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TECHNICAL REPORTS

Organic Compounds in the Environment

Application of Enzyme-Linked Immunosorbent Assay Analysis for Determination of Monensin in Environmental Samples

^a Dep. of Plant and Earth Science, Univ. of Wisconsin-River Falls, 410 S. 3rd St., River Falls, WI 54022
^b Res. and Dev., Metropolitan Water Reclamation District of Greater Chicago, 6001 West Pershing Rd., Cicero, IL 60804
^c Dep. of Soil, Water, and Climate, Univ. of Minnesota, 1991 Upper Buford Circle, St. Paul, MN 55108
^d Veterinary Population Medicine, Univ. of Minnesota, 1333 Gortner Ave., St. Paul, MN 55108

^* Corresponding author (sgupta{at}umn.edu).

Received for publication July 25, 2007.

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results Discussion Conclusions REFERENCES

 
There is growing concern that antibiotic use in livestock production is contributing to contamination of soil and aquatic environments. Monensin, a polyether ionophore antibiotic, accounts for approximately 13% of total subtherapeutic livestock antibiotic usage in the USA and has been widely detected in aquatic environments. Due to insufficient ultraviolet absorbance, liquid chromatography analysis of monensin in environmental samples is limited to equipment with mass spectrometry (e.g., liquid chomatography–mass spectrometry [LC-MS]). However, LC-MS equipment is costly, and extensive sample preparation and clean-up is often required. Rapid, low-cost analytical techniques are needed to monitor for monensin residues in the environment. In this study, a commercially available enzyme-linked immunosorbent assay (ELISA) for detecting monensin in animal feed extracts was evaluated for determination of monensin in water, soil, and manure. The monensin ELISA was highly sensitive, with limits of detection and quantification at 1.5 and 3.0 µg L^–1, respectively. Recovery of monensin in spiked water samples was approximately 100%. Cross-reactivity was not observed with similar polyether ionophores, tetracyclines, macrolides, or sulfonamides. Concentrations of monensin using ELISA were greater than concentrations measured with LC-MS. This is attributed to cross-reactivity of the monensin ELISA toward structurally similar monensin compounds, such as factors (slight naturally produced structural variants) and metabolites. Results from this study showed that ELISA can be a reliable, rapid, and low-cost alternative to LC-MS analysis of environmental samples.

Abbreviations: BSA, bovine serum albumin • ELISA, enzyme-linked immunosorbent assay • LC-MS, liquid chromatography-mass spectrometry • OD, optical density • PBS, phosphate buffered saline

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results Discussion Conclusions REFERENCES

 
MONENSIN (Fig. 1 ) is a polyether ionophore antibiotic used in veterinary medicine. Monensin is produced by Streptomyces spp. and consists primarily of factor A (>90%), with <10% of factors B, C, and D (Elanco Products Company, 1989). Along with treatment and prevention of coccidiosis in poultry and cattle, monensin is used for subtherapeutic (growth promotion) feeding in livestock. Monensin has been shown to increase feed efficiency in cattle by as much as 9% (Tedeschi et al., 2003). Monensin has also been shown to reduce methane emissions, a potent greenhouse gas, from cattle by up to 20% (Tedeschi et al., 2003). Approximately 1.5 million kilograms of monensin is used annually in the USA for subtherapeutic purposes in cattle and poultry production (Mellon et al., 2001). This accounts for approximately 13% of the total subtherapeutic antibiotic usage in animal agriculture (Mellon et al., 2001). Only chlortetracycline is used more than monensin in the USA (Mellon et al., 2001).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Structures of monensin, factors, and major metabolites isolated from cattle manure (Donoho et al., 1978) (adapted from Kiehl et al., 1998).

Environmental levels, fate, and transport of monensin are poorly understood. Compared with tetracyclines and macrolides (K_d = 50–2000 L kg^–1), monensin is not tightly adsorbed to soil (K_d = 9.3 L kg^–1) (Elanco Products Company, 1989; Kumar et al., 2005). However, Carlson and Mabury (2006) concluded that monensin was immobile in soil because it was not detected in soil below 25 cm that had received a manure application containing 1 mg monensin kg^–1 manure and 78 mm of precipitation. In contrast, Kim and Carlson (2006) detected monensin in river water and aquatic sediments in Colorado. The authors suggested that monensin came from feedlots in the watershed. Maximum concentrations of monensin were 0.036 µg L^–1 in river water and 31.5 µg kg^–1 in sediment. Monensin was also detected in approximately 75% of stream samples at concentrations ranging from 0.006 to 1.2 µg L^–1 in southern Ontario (Lissemore et al., 2006). In a small-plot rainfall simulation study, average monensin concentrations in the aqueous and sediment phase of runoff were 1.2 and 10.5 µg L^–1, respectively, which was greater than concentrations of tetracyclines, sulfonamides, and macrolides (Davis et al., 2006). Unlike many antibiotics used in veterinary medicine, monensin is not used in human medicine. As a result, monensin could be used as a tracer to detect agricultural contamination of aquatic environments.

Although antibiotic resistance is a concern when antibiotics are present in the environment, monensin is not considered a public health threat because it is not used in human medicine (Russell and Houlihan, 2003). Because the mechanisms of monensin resistance are poorly understood, it is unclear what threat monensin resistance may pose to animals (Russell and Houlihan, 2003). Monensin toxicity toward microorganisms, plants, animals, and humans may be of concern. For example, monensin concentrations >9 mg L^–1 are lethal to rainbow trout (Oncorhynchus mykiss), bluegill (Lepomis macrochirus), and fathead minnows (Pimephales promelas) (Ramsdell, 2003; Elanco Products Company, 1989). Aquatic microorganisms, such as green algae and ampihods, are even more sensitive, with lethal concentrations of <1 mg L^–1 in water or 1 mg kg^–1 in sediment (Ramsdell, 2003; Elanco Products Company, 1989). Monensin has also been shown to adversely affect plant growth at concentrations >1 mg kg^–1 (Brain et al., 2004). Lethal effects of direct monensin ingestion have also been documented in humans (Kouyoumdjian et al., 2001). It is unclear what effects may occur at sublethal monensin exposure.

Due to environmental contamination and toxicity concerns, it is necessary to monitor and quantify monensin residues in terrestrial and aquatic environments. The conventional analytical technique for antibiotic analysis in environmental samples is high-performance liquid chromatography; however, monensin does not have sufficient ultraviolet absorbance for detection. Derivatization techniques have been developed to overcome weak ultraviolet absorbance (Moran et al., 1994; Dusi and Gamba, 1999); however, limits of quantification are generally not sufficient for trace analysis (<20 µg L^–1 or kg^–1). In general, monensin analysis is limited to expensive equipment with mass spectrometry capabilities (Cha et al., 2005).

Recently, enzyme-linked immunosorbent assay (ELISA) analysis has been used as an alternative analytical technique for rapid, low-cost antibiotic analysis in environmental matrices such as water, soil, and manure (Aga et al., 2003; Kumar et al., 2004; Aga et al., 2005). The basis of ELISA analysis is a highly specific and sensitive antibody antigen reaction. Although generally considered semi-quantitative, ELISA analysis has been shown to be a reliable and highly sensitive analytical tool (Aga et al., 2003; Kumar et al., 2004).

The objective of this study was to evaluate a commercially available ELISA for measuring monensin in water, soil, and manure. Matrix effects, sensitivity, and cross-reactivity were assessed to determine ELISA effectiveness and reliability. In addition, ELISA results were compared with conventional liquid chromatography–mass spectrometry (LC-MS) for verification.

	Materials and Methods

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results Discussion Conclusions REFERENCES

 
ELISA Analytical Procedure
A commercially available monensin ELISA kit was purchased from Immuno-Diagnostic Reagents (Vista, CA). The ELISA kit was designed for analysis of monensin in animal feed and edible tissues. The kit consisted of a 96-well microtitre plate precoated with rabbit antibodies directed against monensin. Analysis was performed as instructed by the manufacturer. After addition of standards and samples (25 µL), monensin-alkaline phosphatase conjugate (100 µL) was added to the plate and allowed to compete with monensin in the sample for binding to the antibodies. After a 40-min incubation period at room temperature, solutions were discarded, and nonbound components were removed by a series of three washings (250 µL) with phosphate-buffered saline (PBS)-Tween. Next, p-nitrophenyl phosphate substrate (100 µL) was added to the plate and incubated at room temperature for 20 min. The reaction was stopped with the addition of 3N NaOH (50 µL), and color intensity (i.e., optical density) was measured at 405 nm using a spectrophotometer (Molecular Devices, Sunnyvale, CA).

Standard/sample dilution buffer solution was not provided with the monensin ELISA kit. Dilution buffer is important for reducing matrix effects and variability. We evaluated several dilution buffers, including nano-pure water and various combinations of bovine serum albumin (BSA) in PBS. The best overall performance was achieved with 0.1% BSA in PBS (data not shown).

Standards were prepared by serially diluting a 1 mg L^–1 solution of pure monensin (supplied with kit) with 0.1% BSA in PBS to obtain the desired concentrations. A six-point (0, 3.125, 6.25, 12.5, 25.0, and 50.0 µg L^–1) standard curve was developed for quantification of monensin in samples. Intra-assay variability was determined by testing samples in triplicate within a plate. Interassay variability was determined on consecutive plates (n = 7) assayed during a 50-d period.

ELISA Matrix Effects
A matrix effect can occur when a substance or substances in the matrix interferes with the assay, thus producing inaccurate results. In general, the more complex the matrix, the more likely a matrix effect will be encountered with ELISA analysis. Matrix effects for the monensin ELISA were assessed for a variety of water, soil, and manure matrices. Preliminary analysis showed that monensin was not present in any of the matrices. Water matrices included three heavily affected urban and agricultural surface waters (Lake Nokomis, Minneapolis, MN; Minnehaha Creek, Minneapolis, MN; agricultural runoff, Lancaster, WI), along with one near-surface ground water matrix (agricultural leachate; Lancaster, WI). After collection, water samples were filtered through a 0.45-µm nonsorptive filter.

Soil matrices included Hubbard sandy loam (Entic Hapludoll; Becker, MN), Rozetta silt loam (Typic Hapludalf; Lancaster, WI), and Webster clay loam (Typic Endoaquoll; Lamberton, MN). Particle size distribution and organic matter contents of these soils were as follows: 78% sand, 12% silt, 10% clay, and 2.2% organic matter for Hubbard sandy loam; 19% sand, 69% silt, 12% clay, and 3.6% organic matter for Rozetta silt loam; and 30% sand, 36% silt, 34% clay, and 4.4% organic matter for Webster clay loam. Manure matrices included turkey and cattle manure obtained from local farms where monensin had not been used therapeutically or subtherapeutically. For soil and manure samples, extraction (1:1 for soil and 1:5 for manure) was performed with nano-pure water. Suspensions were vortexed for 5 min, centrifuged at 2000 g for 20 min, and filtered through a 0.45-µm nonsorptive filter. Water, soil, and manure matrices were evaluated for matrix effects at dilution factors ranging from 2 to 150. Dilution factors referenced in this paper account for dilution from extraction as well as further dilutions performed to assess matrix effects. After extraction, dilutions were performed with 0.1% BSA in PBS. Each sample was run in duplicate.

ELISA Sensitivity
Sensitivity refers to the ability of an analytical procedure to discriminate small differences in concentration of the analyte being measured. Sensitivity of the monensin ELISA kit was evaluated using the water matrices described in the previous section. Water matrices were spiked with pure monensin at concentrations of 6.25, 12.5, 25.0, and 50.0 µg L^–1. For ELISA analysis, samples were diluted 1:1 with 0.1% BSA in PBS. Each sample was run in duplicate.

ELISA Cross-Reactivity
Antibiotics of different classes are often simultaneously administered to livestock with feed; therefore, it is important to evaluate cross-reactivity. Cross-reactivity was evaluated for a similar polyether ionophore, salinomycin. In addition, cross-reactivity was evaluated for tetracyclines (chlortetracycline and doxycycline), macrolides (erythromycin and tylosin), a sulfonamide (sulfamethazine), and a streptogramin (virginiamycin). These represent a wide range of antibiotics that could potentially appear in environmental samples concurrently with monensin. Pure antibiotics were purchased from Sigma Aldrich (St. Louis, MO). Cross-reactivity was evaluated at a concentration of 1 mg L^–1 in 0.1% BSA in PBS.

Verification of ELISA with LC-MS
Several water and manure samples were obtained to verify monensin ELISA results with LC-MS. Water samples were agricultural runoff samples from a field where monensin had been land-applied in cattle manure. Manure samples were obtained from cattle and turkey manure compost and stockpiles. Water samples were filtered through a 0.45-µm nonsorptive filter. Manure samples (5 g) were prepared by liquid–liquid extraction (10 mL followed by 5 mL) with an 80:20 (v/v) methanol:water solution. For each extraction, samples were vortexed for 1 min, shaken on an end-over-end shaker for 15 min at 4°C, and centrifuged at 2000 g for 20 min. Supernatants were combined, centrifuged at 2000 g for 20 min, and filtered through a nonsorptive 0.45-µm filter.

The liquid chromatography system consisted of a Finnigan SpectraSystem P4000 pump connected to a SpectraSystem AS3000 autosampler (Thermo Electron Corp., San Jose, CA). Analyte separation was achieved using a Zorbax Eclipse XDB-C8 column (150 mm x 4.6 mm x 5 µm) (Agilent Technologies, Inc., Santa Clara, CA) in combination with a MetaGuard 4.6 mm C18 guard column (Varian, Inc., Torrance, CA). Mobile phase A was 0.1% formic acid in nano-pure water, and mobile phase B was HPLC-grade methanol (Sigma Aldrich). Separation was achieved using the following gradient: 0 min A/B = 50:50, 1 min A/B = 10:90, 18 min A/B = 50:50. Injection volume was 50 µL, and flow rate was 1 mL min^–1. Retention time for monensin was 5.8 min. Calibration curves were linear between 0 and 1 mg L^–1 (r² > 0.99).

Tandem mass spectrometry was performed using a Finnigan LCQ Duo ion trap mass spectrometer (Thermo Electron Corp., San Jose, CA) equipped with an electrospray ionization source operated in positive mode. The spray needle was operated at a voltage of 4.0 kV, and capillary voltage was 6.0 V. Capillary temperature was set to 225°C. Sheath and auxiliary gas were nitrogen, and the ion trap collision gas was helium.

A T-splitter was used for infusion into the mass spectrometer. Flow rate into the mass spectrometer was approximately 0.25 mL min^–1, with the remaining flow diverted to waste. Isolation width was 3.0 (m/z), and collision energy was 28%. The product ion producing the highest signal was chosen for selected reaction monitoring and quantification. The precursor ion was protonated monensin sodium salt [M + Na]⁺ at 693 (m/z), whereas the product ion with the highest signal was 675 (m/z), corresponding to neutral water loss [M + Na – H₂O].

	Results

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ELISA Analytical Procedure
A typical standard curve for the monensin ELISA is shown in Fig. 2 . The standard curve was log-linear with, a coefficient of determination >0.99. Percent inhibition was calculated from the optical density (OD) using the following equation:

[1]

Percent inhibition ranged from 27% for the 3.125 µg L^–1 standard to 80% for the 50 µg L^–1 standard. The mid-point of the standard curve (50% inhibition) corresponded to approximately 10 µg L^–1. The coefficient of variation for inter-assay variability ranged from an average of 16.5% for the 3.125 µg L^–1 standard to 2.2% for the 50 µg L^–1 standard. Overall, interassay variability averaged 8.1%, whereas intra-assay variability was 5.1%. The limit of detection (3 SD of the OD of the 0 µg L^–1 standard) and the limit of quantification (6 SD of the OD of the 0 µg L^–1 standard) were 1.5 and 3.0 µg L^–1, respectively. The working range of the monensin ELISA kit was approximately 2 to 100 µg L^–1.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Standard curve for the monensin ELISA. Error bars represent standard deviation of the mean. The working range of the monensin ELISA kit was approximately 2 to 100 µg L–1.

[2]

Monensin concentrations were subsequently estimated from the standard curve.

The monensin ELISA was highly sensitive when varying concentrations from 6.25 to 50.0 µg L^–1 in spiked water samples (Table 2 ). Recovery of monensin in spiked water samples ranged from 76 to 117%, with an average recovery of 100% (Table 2; Fig. 3 ). Recovery was similar across the water matrices. The standard deviation of monensin concentration increased as spiked concentration increased (Fig. 3), suggesting better reliability at lower concentrations. Coefficients of variation for intra-assay variability in monensin concentrations of spiked water matrices were <20%.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Relationship between spiked and measured ELISA monensin concentrations for the water matrices.

Verification of ELISA with LC-MS
Water and manure samples were collected and used to compare ELISA and LC-MS results. Monensin concentrations obtained with ELISA were generally in good agreement with concentrations obtained using LC-MS (Table 3 ). In all cases, ELISA concentrations were greater than concentrations obtained with LC-MS. Monensin concentrations in water samples were on average 18.5% greater for ELISA analysis, whereas manure concentrations were on average 25.5% greater (excluding the fresh cattle manure). Because manure samples were analyzed with the monensin ELISA using dilution factors in excess of 500, matrix effects were assumed to be negligible (Table 1). Higher monensin concentrations from the ELISA analysis may be due to cross-reactivity with monensin factors (slight naturally produced structural variants), metabolites, and/or degradation products (Fig. 1). Cross-reactivity of structurally similar compounds has been documented for ELISA analysis. For example, in a tetracycline ELISA, cross-reactivity was found for structurally similar tetracyclines (chloretercylcine, oxytetracycline, doxycycline, etc.) along with transformation products (epitetraycline, anhydrotetracycline, etc.) (Aga et al., 2003; Kumar et al., 2004).

	Discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results Discussion Conclusions REFERENCES

 
The results from this study demonstrate that ELISA analysis can provide an alternative to LC-MS for measuring monensin in environmental samples. The commercially available monensin ELISA kit was highly sensitive (limit of quantification of 3 µg L^–1) and covered a broad concentration range (up to 100 µg L^–1). Using the commercially available monensin ELISA, the analysis could be completed in <1.5 h. The cost of each sample, including associated materials needed for analysis (sample tubes, pipette tips, etc.), was less than $5. Furthermore, analysis did not require extensive sample clean-up procedures, such as solid-phase extraction, which are time consuming and expensive and are commonly needed in LC-MS analysis (Cha et al., 2005).

Limits of detection and quantification in this study were lower than limits reported for a monensin fluoroimmunoassy (Crooks et al., 1998) but were comparable to similar monensin ELISAs (Mount and Failla, 1987; Crooks et al., 1997). The monensin limits of detection and quantification were also considerably lower than those reported for high-performance liquid chromatography using derivatization (Elliott et al., 1998). Comparable or lower limits have been reported for a variety of LC-MS methods, depending on the environmental matrix (Cha et al., 2005; Carlson and Mabury, 2006; Hao et al., 2006).

Matrix effects are commonly encountered in ELISA analysis of environmental samples (Mount and Failla, 1987; Aga et al., 2003). Therefore, for reliable ELISA analysis, it is important to understand the influence of the sample matrix. In general, matrix effects are greater as the complexity of the matrix increases. Matrix effects are more likely to be problematic in matrices where concentrations are expected to be low (e.g., water), which limits the ability to dilute the matrix. In this study, matrix effects were relatively small for water matrices; however, significant effects were noted in manure matrices (Table 1). Because monensin concentrations in manure commonly exceed 1 mg L^–1 (Mount et al., 1996), high dilution factors (>50) should not be problematic. Furthermore, samples with significant matrix effects can be corrected using Eq. 2 as long as the sample matrix effect has been well characterized at a given dilution level.

An additional benefit of the monensin ELISA analysis may be its ability to detect structurally similar factors, metabolites, and/or degradation products. Collectively, monensin factors B, C, and D account for up to 10% of total monensin in animal feed (Elanco Products Company, 1989). In addition, metabolites account for 40 to 90% of total excreted monensin (>90% of administered dosage) in manure depending on animal species (Donoho, 1984). The primary metabolic pathways are O-demethylation and oxidation (Donoho et al., 1978), which results in slight structural deviations (Fig. 1). More than 50 metabolites of monenesin have been detected in animal manure; however, individual metabolites typically do not account for more than 5% of total excreted monensin (Donoho et al., 1978). We speculate that slight structural variations in factors and metabolites exhibit cross-reactivity with the antibodies of the monensin ELISA. Higher ELISA compared with LC-MS concentrations were also obtained for a similar monensin ELISA, which was also attributed to metabolite detection (Crooks et al., 1998). Research-grade or commercially available factors or metabolites were not available for confirmation, and low abundance renders isolation difficult. Where desirable, the ELISA analysis could provide a measure of "total" monensin-related compounds. This ELISA could also be used in conjunction with LC-MS to determine the contribution of the parent compound versus structurally similar compounds. Future research is needed to quantitatively characterize the cross-reactivity of individual factors, metabolites, and degradation products.

	Conclusions

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results Discussion Conclusions REFERENCES

 
In this study, a commercially available ELISA for detecting monensin in animal feed extracts and edible tissues was evaluated for determination of monensin in environmental samples (water, soil, and manure). The monensin ELISA was highly sensitive, with limits of detection and quantification of 1.5 and 3.0 µg L^–1, respectively. Recovery of monensin in spiked water samples averaged 100%. Coefficients of variation were generally <20%. Matrix effects were minimal in water; however, significant matrix effects were observed for soil and manure samples. Cross-reactivity was not observed for other polyether ionophores antibiotics or antibiotics of different classes, such as tetracyclines, macrolides, and sulfonamides. There was strong evidence that the monensin ELISA was capable of detecting structurally similar factors and/or metabolites. Overall, the monensin ELISA proved to be a reliable analytical technique for rapid, low-cost detection of monensin in environmental samples.

	ACKNOWLEDGMENTS

 
This research was supported in part by funds from the USDA-NRI program (grant number 2003-35102-13519) the Rapid Agricultural Response Fund at the University of Minnesota. The authors thank Mr. Tom Krick of the Center for Mass Spectrometry and Proteomics at the University of Minnesota for his analytical assistance.

	NOTES

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