repeatability for glucocorticoids with both the MS/MS and HRMS methods were compared, but in the case of polyether ionophores, slightly better limits of detection and limits of quantification were obtained with the HRMS method because of the high sensitivity obtained when diagnostic ions are used for quantification instead of selected reaction monitoring transitions for these compounds. The two methods have been applied to the analysis of several influent and effluent sewage samples from sewage treatment plants located in the Tarragona region (Catalonia, Spain), showing an excellent correlation between the two methods.

Detection and quantification of ionophore antibiotics in runoff, soil and poultry litter.

Ionophore antibiotics (IPAs) are widely used as coccidiostats in poultry and other livestock industries to promote growth and prevent infections. Because most of the ingested IPAs are excreted in poultry litter, which is primarily applied as grassland fertilizer, a significant amount of IPAs can be released into the litter-soil-water environment. A robust analytical method has been developed to quantify IPAs (monensin (MON), salinomycin (SAL) and narasin (NAR)) in complex environmental compartments including surface runoff, soil and poultry litter, with success to minimize matrix interference. The method for water samples involves solid-phase extraction (SPE) followed by liquid-liquid extraction (LLE) post-clean up steps. The method for solid samples involves bi-solvent LLE. IPAs were detected by HPLC-MS, with optimized parameters to achieve the highest sensitivity. Nigericin (NIG), an IPA not used in livestock industry, is successfully applied and validated as a surrogate standard. The method recoveries were at 92-95% and 81-85% in runoff samples from unfertilized and litter-fertilized fields, respectively. For solids, the method recoveries were at 93-99% in soils, and 79-83% in poultry litter samples. SAL was detected at up to 22mg/kg and MON and NAR at up to 4mg/kg in broiler litter from different farms. Up to 183µg/kg of MON was detected in litter-fertilized soils. All three IPAs were detected in the rainfall runoff from litter-fertilized lands at concentrations up to 9µg/L.