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.

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.

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.

Rapid method for the simultaneous determination of six ionophores in feed by liquid chromatography/mass spectrometry.

A simple and highly sensitive LC/MS method was developed for the simultaneous determination of six ionophores--lasalocid, monensin, laidlomycin, maduramycin, salinomycin, and narasin--in feed. The procedure involved extraction of 1 g of feed with 4 mL of methanol-water (9 + 1, v/v) by shaking on a platform shaker for 45 min. After centrifugation, the extracts were diluted with methanol-water (75 + 25, v/v) and analyzed without any cleanup. The analysis was performed on a Betasil C18 column (150 x 4.6 mm id, 5 pm particle size) connected to an LC/MS system operated in the atmospheric pressure chemical ionization (APCI) mode. We believe this to be the first method that uses the APCI mode for the analysis of ionophores. The mobile phase consisted of 50 mM ammonium acetate as solvent A and acetonitrile-methanol (7 + 3, v/v) as solvent B in a gradient run. Excellent recoveries of 81-120% were found for all compounds at fortification levels of 1-200 microg/g, with RSD < or =15% (except 17% for maduramycin at 2 and 5 microg/g, and 16% for salinomycin at 1 microg/g). At 0.5 microg/g, recoveries of 87-119% were obtained, with RSD < or =20%. However, recovery of lasalocid was 133% and salinomycin 79% in sow and horse feed, respectively. Average RSD values of lasalocid and salinomycin were 22 and 21%, respectively. Finally, proficiency test samples analyzed with the method demonstrated favorable agreement with the certified values.

Quantification of ionophores in aged poultry litter using liquid chromatography tandem mass spectrometry.

Veterinary anticoccidials, biochemically known as ionophores, are widely used in poultry feed at therapeutic levels to treat Coccidiosis and at sub-therapeutic levels for growth- promotion. Commonly used ionophores in the US poultry industry are monensin, salinomycin, lasalocid and narasin. There is an increasing concern regarding the persistence of these anticoccidials in the environment. However, little attention has been directed to methods development for quantitatively measuring ionophores in complex environmental matrices such as poultry litters that are land applied. Here, we describe a rapid and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method developed for simultaneous quantification of monensin, lasalocid, salinomycin, and narasin in aged poultry litter samples. Results show significant level of monensin (97.8 ± 3.2 µg kg⁻¹), lasalocid (19.2 ± 6.6 µg kg⁻¹), salinomycin (70 ± 2.7 µg kg⁻¹) and narasin (57.3 ± 2.6 µg kg⁻¹) in poultry litter stored for over three years at < 5°C. Our findings indicate that even after several years of unmanaged storage of poultry litter, ionophores may continue to persist in this matrix, raising the possibility of prolonged release into the environment.

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.

Development and validation of an UPLC-MS/MS method for the determination of ionophoric and synthetic coccidiostats in vegetables.

In poultry farming, anticoccidial drugs are widely used as feed additives for the prevention and treatment of coccidiosis. Because coccidiostats and veterinary medicines, in general, are often poorly absorbed, manure from treated animals may contain high concentrations of these compounds. Experimental studies have shown that the uptake of veterinary medicines by plants from soil containing contaminated manure may occur. This leads to several questions regarding the impact on the environment, resistance problems, and public health and allergy issues. This work describes the development of a quantification method for coccidiostats in vegetables. Vegetables were spiked at 100 µg kg(-1) (dry weight) with coccidiostats (monensin, narasin, lasalocid A, salinomycin, diclazuril, and nicarbazin) in order to optimize the extraction and clean-up. Possible critical factors (e.g., extraction solvent) were statistically examined by linear regression with the use of Plackett-Burman and full factorial designs. Final extracts were analyzed with ultra-performance liquid chromatography tandem mass spectrometry operating in multiple-reaction monitoring mode. Both the synthetic and ionophoric coccidiostats could be determined in a single run with an analysis time of 5 min. The developed method was validated taking into account the requirements of the Commission Decision 2002/657/EC as a guideline. The method is regarded as applicable for its intended purposes with quantification limits between 0.30 and 2.98 µg kg(-1). This method could be used to establish possible maximum residue limits for coccidiostats in vegetables, as already exist for eggs, meat, and milk.

Li⁺ selective podand-type fluoroionophore based on a diphenyl sulfoxide derivative bearing two pyrene groups.

New podand-type fluoroionophores having two pyrene moieties: 2,2´-bis(1-pyrenylacetyloxy)diphenyl sulfide (3), 2,2´-bis(1-pyrenylacetyloxy)diphenyl sulfoxide (4), and 2,2´-bis(1-pyrenylacetyloxy)diphenyl sulfone (5), have been synthesized by connecting two 1-pyrenecarbonylmethyl groups with the two hydroxy groups of 2,2´-dihydroxydiphenyl sulfide, sulfoxide, and sulfone, respectively. Their complexation behavior toward alkali metal ions was examined by fluorescence spectroscopy. Among these fluoroionophores, compound 4, having a sulfinyl group, showed high selectivity toward Li⁺.

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.

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 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 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.

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.

Construction of new iodide ion-selective electrodes based on new ionophores: comparison of the effect of structures and additives on electrode response.

New iodide ion-selective electrodes based on bis(2-methyl-3-phenyl-propenylidene)1,2-propanediamine)mercury(II) bromide [Hg(BMPPMB)Br2], bis (5-bromo salicylaldehyde) ethylenediimine vanadyl(IV) [VO(5-Br Salen)], and bis(5-bromo salicylaldehyde) ethylenediimine uranyl(VI) [UO2(5-Br Salen)] carriers are described. The electrodes exhibited Nernstian slopes of -58.3 +/- 1.0, -58.5 +/- 1.4, and -59.0 +/- 1.2 mV/decade for Hg(BMPPMB)Br2, VO(5-Br Salen), and UO2(5-Br Salen) iodide ion concentration in the range of 1.0 x 10(-6)-0.1 M, with detection limits of about 0.6, 0.5, and 0.3 microM, respectively. The potentiometric responses of the electrodes were independent of pH over the range 1.7-10.6, 2.2-11.3, and 2.1-11.1, with satisfactory reproducibility. The electrodes had response times of < or =5 s and could be used for at least 84, 80, and 100 days for Hg(BMPPMB)Br2, VO(5-Br Salen), and UO2(5-Br Salen), respectively, without any considerable divergence in their potential responses.

Isolation and partial characterization of four fluorophores formed by nonenzymatic browning of methylglyoxal and glutamine-derived ammonia.

An aqueous solution of L-glutamine (50 mmol/L) and methylglyoxal (100 mmol/L) was incubated at 120 degrees C for 3 h in a 200 mmol/L phosphate buffer (pH 7.4). Four major fluorophores were revealed on the HPLC chromatogram. The same four fluorophores were obtained from the heating of a mixture of ammonia and methylglyoxal. After purification and concentration, they were structurally characterized by electrospray ionization mass spectrometry (ESI-MS) using the high resolution and tandem mass spectrometry capabilities of a quadrupole time-of-flight MS. The accurate mass measurement of their [M+H]+ ions, the MS fragment patterns, and the presence of one to two nitrogen indicate the formation of fluorophores with molecular formulas of C7H7NO3, C8H9NO3, C12H14N2O4, and C12H14N2O5. These results show that, in an aqueous solution, free glutamine undergoes a rapid degradation, leading to the formation of ammonia which reacts with methylglyoxal to form fluorescent heterocyclic Maillard products.

Determination of ionophore coccidiostats in feedingstuffs by liquid chromatography-tandem mass spectrometry Part I. Application to targeted feed.

A new and fit to purpose multi-analyte method for the determination of six coccidiostats (monensin A, salinomycin, narasin composed of its principal component narasin A and its minor component narasin I, lasalocid, semduramicin and maduramicin) in poultry and cattle compound feed by liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed and in-house validated. The concentration level of the target analytes at which the validation experiments have been carried out varied between 1 and 9 mg kg(-1). The method developed involved a simple extraction of the coccidiostats from the feed samples followed by a clean-up by solid-phase extraction prior to chromatographic analysis. The analytes were quantified either by matrix-matched standards or by the standard addition technique, obtaining the following performance profile of the method for the various analyte/matrix combinations. When quantifying against matrix-matched standards, the concentration independent intermediate precision expressed in terms of relative percentage standard deviation varied between 4 and 10% and the relative percentage recovery rates ranged from 86 to 120%, depending on the target analyte and matrix. When using the standard addition technique, the corresponding values for the intermediate precision varied between 2 and 8% and the relative percentage recovery rate ranged from 73 to 115%. The limit of detection (LOD) and limit of quantification (LOQ) were different for the various analyte/matrix combinations but were in all cases below 0.014 and 0.046 mg kg(-1), respectively. Based on the obtained method performance characteristics, the method is considered suitable for the determination of ionophore coccidiostats in target feed. The main field of application of the validated method is to enforce European legislation regarding the authorisation of coccidiostats, focusing on the measurement at the authorised levels and at low level in feed during the withdrawal period at which the coccidiostats must not be added to the feed. Overall, the method proposed appears to be appropriate as a confirmatory method for the monitoring of these six ionophore coccidiostats and can therefore be considered as complementary to the official HPLC-UV methods.

Environmental occurrence and shallow ground water detection of the antibiotic monensin from dairy farms.

Pharmaceuticals used in animal feeding operations have been detected in various environmental settings. There is a growing concern about the impact on terrestrial and aquatic organisms and the development of antibiotic-resistant strains of microorganisms. Pharmaceutical use in milking cows is relatively limited compared with other livestock operations, except for the ionophore monensin, which is given to lactating cows as a feed. By weight, monensin can be the most significant antibiotic used in a dairy farm. This study investigates the potential of monensin to move from dairy operations into the surrounding ground water. Using two dairy farms in California as study sites, we twice collected samples along the environmental pathway-from flush lanes, lagoon waters, and shallow ground water beneath the dairies and beneath its associated manured fields. Monensin concentrations were determined using solid-phase extraction and liquid chromatography-tandem mass spectrometry with positive electrospray ionization. Monensin was detected in all of the flush lane and lagoon water samples. Theoretical maximum concentration estimated from the actual dosing rate and the theoretical excretion rate assuming no attenuation was one order of magnitude greater than observed concentrations, suggesting significant attenuation in the manure collection and storage system. Monensin was also detected, at levels ranging from 0.04 to 0.39 microg L(-1), in some of the ground water samples underneath the production area of the dairy but not from the adjacent manured fields. Concentrations in ground water immediately downgradient of the lagoons were one to two orders of magnitude lower than the concentrations detected in lagoons, suggesting attenuation in the subsurface. The data suggest the possibility of monensin transport into shallow (2-5 m) alluvial ground water from dairy management units, including manure storage lagoons and freestalls occupied by heifers, lactating cows, and dry cows.

Determination of lasalocid sodium in animal feeds and premixes by reversed-phase liquid chromatography: collaborative study.

A liquid chromatographic (LC) method for the analysis of lasalocid sodium in premixes, complete animal feeds, and trace-level feeds was collaboratively studied. The method employs a 0.5% HCI acidified methanol extraction followed by 20 min sonication in a water bath heated to 40 degrees C. Samples are then shaken on a mechanical shaker for 1 h and stored overnight, followed by an additional 10 min shaking the following morning. Sample extracts are diluted if necessary with extractant, filtered, and injected onto an LC system. Determination of all lasalocid homologs is by reversed-phase LC with fluorescence detection at 314 nm excitation and 418 nm emission. Eight samples of drug premixes, medicated feeds, and mineral supplements, along with 2 samples for trace-level analysis were sent to 20 collaborators in the United States, Canada, and The Netherlands. Study data were returned by 17 laboratories. Two additional supplemental trace-level samples and a blank feed were provided to 15 of the collaborating laboratories, and test data were received from all 15 participants. For the drug premixes, medicated feeds, and mineral supplements, RSDr values (within-laboratory repeatability) ranged from 1.2 to 19.9%, RSDR values (among-laboratory reproducibility) ranged from 3.4 to 32.3%, and HorRat values ranged from 0.35 to 3.73. For the trace-level samples, only lasalocid A, the predominant homolog comprising > 90% of the sum of all homolog peak area, was quantified. All laboratories correctly identified the analyte. Although some instrument response was reported by a number of laboratories for the blank feed, all but one laboratory's results were well below the 1 mg/kg limit of quantification. RSDr values for the initial 2 trace-level samples were excessive, ranging from 51.6 to 64.4%. RSDR values ranged from 51.6 to 75.7%, and HorRat values ranged from 3.6 to 4.0. Data for the initial trace-level samples indicated that the test samples were improperly prepared to ensure homogeneity, and a new set of supplemental samples was provided to collaborators, with significantly improved results. RSDr values for the 2 supplemental trace-level samples ranged from 1.6 to 2.5%, RSDR values ranged from 5.6 to 9.2%, and HorRat values ranged from 0.43 to 0.62.

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.