Detection and Quantification of Salinomycin in Chicken Liver Tissue

Chapter 9

Detection and Quantification of Salinomycin in Chicken Liver Tissue Comparison of Enzyme-Linked Immunosorbent Assay and High-Performance Liquid Chromatography Methods M a r k T. Muldoon, Marcel H . Elissalde, Ross C . Beier, and Larry H . Stanker Downloaded by FUDAN UNIV on March 9, 2017 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch009

1

Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 2881 F & Β Road, College Station, T X 77845-9594

Salinomycin is one of the most widely used coccidiostats in the U.S. poultry industry. A rapid and accurate analytical method for this drug would be valuable to producers and users as an effective management tool. We have developed an enzyme-linked immunosorbent assay (ELISA) coupled to a simple aqueous extraction procedure for the analysis of salinomycin in chicken liver, since it is the target tissue in this species. Recoveries from spiked chicken liver homogenates were quantitative in the rangefrom5.0 to 0.05 ppm. The ELISA was used to monitorfractionsfromreversed-phase high performance liquid chromatography (HPLC) in order to characterize non-specific matrix effects on the assay. Resultsfromthe analysis of incurred salinomycin residues in chicken livers obtained by both the ELISA method and a HPLC method were highly correlated (p < 0.0001). The ELISA method could detect 20 ng of drug in a 100 µL sample and has a limit

of quantification of 50 ppb in chicken liver tissue. The limit of quantification was lower with the ELISA method than with the HPLC method. The polyether ionophore antibiotics, produced by various species of Streptomyces, possess broad spectrum anticoccidial activities (7). Members of this class of compounds include salinomycin, monensin, narasin, and lasalocid A (Figure 1). The mode of action of the ionophores is attributed to their ability to form complexes with mono- and divalent cations disrupting cell membrane function (2). In the United States, salinomycin (SAL) is registered for use as a feed additive at concentrations of 44 to 66 ppm to control coccidiosis in broiler chickens. In 1990, it was the most widely used coccidiostat in agriculture (3). SAL toxicity in broilers can occur when 1

Corresponding Author This chapter not subject to U.S. copyright Published 1996 American Chemical Society Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

IMMUNOASSAYS FOR RESIDUE ANALYSIS

Downloaded by FUDAN UNIV on March 9, 2017 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch009

110

Lasalocid A Figure 1. Structures of various polyether ionophore antibiotics.

Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


9. MULDOONETAL.

ELISA and HPLC Detection of Salinomycin

111

birds are fed the drug above the recommended therapeutic levels. However, the therapeutic levels in broilers can be toxic to turkeys and horses (1). Because of its importance in poultry production, rapid and sensitive analytical methods for SAL in animal tissues and feeds would provide useful management tools (See Shimer et al., this volume). The ionophores are commonly used in the form of non-volatile sodium salts, and thus are not amenable to gas chromatographic analysis. In addition, they are not fluorescent and do not possess a useful chromophore. Therefore, they are not readily detected spectrophotometrically without prior derivatization. To circumvent these limitations, analytical methods developed for the detection of the ionophores have utilized either the compounds' biological activity (bioassays), or more commonly, employed derivatization procedures to convert the analyte to a chromophoric or fluorescent species which can be more readily measured. Table I summarizes several methods used for the detection of SAL (and in most cases the method also is used for other ionophores) in various sample matrices. The detection limit of each method is expressed as the amount detected (e.g., μg or ng), as well as the sample concentration. Early analytical methods employed bioautographic detection following thin-layer chromatography (TLC). These methods were very sensitive but extremely time consuming, requiring over 18 h for their completion. Vanillin has been used to convert various ionophores to chromophoric derivatives which absorb in the visible range. This was performed following either T L C or high performance liquid chromatography (HPLC). The advantage of the HPLC methods was the ability to perform a post-column derivatization step on-line. These methods were most commonly applied to the analysis of feed grains where high sensitivity is not critical and performed using a dilute sample extract, hence high sample detection limits are obtained. Useful chromophoric derivatives have been obtained via pre-column pyridinium dichromate oxidation resulting in UV-absorbing species. This method has been employed for the analysis of various animal tissues. Fluorescent derivatives using 9-anthrydiazomethane (ADAM) and l-(bromoacetyl)pyrene were made for the detection of various ionophores in animal tissue and feed grains, respectively. These pre-column derivatization procedures, although sensitive, require extensive sample purification prior to derivatization. Additional purification of the derivatized mixture was necessary prior to separation by HPLC and either U V or fluorescence detection. Recently, we reported the development of a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for S A L (15,16). Salinomycin was coupled to carrier proteins via the carboxylic acid moiety and sixteen monoclonal antibodies were produced which equally recognized both salinomycin and narasin. Neither monensin nor lasalocid A were recognized by the antibodies. Using a homologous indirect ELISA, less than 0.3 ng of SAL could be detected in a 100 uL sample. Preliminary results showed the assay to be suitable for analyzing buffer extracts of chicken livers spiked with SAL at concentrations from 1.25 to 5.0 ppm. The ELISA method was further optimized which lowered the detection limit of the assay to 20 pg SAL (Table I) and made it more suitable for residue monitoring (14). It was applied to the analysis of chicken liver tissues from an incurred residue study. These results were validated using conventional HPLC methodology. This paper summarizes these results and discusses further applications of the method.


112


Table L Analytical Methods for the Determination of Salinomycin in Various Sample Matrices.


Sample Matrix

Method Description

Detection Limit amount / sample concentration

Reference

Various edible rabbit tissues

Organic extraction, TLC, bioautographic detection.

0.5ng/10ppb

4

Chicken liver

Organic extraction, TLC, bioautographic detection

50ng/25ppb

5

Feed grains

Organic extraction, TLC, derivatization, visualization

1.5 μg / 3 ppm

6

Feed grains

Organic extraction, HPLC, postcolumn derivatization, spectrophotometric detection

20 ng / lppm

7

Feed grains


10 ng/0.25 ppm

8

Beef liver tissue

Organic extraction, purification, derivatization, purification, HPLC, fluorometric detection

30ng/150ppb

9

Various chicken tissues

Organic extraction, purification, derivatization, purification, HPLC, spectrophotometric detection

13 ng / 5 ppb

10

Feed grains


10ng / lppm

11

Human plasma

Organic extraction, purification, derivatization, HPLC, spectrophotometric detection

4 ng / 5 ppb

12

Feed grains

Organic extraction, purification, derivatization, purification, HPLC, fluorometric detection

not indicated / 25 ppm

13

Chicken liver


25 ng/100 ppb

14

Chicken liver

Aqueous extraction, immunochemical detection

20 pg/50ppb

14


9. MULDOON ET AL.


113


Methods Competitive Inhibition E L I S A . The ELISA procedure was adapted from the method reported by Elissalde et al. (15) and further modified by Muldoon et al. (14). Briefly, wells of microtiter plates were coated with B S A - S A L in 100 μL of coating buffer (carbonate, pH 9.6), incubated 18 h at 4 °C, and washed (0.05% (v/v) Tween 20). Wells were blocked with 200 μL of blocking buffer (0.5% (w/v) B S A in phosphate buffered saline containing 0.05% (v/v) Tween 20, pH 7.0 incubated 60 min at ambient room temperature, washed, and stored at -20 °C until used. For the ELISA, 100 uL of sample diluted in assay buffer (Tris, pH 7.75) was added to the microtiter plate well and mixed with purified anti-salinomycin monoclonal antibody (2 ng in 100 uL of assay buffer). The mixture was incubated at room temperature for 60 min and then the plate was washed. Horse radish peroxidase labelled goat anti-mouse IgG (100 uL of 1:500 dilution in assay buffer) was added to each well, incubated at room temperature for 60 min, and then the plate was washed. K-Blue enzyme substrate (ELISA Technologies, Lexington, K Y ) (100 uL) was added and plate optical density (OD) measurements (655 nm) were taken at 30 min. High Performance Liquid Chromatography. The HPLC method was adapted from previously described methods (5,77). Modifications and details of the method are described in detail elsewhere (14). Briefly, the HPLC method used a Dionex (Sunnyvale, CA) microbore system with a post-column reaction system, and a variable wavelength detector set at 520 nm. The column was a 5 μιη Supelcosil L C 18 (15 cm X 2.1 mm) from Supelco (Bellefonte, PA). Samples (25 μL) were injected onto the system using a Spectra-Physics (San Jose, CA) SP 8880 autosampler. The isocratic solvent system was 95% methanol/acetic acid (1% v/v, water). The gradient solvent system was 80% methanol/acetic acid (1% v/v, water), maintained for 1 min post injection, a gradient to 100% methanol reached at 4 min post injection and maintained until 18 min. Then, 80% methanol/acetic acid gradient was reached at 20 min post injection. The solvent flow rate was 0.25 mL/min for both solvent systems. The derivatization reagent consisted of 10% (w/v) vanillin in methanol containing 2% (v/v) sulfuric acid delivered at 0.5 mL/min. Aqueous Extraction of Salinomycin from Chicken Liver Tissue. Sixty gram portions of control chicken liver, obtained from a local grocery store, were homogenized using a tissue homogenizes Four grams of the homogenized liver was weighed into a 50 mL polypropylene centrifuge tube. Assay buffer (40 mL) was added and the sample was vortexed to suspend the homogenate. The sample was centrifuged and the supernatant was further diluted in assay buffer prior to ELISA analysis. Methanolic Extraction of Salinomycin from Chicken Liver Tissue. The extraction method was an adaptation of a previously described method used for the analysis of SAL in beef liver tissue (9). Ten grams of homogenized liver were weighed into a 50 mL polypropylene centrifuge tube and extracted twice with 25 mL


114


portions of 80% (v/v) methanol/water. The methanolic liver extract was backextracted with methylene chloride. The methylene chloride extract was evaporated to dryness, the residue redissolved in 1 mL of methanol, filtered, and analyzed by HPLC. Evaluation of the Aqueous Liver Extract on E L I S A . In order to determine the effects of the aqueous liver extract on the ELISA, SAL standard curves made in assay buffer and various dilutions of aqueous liver extracts (1:10, 1:50, and 1:100) were compared. Results were analyzed using either OD values or B/B -transformed data, where Β is the OD value of the sample and B is the OD value of the sample without competitor (assay buffer or the appropriate dilution of unspiked liver extract). IC50 values (concentration of inhibitor which produces a 50% decrease in signal of the no competitor control) were derived using a 4-parameter log-logistic curve fitting function (SOFTmax 2.01, Molecular Devices Corp., Menlo Park, CA) for each standard curve. 0


0

Analysis of Spiked and Non-Spiked Control Liver Extracts by H P L C - E L I S A . Methanolic extracts from 2 aliquots of non-spiked control liver homogenate were obtained as previously described and pooled. In addition, a solvent blank was obtained in which the entire extraction procedure was carried out with the omission of a liver sample. The pooled liver extract sample was split (2 χ 1 mL) and one was spiked to 10 ppm SAL. The 3 samples were analyzed by H P L C using the isocratic solvent system and post-column derivatization (vanillin) with eluent monitored at 520 nm. In addition, samples were injected onto the H P L C system under the same solvent conditions but without post-column derivatization. In this case, 100% methanol was pumped through the post-column reaction solvent pump (0.5 mL/min) and thirty 15 s fractions were collected by hand for each injection. The fractions were evaporated to dryness, reconstituted in assay buffer and analyzed by ELISA. During these runs, H P L C eluant was monitored at 220 nm. Comparison o f E L I S A and H P L C Methods for the Determination o f Salinomycin in Spiked Chicken Liver Tissue. Control liver tissue was homogenized and aliquots (4 g for ELISA; 10 g for HPLC) were spiked with various amounts of SAL to give tissue concentrations of 5.0, 1.0, 0.5, 0.25, 0.1, 0.05, and 0.0 (no SAL) ppm. The samples were extracted and analyzed by either the ELISA or H P L C methods as described above. For the ELISA analysis, several dilutions of each sample extract were made on the plate. Raw OD values were transformed to B / B values (as described above). Concentrations of S A L in the extracts were calculated based on the standard curve analyzed on each plate using the 4-parameter log-logistic curve fitting function in SOFTmax. The lowest extract dilution which resulted in a B / B value in the linear, quantitative region of the standard curve ( B / B = 0.70 to 0.20) was used for determining SAL in the sample. Three sets of samples were prepared and analyzed immediately in triplicate wells of a microtiter plate. For the HPLC analysis, three sets of spiked samples were prepared one set at a time and analyzed immediately. A l l samples were analyzed in duplicate. The isocratic H P L C solvent system was used in this study. Sample concentrations were determined 0

0

0


9. MULDOONETAL.

115



based on S A L standards ranging from 0.41 to 100 ppm in methanol analyzed concurrently with the sample extracts. Comparison o f E L I S A and H P L C Methods for the Determination o f Salinomycin in Chicken Liver Tissues from an Incurred Residue Study. Livers were obtained from chickens which were fed, from 6 weeks of age, either a control diet (no SAL), a diet containing the recommended therapeutic dose of salinomycin (66 ppm), or a diet containing 2 times the therapeutic dose of salinomycin (132 ppm). The birds were maintained on these diets for 2 weeks at which time 5 birds from each treatment group (No Dose, 66 ppm, or 132 ppm) were killed and their livers removed and frozen at -70 °C until processed. The remaining 10 birds in each of the 3 treatment groups were immediately given control feed and sacrificed 18 and 72 hours later (5 birds/group/time period). Livers were thawed at room temperature, homogenized, and stored at 4 °C prior to analysis (within 18 h). Livers were extracted and analyzed by either ELISA or H P L C as described above. For the ELISA, the OD values of the aqueous control liver extract was used for calculating B for data transformation of the samples. For each set of samples (one bird from each treatment group per withdrawal time (9 samples)), the ELISA analysis and extraction of SAL into methylene chloride for subsequent HPLC analysis were performed on the same day. On the next day, samples were further processed and analyzed by HPLC using the gradient solvent system. 0

Results and Discussion E L I S A Optimization. Several modifications were made in the ELISA procedure as reported by Elissalde et al. (15) which resulted in improvements in sensitivity and precision. These are described in detail in Muldoon et al. (14). Most importantly was the coating conditions used for immobilizing the ELISA antigen and the use of purified anti-salinomycin antibody (derived from ascites fluid). In the previous method, antigen was dissolved in distilled water and plates were coated overnight at 37 °C. In the current method we dissolved the coating antigen in carbonate buffer (pH 9.6) and plates were coated overnight at 4 °C. This had the effect of increasing the OD values of the no competitor control samples from a reading of 0.6 units using the previous coating method to 1.3 units using the current coating method. In addition, there was an improvement in well to well variation to below 10%. A l l modifications considered, there was an improvement in assay sensitivity from an IC50 of 3.30 ng/mL (interassay C V = 30.3%) for the previous method to an I C of 0.52 ng/mL (interassay C V = 20.7%) for the current procedure. 5 0

Evaluation of the Aqueous Liver Extract on E L I S A . Salinomycin standard curves prepared in assay buffer and in various dilutions of the aqueous liver extract were evaluated in order to detect any matrix effects on the assay. Maximum optical densities of 1.38, 0.85, 0.72, and 0.66 units were observed when the ELISA was performed in assay buffer or aqueous liver extracts diluted 1:100, 1:50, and 1:10 in assay buffer, respectively. Due to these effects, it was necessary to transform OD


116


measurements to B / B values where B was the mean OD measurement of the no competitor control in buffer for the standards, and the appropriate dilution of control liver extract for the samples. Figure 2 shows the standard curves made in assay buffer and in the various dilutions of aqueous liver extract following B/B -transformation of the OD measurements. The standard curves prepared in buffer and the 1:100 dilution of aqueous liver extract overlapped. The IC50 value obtained from the standard curve made in assay buffer (0.63 ng/mL, C V = 5.65%) was not different from the standard curve made in liver extract diluted 1:100 (0.98 ng/mL, C V = 7.52%) (p > 0.05). Therefore, it was possible to use a B/B -transformed standard curve prepared in buffer for extrapolating B/B -transformed sample data, with the requirement that a valid matrix control be used for the sample B and the samples diluted to a minimum of 1:100. 0

0

0

0

0


0

Analysis of Spiked and Non-Spiked Control Liver Extracts by H P L C - E L I S A . After observing the apparent non-specific interference of control liver extract on the ELISA, we were interested in further characterizing these effects by separating the sample by HPLC and analyzing column fractions by ELISA since this detection system is over 1000 times more sensitive than the spectrophotometric detector (see Table I). Aliquots of control liver extract (spiked and non-spiked), a standard solution containing 10 ppm SAL, and a solvent extract blank were injected onto the H P L C system without post-column derivatization. Column eluent fractions were collected, evaporated, reconstituted in assay buffer, and analyzed by ELISA. Figure 3 shows the HPLC-ELISA chromatogram from the analysis of unspiked control liver extract, control liver extract spiked to 10 ppm SAL (tissue concentration equivalent of 1 ppm), and the 10 ppm SAL standard. No inhibition was observed in any of the fractions from the solvent extract control blank (data not shown). The total SAL equivalents (ng) recovered in the unspiked control liver, the control liver extract spiked to 10 ppm SAL, and the 10 ppm SAL standard were 5.26, 222.3, and 204.8 ng/25 uL, respectively. The amounts which eluted at the same retention time as SAL (as determined by the 10 ppm standard) were 0.83, 217.5, and 204.8 ng/25 uL, respectively. For the control liver extract, this corresponded to a tissue concentration of 3.32 ppb SAL. Most of the inhibition observed with the ELISA in this sample (84.2%) was associated with fractions collected between 3 and 4.5 min and was not attributed to S A L (6 to 8 min). In addition, the U V chromatograms (220 nm) for both the unspiked and spiked extracts were identical (data not shown); each showed multiple peaks with a large, broad peak from 5 to 7 min, where SAL eluted. The U V chromatogram (220 nm) for the 10 ppm SAL standard gave no peaks which was not surprising since post-column derivatization was not performed. It is possible that the inhibition observed with the unspiked control liver extract in the fraction near the retention time of SAL is in fact residual drug, however, since it was associated with high non-specific U V absorbance, it may be non-specific inhibition. Nevertheless, the use of a sample extract dilution of 1:100 for ELISA analysis alleviates the influence of such low background levels of interference on the ELISA measurement. In addition, since the amount is over 10 times less than the lower limit of detection of the H P L C method (approximately 50 ppb) it should not influence the results using this method.


9. MULDOON ET AL.


117

••— Buffer

1.0

• · — Extract 1:100 a—Extract 1:10

0.8

0.6

O CD m

0.4


0.2

0.0 0.001

0.01

0.1

Salinomycin Concentration, ppb

Figure 2. Salinomycin ELISA standard curves made in assay buffer and aqueous liver extract diluted in assay buffer after B / B transformation of OD readings. Mean IC50 values derived from standard curves made in either assay buffer or liver extracts diluted 1:10, 1:50, or 1:100 in assay buffer were 0.63, 0.98, 1.00, and 1.25 ng/mL, respectively. Mean IC50 values derived from standard curves made in liver extract diluted 1:10 and 1:50 were significantly different from the value obtained in assay buffer alone (p > 0.05). (Reprinted with permission from ref. 14). 0

120

n

100-

S c ω >

8 0

60

40

20-

- Control Liver Extract - Control Liver Extract containing 10 ppm SAL -10 ppm SAL Standard

—1— 12

—1— 14

16

Retention Time, minutes

Figure 3. HPLC-ELISA chromatograms of 10 ppm SAL standard, unspiked, and spiked (10 ppm SAL) control chicken liver extracts.


118


Comparison of E L I S A and H P L C Methods for the Determination of Salinomycin in Spiked Chicken Liver Tissue. Chicken liver tissue homogenate was spiked with various levels of SAL and analyzed by ELISA and HPLC. The results from this study are shown in Figure 4. Linear regression showed that for both the ELISA and the HPLC methods, the amount detected was highly correlated to the amount added ( R = 0.999, ρ < 0.0001). The ELISA method was more accurate than the H P L C method, particularly at the lower tissue concentrations. In addition, mean recoveries for the ELISA method were quantitative over the entire range tested. This was not the case with the HPLC method which showed a large deviation from linearity below 100 ppb. Variability between triplicate extraction and analyses by ELISA ( C V = 19.9%) was higher than when the HPLC method was used ( C V = 4.11%). The average C V observed for individual sample determinations was 8.4% (n = 3) for the ELISA, and 4.13% (n = 2) for the H P L C method. The lower limit of quantification by the ELISA method was approximately 50 ppb S A L in liver tissue. Figure 2 shows that the analysis of a 1:100 dilution of aqueous liver extract spiked at a level corresponding to 50 ppb SAL in liver (0.5 ppb) gave B / B values which were within the linear, quantitative range of the assay ( B values between 0.7 and 0.2). Acceptable recoveries (83%) were obtained from control liver samples spiked at this level and analyzed by ELISA. The lower limit of quantification for the HPLC method, defined as 10 times the variance (10σ) (77), was also approximately 50 ppb. However, as shown in Figure 4, there was a large deviation from linearity below 100 ppb resulting in an over estimation of analyte using this method. This effect limited the accuracy of the HPLC method to levels at or above 100 ppb. Therefore, we report the limit of quantification for the HPLC method as 100 ppb. This limit of quantification is similiar to that reported for other instrumental methods developed for the analysis of SAL in animal tissues using either pre- or post- column derivatization (see Table I).


2

0

0

Determination of Salinomycin in Chicken Liver Tissues from the Incurred Residue Study. Table II shows the results from the incurred residue study. Results from the analysis of incurred residue samples were highly correlated (p < 0.0001). When compared to HPLC results (all 45 samples) only 1 false negative and 2 false positives were produced by ELISA. However, these occurred near the limit of quantification for the H P L C method (100 ppb) and also could be interpreted as being 1 false positive and 2 two false negatives by HPLC. Nevertheless, these results indicated that the ELISA method is a reliable screening tool for SAL in chicken liver tissue. There was good agreement between the two methods in the samples where the S A L concentrations obtained by the two methods were above the limits of quantification for the two methods. Results from bird numbers 2 and 3 in the 132 ppm, 2 h withdrawal time group were exceptions, and the concentration estimates by ELISA were underestimated for bird number 2 and overestimated for bird number 3. Inconsistencies such as these can be caused by sample matrix effects which can occur with both methods.


9. MULDOONETAL.


119

Table Π. ELISA and HPLC Results from the Analysis of Salinomycin in Chicken Liver Tissues from an Incurred Residue Study.


Treatment

Bird No.

ELISA Concentration, ppb Ohr

No Dose

66 ppm

132 ppm

1 2 3 A 5

18 hr

72 hr

-

H P L C Concentration, ppb Ohr

18 hr

-

-

72 hr

_

1 2 3 4 5

101.2 459.0 153.0 146.4 155.3

1 2 3 4 5

317.9 181.6 688.0 315.3 251.5

-

-

-

-

-

-

-

339.2

-

-

_

-

-

93.8 168.1 278.0 364.4 157.6 245.5 238.6

_

111.6

-

a

-

Below the limits of quantitation: 50 ppb for ELISA; 100 ppb for HPLC. E L I S A and H P L C results were highly correlated, ρ < 0.0001. (Reproduced with permission from reference 14. Copyright 1995 American Chemical Society).



120


0.01 H 0.01

0.1

1

•

• • •

Salinomycin Spike Level, ppm

Figure 4. Results from HPLC and ELISA analysis of salinomycin in spiked chicken liver homogenates. (Reprinted with permission from ref. 14).

Both methods showed that S A L residues were present immediately following withdrawal from medicated feed and are undetectable after 18 h. These results are similiar to the results reported by Atef et al. (18). It is important to note that either of the methods used alone would have provided the same pharmacokinetic data. Conclusions Most current analytical methods for the determination of SAL require organic solvent extraction followed by sample cleanup, chromatography, and pre- or post-column derivatization. These procedures are laborious and this limits the number of samples that can be effectively processed. We have developed a procedure which uses an aqueous buffer extraction coupled to an ELISA for the analysis of SAL in chicken liver tissue. The method was more sensitive and accurate than a HPLC method. ELISA and HPLC results from the analysis of incurred residue liver samples showed a rapid dissappearance of SAL from the tissue. The advantages of the ELISA method were reduced organic solvent use and increased sample throughput which should save time and expense in residue monitoring. The ELISA was coupled to HPLC as a sensitive detection system. This alleviated the need to derivatize the drug prior to detection. The HPLC-ELISA system was used to characterize non-specific matrix effects, but in the future, it should provide a general analytical strategy in situations where an ultra-sensitive detection system is required. This strategy is particularly interesting for the analysis


9. MULDOONETAL.


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of compounds which otherwise require derivatization for detection and ones in which the required derivatization is particularly difficult. Further work will focus on utilizing this hybrid technology in other applications.


Literature Cited 1. McDougald, L. R.; Roberson, E. L. In Veterinary Pharmacology and Therapeutics; Booth, Ν. H., McDonald, L. E., eds.; Iowa State University Press: Ames, IA, 1988; pp 950-968. 2. Pressman, B. C.; Fahim, M. Ann Rev. Pharmacol.Toxicol.1982, 71, 480-484. 3. SRI International. Chemical Economics Handbook; Dialog Information Services: Menlo Park, CA, 1992, File 359; CEH Accession 201.8002B-2. 4. Heil, K.; Peter, K.; Cieleszky, V. J. Agric. Food Chem. 1984, 32, 997-998. 5. Dimenna, G. P.; Walker, B. E.; Turnbull, L. B.; Wright, G. J. J. Agric. Food Chem. 1986, 34, 472-474. 6. Owles, P. J. Analyst 1984, 109, 1331-1333. 7. Goras, J. T.; LaCourse, W. R. J. Assoc. Off. Anal. Chem. 1984, 67, 701-706. 8. Blanchflower, W. J.; Rice, D. Α.; Hamilton, J. T. G. Analyst 1985, 110, 12831287 9. Martinez, E. E.; Shimoda, W. J. Assoc. Off.Anal.Chem. 1986, 69, 637-641. 10. Dimenna, G. P., Lyon, F. S.; Thompson, F. M.; Creegan, J. Α.; Wright, G. J. J. Agric. Food Chem. 1989, 37, 668-676. 11. Lapointe, M. R.; Cohen, H. J. Assoc. Off.Anal.Chem. 1988, 71, 480-484. 12. Karnes, H. T.; Wei, A. T.; Dimenna, G. P. J. Pharm.Biomed.Anal. 1993, 11, 823-827. 13. Asukabe, H.; Murata, H.; Harada, K.; Suzuki, M.; Oka, H.; Ikai, Y. J. Agric. Food Chem. 1994, 42, 112-117. 14. Muldoon, M. T.; Elissalde, M. E.; Beier, R. C.; Stanker, L. H. J. Agric. Food Chem. 1995, 43, 1745-1750. 15. Elissalde, M. H.; Beier, R. C.; Rowe, L. D.; Stanker, L. H. J. Agric. Food Chem. 1993, 41, 2167-2171. 16. Stanker, L. H.; Elissalde, M. H.; Rowe, L. D.; Beier, R. C.; Nasr, M. I. A. Food Agric. Immunol. 1994, 6, 45-54. 17. Keith, L. H. In Environmental Sampling and Analysis. A Practical Guide; Lewis: Chelsea, MI, 1991. 18. Atef, M; Ramadan, Α.; Youssef, S. A. H.; Abo El-Sooud, K. Res. Vet. Sci. 1993, 54, 179-183. RECEIVED October 6, 1995


Detection and Quantification of Salinomycin in Chicken Liver Tissue

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