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1993 Sulfadimethoxine Analysis in Channel Catfish: A Model Drug Residue Monitoring Program for Aquaculture. Calvin Charles Walker Louisiana State University and Agricultural & Mechanical College

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Sulfadimethoxine analysis in channel catfish: A model drug residue monitoring program for aquaculture

Walker, Calvin Charles, Ph.D.

The Louisiana State University and Agricultural and Mechanical Col., 1993

UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SULFADIMETHOXINE ANALYSIS IN CHANNEL CATFISH: A MODEL DRUG RESIDUE MONITORING PROGRAM FOR AQUACULTURE

A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical college in partial fulfillment of the requirements for the degree of Doctor of Philosophy in

The Interdepartmental Program in Veterinary Medical Sciences

through the Department of Veterinary Physiology, Pharmacology and Toxicology

by Calvin Charles Walker B.S., Mississippi State University, 1973 D.V.M., Auburn University, 1978 December, 1993

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dedicated to

Rosanne, Phillip, and James

ii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS

I would like to express my appreciation to the

graduate faculty members of my doctoral advisory committee for their consultations and guidance:

Steven A. Barker, Ph.D., Chair Jay C. Means, Ph.D. Samuel P. Meyers, Ph.D. Steven S. Nicholson, D.V.M. David H. Swenson, Ph.D. Ronald L. Thune, Ph.D.

I would also like to thank Dr. Gary L. Ter Haar and the Ethyl Corporation for providing financial support for

the Ethyl Corporation Graduate Fellowship in Toxicology. Special gratitude is expressed to Dr. Steven Barker for freely sharing his knowledge of analytical toxicology and for providing the direction and support needed during

this course of study.

Finally, I am thankful for the support and sacrifices of my wife, Rosanne, and my children, Phillip and James,

during this period of study.

iii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS

DEDICATION...... ii ACKNOWLEDGEMENTS ...... iii

LIST OF T A B L E S ...... vii

LIST OF FIGURES...... ix

ABSTRACT ...... xi CHAPTER 1 GENERAL INTRODUCTION ...... 1 BACKGROUND ...... 1 RESIDUE ANALYSIS ...... 2 PURPOSE ...... 9 REFERENCES ...... 10 2 REVIEW OF LITERATURE ...... 13

EXTRACTION AND ANALYSIS OF DRUGS IN AQUATIC S P E C I E S ...... 13 INTRODUCTION ...... 13 DRUG RESIDUE PROBLEMS IN AQUACULTURE . 17 METHODS OF DRUG RESIDUE ANALYSIS IN AQUATIC SPECIES ...... 21 MICROBIAL INHIBITION TESTS...... 28 SWAB TEST ON P R E M I S E S ...... 30 CALF ANTIBIOTIC AND SULFA TEST . . . 31 LIVE ANIMAL SWAB T E S T ...... 32 FAST ANTIBIOTIC SCREEN TEST .... 33 DELVOTEST P ...... 34 BRILLIANT BLACK REDUCTION TEST . . . 35 BIOAUTOGRAPHY ...... 35 COLORIMETRIC METHODS ...... 36 RECEPTOR BASED ASSAYS ...... 37 BACTERIAL CELL RECEPTOR ASSAY . . . 38 RADIO-IMMUNOASSAY...... 41 ENZYME IMMUNOASSAY...... 42 CHROMATOGRAPHIC ANALYSIS ...... 51 THIN LAYER CHROMATOGRAPHY ...... 51 CLASSICAL THIN LAYER CHROMATOGRAPHY...... 5 1 HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY ...... 55 TLC-BIOAUTOGRAPHY ...... 57 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ...... 58 GAS CHROMATOGRAPHY...... 67

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MASS SPECTROMETRY...... 69 OTHER RESIDUE DETECTION METHODS .... 71 METHODS OF ISOLATION ...... 72 EVOLVING METHODS OF RESIDUE ANALYSIS .. . 79 SOLID PHASE EXTRACTION ...... 80 IMMUNOAFFINITY ...... 81 SUPERCRITICAL FLUID EXTRACTION .... 85 MATRIX SOLID PHASE DISPERSION ...... 88 MSPD APPLIED TO AQUATIC RESOURCES .. . 90 DISCUSSION ...... 91 REFERENCES ...... 94 3 EXTRACTION AND ENZYME IMMUNOASSAY OF SULFADIMETHOXINE RESIDUES IN CHANNEL CATFISH (Ictalurus punctatus) MUSCLE ...... 121 INTRODUCTION ...... 121 EXPERIMENTAL ...... 125 REAGENTS AND EXPENDABLE MATERIALS . . 125 SAMPLES ...... 126 EXTRACTION PROCEDURE ...... 127 ELISA PROCEDURES ...... 128 RESULT DETERMINATION ...... 131 RESULTS AND DISCUSSION ...... 133 PRECISION...... 133 TEST PERFORMANCE ...... 140 REFERENCES ...... 155 4 EXTRACTION AND LIQUID CHROMATOGRAPHIC ANALYSIS OF SULFADIMETHOXINE AND 4-N-ACETYLSULFADIMETHOXINE RESIDUES IN CHANNEL CATFISH (Ictalurus punctatus) MUSCLE AND PLASMA ...... 160 INTRODUCTION ...... 160 EXPERIMENTAL ...... 163 REAGENTS AND EXPENDABLE MATERIALS . . 163 EXTRACTION PROCEDURE ...... 166 LIQUID CHROMATOGRAPHIC ANALYSIS . . . 169 DATA AN A L Y S I S ...... 170 RESULTS AND DISCUSSION ...... 173 CHROMATOGRAPHIC SYSTEM ...... 173 LINEARITY OF RESPONSE ...... 174 LIMITS OF DETECTION AND QUANTITATION 184

INTRA-ASSAY VARIABILITY...... 184 INTER-ASSAY VARIABILITY...... 184 RECOVERY OF METHOD ...... 186 REFERENCES ...... 195

V

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5 PLASMA/MUSCLE RATIOS OF SULFADIMETHOXINE RESIDUES IN CHANNEL CATFISH (Ictalurus punctatus) 199 INTRODUCTION ...... 199 EXPERIMENTAL ...... 204 REAGENTS AND EXPENDABLE MATERIALS . . 204 EXPERIMENTAL ANIMALS, DRUG ADMINISTRATION, AND SAMPLING.... 206 EXTRACTION PROCEDURE ...... 207 LIQUID CHROMATOGRAPHIC ANALYSIS .. . 208 DATA A N A L Y S I S ...... 209 RESULTS AND DISCUSSION ...... 210 REFERENCES ...... 225

6 SUMMARY AND CONCLUSIONS ...... 229 FIELD ANALYSIS OF RESIDUES ...... 230 LABORATORY ANALYSIS OF RESIDUES ...... 233 CONCLUSIONS...... 236 APPENDIXES APPENDIX A: OPTICAL DENSITY VALUES OBTAINED WITH THE IDS SULFADIMETHOXINE ONE-STEP ELISA ...... 239 APPENDIX B: OPTICAL DENSITY VALUES OBTAINED WITH THE SIGNAL SULFAMETHAZINEEL ISA...... 242 VITA ...... 245

vi

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LEST OF TABLES

2.1 REPRESENTATIVE EXTRACTION AND LIQUID CHROMATOGRAPHIC METHODS FOR DRUGS USED IN AQUACULTURE...... 59

2.2 COMPARATIVE SOLVENT USE AND WASTE PRODUCTION OF REPRESENTATIVE EXTRACTION METHODS FOR DRUGS USED IN AQUACULTURE...... 74 3.1 INTRA-ASSAY VARIATION (CV) ...... 135

3.2 THE % AGREEMENT BETWEEN OBSERVERS FOR VISUALLY EVALUATED TESTS USING MSPD EXTRACTEDSAMPLES . 137

3.3 INTER-ASSAY VARIATION (CV) ...... 139

3.4 RESULTS & PERFORMANCE FROM THE ANALYSIS OF MSPD EXTRACTED CATFISH MUSCLE ...... 142

3.5 OVERALL PERFORMANCE FROM THE ANALYSIS OF MSPD EXTRACTED CATFISH MUSCLE ...... 143 3.6 CROSS-REACTIVITY WITH E L I S A S ...... 147 3.7 OVERALL PERFORMANCE COMPARISONS OF SINGLE/DUPLICATE WELLS AND OF OPTICAL DENSITY/ % BLANK VALUES FROM THE ANALYSIS OF MSPD EXTRACTED CATFISH MUSCLE ...... 150

3.8 COMPARISONS OF INDIVIDUAL CONCENTRATION RESULTS AND PERFORMANCES FROM THE ANALYSIS OF MSPD EXTRACTED CATFISH MUSCLE USING SINGLE/DUPLICATE WELLS AND OPTICAL DENSITY/% BLANK VALUES . . . 151

4.1 INTRA-ASSAY VARIATION (CV) ...... 185

4.2 INTER-ASSAY VARIATION (CV) ...... 187

5.1 SULFADIMETHOXINE RESIDUES AND PLASMA:MUSCLE RATIOS - 6 HOURS P O S T - D O S E ...... 211

5.2 SULFADIMETHOXINE RESIDUES AND PLASMA:MUSCLE RATIOS - 12 HOURS POST-DOSE...... 212

5.3 SULFADIMETHOXINE RESIDUES AND PLASMA:MUSCLE RATIOS - 24 HOURS POST-DOSE...... 213

5.4 SULFADIMETHOXINE RESIDUES AND PLASMA:MUSCLE RATIOS - 48 HOURS POST-DOSE...... 214

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.5 SULFADIMETHOXINE RESIDUES AND PLASMA:MUSCLE RATIOS - 72 HOURS POST-DOSE...... 215

5.6 SULFADIMETHOXINE RESIDUES AND PLASMA:MUSCLE RATIOS - 96 HOURS POST-DOSE...... 216

Vlll

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3.1 2 X 2 CELL MATRIX ILLUSTRATING THE FOUR POSSIBLE TYPES OF TEST RESULTS AND FORMULAS FOR CALCULATING PERFORMANCE PARAMETERS ...... 141

4.1 LIQUID CHROMATOGRAM OF MSPD EXTRACT FROM CHANNEL CATFISH MUSCLE CONTAINING NO D R U G ...... 175 4.2 LIQUID CHROMATOGRAM OF MSPD EXTRACT FROM CHANNEL CATFISH MUSCLE FORTIFIED WITH SMX AT 1000 ng/g, SDM AT 1000 ng/g, AND N-ACETYL SDM AT 100 ng/g ...... 176

4.3 LIQUID CHROMATOGRAM OF MSPD EXTRACT OF CHANNEL CATFISH MUSCLE FROM ROMET® FED CATFISH; CALCULATED CONCENTRATIONS OF 507 ng SDM/g AND 55 ng N-ACETYL SDM/g; FORTIFIED WITH SMX AS INTERNAL STANDARD AT 1000 ng/g ...... 177

4.4 LIQUID CHROMATOGRAM f'F MSPD EXTRACT FROM CHANNEL CATFISH PLASMA CONTAINING NO D R U G ...... 178 4.5 LIQUID CHROMATOGRAM OF MSPD EXTRACT FROM CHANNEL CATFISH PLASMA FORTIFIED WITH SMX AT 1000 ng/ml, AND N-ACETYL SDM AND SDM AT 400 ng/ml EACH . . 179 4.6 LIQUID CHROMATOGRAM OF MSPD EXTRACT OF CHANNEL CATFISH PLASMA FROM ROMET® FED CATFISH; CALCULATED CONCENTRATIONS OF 1158 ng SDM/ml AND 379 ng N-ACETYL SDM/rnl; FORTIFIED WITH SMX AS INTERNAL STANDARD AT 1000 ng/ml ...... 180 4.7 UV ABSORBANCE SPECTRA OF SDM (- ) AND N-ACETYL SDM (---) STANDARDS IN MOBILE PHASE. LIQUID CHROMATOGRAPHIC ANALYSIS WITH DIODE ARRAY D E T E CTION...... 181 4.8 UV ABSORBANCE SPECTRA OF SDM (- ) AND N-ACETYL SDM (---) IN MSPD EXTRACT OF MUSCLE FROM ROMET® FED CHANNEL CATFISH. LIQUID CHROMATOGRAPHIC ANALYSIS WITH DIODE ARRAY DETECTION...... 182

4.9 UV ABSORBANCE SPECTRA OF SDM (--- ) AND N-ACETYL SDM (---) IN MSPD EXTRACT OF PLASMA FROM ROMET® FED CHANNEL CATFISH. LIQUID CHROMATOGRAPHIC ANALYSIS WITH DIODE ARRAY DETECTION...... 183

IX

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.1 PLASMA:MUSCLE CORRELATION OF TOTAL SDM RESIDUES FROM INDIVIDUAL CHANNEL CATFISH FOLLOWING ADMINISTRATION OF ROMET® MEDICATED FEED. LIQUID CHROMATOGRAPHIC ANALYSIS WITH DIODE ARRAY DETECTION ...... 2 2 0

5.2 PLASMA:MUSCLE CORRELATION OF TOTAL SDM RESIDUES FROM INDIVIDUAL CHANNEL CATFISH FOLLOWING ADMINISTRATION OF ROMET® MEDICATED FEED. MUSCLE CONCENTRATION <2000 ng/g ...... 221 5.3 TOTAL SDM RESIDUE DEPLETION IN CHANNEL CATFISH FOLLOWING ADMINISTRATION OF ROMET® MEDICATED FEED WATER TEMPERATURE 27°C. MEAN VALUES (n=12) AT EACH SAMPLING PERIOD. 2° POLYNOMIAL CURVE FIT . 223 5.4 TOTAL SDM RESIDUE DEPLETION IN CHANNEL CATFISH FOLLOWING ADMINISTRATION OF ROMET® MEDICATED FEED WATER TEMPERATURE 27°C. MEAN VALUES (n=12) AT EACH SAMPLING PERIOD. SEMI-LOG PLOT. LINEAR CURVE FIT...... 224

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT

Analytical methods for extraction, detection, and

quantitation of sulfadimethoxine (SDM) residues in channel catfish muscle and plasma were developed for a tiered

residue monitoring program. The plasma and muscle

concentrations of SDM and its primary metabolite in.

channel catfish, 4-N-acetylsulfadimethoxine (N-acetyl SDM), were determined in SDM medicated fish. Drug extraction methods using matrix solid phase dispersion

(MSPD), drug screening methods using enzyme-linked

immunoassay (ELISA), and drug residue quantitation methods

using high pressure liquid chromatography (LC) are presented. All methods were developed for the simultaneous extraction and analysis of the parent

compound and metabolite. MSPD extracts of muscle or

plasma were reconstituted in mobile phase for LC analysis or in a buffer suitable for use in an ELISA system. The

performances of four commercially available ELISAs as

screening assays were evaluated using MSPD derived extracts of catfish muscle fortified at concentrations of 0, 25, 50, 100, 250 ng/g. Overall sensitivities of 98-

100% and specificities of 71-94% were obtained for the

four ELISAs examined. Cross-reactivity with a number of compounds was examined. All four assays reacted equally well with SDM or N-acetyl SDM. Performance results

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. indicated that MSPD extracts can be used in these

immunoassays for screening catfish muscle for violative SDM residue levels (>100 ng parent and metabolite/g).

Methods for the LC analysis of MSPD derived muscle and plasma extracts are presented and evaluated. Results of

the LC analysis of MSPD extracts of catfish muscle and plasma indicated that these extracts may be used for

quantitation of SDM and N-acetyl SDM residues at

concentrations of 50-1000 PPB. Drug concentration ratios were determined for total SDM residues in channel catfish

plasma and muscle. Fish maintained in 27°C water were

dosed once daily for five days and sampled at intervals of

6, 12, 24, 48, 72, 96 hours after the last dose. The mean plasmaimuscle total SDM residue ratio was 1.8:1. The 95%

confidence interval for individual fish was (1.2:1,2.4:1).

Such a tissue concentration ratio enables one to use a rapid screening method, such as an immunoassay, with a reference body fluid as an indicator of the presence of

violative drug residues in an edible target tissue.

xii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1

GENERAL INTRODUCTION

BACKGROUND

Aquaculture is a large and rapidly growing industry. Seafood consumption has shown tremendous growth in the last 10 years and is expected to continue. This increase in seafood consumption and a decline in landings from wild

fishery stocks has led to an increased demand for aquaculture products [1.1]. Accompanying the increase in

seafood consumption is a heightened concern regarding the safety of this food source from pathogenic bacteria and

viruses, parasites, biotoxins, and chemical residue hazards [1.2-1.4]. However, with the exception of certain shellfish, continuous and systematic seafood inspection is not required in the United States [1.2].

Regulatory authority for seafood safety in the United States is divided among a number of local, state, and

federal agencies. The U.S. National Marine Fisheries

Service offers a voluntary federal seafood inspection

program. However, this voluntary program is primarily a product grading system and plant sanitation inspection service with only a quite limited number of food safety

1

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analyses performed annually. The U.S. Food and Drug Administration has the regulatory authority to require the inspection of any domestic or imported seafood product but such inspection is at the discretion of the agency and is not performed on a systematic or continuous basis [1.2].

Further, analytical methods for only two drugs - chloramphenicol in shrimp and oxolinic acid in salmon - have been validated for regulatory use by these agencies for monitoring drug residues in aquatic food resources [1.5-1.8]. Because of the lack of validated analytical methods for enforcement purposes the FDA is in the process of developing methods for a number of veterinary drugs used in a variety of cultured aquatic species [1.9]. The

growth and increased visibility of the seafood and aquaculture industries and a public perception that the nations seafood supply is not adequately inspected have

led to calls for a mandatory federal seafood inspection

program similar to the ones that now exist for red meat and poultry [1.2-1.3].

RESIDUE ANALYSIS

Pending U.S. federal legislation concerning establishment of such an inspection program for seafood

will require analytical methods that are rapid, accurate,

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sensitive, and specific for determination of chemical residues in food fish [1.10]. Traditional drug residue

screening methods, such as microbial inhibition tests,

thin layer chromatography, and colorimetric assays, as

presently used in poultry and red meat inspection, lack one or more of these characteristics. There is also a

lack of residue screening methods suitable for use in a field setting by an aquaculturist for the pre-harvest detection of violative drug residues, a practice which

would greatly reduce violations and consequent producer

losses from condemnation. A tiered approach to residue monitoring utilizes analytical techniques of varying sensitivity, specificity,

and precision for chemical residue detection, quantitation, and identification. Such methods may be

generally classified as 1) rapid initial screening assays such as microbiological inhibition tests (MIT), thin layer

chromatography, or receptor-based assays, 2) quantitative

methods such as high performance liquid chromatography (LC), gas-liquid chromatography (GLC), high performance

thin layer chromatography, and some MITs, and 3) residue

identity confirmatory methods such as mass spectrometry

and infra-red spectrometry [1.11]. All require varying degrees of sample extraction and manipulation to remove

target analytes. With recent improvements in analytical

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instrumentation and the introduction of new rapid

screening and determinative methods, a major limiting

factor in the efficient utilization of such assays has

been identified as the process for the extraction and isolation of the analyte(s) from tissues.

The classical methods of extraction that have

traditionally been employed for analyte isolation from various tissue matrices involve mechanical homogenization

and repeated sample manipulations. Such techniques are

labor and solvent-use intensive and limit the number of samples that can be practically analyzed in a day. Newer methods of analyte isolation that eliminate many of the

disadvantages of traditional extraction methods are needed

if one is to fully benefit from recent advances in the

determinative aspects of residue analysis. Isolation techniques that have recently become available include

supercritical fluid extraction, solid phase extraction,

and matrix solid phase dispersion (MSPD). Matrix solid phase dispersion, in particular, offers a technique for

greatly decreasing extraction time, solvent volumes, and tissue mass when compared to traditional methods [1.12].

The relative advantages of these methods are reviewed in Chapter 2.

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Sulfonamides are a class of anti-bacterial compounds

widely used for therapeutic and prophylactic purposes in

animal agriculture. However, the use of these compounds

in food animals may result in unwanted sulfonamide residues in edible tissues. The presence of such residues

in animal origin foods may pose a health hazard to

consumers. Chronic consumption of foods containing sub-

therapeutic levels of sulfonamides may lead to the development of plasmid-mediated bacterial drug resistance.

Further, sulfamethazine has been implicated as a potential carcinogen. Because of these concerns, the FDA has set the maximum residue limit for most sulfonamides at 100 ng/g in meat, poultry, and aquaculture products.

Drug residue violations in the United States red meat and poultry industries most commonly occur with approved

drugs not used in accordance with label directions [1.13,1.14]. If a similar trend holds for violations in

the aquaculture industry, then the greatest number of drug residue violations would be expected for approved drugs that have been used in a non-approved manner. Therefore,

efforts to develop methods for drug residue detection for

domestically produced aquaculture products should initially focus on extraction and detection methods for the limited number of currently approved drugs. One such

compound is sulfadimethoxine. Sulfadimethoxine is the

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sulfonamide component of the potentiated sulfonamide

Romet-30®, which is commonly used in channel catfish aquaculture. Sulfadimethoxine may, therefore, serve as a

model for the application of new extraction and detection

technologies to monitoring of residues in food animals and for the design of an overall approach to aquaculture drug monitoring.

One technique with potential as a residue screening method for regulatory laboratory and field use is the

enzyme-linked immunosorbent assay (ELISA). Enzyme-linked immunosorbent assays for detection of chemical residues

are relatively inexpensive, easy to use, require no special equipment, and are commercially available. They

also provide the sensitivity and specificity required for residue monitoring at tolerance [1.15], The sequential use of MSPD for drug extraction and ELISA for drug

detection can accommodate the need for rapid, practical,

and efficient extraction, and also provide the sensitivity and specificity required for a drug residue monitoring or

surveillance program. Application of combined techniques to food animal tissues would result in a more efficient

residue monitoring program by increasing the number of samples analyzed, by decreasing the turnaround time for sample analysis, and by providing a method for pre-harvest residue determination on site.

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However, the simplest residue screening method is one that uses the presence of the drug and/or its

metabolite(s) in a reference biological fluid to indicate

the presence of violative residues in a target tissue(s)

and does not require tissue extraction. Before such a

technique can be implemented, a relatively constant

relationship between the drug concentration in the target tissue and the reference biological fluid must be demonstrated. The correlation between plasma drug

concentration (central compartment) and muscle drug

concentration (peripheral compartment) is the basis for

pharmacokinetic principles and residue depletion time estimation [1.16,1.17]. For compounds where a stable

correlation between plasma/muscle concentrations can be experimentally determined, plasma may be used directly in a screening assay, such as an ELISA, for prediction of

violative drug residues in muscle.

Tolerances for animal drugs, as set by the FDA, are often based on total residue present, including parent drug and all metabolites, in edible tissues [1.18]. A

marker residue, parent compound or metabolite, is often

used to indicate the total residue present in the target tissue of interest. To establish such a marker residue, a

constant relationship between marker residue concentration and total residue in the target tissue(s) over the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. concentration range of interest must be known. Nonetheless, such information is often lacking for drug

behavior in non-approved species or for non-approved drug

uses. In the absence of such information, extraction, detection, quantitation, and confirmatory methods should

be capable of simultaneous extraction or analysis of the

parent drug and its important metabolites.

The primary metabolite of sulfadimethoxine (SDM) in

channel catfish is 4-N-acetylsulfadimethoxine (N-acetyl SDM)[1.19]. However, because the ratio of parent SDM:N-

acetyl SDM over a range of tissue concentrations, including tolerance (100 ng SDM/g), is not known, there is a need for a chromatographic assay to quantify these two

compounds simultaneously about tolerance. Such a

chromatographic system would also enable determination of recovery of both compounds when using MSPD extraction.

Additionally, information concerning the relative degree

of cross reactivity of this metabolite and other compounds with available ELISAs is required to properly interpret test results.

Suspect samples, identified by screening assays

require further confirmation of the concentration and

identity of the drug/metabolite for regulatory action. Quantification and identification of veterinary drug residues is most commonly accomplished with high

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performance liquid chromatography (LC) or gas-liquid

chromatography (GLC) combined with mass spectrometry.

However, there are presently no LC methods available for the simultaneous determination of SDM and N-acetyl SDM in channel catfish.

PURPOSE

In summary, a model residue monitoring program

applying new or improved methods for extraction, screening, quantification, and identification of drug

residues in aquaculture species is needed. Protocols for such a program may include MSPD for drug isolation, ELISAs

for initial detection, and LC methods for quantification

and presumptive identification. Alternately, where a tissue concentration relationship between a biological fluid (plasma) and target tissue (muscle) can be

demonstrated, the use of such a fluid, not requiring

extraction, may be used for initial residue screening. It is the purpose of this research to develop the

analytical methods and basic tissue compartment

concentration relationships required for a drug residue monitoring program for sulfadimethoxine residues in channel catfish. Specific research needs for the creation

of such a program include the development of matrix solid

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phase dispersion techniques for the simultaneous extraction of SOM and N-acetyl SDM in plasma and muscle,

evaluation of MSPD-derived extracts in commercially

available immunoassays, development of LC methods for

plasma and muscle for the simultaneous analysis of SDM and N-acetyl SDM, and a residue depletion study with groups of

fish sacrificed at various time points in order to characterize the relationship of SDM concentrations in channel catfish plasma and muscle. The following chapters

offer a review of the literature concerning historical and

present seafood inspection protocols for chemical residues in aquatic food resources and present the results of

methods development research for development of a model

drug residue monitoring program for aquaculture.

REFERENCES

1.1 Broussard, M.C., Jr. (1991) Aquaculture: Opportunities for the nineties. Journal of Animal Science, 69:4221-4228. 1.2 Seafood Safety, F.E. Ahmed (Editor), National Academy Press, Washington, DC, 1991.

1.3 Johnsen, P.B. (1991) Aquaculture Product quality issues: Market position opportunities under mandatory seafood inspection regulations. Journal of Animal Science, 69:4209-4215.

1.4 Consumer Reports, Is our fish fit to eat? February 1992, pp. 103-114.

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1.5 Montgomery, A.M., "The FDA Office of Seafood - What does it do? How can it benefit the industry at the state level? Regulatory Responsibility?" Presentation made at the National Association of State Aquaculture Coordinators Annual Meeting, Seattle, WA, 1993.

1.6 Plakas, S.M. "Method development for drugs and other chemicals in aquaculture products. The role of tissue distribution and metabolism studies." Proceedings of the Aquaculture Products Safety Forum, B.E. Perkins, (Editor), Auburn, AL, February, 1993, pp. 30-47

1.7 Munns, R.K., Holland, D.C., Stehly, G.R., Plakas, S.M., Roybal, J.E., Storey, J.M., and Long, A.M. (1993) Determination of chloramphenicol residues in shrimp by gas chromatography: interlaboratory study. Journal of the AOAC International, in press. 1.8 Bencsath, A.F., Plakas, S.M., and Long, A.R. (1993) Development of a gas chromatographic/mass spectrophotometric method for confirmation and quantitation of chloramphenicol in shrimp at low parts-per-billion levels using sector instrument and electron capture ionization. Biological Mass Spectrometry, in press.

1.9 Geyer, R.E. (1993) Aquanotes. FDA Veterinarian, 8(5):6-7.

1.10 Mitchell, G.A. (1991) Compliance issues and proper animal drug use in aquaculture. Veterinary and Human Toxicology, 3 3(Supplement 1):9-10.

1.11 Ellis, R.L. "Analytical detection systems: Past and Future", in Proceedings of the Sixth Biennial Symposium on Veterinary Pharmacology and Therapeutics, J.C. Lee, P. Eyre, W.G. Huber, K. Greenlees, H.P. Misra, and D.Ochs (Editors), Blacksburg, VA, 1988, pp. 39-43.

1.12 Walker, C.C., Lott, H.M., and Barker, S.A. (1993) Matrix solid-phase dispersion extraction and the analysis of drugs and environmental pollutants in aquatic species. Journal of Chromatography, 642:225-242.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12 1.13 Van Dresser, W.R., and Wilcke, J.R. (1989) Drug residues in food animals. Journal of the American Veterinary Medical Association, 194(12):1700-1710. 1.14 Paige, J.C. (1993) Analysis of illegal residues in meat and poultry. FDA Veterinarian, 8(2):5-6.

1.15 Golden, C.A. (1991) Overview of the state of the art of immunoassay screening tests. Journal of the American Veterinary Medical Association, 198(5):827-830.

1.16 Dittert, L.W. (1977) Pharmacokinetic prediction of tissue residues. Journal of Toxicology and Environmental Health, 2:735-756.

1.17 Mercer, H.D., Baggot, J.D., and Sams, R.A. (1977) Application of pharmacokinetic methods to the drug residue profile. Journal of Toxicology and Environmental Health, 2:787-801. 1.18 Livingston, R.C. (1985) Antibiotic residues in animal-derived food. Journal of the Association of Official Analytical Chemists, 68(5):966-967. 1.19 Squibb, K.S., Michel, C.M.F., Zelikoff, J.T., 0 /Connor, J.M. (1988) Sulfadimethoxine pharmacokinetics and metabolism in the channel catfish (Ictalurus punctatus). Veterinary and Human Toxicology, 30(Supplement 1):31-35.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 2

REVIEW OF LITERATURE

EXTRACTION AND ANALYSIS OF DRUGS IN AQUATIC SPECIES

INTRODUCTION

Aquatic resources are monitored for the presence of

tissue residues of chemical agents for two main reasons:

1) for food safety— to identify and remove from commercial markets any edible tissues that contain potentially hazardous levels of drug or other chemical residues and 2) for environmental monitoring— to help identify

geographical areas where environmental quality may have

been significantly compromised. This review will focus primarily on the food safety aspects of veterinary drug residues in aquaculture products.

With increasing reliance on aquatic species as a

source of dietary protein there is a strong public interest in the safety of edible aquatic resources. This

interest is based on concerns about potential unacceptable

health risks associated with eating fish containing residues of veterinary drugs, agricultural pesticides, and environmental pollutants [2.1]. Such residues may exist

13

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in fish bought by consumers in commercial markets or in

fish caught for recreational purposes from rivers, lakes,

and oceans. Further, seafood sold in the markets of one country may often have been imported from another with different regulatory policies concerning drug and

pesticide use in aquatic environments. For example,

imports accounted for over 60% of the fish and shellfish

consumed in the United States in 1990 [2.2]. Therefore,

analytical methods are needed for compounds that may be present in domestic or international products. There is

also a need for an international consensus regarding residue levels and concerns.

In this regard, the Joint Food and Agriculture

Organization (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) serves as a scientific advisory body to FAO, WHO, the Codex Committee on Residues of Veterinary Drugs in Foods (CCRVDF), and the Codex

Committee on Food Additives and Contaminants, concerning

the safety of residues of food additives, contaminants,

and veterinary drugs. Recommended acceptable daily intake

and maximum residue level (MRL) for many of these

substances have been proposed by JECFA and are used by

many countries to formulate regulations regarding chemical residues in foods— including aquatic food resources. The analytical needs of an effective residue monitoring

program are in part determined by the MRLs as set by a

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nation's regulatory authorities. Appropriate analytical methods for these programs are recommended by the CCRVDF.

A listing of reports and other documents published by the JECFA is available [2.3]. Although aquatic species are sporadically monitored

for veterinary drugs and various environmental

contaminants, fish products are not required to pass

unified continuous federal inspection. Additionally, some

of the existing seafood inspection efforts are not designed to be of direct use in evaluating many aspects of

seafood safety concerns. This is due in part to the fact that the present voluntary seafood inspection program administered by the National Marine Fisheries Service of the U.S. Department of Commerce is primarily a plant

sanitation and product grade inspection program. It is not designed to be of direct use in evaluating the safety

of aquatic food products in regards to chemical residues,

although a limited number of chemical residue analyses are done [2.4]. The U.S. Food and Drug Administration has regulatory authority under the Food Drug and Cosmetic Act,

the Public Health Service Act, and other acts to assure

seafood product wholesomeness. This authority includes

mandatory inspection of seafood processing plants and

products, and regulation of aquaculture products, drug use, and aquaculture production practices [2.4,2.5]. The

FDA has announced its intention to implement a Hazard

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ensure seafood product safety and has begun testing

domestic and imported aquaculture products for residues of

the veterinary antibacterials chloramphenicol and oxolinic acid [2.6,2.7]. The FDA is also in the process of

developing additional analytical methods for a numbe r of

veterinary drugs used in aquaculture [2.8] Nonetheless, fish products are not at present monitored on a continuous

basis by any regulatory agency for residues of the

veterinary drugs used domestically or internationally in

aquaculture. Further, efforts at drug residue monitoring are hampered by a lack of personnel and a dearth of legally defensible methods of residue analysis [2.4]. As a result of public concern over the safety of

seafood, the failure of environmental and drug monitoring

programs to contribute valuable residue data for human

food analysis, and the fact that present seafood

monitoring and inspection programs lack both the frequency and direction sufficient to ensure effective implementation of current regulatory limits for seafood

safety, several governmental bodies, including the U.S.

government, have declared their intention to develop a new seafood inspection system [2.4,2.5]. There is early

recognition that the key to the success of this new system

will be development and application of more efficient and cost-effective analytical methods.

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Several classes of chemicals that will need to be included in any aquatic food safety program and in

existing and future environmental monitoring programs are

veterinary drugs, agricultural pesticides, and industrial pollutants [2.4,2.9]. This review offers an overview of present methods of veterinary drug residue analysis as

used in domestic farm animals, their potential

applicability to aquatic species, and a summary of

existing methods for the analysis of many of the drugs

used in aquatic species. Several major drawbacks of the

methods are discussed and three relatively new methods that offer solutions to these problems are described.

DRUG RESIDUE PROBLEMS IN AQUACULTURE

Diseases are the single most important cause of

economic loss in intensive aquaculture and necessitate the

use of antibacterial and other therapeutic compounds to

maintain the health and production of cultured species [2.2], Although there is a degree of variability there

are numerous therapeutants which are consistently used

worldwide in aquaculture [2.10-2.13]. These agents belong to a wide range of chemical and therapeutic classes such

as antibacterials (e.g., sulfonamides and potentiated sulfonamides, aminoglycocides, j8-lactams, tetracyclines,

quinolones, macrolides, etc.), parasiticides (e.g.,

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mebendazole and dichlorvos), disinfectants, pisicides, herbicides, algacides, anesthetics, water treatments, and

dyes. This large range of chemical classes presents a

problem for effective residue monitoring, and requires the use of screening tests to adequately detect the presence of illegal residues.

A residue may be defined as "any compound present in edible tissues of the target animal that results from the

use of the sponsored compound, including the sponsored

compound, its metabolites and any other substances formed

in or on food because of the sponsored compound's use"

[2.14,2.15]. Metabolites are considered to be as toxic as the parent compound unless shown otherwise [2.14,2.15].

Under the FDAs general food safety requirements for

metabolism studies, individual metabolite identification, concentration, and persistence should be obtained for metabolites comprising 100 ppb or >10% of the total

residue (whichever is lower) at zero withdrawal [2.16].

For approved veterinary drugs used in accordance with label directions, a marker residue (Rm)(parent compound or

metabolite) is often used to indicate the total residue

level in a target tissues(s). However, for drug residue surveillance in cases of extra-label drug use where such

concentration relationships are lacking, the extraction and analytical methods used must be capable of providing

extraction and analysis for both the parent drug and its

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important metabolites present at or less than MRLs in the

tissues of interest.

However, MRLs are not always static. Toxicological data are always being updated and the JECFA periodically

issues recommendations for MRLs based on such available

toxicology information for selected veterinary drugs,

including those used in aquaculture. Many countries have

established MRLs based on JECFA recommendations for a number of these compounds. A listing of WHO publications

containing JECFA recommended MRLs is available [2.3].

The use of therapeutants in aquaculture not only may result in unacceptable residues in edible tissues but also

in the environment. Drugs used in aquaculture may be directly introduced into the environment, as with

ectoparasiticides, or indirectly introduced in medicated feeds (via non-consumption of the feed, poor bioavailability, and limited biotransformation). The environmental degradation, accumulation, and persistence

of these agents is affected by water temperature, sediment

microenvironment, and factors affecting dispersion [2.17]. It has been estimated that 70 to 80% of orally

administered oxytetracycline remains in the environment [2.18]. Furthermore, there are large variations in the persistence of antibiotics in sediments from fish farms. Furazolidone exhibits a very short half-life (18 hr.)

[2.19] and oxytetracycline a half-life of 32 to 64 days

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. depending on sediment conditions [2.18]. The environmental fates [2.17-2.26] and effects [2.19,2.27-

2.32] of several compounds commonly used in aquaculture have been the subject of recent studies. However, the

environmental metabolism, fate, and effects of most drugs introduced into the aquatic environment is poorly

understood and relatively few methods are available for

the multitude of compounds, environmental matrices, and environmental conditions that are of importance in

assessing the environmental impact of these compounds.

The National Environmental Policy Act of 1969 requires an environmental impact assessment for aquaculture drug approval in the United States [2.33,2.34], and the FDA has

proposed a requirement that each new animal application

include a section on the environmental effects of the use of the drug [2.35]. Additionally, periodic monitoring of

fish farm effluents for drug residues may be required.

Therefore, methods of analysis of therapeutic agents in

environmental samples are now part of the drug approval process and should be part of our continuing environmental concern.

Development of new methods of analysis of therapeutants was identified at a recent joint FDA-U.S.

Department of Agriculture (USDA) sponsored Interregional Research Project Number 4 (IR-4) meeting as a priority

need in aquaculture [2.36]. In general, new methods for

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veterinary drug residue analysis in aquatic species are

needed for screening, quantitating, and confirming tissue

residues of drugs used domestically and those present in imported aquacultural products. The methods should also be suitable for analyzing environmental samples for drug and contaminant residues.

Because efficient, cost-effective, universal methods for the extraction, detection, quantitation, and

confirmation of these residues in aquatic matrices do not exist, the need for a better approach to analysis has

recently been acknowledged. The research and development plans of regulatory agencies, such as the FDA, currently include commitments to increase and improve capabilities

for testing for veterinary drug residues in aquaculture

products [2.8,2.37,2.38].

METHODS OF DRUG RESIDUE ANALYSIS IN AQUATIC SPECIES

Regulatory agencies require practical analytical

methods for detecting, quantifying, and identifying

violative residues that may occur in food animal tissues. These agencies use available methodology for monitoring

and surveillance and for enforcement action. However, practical methods are not available for many compounds of

interest that may occur as tissue residues. Further, the

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reliability of analytical results obtained with some

'official' methods may be questioned. For enforcement of

regulatory limits for chemical residues in any food

animal, analyses employed must withstand legal challenges for reliability and accuracy.

Accordingly, the FSIS categorizes analytical methods into three levels according to their intended use for

screening, quantitation, or confirmation; and within each

level by the method's relative degree of validation, the confidence that may be placed in test results, and it's

suitability for regulatory action. Validation is the

process of assuring that an analytical method is capable of performing as intended with acceptable accuracy and sensitivity. The most widely accepted procedure for

validation is the interlaboratory collaborative study [2.39]. However, interlaboratory validation is often not possible for the multitude of compounds and matrices a

regulatory laboratory must examine. Therefore, standard

evaluation criteria for detection and quantitation of analytes may be used [2.40]. The term 'official

validation' may refer to validation by the Association of

Official Analytical Chemists International (AOAC) or validation by a regulatory agency. The US Department of

Agriculture, Food Safety and Inspection Service (FSIS) classifications for method status are listed as follows in

decreasing order of confidence of result:

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A. AOAC Official Methods. These are methods published in the AOAC publication Official Methods of Analysis

[2.41]. Interlaboratory validation of these methods has

been obtained with five or more participating laboratories. Such methods are considered the most

authoritative and legally defensible methods for residue

analysis [2.39]. Seven methods are listed as AOAC Official Methods of Analysis (AOAC/OMA) for the detection

of antibacterials in milk, but only three official methods are listed for the detection of antibacterial residues in

other food animal tissues, and no methods are listed for veterinary drug residue determination in aquatic food resources [2.41].

B. Validated Methods. These methods have been subjected to an interlaboratory study in two or three laboratories with at least three independent analysts and the results peer-reviewed by government scientists.

C. Federal Register Methods. This group includes

analytical methods published in the Federal Register and later incorporated into the Code of Federal Regulations.

D. Historical Official Methods. Methods that were regarded as the best available at the time of initial

acceptance and are in continued use due to the absence of improved methods.

E. Non-Validated Methods. Methods for quantitation

and/or identity confirmation that have not undergone a

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collaborative study involving at least three independent analysts.

F. Published Methods. These methods have undergone

study by a single analyst or laboratory and the data peer- reviewed by government scientists.

G. Correlated Methods. These methods have not been

subjected to interlaboratory study, but results obtained with the method have been compared to results obtained

with a current method for regulatory enforcement using the

same samples and the data peer-reviewed by government scientists.

A tiered approach to residue monitoring employs methods for screening, quantitation, and confirmation.

The FSIS has classified analytical methods into three levels based on relative combinations of accuracy,

specificity, and practicality. Each of the three levels

may contain methods classified by validation status as

given above.

Level I — These are assays that provide unequivocal data concerning concentration and identity of the analyte at the level of interest. Level I methods have the

highest level of credibility and are often combinations of two or more assays such as gas liquid chromatography/mass spectrometry or high performance liquid chromatography/ mass spectrometry.

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Level II — These are assays used to determine analyte concentration and perhaps provide presumptive

identification based on retention time, UV spectrum, or

biochemical characteristics but do not provide unequivocal results. Level II methods include various chromatographic and microbial inhibition assays.

Level III — This level includes screening methods to

indicate the presence or absence of an analyte or class of compounds in food animal tissues. These assays are used

because of their high throughput or field applicability, but results from these tests require verification by Level I or II methods for regulatory action [2.9]. Alternately,

some Level II methods may be used as screening assays when

they are applied to reference biological fluids to indicate the residue level in the target tissue of interest [2.42].

All official methods for the analysis of drugs in

animal tissues published in the AOAC/OMA [2.41], FDA

Animal Drug Analysis Manual (ADAM) [2.43], or the FSIS

Compound Evaluation and Analytical Capability Manual (CEACM) [2.9] are for use with red meat, poultry, or milk,

although some of the methods could likely be adapted for use with tissues from aquatic species. The FDA's 1973 Food Additives Analytical Manual (FAAM) [2.44] contains methods of residue analysis supplied by the manufacturer

as a part of a new animal drug application - including

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drugs for use in aquaculture. Some FAAM methods have been evaluated in FDA laboratories, but very few have undergone

complete AOAC validation. The FAAM will be inactivated

once the methods listed in the FAAM are incorporated in the ADAM [2.43].

Many of the methods currently being used by

monitoring agencies for residue analysis in red meat and

poultry are based on 'classical' methods of extraction and analysis [2.9]. Some of these methods have undergone

rigorous multi-laboratory calibration studies and work well under certain conditions and for certain purposes. Perhaps the greatest drawback to their continued use is their inefficiency as screening methods. Some of the

methods are sufficiently complex as to not allow the generation of relevant data in time to prevent

contaminated foods from entering the marketplace and

analytical results are often obtained too late to prevent enforceable removal of the contaminated product.

Additionally, the complexity of these methods and the

length of time required to perform them limits the number of samples that can be practically analyzed in a day.

A variety of methods for the analysis of drugs in

aquatic species have been published in the literature. However, a review of the literature for analytical methods used to extract and quantitate residues of therapeutic

agents used in aquatic species reflects the confusion

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currently felt in the field concerning which protocols are

most efficient, accurate, reliable, and cost-effective

(Table 2.1 [2.45-2.97]). Additionally, only one inter­

laboratory validation study for a veterinary drug residue

analytical method for use with aquatic food products was found [2.98] although a second validated method is in press [2.99].

Since there is presently only one validated method

for the analysis of veterinary drugs in aquatic species, an opportunity exists to introduce analytical methods for

this purpose which utilize improved methods of extraction,

screening, and determination rather than simply adapting existing methods used in other inspection programs. A

number of analytical techniques developed in the last decade can potentially be employed in a drug residue monitoring program for food fish.

Recently introduced drug screening methods include

radioimmunoassay, competitive bacterial receptor binding assay, enzyme immunoassay, high performance thin layer chromatography, and several forms of bioautography. At

present such residue screening methods are not validated

by any federal agency [2.100]. The development of rapid

screening tests that are practical and rugged would allow for routine monitoring and surveillance of larger numbers of samples in a shorter time period with greater

sensitivity and selectivity than is often currently

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methodology also allows chromatographic instrumentation to

be reserved for confirming positive samples. The development of such screening tests may soon be required by many governments as a part of the methods package needed for drug approval of new animal drugs [2.14].

Further, present methods of analysis as used by

several regulatory agencies have failed to fully utilize

the dramatic improvements in reliability and sensitivity

that determinative methods such as LC, GLC, and MSD have

undergone in the last decade. Likewise, advances in extraction methodology have not been fully implemented. A review of traditional and recently introduced methods for veterinary drug residue isolation and determination

follows this section.

MICROBIAL INHIBITION TESTS

Antibacterials have historically been detected in animal tissues and fluids by microbial inhibition tests

(MIT) and these tests continue to be in wide use. All

MIT's are based on the inhibition of bacterial growth by residues of antibacterial compound(s) present in a

biological fluid or tissue. Early assays for chlortetracycline and oxytetracycline residues in milk

utilized reduction of methylene blue as an indicator of

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bacterial growth, and therefore, the absence of the drug [2.101,2.102], Numerous MITs for the detection and

quantitation of antibacterials in fish tissues have since

been described [2.103-2.111]. MITs are relatively simple to use, detect many classes of antibacterial compounds,

and selective sensitivity for specific classes of

antibacterials can be obtained by changes in the culture medium, indicator bacteria, or pH [2.112,2.113]. However,

these methods often lack the specificity and sensitivity required for residue detection at MRLs, may be affected by

non-specific inhibitors, do not detect microbiologically

inactive metabolites [2.114], and often have a 20-24 hour incubation time. Imprecision occurs as a result of zone

size differences between replicate plates. Zone size may

vary as a result of differences in agar layer thickness, agar quality, uneven seeding of bacterial spores on the agar surface, or incubator temperature variation [2.115].

Additionally, bacteriostatic drugs such as sulfonamides

result in a diffuse zone while bacteriocidal drugs provide a sharply defined zone of inhibition.

Plate assay MITs are performed by streaking a uniform

suspension of indicator bacterial spores over an agar medium. Swabs or disks soaked in a body fluid are placed on the plate and incubated. A positive control is

provided by a neomycin sensitivity disk. The observed end

point can be a zone of inhibition surrounding a sample or

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a color change resulting from pH changes. MITs currently in use by the USDA-FSIS for screening red meat and poultry

tissues for antibacterial residues are the Swab Test On

Premises (STOP), Live Animal Swab Test (LAST), and the

Calf Antibiotic and Sulfa Test (CAST) [2.116]. The USDA- FSIS is currently evaluating the Fast Antibiotic Screen

Test (FAST) as a replacement for the STOP and CAST methods [2.117]. All of these MITs can potentially be adapted for use with aquatic animal tissues.

SWAB TEST ON PREMISES

The STOP test is used to detect antibiotic residues

in kidney and other tissues of slaughter animals [2.118]. The STOP method is relatively simple and requires only a

few minutes of analyst time [2.119]. A cotton swab is inserted directly into the meat sample, left in place for

thirty minutes, and the cotton tip placed on a test plate

containing Difco Antibiotic Medium No. 5 previously

streaked with a spore suspension of Bacillus subtilis. The plate is incubated at 29°C overnight (16-20h) and

observed for inhibition of bacterial growth surrounding the swab. Johnston et al, reported 94% agreement with results of STOP and standard microbial assays [2.120].

Korsrud and MacNeil [2.112] reported varying sensitivity

with different media using standard solutions of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tetracyclines. With the standard Antibiotic Medium No. 5, limits of detection (LOD's, ppm) were 6.2 (CTC), 3.1

(OTC), and 1.6 (TC). With Antibiotic Medium No. 2, LODs

were 0.06 (CTC) and 1.6 (OTC and TC). Minimal detectable

levels using Antibiotic Medium No. 5 as reported by

Johnston, et al. [2.118], were 0.01 ppm (CTC) and 0.08 ppm

for OTC and TC. In a comparison of STOP, high performance liquid chromatography (LC), MIT, and thin-layer chromatography-bioautography (TLCB) by MacNeil, et al.

[2.120], STOP lacked the sensitivity of LC but had greater

or lesser sensitivity for OTC than TLCB or MIT depending on the growth medium used.

CALF ANTIBIOTIC AND SULFA TEST

The CAST procedure was introduced by the USDA to increase sulfonamide detection sensitivity in bob veal

calves and was the first test available for pre-slaughter

determination of sulfonamide and antibiotic residues. This test is also sensitive to a variety of other

antimicrobials including tetracyclines, and the degree of

inhibition varies with the compound tested [2.121]. The CAST procedure is similar to the STOP and LAST but uses Mueller-Hinton Medium and Bacillus megaterium ATCC 9885 as

the indicator bacteria, and is incubated at 44°C [2.122].

Plates are read as for the STOP procedure and kidney is

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used as the sample tissue. Korsrud and MacNeil [2.112] reported the CAST procedure was more sensitive than the

STOP procedure for standard solutions of 22 antibiotics tested including CTC, OTC, and TC. Minimum detectable levels (ppm) were 0.2 (CTC), 0.8 (OTC), and 0.4 (TC).

LIVE ANIMAL SWAB TEST

The LAST procedure is a modification of the STOP procedure differing only in the amount of B. subtilis used

[2.123]. It is used for preslaughter field screening of residues in urine and for prediction of residues in edible tissues. It was the first on-farm test available for

screening live cattle for possible residues and is based

on the correlation between urine and tissue residue levels. Urine or blood samples may be used [2.123,2.124].

Two sterile swabs are dipped in a urine sample and placed on the LAST plate containing Antibiotic Medium No. 5

streaked with B. subtilis ATCC 6633 spores. A neomycin disc is used as a positive control, and incubation and

test interpretations are carried out as is done for the

STOP procedure. Several reports indicate a high incidence of false positive results using the LAST assay. In one study 75% (15 of 20) of untreated cows showed a positive result [2.125]. Tritschler et al. [2.123], reported 5.4%

positive results and 19.9% questionable results from 221

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untreated dairy cows and heifers. TerHune and Upson [2.126] had varying results for LAST detection of OTC when

compared to standard quantitative OTC MIT procedures.

LAST was 100% accurate when urine OTC concentration was >4.3 iiq/Tnl and 60% accurate when urine OTC concentration

was <4.3 ficf/ml. Some 20% of LAST results were false positive and 20% were false negative. False positive

results were associated with high urine osmolarity and

high urine pH, apparently resulting in inhibition of bacterial growth. False negative samples were associated

with dilute urine. In this study LAST was 100% accurate in detecting OTC in the urine and predicting tissue OTC residues when OTC concentration was at therapeutic levels. However, LAST did not detect OTC in the urine or predict

OTC concentrations of 0.1-0.4 ppm in tissue.

FAST ANTIBIOTIC SCREEN TEST

The Fast Antibiotic Screen Test (FAST) is a new

procedure under evaluation by USDA-FSIS and provides results within 6 hours. It has undergone field trials

involving 10,000 samples for comparison with STOP and CAST for sensitivity. The FAST assay is similar to the CAST procedure but the FAST growth medium contains sugar and a purple dye. Bacterial metabolism of the sugar results in

acid production causing a color change from purple to

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yellow for the pH-sensitive dye used. A sterile cotton swab is saturated with fluid from a tissue sample and

placed on a plate of growth medium streaked with bacterial spores and incubated for 6 hours. A purple zone

surrounding the sample swab indicates the presence of antimicrobial agent(s) [2.117].

DELVOTEST P

This test is a qualitative color reaction test based

on acid production by Bacillus stearothermophilius var.

calidolactis. The lowered pH changes the color of bromocreosol purple to yellow. If antibacterials are

present, bacterial growth is inhibited and the purple

color remains. Delvotest P is an AOAC Official Method for 0-lactams in milk. Sensitivity for 0-lactams is > 0.005

IU/ml milk. 0-lactam residue is confirmed using

penicillinase [2.41]. It will also detect a wide range of antibiotics including TC at 0.2 fig/ml and OTC at 0.3 fig/ml [2.124]. Macaulay and Packard reported 11% false

positives with this test [2.127]. Delvo P is simple to

run and the color change is easily evaluated as blue vs

yellow. A disadvantage is the 2.75 hr analysis time.

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BRILLIANT BLACK REDUCTION TEST

The Brilliant Black Reduction Test is another

qualitative color reaction test and can be used to detect antibiotic residues in milk and tissue. Bacillus

stearothermophilus is the test organism used with an assay

medium containing brilliant black indicator. The assay medium remains blue if bacterial growth is inhibited by antibiotic residues, but if no residues are present the growth of the bacteria reduces the indicator to a yellow

color. Limit of detection of tissue extracts for OTC is 0.1 /jg/ml [2.128].

BIOAUTOGRAPHY

Several residue analysis methods employing a blend of

physicochemical separation procedures and bacterial growth

inhibition techniques have been reported. Microbial inhibition techniques applied to sample extracts separated by various techniques include paper chromatography-

bioautography [2.129], thin layer chromatography-

bioautography (TLCB) [2.120,2.130-2.132], and electrophoresis-bioautography [2.133]. In these techniques developed chromatographic plates or

electrophoresis gels are placed on bacterial growth medium

seeded with B. subtilis. The location of zones of

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inhibition are used to identify specific antibiotic residues. The sensitivity of the method can be adjusted

and antibiotic residue determination is quantitative.

Electrophoresis-bioautography is usually preceded by a set of MITs and antibiotic identification is based on

initial MIT results, electrophoretic migration distance,

and the appearance of the zone of inhibition. This assay

has been applied to milk and meat samples for residue determination and provides qualitative or semi-

quantitative results. It is however, unlikely to allow resolution of related compounds within a drug class

[2.133]. TLCB is discussed under thin layer chromatography.

COLORIMETRIC METHODS

The Bratton-Marshall method, first introduced in

1939, was the mainstay of sulfonamide residue test methods

for several decades [2.134-2.136]. However, because of

relatively high background readings from naturally occurring primary aromatic amines and cross reactivity

with other primary aromatic amines, these methods generally lack the sensitivity and specificity required for residue analysis at tolerance [2.136], As a result, the FSIS has largely replaced these methods with TLC and

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immunoassay for sulfonamide analysis in domestic food animal tissues [2.9].

A number of colorimetric assays based on the Bratton-

Marshall procedure have been published for several compounds used in aquaculture in a variety of species. These assays have also been frequently used in

experimental studies [2.137-2.140], A 1981 review of

methods for sulfonamide residue analysis in animal tissues is offered by Horwitz [2.135,2.136]. Specific

applications of the Bratton-Marshall method used with

aquatic species include the analysis of sulfamerazine in rainbow trout [2.44,2.135], sulfadimethoxine in channel

catfish, salmonid, and lobster tissues [2.141], and

sulfachloropyridazine in channel catfish plasma [2.140]. Bratton-Marshall colorimetric methods have also been

reported for the analysis of the fish anesthetics tricaine methane sulfonate [2.137] and benzocaine [2.138] as

residues in several fish species. The limit of

quantitation of these assays is in the PPM range which limits their usefulness for residue analysis at tolerance [2.136,2.137].

RECEPTOR BASED ASSAYS

Ligand receptor techniques that show promise as

screening methods for aquatic species include immunoassays

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[2.142-2.144], bacterial cell receptor assays [2.145], and radioimmunoassays [2.146,2.147]. These techniques have

great potential applicability to regulatory monitoring programs, but their efficient utilization is often limited by lengthy tissue preparation procedures. To fully

utilize the speed and simplicity of these tests they must

be combined with newer extraction methods, or used with a

reference biological fluid not requiring extraction to indicate the residue level in a target tissue. Still, the

ability of these screening assays to accurately identify positive and negative samples must be evaluated based on

performance parameters such as sensitivity, specificity, cross-reactivity, predictive values (positive and

negative) and efficiency before the test can be included in a residue monitoring program.

BACTERIAL CELL RECEPTOR ASSAY

The Charm II test is a proprietary competitive microbial receptor binding assay that can detect residues of seven classes of antibiotics and is the only AOAC

Official Method of Analysis for sulfonamides and tetracyclines in milk [2.41]. Methods for examination of

other matrices such as serum, plasma, urine, honey and extracts of egg, muscle, liver, or kidney are available

but are not validated by the AOAC. The test instructions

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also contain a method for analysis of sulfadimethoxine

residues in fish muscle [2.145]. The Charm II test

requires 12-15 minutes per individual drug class test

[2.148]. In this assay, microbial cells possessing

specific antibacterial class receptor sites are added to milk or tissue extract containing added 3H or 14C labelled

drug. The radio-labelled drug competes with the residue

of compounds of a drug family that are present in the

sample for the available bacterial receptor sites.

Following centrifugation, the sample is decanted, the

precipitate resuspended, combined with scintillation fluid and its activity measured using a scintillation counter. Sample activity is compared to a decision point obtained

using the mean of six replicate fortified samples ± 15%. The sample level of radioactivity is inversely related to the residue level of the sample. The level of

radioactivity used (0.5 /iCi/jmol, 0.052 /xCi/test) is

exempt from Nuclear Regulatory and Agreement State regulations [2.145]. Limits of detection (ppb) in milk are 3 (CTC), 6 (Democycline), 100 (DC), 4 (MC), 5 (OTC), and 1 (TC). Serum, urine, and egg LODs are 100 for TC [2.145].

Several comparisons of the Charm II with other

methods of residue analysis for milk have been reported. Brady and Katz reported confirmation of Charm II test

results for CTC by a comparison with MIT assays. Nine

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samples positive for TC with the Charm II were confirmed positive by MIT [2.149], However, Collins-Thompson, et

al. reported that 40 of 48 milk samples positive for TC by the Charm II test were negative by disc assay, and 8 showed indistinct zones of inhibition. Increased

sensitivity of the Charm II and a possible unknown

interfering factor were suggested [2.150], Charm and Chi reported a 2.3% incidence of false positives for TC in

milk [2.148], and Senyk et al. reported no false positives for TC in milk [2.151].

Because the bacterial receptors bind a functional group of the drug, rather than a side chain as with

immunoassay tests, the Charm II test provides detection of a class of antibacterial compounds rather than a single

compound. Although this test can detect a number of drugs within a class, the relative sensitivity of the test to

individual drugs varies. The Charm II test sums, although

not in a linear manner, concentrations of various drugs

within the same class in a sample. The Charm II test

performed inconsistently among analysts and laboratories in FDA studies [2.152-2.154]. Although the Charm II has

been proposed as a confirmatory method [2.149,2.155], the non-specific nature of the test, its variable sensitivity for individual drugs within a drug class, and the relatively high rate of false positive results restricts

this test to use as a screening assay.

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RADIOIMMUNOASSAY

Although radioimmunoassays (RIA) are characterized by

high sensitivity and specificity, relatively few methods have been published for RIA detection of drug residues in

fish. In a pharmacokinetic study, Rolf et al. used RIA for gentamicin quantitation in channel catfish plasma [2.146], and Rattenberger et al. used RIA to examine

chloramphenicol depletion in rainbow trout plasma and muscle [2.147]. Additionally, RIA methods for detection

of chloramphenicol residues in several red meat and

poultry products and evaluations of their performance as residue screening tests are available [2.156,2.157]. The FSIS analytical manual lists RIA as a screening method for

residues of zeranol and it's metabolite taleranol in cattle liver and muscle [2.9], and Daeseleire et al.

reported a combined LC and RIA procedure as a screening

method for 19-nortestosterone and methyltestosterone residues in beef muscle tissue [2.158].

In addition to the Charm II Bacterial Receptor Test,

Charm Sciences, Inc. also markets Charm II Antibody Tests

for the detection of several antimicrobials in milk. These RIAs may also be used with serum, plasma, urine, tissue, and eggs. Tissue and eggs however, require a

separate extraction procedure before testing. Available

RIAs from Charm Sciences, Inc. include chloramphenicol,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 42 gentamicin, sulfadimethoxine, sulfamethazine, and tetracycline. In the Charm II Antibody test, ,4C or 3H

labeled drug competes with any drug residue present for a

specific antibody bound to microbial cells. These tests reportedly are rapid (12 minutes per test) and specific

with a level of detection of 10 ng/ml for drug residue in

milk. The principal disadvantages of RIAs when compared with other immunoassays involve the use and disposal of radioactive compounds and the need for scintillation

counting equipment [2.41]. As a result, ELISA and

fluorescent labelled immunoassays are becoming more widely used.

ENZYME IMMUNOASSAY

Immunoassays are widely used to monitor therapeutic drugs and drugs of abuse in human medicine, for dog and

racehorse testing, and on dairy farms and processing

plants to screen milk samples for veterinary drug residues. Nonetheless, they have seen limited use for

analysis of veterinary drug residues in food animal

tissues. The major market for these tests in agriculture has been for dairy products. However, with appropriate extraction methodology many of these assays may be used for residue analysis of food animal tissues, including

food fish. Immunoassay offers a cost-effective and rapid

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alternative to conventional methodology for drug residue screening. Immunoassays can determine within minutes

whether edible tissue contains veterinary drug residues

above or below tolerance, and sample preparation for immunoassay is typically shorter and less demanding than the comparable preparation methods used for

chromatography. The reduction in preparation time and

ability to simultaneously process many samples can greatly increase a laboratory's throughput and provide relevant

residue concentration information in time to allow

retention or removal of potentially harmful products from the food supply [2.159-2.161].

Like radioimmunoassay, enzyme immunoassays typically possess high sensitivity and specificity. The high

specificity of these assays may be advantageous or not

depending on the intended use of the test, i.e., detection of a single analyte or a class of compounds. Although an

assay using antibodies of moderate specificity may detect

a class of compounds, immunoassay does not readily lend itself to multiresidue detection. It is most useful for screening a large sample set for one or a few drugs in a

given matrix. Nonetheless, the high specificity of immunoassays does permit minimal sample cleanup compared to chromatographic methods.

Because of the ability to screen large numbers of

samples and detect analytes in the low parts per billion

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range, early and continuing interest in immunoassays has

centered on veterinary drug residues of greatest toxicological and public health concern. Compounds of

high regulatory concern, as designated by the FDA, include chloramphenicol, nitrofurans, fluroquinolones, quinolones, malachite green, nitroimidazoles, and sulfamethazine

[2.162,2.163], Because of their wide use and toxicity and carcinogenicity concerns the two drugs that have generated the greatest interest in screening methods development are

chloramphenicol and sulfamethazine.

Chloramphenicol can induce an idiosyncratic, dose independent aplastic anemia in man. Of particular concern in seafood is the use of chloramphenicol in shrimp

mariculture [2.6]. A number of ELISA methods for

detection of chloramphenicol residues and evaluations of

their performance in several food animal tissues have been published [2.154,2.157,2.164-2.168]. Sulfamethazine

(sulphadimidine) is widely used in some food animals, is a

potential carcinogen, and is of high regulatory interest. As such, ELISAs were developed early on for this compound

[2.169-2.172]. Other compounds for which ELISA analysis

of drug residues are reported include monensin,

cephalexin, penicillins, sulfathiazole; anabolic agents such as nortestosterone and the /3-agonists clenbuterol,

cimateraol, and salbutamol [2.173-2.180].

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Only three reported applications of enzyme

immunoassay for detection of veterinary drug residues in

fish were found. Kitagawa et al. developed an ELISA for detection and quantitation of the peptide antibiotic

colistin in rainbow trout muscle, liver, kidney, spleen,

serum, and bile. This assay reportedly can detect as

little as 3 ng per well [2.142]. The EZ-SCREEN® test for

sulfadimethoxine was evaluated by Wu et al. for detection of sulfadimethoxine residues in channel catfish muscle and

liver. Homogenization and ten-fold sample dilution

provided detection of sulfadimethoxine at the tolerance of 100 ng/g [2.143]. Walker and Barker evaluated four commercially available ELISAs for detection of

sulfadimethoxine and 4-N-acetylsulfadimethoxine residues at concentrations surrounding tolerance in channel catfish muscle [2.144]. This study is presented in detail in

Chapter 3.

Commercially obtainable enzyme-linked immunosorbent assays (ELISA) are available in kit form for several of the antibacterials used in land- and water-based

agriculture. Included are assays for chloramphenicol,

several /S-lactams, tetracyclines, tylosin, sulfamethazine, and sulfadimethoxine. These test kits utilize enzyme-

linked immunosorbent assays (ELISA) based on the principle of direct competitive solid phase enzyme immunoassay and

compare the relative color development of the sample to a

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 6 negative control as the measurable endpoint. Negative

samples exhibit the greatest color development and

positive samples the least development. The tests are

packaged containing all reagents and components, and test

procedures are described in directions for use included with each kit. Results may be qualitative or semi- quantitative respectively when determined visually or using optical density (OD) values. Test formats, as

determined by the type of solid support used for

immobilization of the antibody, include the MICROTITER®

well, tube tests, and membrane based ELISAs such as the

CITE® cup device, CITE® probe device, SNAP™ test, I.D. Block™, and the Quik-Card® test.

The FSIS compound evaluation and analytical

capability manual currently lists EZ-SCREEN® immunoassay methods for chloramphenicol, gentamicin sulfate, neomycin

sulfate, sulfadimethoxine, sulfamethazine, and tylosin as

non-validated screening methods for red meat and poultry

[2.9]. EZ-SCREEN® is a qualitative colorimetric immunoassay for detection of drug residues in milk, urine, serum, and feed using the QUIK-CARD® format. The QUIK-

CARD® system consists of a solid support card containing two membrane-based test ports; negative control and

sample. Results are obtained within 10 minutes by visual

comparison of relative color development of the two spots.

The lower detection limit of the assay is 10 ng/ml. This

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test is marketed by Environmental Diagnostics, Inc. of

Burlington, NC [2.181]. A recent FDA evaluation of the

EZ-SCREEN® chloramphenicol test for milk indicates that this test performs well when testing fresh milk. However, thirty-three percent of previously frozen milk samples

containing one ng/ml provided false negative results.

The primary problem encountered with the test was an occasional failure of the sample to properly adsorb into

the test membrane [2.154]. In addition to the EZ-SCREEN® test, several other

immunoassays are commercially available for screening food animal tissues for drug residues. One such test is the SIGNAL® Sulfamethazine Detection Test marketed by

SmithKline Beecham Animal Health of Exton, PA. This test

uses the MICROTITER® well format and may be used qualitatively or quantitatively. The SIGNAL® test is marketed for the detection of sulfamethazine in milk,

muscle, serum, urine, and feeds, but also exhibits a

degree of cross reactivity with a number of other sulfonamides [2.144].

The IDS Sulfadimethoxine One-Step ELISA is a

quantitative MICROTITER® well ELISA for the detection of sulfadimethoxine residues in serum, milk, urine, muscle,

liver, kidney, and feed. International Diagnostics

Systems Corporation of St. Joseph, MI also markets a

qualitative membrane ELISA for detection of

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sulfadimethoxine under the tradename Sulfadimethoxine I.D. Block™ Testing Device. Both tests have a lower detection

limit of 10 ng/ml [2.182,2.183]. Other ELISAs available from this company are sulfamethazine, gentamicin, tylosin, and neomycin in MICROTITER® well format, and

sulfamethazine and gentamicin in the I.D. Block™ device.

Also immunoassays for forty-two additional therapeutic drugs or mycotoxins are available from this firm.

IDEXX Laboratories, Inc. of Westbrook, ME markets a

number of ELISAs in one of three membrane-based formats; the CITE® cup, the CITE® PROBE, and the SNAP™. The CITE®

cup device holds a membrane filter containing three discrete test areas and a negative control spot; each test

area is coated with antibody to one sulfonamide. Two

qualitative assays are available in the cup format, the CITE® sulfamethazine assay and the CITE® Sulfa Trio™. The CITE® Sulfa Trio™ is used for the detection of

sulfadimethoxine, sulfathiazole, and sulfamethazine residues in milk.

The CITE® PROBE is competitive immunoassay that visually compares the relative color intensity of a

control spot with a sample spot. It is packaged as a self contained kit and can be easily run on-farm. The CITE®

PROBE utilizes membrane coated antibody spots located on the end of a probe device. The probe is moved through a

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series of four development wells supplied with the kit and the result determined visually or with a densitometer. CITE® PROBE tests available include gentamicin,

/3-lactam, /3-lactam/sulfamethazine Combo®, /3-lactam

/tetracycline Combo®, and tetracycline. The /8-lactam

PROBE test does not use antibodies to specifically bind

the analyte(s) but rather a penicillin-binding protein bound to the membrane matrix [2.184], Other CITE® PROBE tests are however, true ELISAs. The CITE® PROBE

tetracycline test is a multi-residue screening test for

chlortetracycline, oxytetracycline, and tetracycline residues in milk. Assay time is 5 minutes. The limits of

detection in milk are 40 ppb for chlortetracycline and

oxytetracycline, and 20 ppb for tetracycline [2.185].

The SNAP™ /8-lactam test is an enzyme-linked receptor- binding screening test for penicillin G, ampicillin,

cephaprin, amoxicillin, cloxacillin, and ceftiofur

residues in raw whole milk. The level of sensitivity

varies among the several drugs. The SNAP testing device is a single unit containing an antibody coated test area,

control spot, and all necessary reagents. This recently

introduced test requires only three steps, no reagent measuring, and provides results in ten minutes [2.186]. ELISAs utilizing test tubes coated with antibody are

available under the LacTek™ brand name from Idetek, Inc.

of Sunnyvale, CA. LacTek™ screening tests for /8-lactams

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(amoxicillin, ampicillin, cephapirin, cloxacillin, hetacillin, nafcillin, oxicillin, and penicillin G),

ceftiofur, chloramphenicol, gentamicin, sulfamethazine, and tetracylines (tetracycline, chlortetracycline, and

oxytetracycline) in milk are available. For multi-residue assays, the relative level of sensitivity varies among the individual drugs from 4-10 ng/ml. Up to five samples can

be tested simultaneously with an assay time of seven minutes and only two minutes of actual analyst time.

Results are determined using a dual wavelength spectrophotometer [2.187].

Although a number of ELISAs are currently marketed, at present none are certified or approved by any federal

agency. However, the FDA has recently issued calls for a voluntary evaluation of milk residue screening assays, including immunoassays, and is presently evaluating

screening assays for their performance in detecting j8-

lactam antibiotic residues. Results are pending at this

time [2.188]. In several published performance evaluations, proprietary immunoassays have performed well at correctly identifying various tissues as containing

above or below tolerance [2.143,2.144,2.157,2.167,2.168, 2.176,2.184,2.189]. Although immunoassays in kit form were only recently available for residue analysis, the

great potential of these tests to improve the efficiency

of drug residue monitoring programs will clearly result in

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a number of additional assays of this type introduced in the future.

CHROMATOGRAPHIC ANALYSIS

There will be the need at some time to provide a less

equivocable analysis of the residue. Suspect samples identified with screening tests require quantitation and confirmation of the presence of residues exceeding the MRL in the target tissue; hence rapid tissue extraction,

quantitation, and confirmatory methods must be available for regulatory purposes. For many such analyses,

chromatographic methods provide the necessary specificity and sensitivity required for both qualitative and quantitative drug analyses. More recent chromatographic methodologies can complement microbial or immunoassay

tests in that they have short analysis times overall and limits of detection in the same concentration range.

THIN LAYER CHROMATOGRAPHY

CLASSICAL THIN LAYER CHROMATOGRAPHY

Thin layer chromatography (TLC) is one of the simplest and most easily used of the chromatographic

methods and has long been used for separation and analysis

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of drug and other residues. In classical TLC, an aliquot of a biological fluid or tissue extract is added onto a

porous layer of adsorbent material. Each component in the

sample has a characteristic mobility pattern on the TLC

plate for a given procedure. The sample is resolved into individual components by the distinctive migration pattern

of each component. This migration distance is usually

expressed as an Rf value where Rf = (distance traveled by the component) (distance traveled by the solvent front).

An Rf value identical to that for a standard provides

presumptive, but not absolute identity of the component. Visual determination of the migration distance and relative spot size or intensity may be obtained with

naturally colored or naturally fluorescent compounds.

Other compounds may be visualized by spraying the plate with specific reagents, charring, or treatment with vapor

phase reagents. Alternately, TLC plate coatings may

contain a fluorescent indicator with UV absorbing

substances seen as dark areas against the fluorescent

background. A wide variety of TLC adsorbent materials may be used for coating of the TLC plate. Silica gel is most

commonly used, but reversed-phase layers are available with C2, C8, C18, and phenyl phases bonded to silica [2.190].

TLC offers several distinct advantages over other

chromatographic procedures for residue analysis. It's

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ability to analyze several samples simultaneously rather than serially may provide significant time savings over

other methods of chromatography, and solvent system-

detector compatibility required with LC or GLC for multi­

residue analysis is not needed [2.191]. Additionally, TLC

provides reproducible results without the potential LC

problems of column contamination and degradation and TLC is relatively inexpensive in terms of equipment when

compared to other chromatographic methods. Further, the components remain in the absorbent layer and can be

recovered for further analysis.

Several classical TLC techniques are listed in the FSIS /official methods' of analysis manual for determination of several veterinary drug residues in red

meat and poultry [2.9], One such method is the Sulfa on Site (SOS) test. This test was developed by the FSIS to identify swine samples containing violative levels of

sulfamethazine and is commercially available in a kit

format. SOS methods for urine and plasma are calibrated so that test readings predict liver or muscle

concentrations exceeding sulfamethazine tolerance. Sulfonamides are visualized on the TLC plate as areas of

blue-green fluorescence under UV light. Suspect animals identified by the SOS test are retained pending

quantitative analysis by GLC/MS [2.9,2.192]. Other

sulfonamides, including sulfadimethoxine, can also be

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detected with the SOS test. The fluorescent intensity and

characteristic migration location (Rf value) indicates the

approximate concentration and provides presumptive identification. This test is a reasonably simple and accurate method for detecting sulfonamide residues, but does require mastery of some technical and manipulative

skills. It is therefore not suited for on-farm use by lay users with few basic laboratory skills [2.193].

Nevertheless, classical TLC techniques, in general,

lack the precision necessary for residue quantitation of target tissue at tolerance. Few published methods for

classical TLC analysis of drug residues in food fish are available. A method for TLC determination of residues of

the anesthetic tricaine methane sulfonate (MS-222) in fish

tissues was reported by Allen et al. The method limit of detection of 0.2 jug/g and limit of quantitation of 2.0

/jg/g are however above tolerance, making this method less

suitable for residue detection use [2.194]. Squibb et al. used TLC for separation of SDM and metabolites in channel catfish plasma, urine, bile, liver, and muscle in a

pharmacokinetic and disposition study [2.195]. Barron and

James used TLC for separation of radiolabelled sulfadimethoxine and metabolites in lobster tissue

extracts with subsequent quantitation by scintillation counting [2.196].

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Separation and determination of radiolabelled

substances may also be obtained using TLC with direct radioscanning or autoradiography. Because of its speed

and improved instrumentation, direct radioscanning of

developed TLC plates may someday replace scintillation counting for radioisotope detection and quantitation in

drug and metabolism studies. TLC may also be combined with other analytical techniques such as mass spectrometry or infrared spectrometry to provide more definitive

identification of compounds [2.190].

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

A modified planar chromatographic method with

potential use in residue monitoring as a screening and quantitative method is high performance thin-layer chromatography (HPTLC). In this technique, sample and

standard solutions are applied manually or by an automated

spotting device to a silica gel coated HPTLC plate, mobile

phase applied, and computer-assisted densitometer or other determinative readings obtained following development.

Determination may be effected using optimal wavelength scanning at visible light absorbance, UV absorbance, or fluorescence. To facilitate separation or enhance detection of analytes, prechromatographic derivatization

is performed or postchromatographic detection reagents may

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be applied to the plate [2.197]. Using these techniques, HPTLC methods can provide results approaching those

obtained with LC in precision and quantitative accuracy [2.191,2.198].

Although HPTLC offers several distinct advantages, relatively few methods are published for HPTLC

determination of veterinary drug residues. One such HPTLC technique is the AOAC validated method for simultaneous

multi-sulfonamide residue screening applicable to swine,

turkey, and duck tissues. This method provides acceptable

accuracy and precision near the official tolerance for six sulfonamides, but tissue extraction requires

homogenization and multiple liquid-liquid partitioning steps and is labor- and solvent-use intensive

[2.41,2.191]. The FSIS uses this procedure for multi­

sulfonamide detection and quantitation in red meat and poultry [2.9]. Tao published a rapid HPTLC method for the

determination of flumequine residues in rainbow trout meat

using chloroform extraction with quantitative analysis

using UV detection at 254 nm [2.199]. Reimer and Suarez used MSPD for the simultaneous extraction of five

sulfonamides from salmon muscle with quantitation by HPTLC using fluorescence detection following fluorescamine spray. The method detection limit for SDM was 0.13 0 /xg/g

[2 .200].

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TLC-BIOAUTOGRAPHY

Thin layer chromatography-bioautography (TLCB) is

based on selective tissue extraction, separation of components by TLC, and bacterial growth inhibition

techniques. TLCB provides a multiresidue detection method

and can be used to identify individual antibiotics within a class of antibiotics [2.131]. It has been used in

Canada since 1984 for the confirmation of positive in-

plant tests of red meat [2.112]. Neidert et al. reported

minimum detectable amounts in fortified muscle samples

(ng/g) as 15 (chlortetracycline) and 30 (oxytetracycline and tetracycline) as determined by the minimum amount causing visible inhibition zones on 100% of tests at that

level [2.130]. MacNeil et al. reported TLCB lacked the sensitivity of LC for oxytetracycline but was of equal

sensitivity with MIT. STOP had greater or lesser

sensitivity than TLCB depending on the growth medium used [2.120]. In a related study evaluating the performance of

five screening tests for detection of penicillin G residues in calves, TLCB had greater sensitivity than the

STOP or CAST, but was less sensitive than the brilliant black reduction test, Charm II, or LC [2.201].

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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

For many of the drugs used in aquaculture the method of choice would be HPLC with UV detection, using a

variable wavelength or diode array detection system.

High performance liquid chromatography (LC) is the most

commonly reported method for quantitation of veterinary drugs in aquatic species, red meat, and poultry. Although

LC is commonly used, complex extraction procedures and/or sample pretreatment are often needed before injection on

conventional reversed phase analytical columns. Newer analytical columns employing internal surface reverse phase or immunoaffinity packing permit direct injection of plasma and other liquid matrices that cannot ordinarily be

used with conventional LC columns [2.79]. With such improvements LC procedures may approach screening tests in

speed and simplicity [2.202]. As illustrated in Table 2.1 [2.45-2.97], a variety of analytical columns and mobile

phases are reported for a number of drugs. Although many

LC techniques have been described over the years, one must exclude from consideration any strict adherence to LC

methodologies reported prior to 1985. This fact is in

large measure due to the very different nature of the solid supports available today when compared to those of the past. Reverse phase C18 packing is most commonly used

but other packings reported are dimethyl silica,

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(Jl u> Ref. [2.48] [2.49] [2.47] [2.50] [2.46] [2.45]

Limit of Detection of Limit 10 ng/g liver ng/g 10 100 ng/g Liver ng/g 100 80 ng/g OTC ng/g 80 ng/g 5 Muscle ng/g 50 5 ng/g muscle ng/g 5 on column on TC ng/g 90 Serum ng/ml 50 500 ng/g CTC ng/g 500 ng/g 5 20 ng TC, OTC TC, ng CTC 20 ng 60

355 nm 355 10 min 10 15 min 15 min 8 nm 357 @ UV 15 min 15 UV @ 355 nm 355 @UV photodiode array photodiode UV @ 370 nm 370 @UV & Analysis Time Analysis & @

EDTA-MeOH 1:1:8 ACN 9:1

[(5 g DAHP + 5 ml DEA)/810 ml UV @ 365 nm 365 @UV ml DEA)/810 ml 5 + DAHP g [(5 ACN - THF - ACN 81:10:9 81:19:6 0.005 M phosphate buffer - buffer phosphate M 0.005 ACN - DMF - 0.01 oxalic acid oxalic 0.01 - DMF - ACN 27:6:67 water] - ACN - DMF - ACN - water] min 8 col A. 0.05 M phosphate buffer - buffer M phosphate M 0.08 0.05 - A. KH2P04 Mcol 0.04 B. col nm 370 @UV trisodium M 0.1 - ACN min - 8 acid NajEDTA M 0.1 - citric M 0.1 citrate 340:5:5:150

10 pm or pm 10 pm 10 LC-18 DB LC-18 5 pm 5 LiChroCART RP-18 MeOH RP-18 LiChroCART pm 5 Merck Hibar 0.01 M oxalic acid - ACN - ACN - acid Hibar oxalic MMerck 0.01 Shandon pm 5 A. Wako Gel Wako A. (dimethylsilica) gel) Gel Shimadzu B. (polystyrene Hypersil SAS Hypersil Supelcosil* 7 pm 7 73:17:10 ODS Hypersil ODS ODS Spheri 5 pm 5

filtration Bondesif C, or Cl8 or C, Bondesif homogenization Bond Elut Cm Elut Bond liquid / liquid / liquid resin Amberlite XAD2 Amberlite Amberlite XAD2 Amberlite homogenization using SPE SPE using SPE Bond Elute CIS Elute Bond C|8 Sep-Pak using SPE homogenization homogenization homogenization SPE using SPE resin Sample Preparation Analytical Column Mobile Phase Column Mobile Analytical Preparation Sample Method Detection SPE using SPE SPE using SPE

AQUACULTURE fish salmon muscle salmon Matrix fish liver, slime, fish vertebrae muscle, hide, liver, muscle liver, RBT muscle RBT homogenization RBT muscle & muscle RBT liver serum, RBT d. Chlortetracycline Compound(s) Chlortetracycline Tetracycline Tetracycline Oxytetracycline Oxytetracycline Oxytetracycline Oxytetracycline Oxytetracycline TABLE 2.1 REPRESENTATIVE EXTRACTIONAND LIQUID CHROMATOGRAPHICMETHODS FOR DRUGS USED IN Oxytetracycline table table con'

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. < J \ o [2.56] [2.52] [2.57] [2.53] [2.54] [2.55] [2.51] [2.58]

ng/g DFZ ng/g ng on column) on ng 1000 bone, skin bone, (1.25 360 nm 50 ng/g muscle, serum muscle, nm ng/g 360 50 365 nm ng/g 365 50 @ ® UV @ 350 nm 4 ng on column nm on ng 350 @ UV 4 min 6 04 - - 04 nm @350 UV ng/ml 12

min 15 P M oxalic acic UV acic oxalic M j H -0.2

30:9:1 min kidney, 12 liver, ng/g 100 M aqueous oxalic acid - - nm ng/g 355 @ 50 acid UV oxalic aqueous M CTC ng/g 100 M oxalic acid - - acid ng/g 50 array oxalic M photodiode M aqueous aqueous M M oxalic acid - acid oxalic M M phosphate buffer - MeOH UV - buffer phosphate M M monobasic sodium sodium CAP, OTC, nm ng/g 265 @monobasic M UV 50 1:1:3.5 19:6 19:6 min 20 1000:28:2:0.6 ACN - THF - ACN 89:6.3:4.7 min 8.5 MeOH - DMF - MeOH 95:5:5 CHClj- MeOH - HjO - cone each cone nm - ng/g HjO 288 @ UV 50 - MeOH CHClj- 0.025 ACN-THF 0.02 MeOH - ACN nm 365 @ 0.02 0.02 ACN - MeOH octanesulfonic acid at 10 mM 10 at acid containing octanesulfonic ACN - 0.05 phosphate 65:35 min 30 FZD SDM, SMM, 70:27.5:2.5 70:27.5:2.5 0.02 min 6 NH4OH NH4OH min 20

A

Cyano Spheri-5 Cyano MPLC 5 pm 5 C,t pack S &M MCH-10 Nucleosil S Cl8 S Nucleosil p-Porasil

trifluoracetic acid trifluoracetic liquid / liquid / liquid protein ppt using ppt protein homogenization C18 Sep-pak direct LC inj and using and 100 SPE PLRP-S inj LC direct polystyrenc- with MSPDCl( MicroPak cu pre-column extraction S pm S extraction pre-column divinylbenzene SPE using Bakerbond using SPE

serum, liver, serum, skin muscle kidney, bone, kidney, salmon muscle filtration filtration muscle pm salmon 5 liquid pm 5 liquid/ muscle salmon using SPE bream, sea red eel muscle C,s muscle Sep-pak eel RBT, yellowtail, homogenization yellowtail, RBT,

Oxytetracycline plasma RBT Oxytetracycline Oxytetracycline muscle, RBT Oxytetracycline plasma fish Oxytetracycline CAP, SMM, CAP, Oxytetracycline channel catfish channel Oxytetracycline Oxytetracycline Pacific pink homogenization Ultraspherre ODS pink Ultraspherre homogenization Pacific ODS Oxytetracycline pink Ultrasphere homogenization Pacific Oxytetracycline DFZ Sulfadimethoxine catfish muscle, homogenization muscle, catfish Sulfadimethoxine OTC, CTC, OTC, FZD, SDM, & Ormetoprim liver, kidney liquid/ liquid kidney liquid/ liver, Ormetoprim & table con'd.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o\ [2.60] [2.62] [2.64] [2.63] [2.61] [2.65]

ng on column) on ng 15 ng/g SDZ ng/g 15 50 ng/ml SDZ ng/ml 50 TMP ng/g 80 250 ng/ml TMP ng/ml 250 -100 ng/gSDZ -100 150 ng/g SDM ng/g 150 100 ng/ml bile ng/ml 100 66 ng/g SMR SMTng/g 66 ng/g 228 SP ng/g 48 plasma muscle 26 ng/g 26

270 nm 270 @ min min kidney liver, 265 nm 265 10 min 10 12 min 12 11 min 11 UV @ 280 nm nm 280 @ UV each min 30 ng/g 200 (2.59] 10 min plasma min 10 photodiode array 50 ng/g ng/g 50 array photodiode muscle, 265 @UV serum, ng/g 50 @nm270 (1.25 spectrometry mass UV 25 ng/g 25 array photodiode nm 265 @ array photodiode 270nm @ min 57 array photodiode 20 min tissue min 20 ®

- ACN -

65:35 2.8)

04-ACN

P M ammonium acetate ammonium M j triethylamine 0.01 M H3P04-ACN H3P04-ACN M Tissue H M plasma M sodium phosphate with phosphate sodium M Plasma muscle 0.1% M phosphate buffer - ACN - buffer phosphate M 17:10:73 ACN - MeOH - 0.1 M H5P04 M 0.1 - MeOH - ACN 0.017 ACN - - ACN 0.05 65:35 containing ACN 35% aqueous composition gradient (pH sulfonate 0.025 hexane 0.1 % formic acid formic % 0.1 80:20 0.017 73:27 77:23 with with 71:29

Ultrasphere ion-pair C„ YMC-Pack 5 pm 5 Clt MicroPak MCH-10 18-DB LC pm 5 LC 18-DB LC Supelcosil Supelcosil Supelcosil 18-DB LC ODS Hypersil ODS pm 5 narrow-bore

homogenization homogenization Sep-Pak C)8 Sep-Pak SPE using SPE liquid-liquid liquid liquid/ Sep-Pak Alumina B Alumina Sep-Pak

RBT serum, RBT muscle flesh muscle, liver, bile muscle, kidney, muscle, & liver Spin-X filter Spin-X liver & muscle, plasma Sulfadimethoxine channel catfish MSPD catfish channel Ormetoprim muscle using SPE Sulfadimethoxine Sulfadimethoxine chinook salmon homogenization salmon chinook Sulfadimethoxine SDM, SMM, SDM, N-acetyl metabolites Sulfadimethoxine coho salmon coho Sulfadimethoxine Sulfadimethoxine channel catfish MSPD catfish channel Sulfadimethoxine Sulfadiazine coho salmon salmon MSPD coho muscle Sulfadiazine Sulfamerazine homogenization Sulfamethazine salmon, liquid Sulfadimethoxine Sulfapyridine Atlantic liquid/ plasma, RBT Sulfadiazine Trimcthioprim 4-N-acetyl SDM muscle and SDM muscle 4-N-acetyl table con'd.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ] ] 0\ to 66 68 . . 2 2 [ [2.67] [2.70] [2.71] [ [2.69]

NA, PA, FZD PA, NA, ng/g NFS ng/g ng/g SMR, FZD, NA FZD, SMR, ng/g 100 80 ng/g PA, NPN, FMZ NPN, PA, ng/g 80 60 ng/g SMM, SSZ, SMM, 40 ng/g 60 SDM,DFZ

min min 13 min 13 15 min 15 fluorescence OA ng/ml 5 fluorescence OA ng/ml 3 fluorescence 380 nm emission nm 380 262 nm excitation 10 ng/ml FEQ ng/ml 10 excitation nm 262 FEQ ng/ml 5 excitation nm 325 emission nm 360 40

- water OA water nm - ng/g 260 @ UV 20 M oxalic UV @ 295 nm 50 ng/g each nm ng/g 295 @ UV oxalic M 50 0.01 M Na2EDTA& M

H j P 0 4

0.001 min 8 M aqueous oxalic acid - OA - nm ng/g 265 @ UV acid 20 oxalic aqueous M M oxalic acid - ACN - ACN - acid oxalic M M orthophosphoric acid orthophosphoric M 16:84 ACN - water water - ACN nm 400 @ng/g ACN UV 5 min 25 SDM, SMM, ng/g 50 containing containing KNOj M 0.1 55:45 SSZ, 0.005 - ACN - THF 29:1:0.06:69.94 ACN - MeOH - - MeOH - ACN ATH C N- - F gradient composition THF - MeOH gradient 20:15:65 acid acid 3:1:6 0.02 min 8 0.025

(C8)

ODS Hypersil ODS 3 pm 3 ODS Inertsil pm S Nucleosil C|, Nucleosil Nucleosil 3 C„ 3 Nucleosil pm 3 PLRP-S MOS-Hypersil polystyrene- divinylbenzene pm 5 pm3

Bond Elut NH] Elut Bond homogenization SPE using SPE Alumina liquid/liquid Bond Elut™ Cjor polystyrene- Elut™ Bond on-line Baker 10 Amino using SPE 10 Baker Cartridge divinylbenzene SPE using SPE

homogenization

eel,

muscle & liver liquid/ liquid homogenization salmon liquid/ liver & Atlantic muscle using SPE RBT, C,s Elut fish, yellowtail, bream Bond sweet sea red meat RBT using SPE yellowtail liquid eel yellowtail liquid/ homogenization sweetfish, salmon plasma salmon eel, RBT, eel, bream, sea red Atlantic salmon Atlantic yellowtail tissue yellowtail plasma

SMR SDM, SMM, SDM, PA, OA, NA, DFZ, FZD, FZD, NFS, FZD, Furazolidone SSZ, PA, NA, OA, NPN, FMZ, NPN, SSZ, Acid NalidixicAcid Piromidic SDM, SMM, SDM, Oxolinic Acid Oxolinic Flumequine Flumequine Oxolinic Acid Oxolinic Acid Oxolinic table con'd.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

10 ng/g FEQ ng/g 10 using fluorescence using OA ng/g 5 5 ng/g 5 3 ng/g FEQ ng/g 3 2 ng/ml 2

nm 2 ng/g OA nm ng/g 2 260 nm excitation 2 ng/g FEQ ng/g 2 excitation nm 12 min 12 13 min 13 15 min 15 min 12 fluorescence OA ng/g 4 fluorescence fluorescence 380 nm emission muscle and liver and muscle emission nm 380 FEQ ng/g 7 excitation nm 325 @UV min 8 fluorescence 260 365 nm emission nm 365 or excitation nm 325 emission nm 365 excitation nm 260 emission nm 380 excitation nm 324 emission nm 363 excitation nm 245 emission nm 350 lg

H j P+ 0 4

MeOH - ACN - fluorescence - ACN - MeOH ACN - THF fluorescence OA THF ng/g - ACN 0.5 fluorescence ACN - THF - ACN fluorescence

[(3g H j P 0 4-

M M orthopliosphoric acid orthopliosphoric M acid orthophosphoric M M citric acid ' acid citric M 125:200:675 0.002 65:20:15 ACN - THF - THF - ACN 0.02 20:14:66 - THF - ACN 0.02 64:21:15 20:15:65 M 0.002 H,P04 0.1 60:30:5:5 - DMFwater] - ACNml TMAC)/675 THF

PLRP-S polymer PLRP-S divinylbenzene PLRP-S S pm S polystyrene- pm 5 divinylbenzene pm S polymer PLRP-S Nucleosileel plasma, plasma, liquid-liquid polystyrene- pm S Hypersil ODS pm 3 Nucleosileel

liquid/ liquid liquid/ homogenization cyano column cyano homogenization liquid/ liquid dialysis liquid/ on-line divinylbenzene liquid liquid/ SPE using C2 or C2 using SPE on-line SPE using SPE on-line polystyrene- homogenization on line dialysis line on on-line SPE using SPE on-line polystyrene- divinylbenzene liquid homogenization liquid/ homogenization liquid/ liquid liquid/

fish muscle, muscle, homogenization fish liver, hide, liquid/ liquid hide, liquid/ liver, Atlantic salmon Atlantic liver muscle kidney, intestine or intestine kidney, Atlantic salmon Atlantic muscle & liver & muscle salmon & RBT & salmon salmon Atlantic muscle aquaria water aquaria

Flumequine Oxolinic Acid Flumequine Oxolinic Oxolinic Acid Oxolinic Oxolinic Acid Flumequine Oxolinic Flumequine Flumequine Oxolinic Acid Flumequine Oxolinic table con'd.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [2.83] [2.84] [2.78] [2.81] [2.82] [2.80] inj) [2.79] inj)

ng/g 1-2 60 ng/g liver ng/g radioactivity 60 using ng/g ng/g 50 100 ng/ml Muscle 50 kidney serum liver, bile 100 ng/ml 100 bile 50 ng/g 50 muscle ng/g 20

nm 1 ng/ml nm ng/ml 1 260 nm or 10 ng/g each ng/g 10 or nm 260 258 @ 10 min 10 fluorescence UV @ 260 nm 260 @UV UV @ 257 nm 257 @UV radio-label monitoring fluorescence emission nm 369 UV 325 nm excitation nm 325 emission nm 365 327 nm excitation nm 327 min 4 65 min 65 min 65 @ UV 2HjO

min 15

M oxalic M H 20) KH2P04 • • KH2P04 0.01 g/1 M GAAM M GAAM

(7.5 0.05 0.05 MeOH - - MeOH M phosphate buffer - ACN UV @ 278 nm ACN 278 @ UV- buffer phosphate M g/1 Na2HP04 • • Na2HP04 g/1 M Na H2P04 - ACN - H2P04 Na M 2.5 gradient composition gradient composition - MeOH gradient MeOH - - MeOH acid 3:1:6 4:6 0.05 65:35 ACN 0.015 65:35

10 pm 10 pm 10 4 pm 4 + Regis Pinkerton GFF ACN - 0.1 M KH2P04 UV @ 254 nm 10 ng/ml serum (direct KH2P04 C„ M 0.1 (direct Nova-Pak - ACN - serum GFF MeOH nm ng/ml 254 @ UV 10 Pinkerton Regis pm 5 ODS-3 Partisil DEAE-2SW ISRP ISRP 1:9 muscle min 12 liver, ng/g 10 5 pm 5 C)g Versapak ToyoGel ODS L-column Versapak C„ Versapak

inj.

muscle, liver muscle, SPE using SPE direct - serum homogenization liquid/ liquid liquid/ or liquid/ liquid Accell Sep-Pak liquid/ or Cjg Elut Bond liquid/ liquid homogenization liquid/ On Elut Bond homogenization SPE using SPE homogenization using SPE

yellowtail, muscle, liver muscle, RBT serum RBT salmon muscle salmon RBT serum, RBT red sea bream, sea red channel catfish MSPD catfish bile & channel MSPD muscle catfish liver & channel muscle amago &serum, RBT salmon & RBT muscle RBT & eel, muscle, liver, muscle, kidney, bile kidney,

Oxolinic Acid Oxolinic Oxolinic Acid Oxolinic M-l metabolite M-l Oxolinic Acid Oxolinic Oxolinic Acid Oxolinic Miloxacin Nalidixic Acid Nalidixic Nalidixic Acid Nalidixic table con'd.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ] ] o\ ui 86 88 . . 2 2 [2.90] [2.91] [2.85] [ [2.89] [2.92] [2.87] [

1.25 ng 1.25

nm 1 ng/ml serum ng/ml nm 1 nm nm ng/ml 100 289

223 ® each ng/g 10 222 nm nm ng/g 222 30 nm 225

278 nm nm 278 — ® ® ® ® ® 12 min 12 fluorescence ng/g 5 fluorescence fluorescence 5 ng/g enrofloxacin ng/g 5 fluorescence 278 nm excitation using fluorescence using excitation nm 278 emission nm 440 UV UV 440 nm emission nm 440 min 7 emission nm 440 min 8 UV UV 225 & 270 nm 270 & 225 min 30 40 min min 40 column) on ng UV (3 min 20 278 nm excitation nm 278 sarafloxacin ng/g 10 excitation nm 278

H j P 0+ 4

- ACN - UV

H j P 0 4 M M H3P04 - ACN - MeOH - ACN - H3P04 M M H3P04 - ACN - MeOH - ACN - H3P04 M g TMAC)/700 ml water] ml TMAC)/700 g M citric acid citric M 15:85 15:42.5:42.5 15:18 1.5:1.5:7 8:2 8:2 tissue ng/g 1 fluorescence or 0.002 MeOH - water - MeOH 73:19:8 g A CD N M- [(1.13 F- MN a 2HP04- MeOH-0.02 0.002 72:20:8 65:35 0.01 water - MeOH 0.002 0.38 water - ACN - water

PLRP-S polymer PLRP-S PLRP-S polymer PLRP-S 5 pm 5 pm 5 RLRP-S pm 5 ODS Chromatorex polymer ODS C„ Nucleosil ODS-120T pm 5 C„ Nucleosil Spherisoib-5 TSK gel TSK p5 m

serum - serum homogenization liquid/ liquid liquid/ SPE using Cj using SPE muscle & liver - liver & muscle SPE using C„ using SPE using SPE liquid homogenization liquid/ Bond EluteRCj Bond homogenization liquid/ liquid liquid/ liquid liquid/ homogenization SPE using SPE Sep-Pak Florisil Sep-Pak liquid homogenization liquid/ Florisil Sep-Pak liquid/liquid SPE using Florisil using SPE using SPE

fish serum fish Atlantic salmon Atlantic muscle, liver muscle, & RBT serum, RBT & Atlantic salmon liver Atlantic muscle, catfish & RBT African muscle yellowtail tissue yellowtail muscle yellowtail salmon Atlantic yellowtail tissue plasma yellowtail Enrofloxacin Sarafloxacin Enrofloxacin Ciprofloxacin Ampicillin Florfenicol Chloramphenicol Florfenicol Sarafloxacin Thiamphenicol Thiamphenicol table con'd.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o\ o\ [2.97] [2.96] [2.95] [2.93] [2.94]

ng/L chromatic form form chromatic ng/L ng/L leuco form leuco ng/L 10 ng/g liver ng/g 10 5 ng/g 5 2.01 2.83

sulfisozole tetracycline trimethoprim sulfamonomethoxine sulfapyridine nm low ng/g range nm ng/g low 290 SSZ SMM SP TC THFTMAC tetrahydrofuran TMP tetraethylammoniumchloride 17 min 17 fluorescence ng/g 1 fluorescence 470 nm emission nm nm 470 254 @UV or UV @ 210 nm 210 @UV UV @ 615 nm 1 ng/g muscle nm ng/g 615 @ UV 1 min 6 35 min 35 25 min 25 excitation nm 340

- ACN - @ UV H j P 0 4

M Na acetate + nm 618 @ UV acetate Na M silica

N P04 - ACN - THF - ACN - P04 j (0.05 0.05 min 30 SMT sulfamethazine SI SMR sulfamerazine SDZSDM sulfadiazine sulfadimethoxine PARBT piromidic acid rainbow trout OTC oxytetracycline M H M M GAA) M 1:1 MeOH - water - MeOH 85:15 water - ACN - water 75:25 water - - water 6:6:88 49:40:1 MeOH - - MeOH gradient flow rate flow gradient 0.1 0.02

and immobilized and 10 pm 10 pBondapak C,8 pBondapak Supelcosil LC-18 Supelcosil and C, C„ Hypersil Nucleosil each pm 5 reactor enzyme MCH-10 polymer PLRP-S Pb02 and postcolumn reactor postcolumn furamizole flumequine furazolidone nalidixic acid nifurpirinol nifurstyrenate oxolinic acid 5000 & 5000 FEQ FMZ NA NFS OA NPN liquid/ liquid pm liquid/ 5 homogenization MicroPak C,t homogenization MicroPak SPE using SPE Lipidex Lipidex liquid/liquidpm 5 SI and C2 liquid/liquid liquid/ liquid liquid/ homogenization centrifugation using SPE silica Sep-Pak SPE using Bond Elute Bond using SPE muscle, homogenization muscle, diatomaceous earth muscle, diatomaceous fish fat, liver, kidney liver, liver urine plasma, kidney, SPE using SPE kidney, plasma, contents, bowel tap water diol tap 10 Baker 17oi-methyl- muscle RBT homogenization 17oi-methyl- Malachite Green pond & pond Green Malachite Febendazole channel catfish channel Febendazole & muscle RBT Green Malachite Praziquantel Praziquantel testosterone ACN acetonitrile CAP chroramphenicol DAHP diammoniumhydrophosphate GAA glacial acetic acid DMF dimethylformamide Abbreviations (Table 2.1) CTC chlortetracycline FZD DEA diethanolamine DFZ difurazone

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 7

polystyrene gel, ion-pair, polystyrene divinylbenzene, and

Regis Pinkerton internal surface reverse phase. Detection

by UV using fixed or variable wavelength detectors or diode array is most frequently reported, but fluorescence detection gives greater sensitivity for the tetracycline and quinolone antibiotics. Reported limits of detection

for some of the LC methods listed in Table 2.1 are above

the current MRLs of many countries and only one of the

methods has undergone interlaboratory validation studies

[2.80,2.98]. Therefore, legally defensible LC methods validated for use about the MRL are needed for the analysis of veterinary drugs and their important metabolites in edible fish tissues.

GAS CHROMATOGRAPHY

There are a limited number of publications involving

the application of gas liquid chromatography (GLC) to the analysis of veterinary drugs used in aquaculture [2.99,

2.203-2.207]. Many of the compounds used therapeutically in aquaculture are of high molecular weight, relatively

non-volatile, and thermally labile; therefore chemical

derivatization is generally required to obtain sufficient

volatility and stability for GC analysis. Additionally, the methylation of sulfonamides with diazomethane prior to

GLC produces N'-methyl and ring-methyl derivatives. The

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 68

formation ratio of the two isomers is variable and the ring-methyl derivative does not elute from the GLC column.

The incorporation of deuterium or 13C labelled sulfonamides

prior to derivatization and the use of isotopic ratios corrects for variable isomer formation but increases the cost and complexity of the analysis [2.208],

As a result of the extensive sample preparation and cleanup necessary prior to derivatization, GLC is not as

frequently utilized as LC in drug residue analysis. Early

reports by Allen and Sills for the GC analysis of the fish

anesthetic quinaldine used a packed column and an alkali flame detector and had a limit of quantitation of 0.01

Mg/g [2.203,2.204]. A method for determination of sulfamethazine residues in swine muscle is the only AOAC

OMA method for the GLC determination of a veterinary drug residue in animal tissue [2.41]. A GLC-MS method for determination of chloramphenicol residues at the low

parts-per-billion level in shrimp tail muscle has since undergone interlaboratory validation and is in press

[2.99-2.208]. A multi-residue GLC method for thirty-six veterinary drugs in red meat and poultry has been reported

[2.209], A number of these drugs or related compounds are also used in aquaculture and the methods could potentially

be applied to food fish tissues. Additionally, techniques

employing GLC separation and mass spectrometric detection

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 9

of quinolone antibacterials in fish tissue are described in the following section.

HASS SPECTROMETRY

'Absolute' confirmation of the presence of a compound

as required for regulatory action may often be secured using gas liquid chromatography/mass spectrometric

detection and confirmation (GLC/MSD). Although such

methods provide quantitation and identity confirmation, a

GLC/MSD method for sulfamethazine residues in swine tissues is the only AOAC OMA validated method available

[2.41]. Only two methods were found in the literature for GLC/MSD determination of veterinary drug residues in

aquatic animals. Takatsuki reported a GLC/MSD method for oxolinic acid and a similar multi-residue GLC/MSD method for determination of oxolinic acid, nalidixic acid, and

piromidic acid in silver salmon using selected ion

monitoring [2.205,2.206]. In both methods, reduction of oxolinic acid, nalidixic acid, and piromidic acid with tetrahydroborate (sodium borohydride) gives compounds that

are sufficiently volatile for GLC. The method was reported capable of performing quantitative analyses with a detection limit of 0.003 fiq/g, but the classical sample

preparation technique is complex and time consuming.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Liquid chromatography/mass spectrometric detection (LC/MSD) is becoming more available and because of

simplified sample preparation requirements, may someday

exceed GLC for the purpose of confirming many of the polar

drugs used in aquaculture. At present however, only two reported applications of LC/MSD determination of

veterinary drugs in aquaculture products could be found. Pleasance et al. determined sulfadimethoxine residues to

0.025 Mg/g in salmon flesh using LC/MSD and LC/MSD/MSD via

a ion-spray interface [2.62]. A thermospray LC/MSD method

reported by Horie et al. provided quantitation and identification of residues of naladixic acid, oxolinic

acid, and piromidic acid in sweet fish and yellowtail muscle to 0.05 jug/g [2.210]. Thermospray LC/MSD is a

relatively mild ionization method and only molecular weight information from MH+ ions is generally obtained.

Since fragment ions are not produced, the additional

molecular and structural information useful in

identification of organic compounds is lacking. Nonetheless, the combination of molecular weight, chromatographic retention time, and UV spectrum

information should provide sufficient verification for regulatory purposes.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7 1

OTHER RESIDUE DETECTION METHODS

Veterinary drug residues have also been readily

detected by a variety of other means. In this regard, the fluorescence of tetracyclines under UV light has been used as an indicator of previous tetracycline treatment. This

technique has been used for the detection of oxytetracycline residues in bone and injection sites, but

fluorescence is non-specific and persists in bone for an

extended time after treatment [2.120,2.211]. Because of

its persistence in bone, oxytetracycline has been used for long term identification of released fish in stocking studies [2.212].

Experimental methods using radiolabelled drug and

liquid scintillation counting [2.114,2.195,2.196,2.213, 2.214] or whole body autoradiographic studies [2.215- 2.218] have been used to provide information on

pharmacokinetic behavior, metabolite formation, disposition, depletion rate, and extraction efficiencies

for compounds used in aquaculture. These methods are,

however, unsuited for use in routine drug residue analysis.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 72 METHODS OF ISOLATION

Determinative technologies for residue analysis require the use of tissue isolation methods that are

simple, fast, and efficient. Sample preparation,

isolation, and cleanup are becoming the major rate- limiting factors in sample analysis as improvements in

analytical methods proceed [2.219]. This fact is

especially important in light of efforts to introduce

rapid screening tests such as immunoassays. The choice of a sample extraction method for a particular application is dependent upon what the analyst must or needs to

accomplish and is determined by several interrelated

factors. These include the following variables:

a. The sample size available or necessary to obtain a given limit of detection with the analytical

instrumentation available.

b. Matrix characteristics and the availability of

existing methods for analyte isolation from the specific matrix.

c. Specificity requirements of the analysis; i.e.,

isolation of a single compound within a drug class, several compounds within a drug class, or several drug classes from a single sample.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 73 d. Number of samples analyzed daily and turn-around time

requirements.

e. Overall cost of the method including supplies and disposables, time and labor involved, and the costs of the instrumentation that will be applied [2.220].

Several approaches have been used over the years for

the preparation, isolation, and cleanup of veterinary drug, pesticide, and environmental contaminant residues

from aquatic matrices. Historically, the classical

approach to isolation of drugs from tissues has most often

been reported [Table 2.1]. This approach involves tissue homogenization followed by liquid-liquid partitioning of the homogenate, with or without additional cleanup or concentration steps. Liquid-liquid partitioning may also

be used with biological fluids. Homogenization and liquid-liquid partitioning methods may provide adequate

separation of the drug from matrix but are often expensive

in terms of time, labor, material use, and organic solvent disposal costs. Such approaches also tend to be highly

nonspecific in their isolation of the target drug(s). Furthermore, these traditional isolation methods may

be generating more contamination than they are

satisfactorily removing. Table 2.2 provides an indication of the problem by listing the volumes of solvent required and waste solvent produced for extraction of various drugs

from aquatic animal tissues.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

[2.50] [2.49] [2.54] [2.47] [2.46] 47 [2.48] 90 -0--0- [2.51] 430 350 [2.45] 50 (serum) 100 100 (tissue) 8 5 -0- 5 5 10 11 9 82 16 29 20 -0- 825 750 100 -0- (ml) (ml) Used (ml) Waste SPE using Amberlite XAD2 SPE using Bond Elute Cl8 SPE using Amberlite XAD2 SPE using Sep-Pak C,8 Homogenization liq / liq SPE using Bond Elut Cl8 Bondasil C8 or Bondasil C8 C18 MSPD homogenization homogenization homogenization trifluoracetic acid homogenization SPE using protein precipitation using fish homogenization RBT muscle & liver fish liver, muscle, RBT muscle salmon muscle RBT serum, liver, and muscle RBT plasma slime, hide, vertebrae METHODS FOR DRUGS USED INAQUACULTURE Compounds Matrix Sample Preparation Organic Solvent Organic Waste Aqueous Reference tetracycline oxytetracycline chlortetracycline tetracycline oxytetracycline oxytetracycline chlortetracycline oxytetracycline oxytetracycline oxytetracycline oxytetracycline channel catfish muscle oxytetracycline TABLE 2.2 COMPARATIVE SOLVENT USE AND HASTE PRODUCTION OF REPRESENTATIVE EXTRACTION table con'd.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ui ] 58 . 2 - - [ 0 - 9

5 5 5 19 [2.71] 10 16 16 8 -0- [2.60] 10 10 10 6 [2.70] 10 10 10 50 [2.74] 25 57 5 22 10 [2.59] 30 [2.66] 65 55 -0- [2.69] 367 95 70 [2.68] 2 liq / liq liq /liq SPE using Sep-Pak C,8 MSPD SPE using Bond Elut NH2 homogenization homogenization SPE using Alumina homogenization SPE using Baker 10 Amino liq / liq cartridge liq / liq homogenization homogenization SPE using Bond Elute C homogenization on-line dialysis on-line SPE using polystyrene-divinylbenzene liq / liq on-line dialysis homogenization on-line SPE using polystyrene-divinylbenzene catfish muscle, liver, chinook salmon muscle meat channel catfish muscle Atlantic salmon muscle & liver eel, RBT, sweetfish, red sea bream, salmon plasma eel, yellowtail, RBT yellowtail tissue Atlantic salmon liver Atlantic salmon muscle sulfadimethoxine sulfadimethoxine sul fadimethoxine sul furazolidone ormetoprim kidney OA, NA, PA FZD, DFZ, NPN, FMZ, SDM, SMM, OA, NA, PA, ormetoprim flumequine SSZ, SMR flumequine oxolinic acid flumequine oxolinic acid oxolinic acid table con'd.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ] 86 . 2 [2.85] [2.82] [2.83] [ [2.87] [2.76] [2.77] [2.78] [2.79] [2.80] [2.81] - - - - ; 1 8 0 3 0 0 4 9 4 0 0 30 75 - - - - - 300 - 1 8 8 2 2 2 0 5 11 60 42 -0- - -0- rCF 4 4 2 2 10 16 16 11 43 26 61 JQ- 4.2 260 liq / liq liq / liq MSPD SPE using C„ liq / liq SPE using Sep-Pak Accell or liq / liq homogenization direct injection homogenization SPE using Bond Elute C18 homogenization MSPD liq / liq SPE liq / liq SPE liq / liq homogenization homogenization homogenization Atlantic salmon muscle RBT serum fish serum muscle, liver salmon muscle eel plasma, aquaria water RBT serum Atlantic salmon & RBT channel catfish muscle, bile channel catfish muscle, liver RBT & Amago salmon serum muscle, liver Atlantic salmon muscle, liver serum, muscle, liver, kidney, bile flumequine flumequine oxolinic acid oxolinic acid oxalinic acid sarafloxacin enrofloxacin sarafloxacin oxolinic acid nalidixic acid enrofloxacin naladixic acid table con'd.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3 3 0.6 -0- [2.88] 2 2 -0- [2.95] 49 12 7 [2.93] 134 134 130 -0- [2.89] SPE using Sep-Pak Florisil liq / liq SPE using diatomaceous liq / liq SPE using Baker diol 10 homogenization homogenization earth RBT & African catfish plasma yellowtail tissue channel catfish plasma, kidney, fat, muscle, bowel contents, urine pond & tap water fenbendazole ciprofloxacin ampicillin malachite green See Table 2.1 for list of abbreviations

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 78

Many of these solvents are of greater toxicological and

environmental concern than the compounds they are used to isolate. During extraction and isolation procedures, much of this solvent volume is evaporated into the atmosphere, which contaminates millions of cubic feet of air, and

solvents are often not disposed of properly, which leads

to further contamination of the atmosphere, aquifers, and

aquatic habits and resources.

As previously mentioned, a further problem with excessive use of solvents is that they make these methods

very expensive to perform. The purchase price and subsequent disposal costs of organic solvents and wastes can be limiting factors in analyses performed by

government agencies operating on a restricted budget.

Employee costs can also be a limiting factor in residue analysis. Present official methods generally require

extensive training of laboratory technicians in order to guarantee consistent, reliable results and most such

methods are not amenable to automation. Therefore, costs for materials and labor can limit the number of samples

which can be consistently analyzed to provide adequate

regulatory enforcement and consumer protection from illegal residues and to provide a statistically sound evaluation of the magnitude of contamination. Any method for the isolation of drugs or other

chemical contaminants must take these factors into

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consideration. Thus, extraction methods that use large sample sizes and, thus, require large volumes of organic

solvent to adequately perform residue isolation are becoming increasingly unacceptable. All of the above problems indicate that the use of classical

homogenization/extraction and determinative methods for

screening purposes should be severely curtailed and phased out as new more appropriate methods are developed and

validated for sample screening.

EVOLVING METHODS OF RESIDUE ANALYSIS

Recent advances in the field of residue analysis

offer several promising techniques as possible solutions

to the problems caused by outmoded and complex analytical methods. Four techniques, solid phase extraction (SPE),

immunoaffinity, supercritical fluid extraction (SFE), and

matrix solid phase dispersion (MSPD), are receiving

particular attention because they have the potential to greatly reduce analysis costs and reduce analyst-generated waste and pollution. All of these methods may prove

useful for a given application.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 80 SOLID PHASE EXTRACTION

In the SPE process, a compound is isolated from a

liquid sample based on its relative solubility in the liquid mobile phase compared to its solubility in a solid

support-bound liquid stationary phase of differing

polarity or its affinity to a solid support stationary phase of differing polarity. Isolation is accomplished by

passing the analyte dissolved in solvent (organic or aqueous) through a column containing the stationary phase

with subsequent elution using an appropriate solvent.

Several solid phase extraction methods have been developed to facilitate the extraction and cleanup of biological liquid and tissue samples.

For plasma, acceptable residue recovery may be

obtained using protein precipitation and direct LC injection without prior cleanup with SPE [2.51]. However,

the many impurities present can affect the chromatogram

and accumulate on the LC analytic column, thus resulting in increasing back pressure and decreased column life. SPE cleanup helps avoid these problems and works well with

biological fluids such as serum, plasma, urine, and cerebral spinal fluid. In addition, SPE extraction and

analysis can be automated and done on-line [2.70,2.71, 2.74] and/or with on-line dialysis and column switching.

However, before SPE can be used with solid tissue (e.g.,

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muscle and liver), a separate homogenization step and

often multiple filtration, sonication, centrifugation, and

liquid-liquid cleanup steps are required. While SPE may

improve cleanup of these solid tissue samples, the additional labor and materials costs make SPE less suitable, in some cases.

Solid phase extraction methods published for fish tissues are often combinations of SPE with other methods

such as homogenization, liquid-liquid partition,

filtration, sonication, and centrifugation (Table 2.1).

Because choice of SPE column depends on the matrix and on the particular compound of interest, a wide range of solid phase columns of differing polarities have been used for drug extraction in fish and include C2, C8, C18, NH2,

amberlite resins, and PLRP (polystyrene-divinylbenzene) polymers (Table 2.1).

IMMUNOAFFINITY

The simplest method of extraction however, is one

that requires minimal or no sample manipulation. These

are the methods that /extract' the drug directly from the sample matrix by means of specific or selective antibodies

or receptors. High performance immunoaffinity

chromatography (HPIAC) is one such method that combines immunoassay and LC. Immunoaffinity techniques may be

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either single or multi-residue methods depending on the specificity of the antibody and the number of specific

types of antibodies immobilized on the beads. These

techniques may be categorized as 1) single antibody/single analyte, 2) single antibody/multi-analyte, or 3) multi­ antibody/multi-analyte [2.221,2.224].

In HPIAC, a flow-through cartridge containing antibody immobilized on glass or porous beads is used in place of an LC analytical column. Following sample

injection, the drug (antigen) is captured on antibody-

coated beads, the cartridge washed to remove unbound material, and the drug eluted from the cartridge.

Detection may be by UV or other LC detection methods. The

cartridge is regenerated by passage of several volumes of

equilibrium buffer. Assay formats other than the on/off format that may be used include sequential, competitive, or sandwich assays. The sandwich assay format uses a second enzyme-tagged antibody to form a three-part layer

consisting of tagged antibody, bound antibody, and immobilized antibody. The limits of detection of the on/off, competitive, and sandwich assay are 100-500 ng, 10

ng, and 10 pg respectively [2.221]. These methods are most applicable to aqueous solutions of the drugs, such as urine, serum, plasma, or tissue homogenate supernatant.

Immunoaffinity isolation and chromatography provides

great specificity for the target analyte(s) and the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 83 ability to concentrate the analyte(s) to the level required for detection and measurement. The specificity

of antigen-antibody bonding, although not absolute, is

much greater than physico-chemical isolation and separation systems now available. While immunochemistry

methods typically possess high specificity, cross­

reactivity with other drugs or their metabolites or naturally occurring compounds in the sample can occur and

lead to false positive results. A further concern is that the drug may remain bound to sample proteins or be in

complexes that do not bind with a given antibody, leading to false negative results or a reduced response. This is

especially of concern for drugs like the tetracyclines

that possess both high protein binding and complex formation potential [2.220].

Immunoaffinity isolation techniques have most commonly been used for sample cleanup prior to

conventional analysis. One such clean-up method uses a

small syringe barrel or column containing antibody-coated

beads or Sepharose™. The sample is applied to the column to isolate and concentrate the target analyte and the

column washed to remove unbound sample material. Following elution, the isolate may be analyzed off-line by GC-MS [2.225,2.226] or another determinative method

[2.222]. Such columns may also be fashioned in a co­

column arrangement with SPE, MSPD or other methods of

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isolation to provide a highly selective extraction process. Applications of immunoaffinity isolation or

chromatography to veterinary drug residue analysis have

been limited but techniques are reported for nortestosterone and methyltestosterone in beef muscle

[2.225]; the jS-agonists clenbuterol, salbutamol,

terbutaline, cimaterol, and mabuterol in bovine and porcine urine and liver [2.226]; the anabolic agents

zeranol and its metabolite /3-zearalanol in calf urine

[2.227]; and chloramphenicol residues in milk, eggs, and swine muscle [2.228,2.229].

Although the application of immunoaffinity techniques to the isolation and analysis of veterinary drug residues

is a recent development, this technique has great potential for improving single or multi-residue drug analysis in aquatic and other species. High performance

liquid chromatography compatible cartridges containing

"flow through" porous beads coated with Protein A or

Protein G for antibody immobilization have recently become commercially available [2.221,2.230]. The main limitation to greater use of immunochemical techniques for residue

isolation and analysis is a lack of commercially

availability antibodies to many of the veterinary drugs of interest as residues [2.223].

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SUPERCRITICAL FLUID EXTRACTION

With the supercritical fluid extraction (SFE) process, supercritical fluids (usually supercritical carbon dioxide [SC-C02]) are used in place of organic

solvents to extract residues. The solubility of a

compound in a supercritical fluid is determined in part by the density of the extracting fluid. Therefore, the

solubility of compounds in SC-C02 can be varied by changes

in temperature and pressure or the addition of organic

solvent modifiers [2.231]. Carbon dioxide becomes a supercritical fluid if handled above its critical temperature and pressure. Although several other

compounds have suitable critical points and could be used for SFE, C02 is naturally occurring, easily obtainable,

inexpensive, non-flammable, non-toxic and can be vented to the atmosphere. Therefore, the use of SC-C02 as an

extracting fluid or chromatography mobile phase eliminates the need for large volumes of organic solvents [2.232,2.233].

The majority of reported SFE applications have been

for lipophilic environmental pollutants such as pesticides

and polycyclic aromatic hydrocarbons which are readily soluble in SC-C02 [2.233,-2.236]. However, various other

lipophilic chemicals and associated tissue lipids are also

soluble in the non-polar liquid SC-C02, and are co­

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extracted and concentrated once the pressurized C02 is

brought back to atmospheric pressure. Because the

extracts contain contaminating lipid-soluble materials, a

pre- or post-extraction cleanup step is usually needed

before samples can be injected onto analytical instruments

such as LC or GLC apparatuses. Cleanup is usually accomplished with gel permeation chromatography or Florisil adsorption chromatography [2.233]. In-line

cleanup could be conducted by using disposable or reusable

SPE cartridges or newer disc SPE technologies and changing

the pressures of the supercritical fluid. Coupling this

system directly to an LC/MSD type interface or a GLC/MSD interface could provide a complete analytical process for

the desired analysis [2.237]. A newly introduced

automated system combining SFE and supercritical fluid chromatography (SFC) is also available. Detection for SFC

is by one of the several types of gas or liquid phase detectors.

The application of SFE to the isolation of polar

drugs from animal tissues poses an additional problem.

Extraction of these compounds from animal tissues is

complicated by the fact that polar drugs are likely to have greater solubility in the aqueous phase of the tissue than in the non-polar SC-C02 extracting fluid. Thus, a

decreased extraction efficiency of polar compared to non­

polar compounds results. The solubility of polar analytes

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in SC-C02 may however be enhanced by the addition of co­

solvents such as methanol or ethanol to the SC-C02

extracting fluid [2.232,2.238]. Unfortunately, the addition of such extraction modifiers to SC-C02 also

enhances the recoveries of lipids and other non-polar

compounds, thus decreasing the selectivity of the

extraction [2.235-2.236]. Only four reports of SFE/SFC of polar veterinary

drugs used in food animals could be found. Ramsay et al.

isolated sulfamethazine, trimethoprim, and three steroids

from homogenized and freeze-dried fortified swine kidney using SFE with a gradient mixture of methanol(0-20%)/SC- C02. SFC/tandem mass spectrometry was carried out using a

moving belt interface. The detection limits of the assay

were above tolerances for these compounds [2.237]. Perkins et al. examined the use of SFC for the

simulatanous analysis of nine sulfonamides in standard

solutions. Sulfamethazine analysis was also examined in

porcine kidney tissue fortified at 3.3 pg/g. The method of detection was UV absorbance or MS. Interface of the LC

with MS was accomplished by moving belt or thermospray

techniques [2.238]. In a similar study, the same authors examined SFC-MS analysis of standard solutions of four

veterinary drugs - chloramphenicol, furazolidone, levamisole, and lincomycin [2.239]. Cross et al. studied

the solubility of six sulfonamides in various SFE media

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and their extraction from chicken liver and swine muscle following blending of the tissues with sand. LC analysis

of extracts indicated highly variable recoveries among

sulfonamides and ranged from 27-97% [2.232],

More work will be necessary to further develop the

SFE/SFC process. The application of these techniques to

fish tissues [2.234] has been quite limited. However, these processes have the potential to provide a near

solventless, in-line, automated process for the rapid

analysis of chemical residues from edible aquatic resources.

MATRIX SOLID PHASE DISPERSION

Of the three techniques being considered, matrix solid phase dispersion, in particular, has the strongest potential to meet the demands of future residue monitoring

of aquatic resources for drugs and environmental

pollutants. In general terms, the process involves blending a tissue sample (0.1-1.0 g) with lipophilic polymer-derivatized silica particles (e.g., octadecylsilyl

[ODS]-derivatized silica [C,8]), which simultaneously

disrupts and disperses the sample. This blend of C18 and

tissue becomes part of a potentially multiphasic column that possesses unique chromatographic character. Elution

of the MSPD column with a solvent or solvent sequence can

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provide a high resolution fractionation of target analytes that can be further purified by simultaneous use of co­

columns of Florisil, silica, alumina, C18 or other silica-

bonded phases. The final eluate can, in most cases, be

directly analyzed or further concentrated or manipulated to meet the demands of the individual analysis. The

extracts obtained from these methods are most often detected by LC (in the case with drugs) or GLC with

electron capture detection (in the case with pesticides)

or mass spectrometry. Additionally, when reconstituted in

an appropriate buffer, the extract can also be used in immuno- [2.144,2.172] or other receptor assays. Additionally, the MSPD process is generic and can be

modified for a particular application by 1) a change in

the eluting solvent or solvent sequence, 2) use of a

different polarity polymer or solid support, and 3) blending of the Ci8/tissue in the presence of modifiers

such as chelators, acids, bases, etc.

MSPD could also be used in conjunction with SFE. The water in biological matrices often interferes with the SFE

extraction process and analysts have used samples blended

with diatomaceous earth to remove water from the sample [2.233]. However, blending samples first with polymer- coated silicas, as is done in the MSPD process, would

remove water and provide an initial stage of fractionation

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at the point of elution of the analytes with supercritical fluid and modifiers.

In general, the three main advantages of MSPD are 1) it allows for rapid turnover of samples and hence, access

to timely data on residue levels present in samples, 2) because of its required small sample size, it considerably

decreases solvent use compared to the classical methods, which in turn decreases environmental contamination and

increases worker safety, and 3) it is suitable for robotics automation. Therefore, MSPD has the potential to

meet the future demands for conducting drug and pesticide analysis for large numbers and varieties of samples.

MSPD APPLIED TO AQUATIC RESOURCES

As seen in Table 2.1, MSPD has been used to provide

for single or multiresidue analysis of various veterinary

drugs in several aquatic matrices. Veterinary drugs

isolated from aquatic animal tissues by this method include oxytetracycline [2.54], sulfadimethoxine and 4-N- acetylsulfadimethoxine from fortified channel catfish

muscle [2.60,2.65,2.144], and oxolinic acid as an incurred residue from channel catfish muscle and bile [2.81]. Reimer and Suarez reported a multi-residue method for MSPD isolation with HPTLC or LC analysis of five sulfonamides

in fortified salmon muscle [2.63,2.200]. Jarboe et al.

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[2.82] have demonstrated its applicability to the

isolation of incurred residues of nalidixic acid from

channel catfish muscle and liver. Walker and Barker evaluated the performance of several enzyme immunoassays

for the detection of sulfadimethoxine residues using MSPD extracts of fortified channel catfish muscle as well

[2.144]. Other compounds used in aquaculture or related compounds have been extracted from various non-aquatic

matrices using MSPD methods [2.172,2.240] and these methods could potentially be applied to aquatic matrices.

Pesticides extracted and isolated from aquatic animal tissues by this method include 14 chlorinated hydrocarbon pesticides from fortified whole oyster homogenate and

crayfish hepatopancreas [2.241,2.242], 9 chlorinated pesticides from fortified catfish muscle [2.243], and a variety of agricultural chemicals from natural fish kills

[2.244]. These isolation methods are a significant

advance in the ability to screen more samples because of

their simplicity and efficiency.

DISCUSSION

Methods development for residue determination should focus on rapid screening tests, multiresidue capabilities,

metabolite detection, and improved sensitivity [2.245].

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Further, the use of determinative methods generally

requires a method of isolating the compound(s) of interest

from edible or marker tissues that is rapid, inexpensive, and does not generate large volumes of organic solvents.

Classical isolation methods using homogenization and/or

liquid-liquid partitioning of biological tissues and fluids may be sufficient for some applications but are poor for screening purposes because they are often

lengthy, involving multiple steps and use large volumes of solvents (Table 2.2). Solvent disposal is becoming

increasingly expensive and environmentally unsound.

Therefore, methods using low solvent volumes are desirable. A main purpose of this review was to present a case for phasing out existing official methods in favor of

newer technologies that require less sample, less solvent, less employee time, and less cost per sample. Newer

techniques such as supercritical fluid extraction, solid

phase extraction, immunoaffinity, and MSPD offer alternative isolation strategies. When compared to the classical methods, these new methods greatly reduce labor and solvents costs and improve throughput. There are a

few drawbacks to the new methods and more work is needed

to further develop SPE, SFE, immunoaffinity, and MSPD for

use with the many different types of matrices that may

contain residues of chemical contaminants. However, of

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the three new methods, MSPD shows tremendous potential for integration into a residue monitoring program.

MSPD methods have been published for the isolation of a wide range of compounds in a variety of matrices

indicating this approach may provide a generic technique

for single or multiresidue extraction of drugs,

environmental pollutants, and their metabolites. In particular, MSPD has already been used to provide a two-

step process for the single or multiresidue analysis of various veterinary drugs and agricultural pesticides in

several aquatic matrices. This process, when compared to

classical methods, has been estimated to reduce solvent use by up to 98% and analysis time by 97%. Furthermore,

once the MSPD column is prepared, the process of solvent

elution, collection, and analysis can be automated by the use of robotics. Cost of analysis is decreased because less solvent is needed and fewer laboratory technicians

need to undergo training. Safety and environmental

protection are increased because less solvent is needed. Finally, data is generated more quickly because of the ease of the process and its potential to be automated.

These features of MSPD make it a general and perhaps significantly useful method in designing future residue

analysis screening programs for aquatic as well as other food animal resources.

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2.42 Dittert, L.W. (1977) Pharmacokinetic prediction of tissue residues. Journal of Toxicology and Environmental Health, 2:735-756.

2.43 Animal Drug Analytical Manual, J.R. Markus and J. Sherma (Editors), Center for Veterinary Medicine, United States Food and Drug Administration, Association of Official Analytical Chemists, Arlington, VA (Publisher), 1985.

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2.46 Reimer, G.J., and Young, L.M. (1990) Validation of a method for determination of tetracycline antibiotics in salmon muscle tissue. Journal of the Association of Official Analytical Chemists, 73(5):813-817. 2.47 Murray, J., McGill, A.S., and Hardy, R. (1987) Development of a method for the determination of oxytetracycline in trout. Food Additives and Contaminants, 5(l):77-83.

2.48 Nordlander, I., Johnsson, H., and Osterdahl, B. (1987) Oxytetracycline residues in rainbow trout analyzed by a rapid HPLC method. Food Additives and Contaminants, 4(3):291-296.

2.49 Bjorklund, H. (1988) Determination of oxytetracycline in fish by high-performance liquid chromatography. Journal of Chromatography, 432:381-387.

2.50 Rogstad, A., Hormazabal, V., and Yndestad, M. (1988) Optimization of solid phase extraction of oxytetracycline from fish tissue and its determination by HPLC. Journal of Liquid Chromatography, 11(11):2337-2347.

2.51 Iversen, B., Aanesrud, A., and Kolstad, A.K. (1989) Determination of oxytetracycline in plasma from rainbow trout using high-performance liquid chromatography with ultraviolet detection. Journal of Chromatography, 493:217-221.

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2.52 Ueno, R., Uno, K., Kubota, S.S., and Horiguchi, Y. (1989) Determination of oxytetracycline in fish tissues by high performance liquid chromatography. Nippon Suisan Gakkaishi, 55(7):1273-1276.

2.53 Eivindvik, K., and Rasmussen, K.E. (1989) Determination of oxytetracycline in fish plasma by high-performance liquid chromatography and column- switching. Journal of Liquid Chromatography, 12(15):3061-3071.

2.54 Long, A.R., Hsieh, L.C., Malbrough, M.S., Short, C.R. and Barker, S.A. (1990) Matrix solid phase dispersion isolation and liquid chromatographic determination of oxytetracycline in catfish muscle tissue. Journal of the Association of Official Analytical Chemists, 73(6):864-867. 2.55 Aoyama, R.G., McErlane, K.M., Erber, H., Kitts, D.D., and Burt, H.M. (1991) High-performance liquid chromatographic analysis of oxytetracycline in chinook salmon following administration of medicated feed. Journal of Chromatography, 588:181-186. 2.56 Carignan, G., Carrier, K., and Sved, S. (1993) Assay of oxytetracycline residues in salmon muscle by liquid chromatography with ultraviolet detection. Journal of AOAC International, 76(2):325-328. 2.57 Horie, M., Hoshino, Y., Nose, N., Iwasaki, H. & Nakazawa, H. (1985) Simultaneous determination of antibiotics and synthetic antibacterials in fish by high performance liquid chromatography. Eisei Kagaku 31(6):371-376.

2.58 Weiss, G., Duke, P.D., and Gonzales, L. (1987) HPLC method for the simultaneous analysis of sulfadimethoxine and ormetoprim in tissues and blood of cattle, chickens, and catfish. Journal of Agricultural and Food Chemistry, 35(6):905-909. 2.59 Walisser, J.A., Burt, H.M., Valg, T.A., Kitts, D.D., and McErlane, K.M. (1990) High-performance liquid chromatographic analysis of Romet-30* in salmon following administration of medicated feed. Journal of Chromatography, 518:179-188. 2.60 Long, A.R., Hsieh, L.C., Malbrough, M.S., Short, C.R. and Barker, S.A. (1990) Matrix solid phase dispersion isolation and liquid chromatographic determination of sulfadimethoxine in catfish

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(Ictalurus punctatus) muscle tissue. Journal of the Association of Official Analytical Chemists, 73(6):868-871.

2.61 Ueno, R., Uno, K., Kato, M., Kubota, S.S., and Aoki, T. (1991) Simultaneous determination of sulfamonomethoxine, sulfadimethoxine and their N4- acetylated forms in fish tissues by high performance liquid chromatography. Nippon Suisan Gakkaishi, 57(3):549-554.

2.62 Pleasance, S., Blay, P., Quilliam, M.A., and O'Hara, G. (1991) Determination of sulfonamides by liquid chromatography, ultraviolet diode array detection and ion-spray tandem mass spectrometry with application to cultured salmon flesh. Journal of Chromatography, 558:155-173 2.63 Reimer, G.J. and Suarez, A. (1992) Liquid chromatographic confirmatory method for five sulfonamides in salmon muscle tissue by matrix solid phase dispersion. Journal of the AOAC International, 75(6):979-981.

2.64 Hormazabal, V., and Rogstad, A. (1992) Simultaneous determination of sulphadiazine and trimethoprim in plasma and tissues of cultured fish for residual and pharmacokinetic studies. Journal of Chromatography, 583:201-207

2.65 Walker, C.C., and Barker, S.A. (1993) Extraction and liquid chromatographic analysis of sulfadimethoxine and 4-N-acetylsulfadimethoxine residues in channel catfish (Ictalurus punctatus) muscle and plasma. Journal of the AOAC International, in press. 2.66 Samuelsen, O.B. (1990) High-performance liquid chromatographic determination of furazolidone in Atlantic salmon (Salmo salar) tissue. Journal of Chromatography, 528:495. 2.67 Horie, M., Saito, K., Hoshino, Y., Nose, N., Nakazawa, H., and Yamane, Y. (1991) Simultaneous determination of residual synthetic antibacterials in fish by high-performance liquid chromatography. Journal of Chromatography, 538:484-491. 2.68 Nose, N. Hoshino, Y., Kikuchi, Y., Horie, M., Saitoh, K., Kawachi, T., and Nakazawa, H. (1987) Simultaneous liquid chromatographic determination of residual synthetic antibacterials in cultured fish.

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2.69 Ikai, Y., Oka, H., Kawamura, N., Yamada, M. , Harada, K-I., Suzuki, M., and Nakazawa, H. (1989) Improvement of chemical analysis of antibiotics XVI. Simple and rapid determination of residual pyridone- carboxylic acid antibacterials in fish using a prepacked amino cartridge. Journal of Chromatography, 477:397-406.

2.70 Rasmussen, K.E., Tonnesen, F., Thanh, H.H., Rogstad, A., and Aanesrud, A. (1989) Solid-phase extraction and high-performance liquid chromatographic determination of flumequine and oxolinic acid in salmon plasma. Journal of Chromatography, 496:355-364.

2.71 Andresen, A.T., and Rasmussen, K.E. (1990) Automated on-line dialysis and column-switching HPLC determination of flumequine and oxolinic acid in fish liver. Journal of Liquid Chromatography, 13(20):4051-4065.

2.72 Samuelsen, O.B. (1990) Simple and rapid method for the determination of flumequine and oxolinic acid in salmon (Salmo salar) plasma by high-performance liquid chromatography and fluorescence detection. Journal of Chromatography, 530:452-457. 2.73 Rogstad, A., Hormazabal, V., and Yndestad, M. (1989) Simultaneous extraction and determination of oxolinic acid and flumequine in fish tissues by high performance liquid chromatography. Journal of Liquid Chromatography, 12(15):3073:3086. 2.74 Thanh, H.H., Andresen, A.T., Agascster, T., and Rasmussen, K.E. (1990) Automated column-switching high-performance liquid chromatographic determination of flumequine and oxolinic acid in extracts from fish. Journal of Chromatography, 532:363-373.

2.75 Steffenak, I., Hormazabal, V., and Yndestad, M. (1991) Rapid assay for the simultaneous determination of residues of oxolinic acid and flumequine in fish tissues by high-performance liquid chromatography. Journal of Liquid Chromatography, 14(1):61-7 0.

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2.76 Samuelsen, O.B. (1989) Determination of flumequine in fish by high-performance liquid chromatography and fluorescence detection. Journal of Chromatography, 497:355-359.

2.77 Boon, J.H., Nouws, J.M.F., van der Heijden, M.H.T., Booms, 6.H.R., and Degen, M. (1991) Disposition of flumequine in plasma of European eel (Anguilla anguilla) after a single intramuscular injection. Aquaculture, 99:213-223.

2.78 Hustvedt, S.O., Salte, R., and Benjaminsen, T. (1989) Rapid high-performance liquid chromatographic method for the determination of oxolinic acid in fish serum employing solid-phase extraction. Journal of Chromatography, 494:335-339.

2.79 Bjorklund, H.V. (1990) Analysis of oxolinic acid in fish by high-performance liquid chromatography. Journal of Chromatography, 530:75-82. 2.80 Larocque, L., Schnurr, M., Sved, S., and Weninger, A. (1991) Determination of oxolinic acid residues in salmon muscle tissue by liquid chromatography with fluorescence detection. Journal of the Association of Official Analytical Chemists, 74(4):608-611.

2.81 Jarboe, H.H., and Kleinow, K.M. (1992) Matrix solid- phase dispersion isolation and liquid chromatographic determination of oxolinic acid in channel catfish (Ictalurus punctatus) muscle tissue. Journal of the Association of Official Analytical Chemists, 75(3):428-432. 2.82 Jarboe, H.H., Toth, B.R., Shoemaker, K.E., Greenlees, K.J., and Kleinow, K.M. (1993) Pharmacokinetics and disposition of nalidixic acid in the rainbow trout. The Toxicologist, 13:376. 2.83 Uno, K., Kato, M., Aoki, T., Kubota, S.S., and Ueno, R. (1992) Pharmacokinetics of naladixic acid in cultured rainbow trout and amago salmon. Aquaculture, 102:297-307.

2.84 Horie, M., and Nakazawa, H. (1992) Determination of miloxacin and its metabolite in fish by high performance liquid chromatography with fluorescence and UV detection. Journal of Liquid Chromatography, 15(12):2057-2070.

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2.85 Rogstad, A., Hormazabal, V., and Yndestad, M. (1991) Extraction and high performance liquid chromatographic determination of enrofloxacin in fish serum and tissues. Journal of Liquid Chromatography, 14(3):521-531.

2.86 Steffenak, I., Hormazabal, V., and Yndestad, M. (1991) A rapid assay for the determination of sarafloxacin (A-55620) in fish serum by high performance liquid chromatography. Journal of Liquid Chromatography, 14(10):1983-1988.

2.87 Hormazabal, V., Rogstad, A., Steffenak, I., and Yndestad, M. (1991) Rapid assay for monitoring residues of enrofloxacin and sarafloxacin in fish tissues by high performance liquid chromatography. Journal of Liquid Chromatography, 14(8):1605-1614.

2.88 Nouws, J.F.M., Grondel, J.L., Schutte, A.R., and Laurensen, J. (1988) Pharmacokinetics of ciprofloxacin in carp, African catfish and rainbow trout. The Veterinary Quarterly, 10(3):211-216. 2.89 Nagata, T., and Saeki, M. (1986) Determination of ampicillin residues in fish tissues by liquid chromatography. Journal of the Association of Official Analytical Chemists, 69(3):448-450.

2.90 Otsuka, K., Horibe, K., Sugitani, A., and Yamada, F. (1986) Determination of residual thiamphenicol in yellow tail by high performance liquid chromatography. Journal of the Food Hygienic Society of Japan, 27 (3) *.263-266.

2.91 Nagata, T., and Saeki, M. (1992) Simultaneous determination of thiamphenicol, florfenicol, and chloramphenicol residues in muscles of animals and cultured fish by liquid chromatography. Journal of Liquid Chromatography, 15(12):2045-2056. 2.92 Martinsen, B., Horsberg, T.E., Varma, K.J., and Sams, R. (1993) Single dose pharmacokinetic study of florfenicol in Atlantic salmon (Salmo salar) in seawater at 11°C. Aquaculture 112:1-11.

2.93 Kitzman, J.V., Holley, J.H., Huber, W.G., Koritz, G.D., Davis, L.E., Neff-Davis, C.A., Bevill, R.F., Short, C.R., Barker, S.A., and Hsieh, L.C. (1990) Pharmacokinetics and metabolism of fenbendazole in channel catfish. Veterinary Research Communications, 14:217-226.

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2.94 Hormazabal, V., Steffenak, I., and Yndestad, M. (1992) A time and cost-effective assay for the determination of residues of malachite green in fish tissues by HPLC. Journal of Liquid chromatography, 15(12):2035-2044.

2.95 Allen, J.L., Meinertz, J.R., and Gofus, J.E. (1992) Determination of malachite green and its leuco form in water. Journal of AOAC International, 75(4):646-649.

2.96 Rogstad, A., Hormazabal, V., and Yndestad, M. (1987) Extraction of praziquantel from fish tissue and its determination by high-performance liquid chromatography. Journal of Chromatography, 391:328-333. 2.97 Cravedi, J.P., and Delous, G. (1991) Use of an immobilized enzyme reactor for the analysis of residues of 17a-methyltestosterone in trout by high- performance liquid chromatography. Journal of Chromatography, 564:461-467. 2.98 Carignan,G., Larocque, L., and Sved, S. (1991) Assay of oxolinic acid residues in salmon muscle by liquid chromatography with fluorescence detection: Interlaboratory study. Journal of the Association of Official Analytical Chemists, 74(6):906-909. 2.99 Munns, R.K., Holland, D.C., Stehly, G.R., Plakas, S.M., Roybal, J.E., Storey, J.M., and Long, A.M. (1993) Determination of chloramphenicol residues in shrimp by gas chromatography: interlaboratory study. Journal of the AOAC International, in press.

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2.102 Schipper,I.A., and Petersen, W.E. (1954) Assay of antibiotics by use of methylene blue milk. American Journal of Veterinary Research, 15:475-476. 2.103 Fribourgh, J.H., Robinson, J.A., and Meyer, F.P. (1969) Oxytetracycline residues in tissues of blue and channel catfishes. Technical Papers, No. 38,

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Bureau of Sport Fisheries and Wildlife, Fish and Wildlife Service, United States Department of the Interior, Washington, DC.

2.104 McCracken, A., Fidgeon, S., O'Brien, J.J., and Anderson, D. (1976) An investigation of antibiotic and drug residues in fish. Journal of Applied Bacteriology, 40:61-66. 2.105 Salte, R., and Liestrol, K. (1983) Drug withdrawal from farmed fish: Depletion of oxytetracycline, sulfadiazine, and trimethoprim from muscular tissue of rainbow trout (Salmo gairdneri). Acta Veterinaria Scandinavica, 24:418-430.

2.106 Grondel, J.L., Nouws, J.F.M., Schutte, A.R., and Driessens, F. (1989) Comparative pharmacokinetics of oxytetracycline in rainbow trout (Salmo gairdneri) and African catfish (Clarias gariepinus). Journal of Veterinary Pharmacology and Therapeutics, 12:157-162. 2.107 Moffitt, C.M., and Schreck, J.A. (1988) Accumulation and depletion of orally administered erythromycin thiocynate in tissues of Chinook salmon. Transactions of the American Fishery Society, 117:394-400.

2.108 Bruno, D.W. (1989) An investigation into oxytetracycline residues in Atlantic salmon, Salmo salar L. Journal of Fish Diseases, 12:77-86. 2.109 Kusser, W.C., and Newman, S.G. (1990) Detection of oxytetracycline residues in fish tissues using a sensitive bioassay. Journal of Fish Diseases, 13:545-548.

2.110 Plakas, S.M., DePaola, A., and Moxey, M.B. (1991) Bacillus stearothermophilus disk assay for determining ampicillin residues in fish muscle. Journal of the Association of Official Analytical Chemists, 74(6):910-912.

2.111 Bowser, P.R., Wooster, G.A., St Leger, J., and Babish, J.G. (1992) Pharmacokinetics of enrofloxacin in fingerling rainbow trout (Oncorhynchus mykiss). Journal of Veterinary Pharmacology and Therapeutics, 15:62-71.

2.112 Korsrud, G.O., and Macneil, J.D. (1988) Evaluation of the swab test on premises, the calf antibiotic

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and sulfa test, and a microbial inhibitor test with standard solutions of 22 antibiotics. Journal of Food Protection, 51(l):43-46.

2.113 Korkeala, H., Sorvettula, 0., Maki-Petays, 0. and Hirn, J. (1983) Comparison of different agar diffusion methods for the detection of residues in the kidneys of pigs treated with antimicrobial drugs. Meat Science, 9:291-304.

2.114 Katae, H., Kouno, K., Sekine, Y., Hashimoto, M., and Shimizu, M. (1979) Distribution, excretion and biotransformation of 14C-piromidic acid in goldfish Carassius auratus (L.). Journal of Fish Diseases, 2:529-542.

2.115 Brady, M.S., and Katz, S.E. (1987) Simplified plate diffusion system for microbial assays of antibiotics. Journal of the Association of Official Analytical Chemists, 70(4):641-646. 2.116 Sundloff, S.F. (1989) Drug and chemical residues in livestock. Veterinary Clinics of North America: Food Animal Practice, 5(2):411-449.

2.117 Food Safety Update (1992) FASTer residue test holds promise. Journal of the American Veterinary Medical Association, 200(7):886-887. 2.118 Johnston, R.W., Reamer, R.H., Harris, E.W., Fugate, H.G., and Schwab, B. (1981) A new screening method for the detection of antibiotic residues in meat and poultry tissues. Journal of Food Protection, 44(11)828-831.

2.119 Masztis, P.S. (1984) Antibiotic residue testing in a beef slaughterhouse. Canadian Veterinary Journal, 25:329-330.

2.120 MacNeil, J.D., Korsrud, G.O., Naylor, J.M., and Yates, W.D.G. (1989) Bioassay techniques and high- performance liquid chromatography for detection of oxytetracycline residues in tissues from calves. American Journal of Veterinary Research, 50(1):72-74.

2.121 Wilson, D.J., Franti, C.E., and Norman, B.B. (1991) Antibiotic and sulfonamide agents in bob veal calf muscle, liver, and kidney. American Journal of Veterinary Research, 52(8):1383-1387.

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2.122 Performing the Calf Antibiotic Sulfa Test, Product Instructional Guide, Environmental Diagnostics, Inc., Burlington, NC, 1990.

2.123 Tritschler II, J.P., Duby, R.T., Oliver, S.P., and Prange, R.W. (1987) Microbiological screening tests to detect antibiotic residues in cull dairy cows. Journal of Food Protection, 50(2):97-102. 2.124 Jones, G.M., and Seymour, E.H. (1988) Cowside antibiotic residue testing. Journal of Dairy Science, 71:1691-1699.

2.125 Seymour, E.H., Jones, G.M., and McGilliard, M.L. (1988) Comparisons of on-farm screening tests for detection of antibiotic residues. Journal of Dairy Science, 71:539-544.

2.126 TerHune, T.N., and Upson, D.W. (1989) Factors affecting the accuracy of the live animal swab test for detecting urine oxytetracycline and predicting oxytetracycline residues in calves. Journal of the American Veterinary Medical Association, 194(7):918-921.

2.127 Macaulay, D.M., and Packard, V.S. (1981) Evaluation of methods used to detect antibiotic residues in milk. Journal of Food Protection, 44(9):696-698.

2.128 Lloyd, D.N., and Van der Merwe, D. (1987) The use of a diffusion test for the detection of antibiotics in the tissues of slaughter stock. Journal of the South African Veterinary Association, 58(4):183-186.

2.129 Stiffey, A.V. and Williams, W.L. (1955) The separation of Aureomycin™ chlortetracycline and oxytetracycline in feeds by paper chromatography. Journal of the Association of Official Agricultural Chemists, 38(4):870-874.

2.130 Neidert, E., Saschenbrecker, P.W., and Tittiger, F. (1987) Thin layer chromatographic/bioautographic method for identification of antibiotic residues in animal tissues. Journal of the Association of Official Analytical Chemists, 70(2):197-200. 2.131 Kondo, F. (1988) A simple method for the characteristic differentiation of antibiotics by TLC-bioautography in graded concentration of ammonium chloride. Journal of Food Protection, 51(10):786-789.

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2.132 Herbst, D.V. (1980) Applications of TLC and bioautography to detect contaminated antibiotic residues: Tetracycline identification scheme. Journal of Pharmaceutical Sciences, 69(5):616-618. 2.133 Lott, A.F., Smither, R., and Vaughan, D.R. (1885) Antibiotic identification by high voltage electrophoresis bioautography. Journal of the Association of Official Analytical Chemists, 68(5):1018-1020.

2.134 Bratton, A.C., and Marshall, E.K., Jr. (1939) A new coupling component for sulfanilamide determination. Journal of Biological Chemistry, 128:537-550.

2.135 Horwitz, W. (1981) Analytical methods for sulfonamides in foods and feeds. I. Review of methodology. Journal of the Association of Official Analytical Chemists, 64(1):104-130. 2.136 Horwitz, W. (1981) Analytical methods for sulfonamides in foods and feeds. II. Performance characteristics of sulfonamide methods. Journal of the Association of Official Analytical Chemists, 64(4):814-824.

2.137 Walker, C.R., and Schoettger, R.A. (1967) Method for determining MS-222 residues in fish. Investigations in Fish Control, No. 14, Bureau of Sport Fisheries and Wildlife, United States Department of the Interior, Washington, DC. 2.138 Allen, J.L. (1988) Residues of benzocaine in rainbow trout, largemouth bass, and fish meal. The Progressive Fish-Culturist, 50:59-60.

2.139 James, M.O., and Barron, M.G. (1988) Disposition of sulfadimethoxine in the lobster (Homarus americanus) Veterinary and Human Toxicology, 30(Supplement 1):36-40.

2.140 Alavi, F.K., Rolf, L.L., and Clarke, C.R. (1993) The pharmacokinetics of sulfachlorpyridazine in channel catfish, Ictalurus punctatus. Journal of Veterinary Pharmacology and Therapeutics, 16:232-236. 2.141 Tissue residue method for sulfadimethoxine (Hoffmann-LaRoche), Food Additives Analytical Manual, Revised Edition, United States Food and Drug Administration, Rockville, MD, 1973.

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2.142 Kitagawa, T., Ohtani, W., Maeno, Y., Fujiwara, K., and Kimura, Y. (1985) Sensitive enzyme immunoassay of colistin and its application to detect residual colistin in rainbow trout tissue. Journal of the Association of Official Analytical Chemists, 68(4):661-664.

2.143 Wu, C.C., Brake, N.P., Lin, T.L., Heitz, J., and Purchase, H.G. (1992) Rapid screening test for sulfadimethoxine residues in channel catfish. Abstracts of the Annual Meeting of the Animal Disease Research Workers of the Southern States, Starkville, MS. 2.144 Walker, C.C. and Barker, S.A. (1993) Extraction and enzyme immunoassay of sulfadimethoxine residues in channel catfish (Ictalurus punctatus) muscle. Journal of AOAC International, Accepted for publication, July, 1993.

2.145 Charm II Test Operator's Manual, Charm Sciences, Inc., Malden, MA, 1991.

2.146 Rolf, L.L. Jr., Setser, M.D., and Walker, J.L. (1986) Pharmacokinetics and tissue residues in channel catfish, Ictalurus punctatus, given intracardiac and intramuscular injections of gentamicin sulfate. Veterinary and Human Toxicology, 28(Supplement l):25-30.

2.147 Rattenberger, E., Dangschat, H., Schwarzer, C.f and Bauer, K. (1986) Chloramphenicol residues in trout measured by radioimmunoassay. Berliner und Munchener Tierarztliche Wochenschrift, 99(7):243-246. 2.148 Charm, S.E., and Chi, R. (1988) Microbial receptor assay for rapid detection and identification of seven families of antimicrobial drugs in milk: Collaborative study. Journal of the Association of Official Analytical Chemists, 71(2):304-316. 2.149 Brady, M.S., and Katz, S.E. (1989) A microbial assay system for the confirmation of residues of receptor assays for antibiotic residues in milk. Journal of Food Protection, 52(3):198-201.

2.150 Collins-Thompson, D.L., Wood, D.S., and Thomson, I.Q. (1988) Detection of antibiotic residues in consumer milk samples in North America using the Charm Test II procedure. Journal of Food Protection, 51(8):632-633.

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2.152 Righter, H.F. (1992) Assessment of the occurrence of violative residues of FDA approved tetracyclines in milk and the accuracy of a multi-residue screening test at detecting them. Study number 305.31, Division of Veterinary Medical Research, Center for Veterinary Medicine, United States Food and Drug Administration, Beltsville, MD 2.153 Settepani, J.A. (1992) An evaluation of the Charm II test for residues of sulfa drugs in milk. Office of Science, Center for Veterinary Medicine, United States Food and Drug Administration, Beltsville, MD

2.154 Adams, L.A. (1992) Evaluation of screening tests for chloramphenicol in milk. Study number 319.01, Division of Residue Chemistry, Center for Veterinary Medicine, United States Food and Drug Administration, Beltsville, MD 2.155 Carlsson, A., and Bjorck, L. (1991) Charm Test II for confirmation of inhibitory substances detected by different microbial assays in herd milk. Journal of Food Protection, 54(l):32-36. 2.156 Arnold, D., and Somogyi, A. (1985) Trace analysis of chloramphenicol residues in eggs, milk, and meat: comparison of gas chromatography and radioimmunoassay. Journal of the Association of Official Analytical Chemists, 68(5):984-990.

2.157 Beck, M., Martlbauer, E., and Terplan, G. (1987) Detection of chloramphenicol residues in eggs: comparison of a radioimmunoassay (RIA) with an enzyme immunoassay (ELISA). Archiv fur Lebensmittelhygiene, 38(4):99-103.

2.158 Daeseleire, E., De Guesquiere, A., and Van Peteghem, C. (1991) Combined high-performance liquid chromatography and radioimmunoassay for the screening of 19-nortestosterone and methyltestostsrone residues in meat samples. Journal of Chromatography, 564:445-449. 2.159 Newsome, H. (1986) Potential and advantages of immunochemical methods for analysis of foods.

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2.160 Van Emon. J.M., Seiber, J.N., and Hammock, B.D. (1989) Immunoassay techniques for pesticide analysis, in Analytical Methods for Pesticides and Plant Growth Regulators, Vol. XVII, J. Sherma (Editor), Academic Press, Inc., San Diego, CA. 2.161 Golden, C.A. (1991) Overview of the state of the art of immunoassay screening tests. Journal of the American Veterinary Medical Association, 198(5):827-830. 2.162 Guest, G.B. (1992) Regulating drug use in aquaculture. FDA Veterinarian, 7(3):3-5.

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2.166 Van De Water, C., and Haagsma, N. (1990) Sensitive streptovidin-biotin enzyme-linked immunosorbent assay for rapid screening of chloramphenicol residues in swine muscle. Journal of the Association of Official Analytical Chemists, 73(4):534-540. 2.167 Van De Water, C., and Haagsma, N. (1991) Analysis of chloramphenicol residues in swine tissues and milk: comparative study using different screening and quantitative methods. Journal of Chromatography, 566:173-185.

2.168 Keukens, H.J., Aerts, M.M.L., Traag, W.A., Nouws, J.F.M., De Ruig, W.G., Beek, W.M.J., and Den Hartog, J.M.P. (1992) Analytical strategy for the regulatory control of residues of chloramphenicol in meat:

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 112 preliminary studies in milk. Journal of AOAC International, 75(2):245-256.

2.169 Dixon-Holland, D.E., and Katz, S. (1988) Competitive direct enzyme-linked immunosorbent assay for detection of sulfamethazine residues in swine urine and muscle tissue. Journal of the Association of Official Analytical Chemists, 71(6):1137-1140. 2.170 Dixon-Holland, D.E., and Katz, S. (1989) Direct competitive enzyme-linked immunosorbent assay for sulfamethazine residues in milk. Journal of the Association of Official Analytical Chemists, 72(3):447-450. 2.171 Ram, B.P., Singh, P., Martens, L., Brock, T., Sharkov, N., and Allison, D. (1991) High-volume enzyme immunoassay test system for sulfamethazine in swine. Journal of the Association of Official Analytical Chemists, 74(l):43-46. 2.172 Renson, C., Degand, G., Maghuin-Rogister, G.# Delahaut, P. (1993) Determination of sulphamethazine in animal tissues by enzyme immunoassay. Analytica Chimica Acta, 275:323-328.

2.173 Rohner, P., Schallibaum, M., and Nicolet, J. (1985) Detection of penicillin G and its benzylpenicilloyl (BPO)-derivatives in cow milk and serum by means of an ELISA. Journal of Food Protection, 48(1):59-62. 2.174 Heitzman, R.J., Carter, A.P., and Cottingham, J.D. (1986) An enzyme linked immunosorbent assay (ELISA) for residues of monesin in plasma of cattle. British Veterinary Journal, 142(6):516-523. 2.175 Kitagawa, T., Gotoh, Y., Uchihara, K., Kohri, Y., Kinoue, T., Fujiwara, K., and Ohtani, W. (1988) Sensitive enzyme immunoassay of cephalexin residues in milk, hen tissues, and eggs. Journal of the Association of Official Analytical Chemists, 71(5):915-920.

2.176 Chesham, J., Gordon, J., and Jackman, R. (1989) A rapid test for penicillin G in raw milk. Dairy Industries International, 54(12):23-24. 2.177 Sheth, H.B., and Sporns, P. (1990) Enzyme immunoassay for sulfathiazole in honey. Journal of the Association of Official Analytical Chemists, 73(6):871-874.

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2.173 Ploum, M.E., Haasnoot, W., Paulussen, R.J.A., Van Bruchem, G.D., Hamers, A.R.M., Schilt, R., and Huf, F.A. (1991) Test strip enzyme immunoassays and the fast screening of nortestosterone and clenbuterol residues in urine samples at the parts per billion level. Journal of Chromatography, 564:413-427.

2.179 Van Look, L.J., Jansen, E.H.J.M., Van den Berg, R.H., Zomer, G., Vanoosthuyze, K.E., and Van Peteghem, C.H. (1991) Development of a competitive enzyme immunoassay for 17a-19-nortestosterone. Journal of Chromatography, 564:451-459. 2.180 Meyer, H.H.D., Rinke, L., and Diirsch, I. (1991) Residue screening for the j8-agonists clenbuterol, salbutamol, and cimaterol in urine using enzyme immunoassay and high-performance liquid chromatography. Journal of Chromatography, 564:551-556.

2.181 EZ-SCREEN®: Sulfadimethoxine Test, Information and Instruction Sheet, Environmental Diagnostics, Inc., Burlington, NC, December, 1991.

2.182 Sulfadimethoxine I.D. Block™ Testing Device. Product information and test procedure sheet, International Diagnostic Systems Corporation, St. Joseph, MI, May, 1992.

2.183 SULFADIMETHOXINE ONE-STEP ELISA. Product information and test procedure sheet, International Diagnostic Systems Corporation, St. Joseph, MI, June, 1992.

2.184 Cullor, J.S. (1992) Tests for identifying antibiotic residues in milk: how well do they work? Veterinary Medicine, 87(12):1235-1241. 2.185 CITE PROBE Tetracycline Test. Information and Instruction Sheet, Idexx Corporation, Portland, ME. 2.186 SNAP™ Beta-Lactam Test. Information and Instruction Sheet, Idexx Corporation, Portland, ME, 1993.

2.187 LacTek™ Animal Drug Residue Detection in Dairy Products. Product information sheet, Idetek, Inc., Sunnyvale, CA, July, 1992.

2.188 Settepani, J.A. (1992) CVM's program to evaluate milk residue screening assays. FDA Veterinarian, 7(2):10.

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2.189 Medina, M.B., Barford, R.A., Palumbo, M.S., Rowe, L.D. (1992) Evaluation of commercial immunochemical assays for detection of sulfamethazine in milk. Journal of Food Protection, 55(4):284-290. 2.190 Touchstone, J.C. (1993) New developments in planar chromatography. LC»GC, 11(6):404-411. 2.191 Thomas, M.H., Soroka, K.E., and Thomas, S.H. (1983) Quantitative thin layer chromatographic multi­ sulfonamide screening procedure, Journal of the Association of Official Analytical Chemists, 66(4):881-883. 2.192 Performing the Sulfa On Site test (SOS), product instruction guide EDI 187, Environmental Diagnostics, Inc., Burlington, NC, 1989.

2.193 McKean, J.D. (1987) A veterinary approach to residue testing: A look at the tools available. Large Animal Veterinarian, 42(7):38-42.

2.194 Allen, J.L., Luhning, C.W., and Harman, P.D. (1970) Identification of MS-222 in selected fish tissues by thin-layer chromatography. Investigations in Fish Control, No. 41, Bureau of Sport Fisheries and Wildlife, United States Department of the Interior, Washington, DC.

2.195 Squibb, K.S., Michel, C.M.F., Zelikoff, J.T., O'Connor, J.M. (1988) Sulfadimethoxine pharmacokinetics and metabolism in the channel catfish (Ictalurus punctatus). Veterinary and Human Toxicology, 30(Supplement 1):31-35.

2.196 Barron, M.G., and James, M.O. (1988) Fate of sulfadimethoxine in the lobster, Homarus americanus. Marine Environmental Research, 24:85-88. 2.197 Sherma, J. (1992) Modern thin-layer chromatographic pesticide analysis using multiple development. Journal of AOAC International, 75(1):15-17.

2.198 Sherma, J. (1991) Comparison of thin layer chromatography and liquid chromatography. Journal of the Association of Official Analytical Chemists, 74(3):435-437.

2.199 Tao, S.H., and Poumeyrol, M. (1989) Quick method of identification of flumequine in trout meat. Revue de Medecine Veterinaire, 140(4):321-323.

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2.200 Reimer, G. J., and Suarez, A. (1991) Development of a screening method for five sulfonamides in salmon muscle tissue using thin-layer chromatography. Journal of Chromatography, 555:315-320.

2.201 MacNeil, J.D., Korsrud, G.O., Boison, J.O., Papich, M.G., and Yates, W.D.G. (1991) Performance of five screening tests for the detection of penicillin G residues in experimentally injected calves. Journal of Food Protection, 54(1):37-40.

2.202 Moats, W.A. (1990) Liquid chromatographic approaches to antibiotic residue analysis. Journal of the Association of Official Analytical Chemists, 73 (3):343-346. 2.203 Allen, J.L., and Sills, J.B. (1970) GLC determination of quinaldine residues in fish. Journal of the Association of Official Analytical Chemists, 53(l):20-23. 2.204 Allen, J.L., and Sills, J.B. (1970) UV identification and quantitative measurement of quinaldine residues in fish. Journal of the Association of Official Analytical Chemists, 53(6):1170-1171.

2.205 Takatsuki, K. (1991) Gas chromatographic-mass spectrometric determination of oxolinic acid in fish using selected ion monitoring. Journal of Chromatography, 538:259-267. 2.206 Takatsuki, K. (1992) Gas chromatographic/mass spectrometric determination of oxolinic, naladixic, and piromidic acid in fish. Journal of the AOAC International, 75(6):982-987.

2.207 Bencsath, A.F., Plakas, S.M., and Long, A.R. (1993) Development of a gas chromatographic/mass spectrophotometric method for confirmation and quantitation of chloramphenicol in shrimp at low parts-per-billion levels using sector instrument and electron capture ionization. Biological Mass Spectrometry, in press.

2.208 Feil, V.J., Paulson,G.D., and Lund, L.L. (1989) Diazomethane derivitization of sulfamethazine: formation of isomeric products. Journal of the Association of Official Analytical Chemists, 72(3):515-516.

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2.209 Mineo, H., Kaneko, S., Koizumi, I., Asida, K., and Akahori, F. (1992) An analytical study of antibacterial residues in meat: the simultaneous determination of 23 antibiotics and 13 drugs using gas chromatography. Veterinary and Human Toxicology, 34(5):393-397.

2.210 Horie, M., Saito, K., Nose, N., Tera, M., and Nakazawa, H. (1993) Confirmation of residual oxolinic acid, naladixic acid and piromidic acid in fish by thermospray liquid chromatography-mass spectrometry. Journal of Liquid Chromatography, 16(7):1463-1472.

2.211 Petzer, I-M., Van Staden, J.J., and Giesecke, W.H. (1984) Tissue reaction and residues in slaughter cattle after administration of longacting oxytetracycline formulations. Journal of the South African Veterinary Association, 55(3):107-111.

2.212 Herman, R.L. (1969) Oxytetracycline in fish culture- A review. Technical Papers, No. 31, Bureau of Sport Fisheries and Wildlife, Fish and Wildlife Service, United States Department of the Interior, Washington, DC.

2.213 Plakas, S.M., McPhearson, R.M., and Guarino, A.M. (1988) Disposition and bioavailability of 3H- tetracycline in the channel catfish (Ictalurus punctatus). Xenobiotica, 18(1):83-93. 2.214 Droy, B.F., Goodrich, M.S., Lech, J.J., and Kleinow, K.M. (1990) Bioavailability, disposition and pharmacokinetics of I4C-ormetoprim in rainbow trout (Salmo gairdneri). Xenobiotica, 20(2):147-157.

2.215 Bergsjo, T., Nafstad, I., and Ingebrigtsen, K. (1979) The distribution of 35S-sulfadiazine and 14C- trimethoprim in rainbow trout, Salmo gairdneri. Acta Veterinaria Scandinavica, 20:25-37. 2.216 Ingebrigtsen, K., Nafstad, I., and Maritim, A. (1985) The distribution of 3H-tetracycline after a single oral dose in the rainbow trout (Salmo gairdneri) as observed by whole body autoradiography. Acta Veterinaria Scandinavica, 26:428-430.

2.217 Hoy, T., Horsberg, T.E., Nafstad, I., and Berge, G.N. (1989) The absorption and distribution of 14C- bacitracin in rainbow trout (Salmo gairdneri) after

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a single oral dose, as observed by whole body autoradiography. Pharmacology & Toxicology, 64:262-265.

2.218 Czerwinska, K., and Sund, R.B. (1990) Drug uptake and disposition in fish: an autoradiographic study with dihydrostreptomycin in the rainbow trout. Acta Pharmaceutica Nordica, 2(6):377-386.

2.219 Guyer, C.G. "Multiresidue Methods: Chemical Assays", in Proceedings of the Sixth Biennial Symposium on Veterinary Pharmacology and Therapeutics, J.C. Lee, P. Eyre, W.G. Huber, K. Greenlees, H.P. Misra, and D.Ochs (Editors), Blacksburg, VA, 1988, pp. 50-51. 2.220 Barker, S.A., and Walker, C.C. (1992) Chromatographic methods for tetracycline analysis in foods. Journal of Chromatography, 624:195-209.

2.221 Afeyan, N.B., Gordon, N.F., and Regnier, F.E. (1992) Automated real-time immunoassay of biomolecules. Nature, 358:603-604. 2.222 Katz, S.E., and Brady, M.S. (1990) High-performance immunoaffinity chromatography for drug residue analysis. Journal of the Association of Official Analytical Chemists, 73(4):557-560.

2.223 Katz, S.E., and Siewierski, M. (1992) Drug residue analysis using immunoaffinity chromatography. Journal of Chromatography, 624(l-2):403-409. 2.224 van Ginkel, L.A. (1991) Immunoaffinity chromatography, its applicability and limitations in multi-residue analysis of anabolizing and doping agents. Journal of Chromatography, 564(2):363-384.

2.225 van Ginkel, L.A., Stephany, R.W., van Rossum, H.J., Steinbuch, H.M., Zomer, G., van de Heeft, E., and De Jong, A.P. (1989) Multi-immunoaffinity chromatography: a simple and highly selective clean­ up method for multi-anabolic residue analysis of meat. Journal of Chromatography, 489(1):111-120. 2.226 van Ginkel, L.A., Stephany, R.W., and van Rossum, H.J. (1992) Development and validation of a multiresidue method for /3-agonists in biological samples and animal feed. Journal of AOAC International, 75(3):554-560.

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2.227 Bagnati, R., Oriundi, M.P., Russo, V., Danese, M., Berti, F., Fanelli, R. (1991) Determination of zeranol and /S-zearalanol in calf urine by immunoaffinity extraction and gas chromatography- mass spectrometry after repeated administration of zeranol. Journal of Chromatography, 564(2):493-502.

2.228 van de Water, C., and Teffal, D., and Haagsma, N. (1989) Monoclonal antibody-mediated clean-up procedure for the high-performance liquid chromatographic analysis of chloramphenicol in milk and eggs. Journal of Chromatography, 478(1):205-215. 2.229 Moretti, V.M., van de Water, C., and Haagsma, N. (1992) Automated high-performance liquid chromatographic determination of chloramphenicol in milk and swine muscle tissue using on-line immunoaffinity sample cleanup. Journal of Chromatography, 583(1):77-82.

2.230 ImmunoDetection™ Product Information Brochure, Publication Number ID001 8/92, PerSeptive Biosystems, Inc., Cambridge, MA. 2.231 Games, D.E., Hernandez, H., Perkins, J.R., Ramsey, E.D., and Thomas, D. (1991) Supercritical fluid chromatography, supercritical fluid extraction and their combination with mass spectrometry for the analysis of pesticides and veterinary drugs. Acta Veterinaria Scandinavica, 87(Supplement):471-472. 2.232 Cross, R.F., Ezzell, J.L., and Richter, B.E. (1993) The supercritical fluid extraction of polar drugs (sulphonamides) from inert matrices and meat animal products. Journal of Chromatographic Science, 31:162-169. 2.233 Hopper, M.L., and King, J.W. (1991) Enhanced supercritical fluid carbon dioxide extraction of pesticides from foods using pelletized diatomaceous earth. Journal of the Association of Official Analytical Chemists, 74(4):661-666.

2.234 Nam, K.S., Kapila, S., Pieczonka, G., Clevenger, T.E., Yanders, A.F., Viswanath, D.S., and Mallu, B. (1988) in Proceedings of the International Symposium on Supercritical Fluids, M. Perrut (Editor), French Chemical Society, Institute National Polytechnique de Lorraine, Paris Cedex France, Tome 2, pp. 743-750.

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2.235 Hawthorne, S.B., and Miller, D.J. (1986) Extraction and recovery of organic pollutants from environmental solids and Tenax-GC using supercritical C02. Journal of Chromatographic Science, 24:258-264. 2.236 Hawthorne, S.B., and Miller, D.J. (1987) Extraction and recovery of polycyclic aromatic hydrocarbons from environmental solids using supercritical fluids. Analytical Chemistry, 59:1705-1708.

2.237 Ramsay, E.D., Perkins, J.R., Games, D.E., and Startin, J.R. (1989) Analysis of drug residues in tissue by combined supercritical-fluid extraction- supercritical-fluid chromatography-mass spectrometry-mass spectrometry. Journal of Chromatography, 464:353-364.

2.238 Perkins, J.R., Games, D.E., Startin, J.R., and Gilbert, J. (1991) Analysis of sulphonamides using supercritical fluid chromatography and supercritical fluid chromatography-mass spectrometry. Journal of Chromatography, 540:239-256. 2.239 Perkins, J.R., Games, D.E., Startin, J.R., and Gilbert, J. (1991) Analysis of veterinary drugs using supercritical fluid chromatography and supercritical fluid chromatography-mass spectrometry. Journal of Chromatography, 540:257-270. 2.240 Barker, S.A., and Long, A.R. (1992) Tissue drug residue extraction and monitoring by matrix solid phase dispersion (MSPD)-HPLC analysis. Journal of Liquid Chromatography, 15(12):2071-2089.

2.241 Lott, H.M., and Barker, S.A. (1993) Matrix solid- phase dispersion extraction and gas chromatographic screening of 14 chlorinated pesticides in oysters (Crassostrea virginica). Journal of AOAC International, 76(1):67-72.

2.242 Lott, H.M., and Barker, S.A. (1993) Extraction and gas chromatographic screening of 14 chlorinated pesticides in crayfish (Procambarus clarkii) hepatopancreas. Journal of AOAC International, in press.

2.243 Long, A.R., Crouch, M.D., and Barker, S.A. (1991) Multiresidue matrix solid phase dispersion (MSPD) extraction and gas chromatographic screening of nine

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 120 chlorinated pesticides in catfish (Ictalurus punctatus) muscle tissue. Journal of the Association of Official Analytical Chemists, 74:667-670.

2.244 Lott, H.M., and Barker, S.A. (1993) Comparison of a new multiresidue extraction technique to classical extraction methodologies for the determination of chlorinated hydrocarbon pesticide residues in fish muscle. Environmental Monitoring and Assessment, In press, 1993.

2.245 Norcross, M.A., Post, A.R., and Miller, W.R. "Analytical methodologies for establishing residue control", in Proceedings of the Sixth Biennial Symposium on Veterinary Pharmacology and Therapeutics, J.C. Lee, P. Eyre, W.G. Huber, K. Greenlees, H.P. Misra, and D.Ochs (Editors), Blacksburg, VA, 1988, pp. 44-49.

2.246 Barker, S.A., Long, A.R., and Short, C.R. (1989) Isolation of drug residues from tissues by solid phase dispersion. Journal of Chromatography, 475:353-361.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 3

EXTRACTION AND ENZYME IMMUNOASSAY OF SULFADIMETHOXINE RESIDUES IN CHANNEL CATFISH {Ictalurus punctatus) MUSCLE

INTRODUCTION

Disease is the most important limiting factor in

intensive aquaculture and necessitates the use of

antibacterial and other therapeutic agents to maintain the health and production of cultured species [3.1]. Few

drugs are approved for food fish use in the US [3.2] but a number of chemicals not approved in the US are widely

employed in other countries [ 3.3,3.4]. Large amounts of

imported cultured seafood are consumed in the US but are not required to pass unified, continuous federal

inspection [3.5,3.6]. The US Food and Drug Administration has begun monitoring imported and domestic aquaculture

products for selected drugs [3.7], but a lack of

appropriate analytical methods limits residue monitoring efforts. There is presently little information concerning

the level of drug residues in domestic or imported

cultured fish or the magnitude of human exposure to these

residues. Public concern over exposure to potentially harmful residues and an increase in the consumption of seafood has led to calls for a seafood inspection program

121

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similar to those that exist for red meat and poultry [3.5,3.6].

Present and pending seafood inspection programs require analytical methods for identification of fish

containing illegal chemical residues in edible tissues. Accordingly, regulatory agencies will need methods that

are sensitive, specific, and practical for this purpose [3.8]. Traditional residue screening methods, such as

microbial inhibition tests and chromatographic methods as

presently used in poultry and red meat inspection, lack one or more of these characteristics. There is also a

lack of practical residue screening methods available for

use by the producer in a field setting for the pre-harvest detection of violative drug residues. Field testing could

reduce the condemnation of harvested fish and subsequent producer losses and impact in a positive manner the function of a food fish drug monitoring program by a

reduction in the marketing of fish with over-tolerance residue levels. However, there are no such methods available for field use at present.

Screening tests may be directed to the detection of a

class of compounds or to a specific compound. The limited

number of drugs approved for use in the US for aquaculture permits residue detection programs for domestically reared catfish to focus on a small number of therapeutic

compounds. The high specificity of immunochemical assays,

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such as enzyme-linked immunosorbent assays (ELISA), make them well suited for the detection of a single compound of

interest. The recent commercial availability of several ELISA-based tests for sulfadimethoxine (SDM) provides an

opportunity for their application to the detection of SDM residues in various food animal tissues. These rapid,

easy to use, relatively inexpensive, and sensitive tests

may be used, depending on test format, in field settings

or regulatory laboratories. Thus, application of

appropriate extraction and determinative techniques to

food animal tissues would allow for a more efficient monitoring program by decreasing the turnaround time,

increasing the number of samples analyzed, and providing a method for preharvest residue determination on site.

Sulfadimethoxine (SDM) is the sulfonamide component

of the potentiated sulfonamide Romet-30® and is commonly used in channel catfish aquaculture. It is one of only

two systemic antibacterial agents available in the U.S. for use in food fish. The withdrawal period of this drug for catfish, when used according to label instructions, is

three days [3.9]. However, a number of factors can affect

the ultimate SDM residue levels in muscle tissue of treated fish and include water temperature and medicated

feed intake variation [3.10-3.12]. Off-label use, such as prophylactic administration, may also result in violative

SDM residues in marketed catfish.

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Recently introduced technology, utilizing matrix

solid phase dispersion (MSPD) for extraction [3.13,3.14]

and enzyme immunoassay for detection and quantitation

[3.15], may accommodate the need for rapid, practical, and

efficient extraction and the sensitivity and specificity requirements of detection needed in a screening assay.

Drug extraction from animal tissues is a limiting factor in the utility of a screening test for field use, and is a labor, solvent, and time cost factor in a regulatory

screening laboratory. MSPD, when compared to classical

extraction methods, greatly reduces extraction time and greatly reduces the tissue mass and hence the solvent

volumes needed for drug isolation. The MSPD procedure and its application to the isolation of a variety of drugs,

including several sulfonamides, from various matrices have been reported [3.16-3.19].

This study examines the use of MSPD derived extracts

of SDM fortified channel catfish muscle in four enzyme

immunoassays for the detection of SDM residues at tissue concentrations of 0, 25, 50, 100, and 250 ng/g. This paper presents the first application of MSPD technology

for the isolation, with subsequent determination by immunoassay, of SDM as a residue in channel catfish muscle.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 2 5 EXPERIMENTAL

REAGENTS AND EXPENDABLE MATERIALS

(a) SOLVENTS.— Liquid chromatography (LC) grade

solvents from commercial sources were used without further purification.

(b) CHEMICALS.— Note: Wear rubber gloves and protective clothing when handling. Sulfachloropyridazine,

sulfadiazine, sulfadimethoxine (SDM), sulfamerazine, sulfamethazine, sulfapyridine, sulfaquinoxaline, sulfathiazole, p-aminobenzoic acid, folic acid,

tetracycline, chlortetracycline, oxytetracycline, erythromycin, lincomycin, flumequine, oxolinic acid,

nalidixic acid, thiamphenicol, novobiocin, and ampicillin were obtained from Sigma Chemical Co., (St. Louis, MO).

Enrofloxacin was obtained as Baytril® Injection

(Haver/Diamond Scientific, Shawnee, KS)/ Ormetoprim was graciously supplied by Dr. Ronald Thune.

(c) 4-N-ACETYLSULFADIMETHOXINE.— synthesized

according to the procedure of Nielsen [3.20]. Identity

confirmed with high performance liquid chromatography and

direct probe, chemical ionization, positive ion mass spectrometry.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 126

(d) MSPD COLUMN MATERIAL — preparative grade C18, 40

/tin, 18% carbon load, endcapped; obtained from Analytichem

International (Harbor City, CA). The C18 was cleaned prior

to use with sequential washings of 2 volumes each of hexane, dichloromethane, and methanol; then vacuum

aspirated until dry. Stored at room temperature in a

closed container until use.

(e) SAMPLE COLUMNS — 10 ml disposable plastic syringe barrels (Becton Dickinson Co., Franklin Lakes, N.J.) were washed with hot soapy water, triple rinsed with

each of tap water, double distilled water, and methanol; air dried prior to use. Whatman no. 1, 1.5 cm paper

filters were placed proximally and distally to the tissue-

Cjg blend to secure this material in place within the

column. A syringe plunger, modified by removal of the

rubber tip, was used to pack material in the column. A modified plastic pipet tip (100 fil) was placed on the

syringe tip to direct and control flow rate through the column.

SAMPLES

Channel catfish, with no history of drug administration, were obtained from an indoor recirculating water culture system. Skinless fillets were stored frozen

(-20°C) and used within 1 month. Samples had

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concentrations of SDM less than 25 ng/g as determined by LC analysis of MSPD extracts [3.19]. A total of 60

fortified muscle samples (dorsal epaxial muscle group)

were extracted and evaluated in each of four ELISA test systems. Three fish were used; four samples from each

fish were analyzed at each of the five SDM concentrations tested.

EXTRACTION PROCEDURE

A 0.5 g portion of catfish fillet was placed in a clean polystyrene weighing boat and injected with 5 fil of SDM stock solution (concentrations of 0, 2.5, 5, 10, 25 /xg

SDM/ml methanol) to give tissue concentrations of 0, 25,

50, 100, and 250 ng/g. The samples were allowed to stand 2 minutes, placed in a glass mortar containing 2 g of C18,

and blended using a glass pestle until a homogenous

mixture was obtained (-1 min.). The resulting blend was

then placed in a column previously fitted with a 1.5 cm

disc paper filter frit, another paper filter was placed on top of the packing, and the packing was compressed to a

volume of -4.5 ml using a modified syringe plunger.

The tissue-Clg blend was washed with 8 ml of hexane

and the wash was discarded. The column was then placed over a clean 12 ml glass vial and the SDM eluted using 8

ml of dichloromethane. The DCM extract was placed in a

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35°C water bath and evaporated to dryness under nitrogen.

The eluent was reconstituted by the addition of 100 p.1 of

methanol and vortexed. Four hundred fil of a buffer appropriate for the test was added to give a final volume

of 500 fil, and vortexed. Depending on the assay level of sensitivity, this solution was either used directly or

further diluted 10-fold with buffer.

ELISA PROCEDURES

MSPD extracts of SDM fortified catfish muscle and sulfonamide standards were analyzed by four commercially available ELISAs. These test kits are based on the

principle of direct, competitive solid phase enzyme

immunoassay and comparison of the relative color development of the sample with a negative control.

Negative samples exhibit the greatest color development

and positive samples the least development. The tests were packaged containing all reagents and components, and test procedures were described in directions for use included with each kit. Results may be qualitative or

quantitative, respectively, when determined visually or

using optical density (OD) values. Test kits were stored

at 4°C when not in use; all reagents and components were

allowed to come to room temperature (23°C-29°C) prior to

use.

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(a) SIGNAL® SULFAMETHAZINE DETECTION TEST (SIGNAL) — (SmithKline Beecham Animal Health, Exton, PA); a

quantitative MICROTITER® well format. The MSPD extract in SIGNAL buffer was used without further dilution. The SIGNAL test is marketed for the detection of

sulfamethazine in milk, muscle, serum, urine, and feeds

but also exhibits a degree of cross-reactivity with SDM and several other sulfonamides. Test Procedure: 20 fil of

sample was placed in the bottom of a test well and 100 fil

sulfamethazine enzyme conjugate added. The solutions were

mixed by gentle tapping and incubated at room temperature for 20 minutes. The sample wells were emptied and washed

3 times using the supplied wash solution. 150 fil of

substrate was then added to each well and incubated at

room temperature for 30 minutes and OD values were obtained.

(b) IDS SULFADIMETHOXINE ONE-STEP ELISA (IDS)—

(International Diagnostics Systems Corp., St. Joseph, MI);

a quantitative MICROTITER® well ELISA for the detection of SDM residues in serum, milk, urine, muscle, liver, kidney, and feed. Same procedure as for the SIGNAL test except

the substrate incubation time was 10 minutes prior to

reading. This test has a lower detection limit of 10 ng SDM/ml, therefore the MSPD extract and SDM standards were

diluted 1:9 with supplied buffer to obtain an appropriate

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test concentration for the detection of tissue concentrations of 100 ng SDM/g.

(c) CITE® SULFA TRIO™ (CITE)— (IDEXX Laboratories,

Inc., Westbrook, ME); a qualitative assay for the detection of sulfdimethoxine, sulfathiazole, and

sulfamethazine residues in milk by use of a CITE cup

device. This device contains a membrane filter with a negative control spot and three discrete test areas, each

coated with antibody to one sulfonamide. The MSPD extract was used without further dilution. Test procedure: The

CITE device is wetted using 15 drops of supplied wash solution and allowed to completely absorb. Sample (240

/xl) and conjugate solution (240 fil) were combined, poured into the CITE device, and incubated for 3 minutes at room

temperature. The prefilter was discarded, the membrane washed using -2 ml of wash solution, and 3 drops of substrate solution were applied to the test spots. The

device was then incubated at room temperature for 2

minutes, 15 drops of stop solution were added, and the result was visually determined.

(d) EZ-SCREEm:SULFADIMETHOXINE (EZ-SCREEN) — (Environmental Diagnostics, Inc., Burlington, NC); a

qualitative assay for detection of SDM residues in milk, urine, serum, and feed using the QUIK-CARD® format. The

QUIK-CARD® system consists of a solid support card

containing two membrane-based test ports; negative control

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and sample. Results are obtained within 10 minutes by visual comparison of relative color development of the two

spots. The lower detection limit of the assay is 10 ng SDM/ml. Therefore, the MSPD tissue extract was diluted

1:9 with supplied buffer to obtain an appropriate test

concentration for the detection of tissue SDM concentrations of 100 ng/g or greater. Test procedure:

One drop of diluted extract was applied to the sample spot and one drop of negative control solution to the control

spot and allowed to absorb. One drop of enzyme solution

was applied to all test sites and allowed to absorb. One drop of negative control was then applied to all test

sites, absorbed, and excess fluid cleaned from around test sites. Two drops of substrate solution were then applied

to all test sites, incubated at room temperature for 5 minutes, and results were evaluated visually.

RESULT DETERMINATION

The ability of an assay to accurately identify

samples containing SDM above tolerance is essential for

its use as a screening test. The United States Food and Drug Administration (FDA) tolerance for total SDM residues in channel catfish muscle is 100 ng SDM/g [3.21].

Therefore, samples containing SDM concentrations less than

100 ng/g were considered negative and those samples

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containing 100 ng SDM/g or greater were considered

positive in this study.

SIGNAL and IDS analyses of extracted fortified muscle samples and standards were performed with a Dynatech

MR5000 ELISA plate reader (Dynatech Laboratories, Alexandria, VA); absorbance was measured in dual

wavelength mode, test filter of 630 nm and reference filter of 490 nm. The mean of duplicate wells was used for determination of test results.

A decision point for the determination of muscle

samples containing greater than or equal to 100 ng SDM/g tissue was developed using the upper end of an interval

expected to contain 95% of the OD sample/OD blank

observations for muscle samples containing 100 ng SDM/g. This interval was obtained using the 95% t distribution of

mean OD sample/OD blank values for MSPD extracted muscle samples containing 100 ng SDM/g. This interval was

calculated using (mean ± t*(std. dev.)] where t* is the a/2

t value for 11 degrees of freedom and alpha is equal to

0.05. The higher end (lower concentration) of this interval was used as a decision point such that OD

sample/OD blank values less than or equal to the decision point were called positive and values greater than the decision point were negative.

CITE and EZ-SCREEN test results were evaluated

visually by 2 independent evaluators in a single blind

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manner. Evaluation criteria for the determination of

positive or negative results were used as given in the

product information sheet for the EZ-SCREEN test. The criteria for the CITE test as furnished in the product information sheet indicated a sample is positive if the

SDM spot is lighter in color than the negative control

spot. However, when using MSPD extracts of muscle samples

containing no SDM, the control spot was often darker in

color than the SDM spot. Therefore, the procedure and evaluation criteria used for the CITE test were modified

by using undiluted extract and declaring only samples that exhibited no color development as positive responses.

Performance as indicated by sensitivity, specificity,

efficiency, and positive and negative predictive values was determined from these results.

RESULTS AND DISCUSSION

PRECISION

Precision was evaluated using intra-assay and inter­

assay variation of the two quantitative MICROTITER® well test systems. Percent agreement of results between the two evaluators was used as an indicator of the variability

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of response for the two qualitative membrane-based visual tests.

INTRA-ASSAY VARIATION — The within-run precision or the precision of duplicate wells of the same sample run in the same assay under the same conditions. It represents

the variability inherent in the test that results from

variations in test components and analytical instrumentation. Intra-assay variation is defined as the coefficient of variation (CV) of duplicate wells and is

calculated for each of five concentrations examined and

overall. The CV of optical density values at each concentration, both MSPD extracted samples and standards,

was calculated using the mean OD value for the

concentration and an estimate of the standard deviation (SD) of duplicates made by pooling the results of

duplicate wells of the individual samples [3.22]. The

estimate of SD was made by: SD = v (ED2) /N , where D =

difference between duplicates, and N = number of

observations (wells). Overall intra-assay variation for the assay is the mean CV of the five individual

concentrations. Results are presented in Table 3.1 with

overall intra-assay variability of 5.6% and 7.7% respectively for the SIGNAL and IDS tests using MSPD

extracted samples and 3.6% and 7.1% for SDM standards.

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TABLE 3.1 INTRA-ASSAY VARIATION (CV)

MSPD extracted SDM standards samples SDM concentration (ng/g) SIGNAL IDS* SIGNAL1’ IDS* 0 5.2 6.0 6.0 5.2 25 5.4 7.3 --- 6.3 50 3.7 6.8 2.6 9.9 100 9.8 10.5 2.2 7.6 250 3.9 7.7 3.5 6.5 Overall CV 5.6 7.7 3.6 7.1 (mean ± SD) ± 2.5 ± 1.7 ± 1.7 ± 1.8

* N = 24 wells / cone t N = 6 wells / cone $ N = 10 wells / cone

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This variability between duplicates is less than 10% as

recommended by Feldcamp and Smith [3.23].

EVALUATOR VARIABILITY — This is expressed as the

percent agreement of the two evaluators for the two visually determined tests (CITE, EZ-SCREEN) for 60 MSPD extracted samples. The formula for calculating %

agreement was: % agreement = (# samples with same result/total # samples)

X 100. The results presented in Table 3.2 show an overall

evaluator agreement rate of 77% for the CITE test and 95%

for the EZ-SCREEN test. There was considerable variation in agreement depending on tissue concentration examined. The CITE test exhibited variable color development of the SDM test spot when testing samples containing less than

100 ng SDM/g. This was especially noticeable at

concentrations of 25 and 50 ng SDM/g tissue as indicated by a higher number of evaluator disagreements at these concentrations. The EZ-SCREEN test had 100% agreement

between evaluators at concentrations of 0, 25, and 250 ng SDM/g and 3 disagreements/24 samples at the borderline concentrations of 50 and 100 ng SDM/g. Both tests had

100% agreement in evaluator results for samples containing 250 ng SDM/g.

INTER-ASSAY VARIATION — This represents the variation among different samples of the same concentration and

includes variables associated with intra-assay variation,

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TABLE 3.2 THE % AGREEMENT BETWEEN OBSERVERS FOR VISUALLY EVALUATED TESTS USING MSPD EXTRACTED SAMPLES

Test* Tissue CITE EZ-SCREEN concentration (ng/g) 0 92 (1) 100 (0) 25 58 (5) 100 (0) 50 42 (7) 83 (2) 100 92 (1) 92 (1) 250 100 (0) 100 (0) overall* 77 (14) 95 (3)

* () Number disagreed upon/12 samples $ () Number disagreed upon/60 samples

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effect can be normalized by transforming the data using

sample OD/Blank OD values. Inter-assay variation for each of the 5 concentrations was determined for both MSPD extracted samples and standards using OD and sample

OD/blank OD values. Inter-assay variation for each concentration is defined as the CV of twelve individual

samples extracted and assayed at different times. It was calculated for each concentration using the mean and SD of

duplicate well means. Overall inter-assay variation was obtained by averaging the individual concentrations' CV's. The inter-assay variation of the SIGNAL and IDS assays is presented in Table 3.3 for MSPD extracted

samples and standards using both optical density and

sample OD/blank OD values. The inter-assay variation increases with increasing concentration. This is the

result of a decreasing mean OD or sample OD/blank OD value

that occurs with increasing concentration, while the SD of these means remains relatively constant over all

concentrations. This is likely representative of the

inherent variability of extraction, measurement, and the

assay itself. The repeatability of the assays for extracted samples, as indicated by the overall inter-assay

variation of sample OD/blank values, was 7.9 ± 2.4% for

SIGNAL and 16.6 ± 7.0% for IDS. This range is typical of

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u> id 6.4 9.9 8.6 -- 5.6 ± ± 3.3 (CV) 4.3 4.2 4.0 4.2 12.6 -- ± ± 0.1 SIGNAL IDS SampleOD/Blank OD

19.0 16.3 11.5 ± ± 3.6 (CV)

3.0 2.4 2.8 16.3 -- ± ± 0.6 Optical density

16.6 ± ± 7.0 -- (CV)

5.6 11.4 3.8 14.0 7.9 9.3 21.8 6.1 9.8 -- 10.5 23.4 3.0 20.5 ± ± 2.4 SIGNAL IDS SIGNAL IDS Sample OD/BlankOD

IDS 13.5 31.9 16.1 14.9 23.6 20.0 ± ± 7.7 MSPD MSPD extracted samples* Standards1* (CV)

17.7 14.4 ± ± 1.9 SIGNAL Optical density

SD) ± 0 13.7 50 13.6 25 14.1 100 13.1 250 (ng/g) N = wells/concentration 6 SIGNAL for wells/concentration= N 10 for IDS N = 12 N = samples/concentration; 12 duplicate wells means Tissue SDM Overall CV (mean (mean concentration t $ * TABLE 3.3 TABLE 3.3 VARIATION INTER-ASSAY (CV)

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ELISAs [3.24-3.26]. Inter-assay variation of standards

using sample OD/blank OD values was about one-half of the

variation for the extracted samples and represents the between run variation of the assay itself. Optical

density values of individual MICROTITER® wells obtained using MSPD extracts of fortified catfish muscle are

presented in Appendixes A and B for the IDS and Signal assays respectively.

TEST PERFORMANCE

The criteria used to evaluate test performance of the four assays were sensitivity, specificity (including

cross-reactivity), efficiency, positive predictive value, and negative predictive value. Sensitivity and

specificity are fundamental parameters of test performance

while efficiency and predictive values provide an

estimation of the likelihood of a test result being correct [3.27]. Formulas used to calculate performance

parameters are given in Figure 3.1. Test performances for

individual concentrations and overall are given in Tables 3.4 and 3.5.

SENSITIVITY — Sensitivity is the ability of a test to

correctly identify samples containing greater than or equal to 100 ng SDM/g tissue. It is the most important

criteria for a screening test. For an assay to be useful

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SDM > 100 ng/g present______absent positive true positive false positive Test (TP) (FP) Result negative false negative true negative (FN) (TN)

Sensitivity [TP/ (TP + FN) ] X 100 Specificity [TN/ (TN + FP) ] X 100 Efficiency [(TP + TN)/total # tests] X 100 Predictive value (+) [TP/ (TP + FP) ] X 100 Predictive value (-) [TN/(TN + FN)] X 100

FIGURE 3.1 2 X 2 CELL MATRIX ILLUSTRATING THE FOUR POSSIBLE TYPES OF TEST RESULTS AND FORMULAS FOR CALCULATING PERFORMANCE PARAMETERS.

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TABLE 3.4 RESULTS & PERFORMANCE FROM THE ANALYSIS OF MSPD EXTRACTED CATFISH MUSCLE*

E L I S A result tissue True False True False Sensitivity Specificity concentration Test1* Negative Positive Positive Negative % % ng/g

Blank SIGNAL 12 0 100

IDS 12 0 100

CITE 21 3 88

EZ-SCREEN 24 0 100

25 SIGNAL 12 0 100

IDS 12 0 100

CITE 19 5 79 EZ-SCREEN 24 0 100 50 SIGNAL 9 3 75 IDS 10 2 83 CITE 11 13 46 EZ-SCREEN 20 4 83 100 SIGNAL 12 0 100 IDS 12 0 100 CITE 23 1 96 EZ-SCREEN 23 1 96 250 SIGNAL 12 0 100 IDS 12 0 100 CITE 24 0 100 EZ-SCREEN 24 0 100

12 samples/concentration Signal, IDS; duplicate wells using sample/blank optical density values with result determined using 95 % t distribution decision point CITE, EZ; 2 independent visual determinations/sample

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TABLE 3.5 OVERALL PERFORMANCE FROM THE ANALYSIS OF MSPD EXTRACTED CATFISH MUSCLE*

Predictive value Sensitivity Specificity Efficiency + test - test Testtt % % % % % SIGNAL 100 92 95 89 100 IDS 100 94 97 92 100 CITE 98 71 82 69 98 EZ- 98 94 96 92 99 SCREEN

* 60 samples/test; 12 at each of 5 concentrations (0,25,50,100,250 ng/g) t Signal, IDS; duplicate wells using mean sample/blank optical density values; result determined using 95 % t distribution decision point $ CITE, EZ; 2 independent visual determinations/sample

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as a screening test it must identify such samples with a

high degree of accuracy. Sensitivity is defined as the

percent of correctly identified positive samples (true

positives) of the total number of known positive samples tested. Performance results show all four assays had

overall sensitivities of 98-100% for MSPD extracted samples containing violative levels of SDM and 100%

sensitivity for samples containing 2.5 times (250 ng

SDM/g) the FDA tolerance. This indicates that these

assays, using MSPD extracts, can be used to accurately

identify catfish muscle samples that contain violative SDM residues.

SPECIFICITY — The ability of a test to correctly

identify negative samples. Muscle samples containing SDM at concentrations below the FDA tolerance of 100 ng SDM/g

were considered negative in this study and included concentrations of 0, 25, and 50 ng SDM/g muscle.

Specificity was calculated as the percent of true negative samples of the total number of known negative samples

examined. The SIGNAL, IDS, and EZ-SCREEN tests correctly

identified all samples containing 0 and 25 ng SDM/g as

negative. For samples containing half of tolerance (50 ng

SDM/g) these assays had specificities of 75%, 83%, and 83% respectively. Difficulty in evaluating negative samples (<100 ng SDM/g) with the CITE test, as a result of

variable color development of the SDM test spot at these

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concentrations, resulted in a higher rate of false

positive results than with the other tests. CITE

specificities were 88%, 79%, and 46% for concentrations of 0, 25, and 50 ng SDM/g. The specificity of a test is important in reducing the number of confirmatory tests needed as a result of

false positive results. The specificity of antibiotic

screening tests for milk has been demonstrated to vary

considerably among assay kits and has been related to the presence of inflammatory conditions of the mammary gland

[3.28,3.29]. What effect the inflammatory process has on the specificity of drug screening tests used with tissues from sick fish is unknown.

CROSS-REACTIVITY — The specificity of an assay may be

influenced by its cross-reactivity with other compounds. Cross-reactivity was examined using standard solutions in

test buffer at concentrations of 0.1, 1, 10, and 100 /xg/ml

for eight sulfonamides and fifteen other therapeutic compounds that are used in aquaculture in the US and

worldwide or that may be present as feed contaminants. The N-acetyl metabolite of SDM may comprise a large

portion of the total SDM residue present in edible tissues. Therefore, to accurately assess total tissue SDM residues, a SDM residue screening assay should also detect

N-acetyl SDM present in edible tissues or in biological

fluids used as reference tissues. The relative

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contribution of this metabolite to total SDM residue varies among species and tissues. In channel catfish, SDM

is present in bile almost entirely in the N-acetyl form (>90% of total SDM residues) but SDM is present in plasma and muscle primarily (>95%) as parent compound [3.30]. In

rainbow trout, however, N-acetyl SDM accounts for over

half of the total SDM residue in muscle [3.31]. In this study, all four assays detected the N-acetyl metabolite equally as well as the parent compound.

Cross-reactivity for the quantitative tests (SIGNAL and IDS) was evaluated by a comparison of concentrations causing a fifty percent inhibition in color development of sample wells compared to blank wells when using standard

solutions (EC-50). Cross-reactivity for the qualitative tests (CITE and EZ-SCREEN) was determined visually over a range of concentrations. Results are presented in Table 3.6.

The SIGNAL test is marketed for the detection of

sulfamethazine residues in several biological matrices but was found to cross-react to varying degrees with a number of other sulfonamides. This test should be considered a

sulfonamide class assay rather than one specific for sulfamethazine. The sulfonamides demonstrating the highest level of reactivity in this test were sulfamethazine, sulfamerazine, SDM, and N-acetyl SDM.

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TABLE 3.6 CROSS-REACTIVITY WITH ELISAS*

EC-50 Minimum detectable (fig/ml) concentration (fig/ml) Sulfonamide SIGNAL IDS CITE* EZ-SCREEN Su1fadimethoxine 0.25 .004 .025* .01 N-acetyl SDM 0.25 .004 .025* .01 Sulfachlorpyridazine 8 76 10 Sulfadiazine 5 — —

Sulfamerazine <0.1 — —

Sulfamethazine <0.1 — —

Sulfapyridine 10 — —

Sulfaquinoxaline 45 100 — Sulfathiazole 7.6 — —

p-Aminobenzoic Acid — — —

Folic Acid —— —

Ampicillin —— — Ormetoprim — 86 100

Erythromycin — — —

Lincomycin —— —

Enrofloxacin —— —

Flumequine — — —

Oxolinic Acid —— —

Nalidixic Acid —— —

Chlortetracycline —— —

Oxyt et racyc 1 ine — — —

Tetracycline — — —

Novobiocin — — —

Thiamphenicol — — —

* Cross-reactivity determined using standardsolutions in test buffer t cross-reactivity for SDM spot only $ lowest concentration examined — EC-50 greater than 100 pg/ml or no reactivity up 100 pg/ml

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The IDS test was quite sensitive to SDM and N-acetyl

SDM with EC-5Os for these compounds of 4 ng SDM/ml. The

IDS test had no appreciable reactivity with other sulfonamides. The CITE and EZ-SCREEN tests were also

quite sensitive and specific havinq no substantial cross- reactivity for the compounds tested.

Fourteen of the non-sulfonamide compounds examined showed no cross-reactivity up to concentrations of 100

lig/ml. Ormetoprim exhibited no reactivity in the SIGNAL and CITE tests but had a low level of reactivity in the

IDS assay with a EC-50 of 86 Atg/ml and in the EZ-SCREEN test with a minimum detectable level of 100 /xg/ml. Romet® medicated feed for catfish contains ormetoprim at a concentration of 420-1650 \igjg feed depending on feeding

rate [3.32]. This level may confound SDM test results for feed extracts of ormetoprim contaminated or medicated

feeds. The expected therapeutic level of ormetoprim in

catfish muscle (0.8 ftgfg) is well below the minimum detectable levels of these tests for ormetoprim [3.33].

Cross-reactivity of a test for compounds within a class of drugs is advantageous for a test intended as a

drug class detection test. However, to accurately

interpret test results the relative level of cross­ reactivity of the test with compounds within the class of drugs must be known. Additionally, tests marketed for the

detection of a single compound (i.e. sulfamethazine in

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milk) often do not include information on cross-reaction. While cross-reactivity does not invalidate the use of a

single compound detection test, it may cause

misinterpretation of a positive result if a cross-reacting

compound(s) is present and the level of cross-reaction is not known.

EFFICIENCY — is the percentage of all test samples that were correctly identified for a given population.

Efficiencies of 82-97% are shown in Table 3.5.

POSITIVE PREDICTIVE VALUE AND NEGATIVE PREDICTIVE

VALUE — indicates the probability of positive or negative

results being correct. Positive predictive values ranged from 69% for the CITE to 92% for IDS and EZ tests. Negative predictive values were 98-100% for all tests

indicating a high level of confidence can be placed in

negative results for these tests. Predictive value results and comparisons of performance using single or

duplicate wells and OD or % blank values are presented in Tables 3.7 and 3.8.

The purpose of the present study was to evaluate the

performance of commercially available immunoassays for the

detection of violative levels of SDM residues in channel catfish muscle using MSPD derived extracts. To fully

utilize the speed and simplicity of newer determinative

methods such as immunoassays in a drug monitoring program, rapid and simple drug isolation methods are needed.

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TABLE 3.7 OVERALL PERFORMANCE COMPARISONS OF SINGLE/DUPLICATE NELLS AND OF OPTICAL DENSITY/% BLANK VALUES FROM THE ANALYSIS OF MSPD EXTRACTED CATFISH MUSCLE*

Predictive Value Test Sensitivity Specificity Efficiency + test - test % % % IDS single well optical 98 93 95 90 99 density % blank 100 90 94 87 100

duplicate wells optical 96 92 93 88 97 density % blank 100 94 97 92 100

Sicraal single well optical 100 58 75 62 100 density % blank 100 72 83 71 100

duplicate wells optical 100 58 75 62 100 density % blank 100 92 95 89 100

60 samples/test; result determined using 95 % t distribution decision points

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TABLE 3.8 COMPARISONS OF INDIVIDUAL CONCENTRATION RESULTS AND PERFORMANCES FROM THE ANALYSIS OF MSPD EXTRACTED CATFISH MUSCLE* USING SINGLE/DUPLICATE HELLS AND OPTICAL DENSITY/ % BLANK VALUES

ELISA result* tissue True False Specificity concentration Test* Negative Positive % ng/g bl a n k Sitmal single well optical 19 5 79 density % blank 24 0 100 duplicate wells optical 10 2 83 density % blank 12 0 100

IDS single well optical 24 0 100 density % blank 24 0 100 duplicate wells optical 12 0 100 density % blank 12 0 100 t Signal, IDS; duplicate wells using sample/blank optical density values with result determined using 95 % t distribution decision point

table con'd

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ELISA result* tissue True False Specificity concentration Test* Negative Positive % ng/g 2 5 Sicmal single well optical 13 11 54 density % blank 19 5 79 duplicate wells optical 6 6 50 density % blank 12 0 100

IDS single veil optical 24 0 100 density % blank 24 0 100

duplicate wells optical 12 0 100 density % blank 12 0 100 t Signal, IDS; duplicate wells using sample/blank optical density values with result determined using 95 % t distribution decision point

table con'd

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ELISA result* tissue True False Specificity concentrat ion Test* Negative Positive % ng/g 50 Signal single well optical 10 14 42 density % blank 9 15 38 duplicate wells optical 5 7 42 density % blank 9 3 75

IDS single well optical 19 5 79 density % blank 17 7 71

duplicate wells optical 9 3 75 density ______% blank______10______2______83

t Signal, IDS; duplicate wells using sample/blank optical density values with result determined using 95 % t distribution decision point

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Classical methods for the isolation of sulfonamide

residues from animal tissues are complex and lengthy

procedures that require multiple steps involving homogenization of the sample in aqueous or organic

solvents, protein precipitation, centrifugation, pH

adjustments, transfer of the supernatant, counter-current extraction, back-extraction, and/or solid phase isolation.

The primary disadvantages of these traditional methods are

that they are costly in terms of labor and solvent use, and they limit the number of samples analyzed due to their

complexity and the time needed to complete the extraction. MSPD eliminates many of the steps and overcomes many of

the difficulties associated with classical extraction techniques. The MSPD theory and process have been the

subject of recent reviews [3.13,3.14]. In general, its

main advantages are that it is a relatively rapid method

that allows for the analysis of a larger numbe r of

samples, it considerably decreases solvent use because of

the small sample size (0.5g) required, and is amenable to automation.

The performance results obtained using fortified

samples indicate MSPD extracts of catfish muscle could be used in the SIGNAL, IDS, CITE, or EZ-SCREEN tests for screening catfish muscle samples for violative SDM residues. The SIGNAL and IDS tests are well suited for

screening large numbers of samples in a properly equipped

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laboratory. The IDS tests' large difference in color

between positive and blank samples might also permit its

use as a visually determined qualitative test but requires further evaluation. Either the CITE or EZ-SCREEN tests could be used in a laboratory for regulatory purposes or

used on-site to determine the advisability of harvesting

treated fish that possibly contain violative levels of SDM. The CITE test, as used in this study, was less

desirable because of the higher number of false positive

results. This may be a matrix-specific effect and could

possibly be rectified by sample dilution and a change in

result evaluation criteria. It should be emphasized that the performances of these tests using muscle extracts

should not be extended to milk or other matrices. Further studies of the applicability of these

procedures to catfish muscle containing incurred SDM residues are planned but were outside the scope of present

methods development research.

REFERENCES

3.1 Meyer, F.P. (1991) Aquaculture disease and health management. Journal of Animal Science, 69:4201-4208.

3.2 Meyer, F.P. (1989) Solutions to the shortage of approved fish therapeutants. Journal of Aquatic Animal Health, 1:78-80.

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3.3 Sano, T., and Fukuda, H. (1987) Principal microbial diseases of mariculture in Japan. Aquaculture, 67:59-69.

3.4 Grave, K., Engelstad, M., Soli, N.E., and H&stein, T. (1990) Utilization of antibacterial drugs in salmonid farming in Norway during 1980-1988. Aquaculture, 86:347-358.

3.5 Ahmed, F.E. (Editor) (1991) Seafood Safety, National Academy Press, Washington, DC

3.6 Johnsen, P.B. (1991) Aquaculture Product quality issues: Market position opportunities under mandatory seafood inspection regulations. Journal of Animal Science, 69:4209-4215. 3.7 Plakas, S.M., "Methods development for drugs and other chemicals in aquaculture products. The role of tissue distribution and metabolism studies." Proceedings of the Aquaculture Products Safety Forum, B.E. Perkins, (Editor), Auburn, AL, February, 1993, pp. 30-47. 3.8 Mitchell, G.A. (1991) Compliance issues and proper animal drug use in aquaculture. Veterinary and Human Toxicology, 33(Supplement 1):9-10. 3.9 United States Food and Drug Administration, New Animal Drugs for use in Animal Feed, Code of Federal Regulations (1988) Title 21, Part 558:575, Washington, D .C.

3.10 Brown, L.A. (1989) Effect of water temperature on withdrawal periods for a potentiated sulphonamide used in Channel Catfish, in Current trends in fish therapy, Proceedings of a joint WAVSD and DVG meeting. Deutsche Veterinarmedizinische Gesellschaft, pp. 116-128.

3.11 Gable D., and Craigmill, A. (1991) Current issues affecting aquaculture drug clearance. Veterinary and Human Toxicology, 33(Supplement 1):60-70. 3.12 Sundlof, S., and Ringer, R. (1991) Focus on metabolism studies and data requirements for aquaculture species. Veterinary and Human Toxicology, 33(Supplement l):71-80.

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3.13 Barker, S.A., Long, A.R., and Short, C.R. (1989) Isolation of drug residues from tissues by solid phase dispersion. Journal of Chromatography 475:353-361.

3.14 Barker, S.A., and Long, A.R. (1992) Tissue drug residue extraction and monitoring by matrix solid phase dispersion (MSPD)-HPLC analysis. Journal of Liquid Chromatography, 15(12):2071-2089.

3.15 Golden, C.A. (1991) Overview of the state of the art of immunoassay screening tests. Journal of the American Veterinary Medical Association, 198(5):827—830.

3.16 Long, A.R., Hsieh, L.C., Malbrough, M.S., Short, C.S., and Barker, S.A. (1989) A multiresidue method for the isolation and liquid chromatographic determination of seven sulfonamides in infant formula. Journal of Liquid Chromatography, 12(9):1601-1612

3.17 Long, A.R., Short, C.S., and Barker, S.A. (1990) Method for the isolation and liquid chromatographic determination of eight sulfonamides in milk. Journal of Chromatography, 502:87-94

3.18 Long, A.R., Hsieh, L.C., Malbrough, M.S., Short, C.S., and Barker, S.A. (1990) Multiresidue method for the determination of sulfonamides in pork tissue. Journal of Agricultural and Food Chemistry, 38:423-426

3.19 Long, A.R., Hsieh, L.C., Malbrough, M.S., Short, C.R. and Barker, S.A. (1990) Matrix solid phase dispersion isolation and liquid chromatographic determination of sulfadimethoxine in catfish (Ictalurus punctatus) muscle tissue. Journal of the Association of Official Analytical Chemists, 73(6):868-871.

3.20 Nielsen, P. (1973) The metabolism of four sulphonamides in cows. Biochemical Journal (London), 136:1039-1045. 3.21 United States Food and Drug Administration, Code of Federal Regulations (1988) Title 21, Part 556:640, Washington, D.C.

3.22 Reed, A.H., and Henry, R.J. (1974) ''Accuracy, Precision, Quality Control, and Miscellaneous Statistics" in Clinical Chemistry: Principles and

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Technics, R.J. Henry, D.C. Cannon, and J.W. Winkelman (Editors), Harper & Row, Hagerstown, MD, pp. 287-341.

3.23 Feldcamp, C.S., and Smith, S.W. (1987) in Immunoassay: A Practical Guide, D.W. Chan, and M.T. Perlstein (Editors), Academic Press, Orlando, FL, pp. 49-95.

3.24 Medina, M.B., Barford, R.A., Palumbo, M.S., and Rowe, L.D. (1992) Evaluation of commercial immunochemical assays for detection of sulfamethazine in milk. Journal of Food Protection, 55(4):284-290. 3.25 Dixon-Holland, D.E., and Katz, S. (1988) Competitive direct enzyme-linked immunosorbent assay for detection of sulfamethazine residues in swine urine and muscle tissue. Journal of the Association of Official Analytical Chemists, 71(6):1137-1140.

3.26 Kitagawa, T., Ohtani, W., Maeno, Y., Fujiwara, K., and Kimura, Y. (1985) Sensitive enzyme immunoassay of colistin and its application to detect residual colistin in rainbow trout tissue. Journal of the Association of Official Analytical Chemists, 68(4):661-664.

3.27 Zweig, M.H., and Robertson, E.A. (1987) ’’Clinical validation of immunoassays: A well designed approach to a clinical study", in Immunoassay: A Practical Guide, D.W. Chan, and M.T. Perlstein (Editors), Academic Press, Orlando, FL, pp. 97-127. 3.28 Cullor, J.S., Van Eenennaam, A., Dellinger, J., Perani, L., Smith, W., and Jensen, L. (1992) Antibiotic Residue assays: Can they be used to test milk from individual cows. Veterinary Medicine, 87:477-494.

3.29 Tyler, J.W., Cullor, J.S., Erskine, R.J., Smith, W.L., Dellinger, J., and McClure, K. (1992) Milk antimicrobial drug residue assay results in cattle with experimental, endotoxin-induced mastitis. Journal of the American Veterinary Medical Association, 201:1378-1384. 3.30 Squibb, K.S., Michel, C.M.F., Zelikoff, J.T., and O'Connor, J.M. (1988) Sulfadimethoxine pharmacokinetics and metabolism in the channel catfish (Ictalurus punctatus). Veterinary and Human Toxicology, 30(Supplement 1):31-35.

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3.31 Hara, T. and Inoue, S. (1967) Concentrations of various sulfonamides in the tissues of rainbow trout after oral dosing. Bulletin of the Japanese Society of Scientific Fisheries 33 (7):618-623. 3.32 Robinson, E.H., Brent, J.R., Crabtree, J.T., and Tucker, C.S. (1990) Improved palatability of channel catfish feeds containing Romet-30®. Journal of Aquatic Animal Health, 2:43-48. 3.33 Plakas, S.M., Dickey, R.W., Barron, M.G., and Guarino, A.M.(1990) Tissue distribution and renal excretion of ormetoprim after intravascular and oral administration in the channel catfish (Ictalurus punctatus). Canadian Journal of Fisheries and Aquatic Sciences, 47:766-771.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 4

EXTRACTION AND LIQUID CHROMATOGRAPHIC ANALYSIS OF SULFADIMETHOXINE AND 4-N-ACETYLSULFADIMETHOXINE RESIDUES IN CHANNEL CATFISH {Ictalurus punctatus) MUSCLE AND PLASMA INTRODUCTION

The demand for fisheries products has greatly

increased in the last 10 years and continued growth is

expected [4.1,4.2]. As a result aquaculture is a large and rapidly growing industry and will play an increasingly

important role in meeting the global demand for edible aquatic resources as wild fishery landings approach their biological limits [4.2]. The use of antibacterial drugs

in intensive fish culture systems is commonly practiced

and is necessary for control of infectious bacterial disease outbreaks [4.3]. However, the use of

antibacterials creates the potential for drug residue

problems in cultured fish. In the U.S., fish products are not required to pass continuous federal inspection.

Regulatory authority for seafood safety is divided among a

number of local, state, and federal agencies [4.4]. Public perceptions that uninspected seafood may contain

harmful chemicals [4.1], the increase in seafood

consumption, and the growth of aquaculture have led to calls for the establishment of a federal seafood

160

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inspection program similar to the ones that now exist for red meat and poultry [4.4,4.5].

Pending U.S. federal legislation concerning establishment of such an inspection program for seafood

will require analytical methods for extraction, screening, quantitation, and confirmation of chemical residues in

food fish [4.6]. At present there are few analytical

methods that have been validated for use in seafood for

detection of violative residues of the many therapeutic

compounds used in aquaculture domestically or worldwide [4.5]. Therefore, methods are needed that are practical,

sensitive, and specific for enforcement of the nation's present and anticipated future regulatory limits for chemical residues in aquatic food resources.

Drug residue violations in the United States red meat and poultry industries most frequently result from a failure by the producer to observe the stated withdrawal

time for an approved drug [4.7,4.8]. If a similar trend holds for aquaculture, the greatest number of residue

violations would be expected for approved drugs that have

been used by the producer in a manner not in accordance

with label directions. Therefore, methods development

efforts for extraction and detection of drug residues in domestically produced aquaculture products should focus

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initially on the limited number of approved compounds used in cultured fish in the US.

There are presently only three systemic antibacterial agents that are approved for use in aquaculture in the

United States; and only two of these are currently marketed. One of these, Romet-30®, is a potentiated

sulfonamide containing sulfadimethoxine (SDM) and

ormetoprim (OMP) in a 5:1 ratio. The channel catfish is

the major aquaculture species in the US accounting for 56%

of the total US aquacultural production in 1988 [4.2] and

360 million pounds produced in 1990 [4.9] and Romet-30® is frequently used in this species for the control of enteric septicemia of catfish. Therefore, practical methods for

detection, quantitation, and confirmation of SDM and OMP residues in channel catfish are urgently needed.

A number of analytical methods have been reported for SDM and OMP residues in edible aquatic tissues [4.10-

4.21], However, no methods are available for the simultaneous quantitation of SDM and its primary

metabolite, 4-N-acetylsulfadimethoxine (N-acetyl SDM), in

channel catfish muscle. Further, several of the methods

lack the sensitivity needed for detection of SDM residues

at tolerance. Methods employing classical extraction

techniques involving homogenization, liquid-liquid partitioning, and/or other sample preparation steps are

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lengthy and use relatively large volumes of organic solvent. One purpose of this study was to develop a

practical, accurate, and precise method for rapidly

extracting and quantifying SDM and N-acetyl SDM residues in channel catfish muscle and plasma at tissue concentrations about the FDA tolerance of 100 ng/g.

Additionally, a method using small volumes of solvent was desired. This paper presents a simplified matrix solid phase dispersion (MSPD) extraction procedure for use with

biological fluids and the first application of MSPD and

microbore analytical column technology for the simultaneous extraction and high performance liquid

chromatographic (LC) analysis of SDM and its primary metabolite as residues in channel catfish muscle and plasma.

EXPERIMENTAL

REAGENTS AND EXPENDABLE MATERIALS

(a) SOLVENTS — Liquid chromatography grade from

commercial sources was used without further purification. Phosphate buffer (0.017M) for LC mobile phase was prepared

by placing 1.143 ml of orthophosphoric acid (44.6N) in

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triple-distilled water sufficient to make 1 L total volume.

(b) WATER — For LC analyses triple-distilled and

passed through a Modulab™ Polisher I water purification

system (Continental Water Systems Corp., San Antonio, TX) (c) CHEMICALS — Note: Wear rubber gloves and

protective clothing when handling. Sulfadimethoxine (SDM) and Sulfamethoxazole (SMX) were obtained from Sigma Chemical Co., (St. Louis, MO).

(d) 4-N-ACETYLSULFAJDIMETHOXINE. — synthesized according to the procedure of Nielsen [4.22]. Identity

confirmed with high performance liquid chromatography and direct probe, chemical ionization, positive ion mass spectrometry.

(e) STANDARD SOLUTIONS — Separate stock solutions of

SDM, N-acetyl SDM, and SMX were prepared by dissolving 40 mg of compound in 50 ml methanol. Fortification solutions

at concentrations of 5, 10, 20, 40, & 100 ngj ml were

prepared for each compound by diluting stock solutions in methanol and were stored in the dark at 4°C

(f) MSPD COLUMN MATERIAL — preparative grade C18/ 40

frn, 18% carbon load, end capped (Analytichem Bondesil™, Part No. 1221-3013)? obtained from Varian Associates (Harbor City, CA). The C18 was cleaned prior to use by

washing with 2 volumes of dichloromethane; then vacuum

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aspirated to remove excess solvent. Stored at room

temperature in a closed container until use.

(g) MSPD EXTRACTION COLUMNS

MUSCLE EXTRACTIONS — 10 ml disposable plastic syringe barrels (Becton Dickinson Co., Franklin Lakes, NJ) were washed with hot soapy water, triple rinsed with each of

tap water, double distilled water, and methanol; air dried

prior to use. Whatman no. 1, 1.5 cm paper filters were placed proximally and distally to the tissue-C18 blend to

secure this material in place within the column. A

syringe plunger, modified by removal of the rubber tip, was used to compact material in the column.

PLASMA EXTRACTIONS --0.8 X 4 cm disposable

polypropylene chromatography columns containing a 35 p polyethylene frit were used as supplied without further cleanup (Poly-Prep column, cat. no. 731-1550, Bio-Rad Laboratories, Richmond, CA).

A plastic pipet tip (100 /xl), modified by removing -1

cm from the proximal and distal ends, was placed on the syringe or column tip to direct and control flow rate through the column.

(h) SAMPLES

MUSCLE — Drug-free channel catfish were obtained from an indoor recirculating water culture system. Skinless fillets were stored frozen (-20°C).

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PLASMA — Market sized channel catfish, with no

history of drug administration, were captured by seining

from a local commercial catfish farm. Blood samples were taken by venipuncture of the caudal vein. Heparinized

blood samples were centrifuged and plasma stored at -20°C

in glass tubes.

Muscle and plasma samples were found to be free of

SDM concentrations greater than 25 ng/g as determined by LC analysis of MSPD extracts [4.19]. Three fish were used

for muscle and plasma fortification studies; two samples from each fish were analyzed at each of the five SDM and N-acetyl SDM concentrations tested. All samples were used within 1 month.

EXTRACTION PROCEDURE

(a) MUSCLE — 0.5 g of catfish fillet was placed in a clean polystyrene weigh boat, allowed to reach ambient

temperature, and injected with 5 pil each of separate SDM

and N-acetyl SDM fortification solutions (concentrations of 0, 5, 10, 20, 100 pig SDM or N-acetyl SDM/ml methanol)

appropriate to achieve tissue concentrations of 0, 50,

100, 200, and 1000 ng/g of each compound. SMX was added as an internal standard by the injection of 5 pi of a 100

pig SMX/ml methanol fortification solution to give a tissue

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concentration of 1000 ng/g. The samples were allowed to

stand 2 minutes, placed in a glass mortar containing 2 g

of Cjg, and blended using a glass pestle until a homogenous

mixture was obtained (-1 min.). The resulting blend was

then placed in a column fitted with a 1.5 cm disc paper

filter frit, another paper filter placed on top of the packing, and the packing compressed to a volume of ~4.5 ml

using a modified syringe plunger.

The tissue-CIg blend was washed with 8 ml of hexane

and the wash discarded. The column was placed over a clean 12 ml glass vial and the three sulfonamides eluted using 8 ml of dichloromethane (DCM). The DCM extract was placed in a 35°C water bath and evaporated to dryness

under nitrogen. The eluent was reconstituted by the

addition of 500 fil of mobile phase, vortexed, and

sonicated (Bransonic® 1200, Branson Ultrasonic Corp.,

Danbury, CT) for 7 minutes. The sample extract was

transferred to a 1.5 ml polypropylene test tube (Micro Tube, cat. no. 72.690.475, Sarstedt, Inc., Newton, NC), centrifuged for 5 minutes at 15,600 X g (Centra-M, International Equipment Co., Needham Hts., MA), and the

supernatant was filtered using a 0.45 jLim syringe filter

(Nalgene®, cat. no. 176-0045, Rochester, NY) into an LC autosampler vial.

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(b) PLASMA — 0.5 ml aliquots of catfish plasma were

placed in 1.5 ml polypropylene test tubes and 5 pi each of

SDM and N-acetyl SDM fortification solutions (concentrations of 0, 5, 10, 20, 40, 100 fig/ml methanol) were added to achieve plasma concentrations of 0, 50, 100,

200, 400, and 1000 ng/g of each compound. 5 /il of a 100 pg SMX/ml methanol fortification solution was added to each tube as an internal standard and vortex mixed.

Plasma samples were extracted using a modification of

the MSPD procedure. The C18 was weighed (400 mg), placed

in a Poly-Prep column, and 100 pi of plasma added. The plasma and C18 were vortex mixed for 30 sec; then packed to

a volume of -0.8 ml using a glass rod. The snap-off tip

was removed from the column, a modified pipette tip placed on the tip, and the column placed over a 6 ml glass tube.

The plasma-C18 blend was washed with 2 ml of hexane and the

wash discarded. The column was then placed over a clean 6

ml glass tube and the three sulfonamides eluted using 2 ml of DCM. The DCM extract was placed in a 35°C water bath

and evaporated to dryness under nitrogen. The eluent was reconstituted by the addition of 250 pi of mobile phase, then processed as with the muscle extracts.

(c) SOLID PHASE EXTRACTION — 100 pi of catfish plasma

fortified with SMX at 1000 ng/ml, and SDM and N-acetyl SDM at 200 ng/ml each was diluted to 2 ml with 0.017 M aqueous

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phosphoric acid and applied to the top of a Poly-Prep

column containing 400 mg of Clg. Positive pressure was

applied to the head of the column using a bulb syringe to

achieve a flow of -1 ml/min through the packing bed. Wash

and elution solvents and sample processing were performed as with the plasma MSPD procedure.

LIQUID CHROMATOGRAPHIC ANALYSIS

(a) APPARATUS — All analyses were performed using a

Hewlett Packard 1090 liquid chromatograph equipped with a variable volume auto-injector and a photodiode array

detector set at 265 nm (20 nm bandwidth), reference

spectrum of 450 nm (100 nm bandwidth), and spectrum range

of 210-320 nm. LC control, data acquisition, and peak integration were performed using Hewlett-Packard HPLC30

ChemStation Software (DOS Series)(Part No. G1300-900006).

A reversed phase narrow-bore column packed with octadecylsilyl (ODS) derivatized silica (200 X 2.1 mm, ODS Hypersil, 5 (m, Part No. 799160D-572, Hewlett-Packard,

Wilmington, DE) maintained at ambient temperature was used for all analyses.

(b) SOLVENT — The mobile phase was a mixture of 0.017

M aqueous H3P04 (pH 2.4)(PB) and acetonitrile (ACN)

delivered at an isocratic flow rate of 0.4 ml/min.

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Solvent ratios of 71:29 (PB:ACN) for muscle analysis and 73:27 for plasma analysis were used. The mobile phase was

filtered over a 0.45 pm membrane filter (FP Vericel™,

Prod. No. 66480, Gelman Sciences, Inc., Ann Arbor, MI) and

degassed with helium before use. A 10 /il aliquot of

muscle extract or a 25 pi aliquot of plasma extract was injected for LC analysis.

DATA ANALYSIS

(a) LINEARITY OF RESPONSE — Five-point calibration curves for SDM and N-acetyl SDM for standards and

extracted samples were constructed by plotting the peak

height ratio of analyte to internal standard (SMX) using data derived from duplicate sample injections. (b) LIMITS OF DETECTION AND QUANTITATION— The method detection limit (MDL) is defined as the minimum

concentration that can be reliably identified with 99%

confidence that the analyte concentration is greater than zero. The MDL was estimated by determining the concentration that corresponds to Student's value

multiple of the standard deviation of instrument values for replicate samples containing 1-5 times the estimated MDL. The MDL was computed using the following equation:

MDL = t' (S)

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where t* is the Student's t value for n-1 degrees of

freedom (n = 12) and a = 0.01; S = pooled standard

deviation of peak height ratios for MSPD extracts of samples containing 50 and 100 PPB analyte [4.23]. The MDL peak height ratio value was converted to tissue

concentration in PPB by linear regression using three

point calibration curves developed with MSPD extracts of

muscle or plasma samples. The practical limit of

quantitation was estimated as the concentration equal to two times the MDL.

(c) INTRA-ASSAY VARIABILITY — The within-run precision or the precision of replicate injections of the

same sample run under identical chromatographic

conditions. Intra-assay variation was calculated for both

MSPD extracted samples and standards for each of five concentrations examined and overall. The intra-assay

variation at each concentration is defined as the coefficient of variation (CV) of triplicate injections of

the same sample (n=6 samples/concentration), and was

calculated using the mean and standard deviation (SD) of peak height ratios for these injections. The CVs of the

six samples were averaged to obtain a CV for each

concentration. Overall intra-assay variability of the method, at the concentrations examined in this study, was

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determined by averaging the CVs for the five concentrations examined.

(d) INTER-ASSAY VARIABILITY — The variation among

samples containing the same concentration of analyte and

internal standard; extracted and analyzed under identical conditions on different days. Inter-assay variation for

each of five concentrations was determined for both MSPD

extracted samples and standards using mean peak height ratio values of duplicate injections. Inter-assay

variation at each concentration is defined as the CV of

six samples extracted and assayed on six different days. Overall inter-assay variation (± SD) of the method was obtained by averaging the individual concentrations' CVs.

(e) RECOVERY — Recovery was evaluated by two methods. Absolute recovery was determined for SDM with tritium labelled SDM (Charm Sciences, Inc., Madden, MA) using liquid scintillation counting data (Packard TRI-CARB®

4640, Packard Instrument Co., Inc., Downers Grove, IL). Relative recovery of SDM and N-acetyl SDM was determined

by a comparison of analyte:internal standard peak height

ratios of MSPD extracted fortified samples to peak height ratios of standard solutions.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 7 3 RESULTS AND DISCUSSION

CHROMATOGRAPHIC SYSTEM

The mobile phase (MP) composition reported by Long et

al. [4.19] of phosphate buffer and acetonitrile at a ratio of 65:35 did not provide adequate resolution of the three

sulfonamides when used with a narrow-bore column containing hypersil ODS. Additionally, endogenous

compounds in muscle and plasma extracts eluted very

closely to the sulfonamides and interfered with peak resolution. Modification in the MP solvent ratio from 29:71 to 27:73 (ACN:PB) resulted in adequate separation of

the sulfonamide and interfering peaks such that they did not affect quantitation for muscle or plasma extracts.

SMX was chosen as the internal standard because of its

favorable elution time and a UV absorbance maximum similar

to those of the analytes. SDM was the latest eluting of the sulfonamides at ~5.8 minutes for plasma samples and

~5.2 min for muscle extracts. A late eluting endogenous compound required run times of 12 minutes for plasma and

muscle extracts. Representative liquid chromatograms of

MSPD derived extracts of blank and sulfonamide fortified channel catfish muscle and plasma, and MSPD extracts of

muscle and plasma from channel catfish fed Romet®-30

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 7 4 medicated feed are shown in Figures 4.1-4.6. Figures 4.7- 4.9 show the UV absorbance spectra of SDM and N-acetyl SDM

in non-extracted standards and in MSPD extracts of incurred muscle and plasma. The absorbance maxima of SDM

and N-acetyl SDM were 265 and 270 nm, respectively. A comparison of the UV spectra of standards to those

obtained with MSPD extracts indicates excellent peak purity.

LINEARITY OF RESPONSE

A linear response was observed for daily five point calibration curves for standards and MSPD extracts of muscle and plasma using mean peak height ratio values of duplicate injections (concentrations of 50-1000 PPB). Correlation coefficients > 0.999 were obtained for all

calibration curves using standard solutions. Daily

standard curves for MSPD extracted muscle and plasma samples had correlation coefficients ranging from 0.993 -

1.0 with average daily values of 1.0 for SDM in both matrices and 0.997 and 0.998 for N-acetyl SDM in muscle and plasma.

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mln 10 2 6 4 0 8 55 55 ng N-ACETYL SDM/g; FORTIFIEDWITH SMXAS INTERNAL STANDARDAT 1000 ng/g. FED CATFISH; CALCULATED CONCENTRATIONS OF 507 ng SDM/gAND mAU 3 3 LIQUID CHROMATOGRAMOF MSPD EXTRACT OF CHANNEL CATFI8HMU8CLE FROMROMET® FIGURE 4 i I

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m l n 10 2 0 4 8 8 WITH SMXAT 1000 ng/ml, ANDN-ACETYL SDMAND SDMAT 400 ng/ml EACH. mAO FIGURE 4.5 LIQUIDCHROMATOGRAM OF M8PD EXTRACT FROM CHANNEL CATFISH PLASMA FORTIFIED

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mini

0 -■ 0 ng N-ACETYL SDM/ml; FORTIFIEDWITH SMXAS INTERNAL STANDARDAT 1000 ng/ml. FED CATFISH; CALCULATED CONCENTRATIONS OF 1158 ng 8DM/ml AND 379 mAU 6 6 LIQUID CHROMATOGRAMOF MSPD EXTRACT OF CHANNEL CATFISH PLASMAFROM ROMET® FIGURE 4. \

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-10

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Reproduced with permission of the copyright owner Further reproduction prohibited without permission. 182 nm ) MSPD) IN EXTRACT --- 450 600 ) AND) N-ACETYL 8DM ( --- 260 300 07 MUSCLE 7ROMROMET® 7ED CHANNEL CAT7ISH. LIQUIDCHROMATOGRAPHIC ANALYSIS WITH DIODEARRAY DETECTION. 2 - 10 40 30 60 60 70 - 70 FIGURE 4.8 UVABSORBANCE SPECTRA07 SDM (

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nm ) INMSPD) EXTRACT --- 450 ) ANDN-ACETYL ) SDM < --- 360 OF PLASMAFROM ROMET® FED CHANNEL CATFISH. LIQUID CHROMATOGRAPHICANALYSIS WITH DIODE ARRAY DETECTION. - 2 0 60 40 - 40 80 Norm.. FIGURE 4.9 UVABSORBANCE SPECTRA OF SDM (

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LIMITS OF DETECTION AND QUANTITATION

The MDL estimates were 26 ng/g for SDM in channel catfish muscle and 33 ng/ml in plasma. The MDLs for N- acetyl SDM were 26 ng/g and 11 ng/ml respectively for

muscle and plasma. Practical limits of quantitation were

52 ng/g for SDM and N-acetyl SDM in muscle, 66 ng SDM/ml plasma, and 22 ng N-acetyl SDM/ml plasma.

INTRA-ASSAY VARIABILITY

Current recommendations for the precision of animal drug residue assays are for the repeatability of a method

not to exceed 10% at concentrations greater than or equal

to tolerance (100 PPB) or 20% at concentrations less than tolerance [4.24]. The repeatability of the method as

determined by the CV of triplicate injections was <10% for

SDM and N-acetyl SDM at concentrations of 50-1000 PPB for all muscle and plasma extracts and standards (Table 4.1).

INTER-ASSAY VARIABILITY

The reproducibility of the method varied from -25% for SDM and N-acetyl SDM at concentrations of 50 ng/g muscle to 5% for muscle containing 400 ng N-acetyl SDM/g

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ul 00 1.7 6.5 6.6 3.5 N-acetyl SDM Plasma 0.74.3 0.6 3.8 9.1 Extracts1, MSPD 1.1 4.9 5.6 ± ± 2.1 ± 3.4 ± 2.7 Muscle 1.74.0 1.4 2.2 1.4 0.9 0.4 8.4 SDM N-acetyl SDM SDM 2.3 3.7 4.9 2.8 5.1 0.5 0.90.7 3.9 5.8 ±2.2 ± 3.0 N-acetyl SDM Standards* 1.4 0.6 0.8 2.5 SDM ± ± 2.2 50 5.3 100 4.2 200 400 SDM 1000 (ng/g) Overall CV (mean ± ± SD) (mean concentration f f N = extractions/cone. 6 injections) (triplicate * * N = 6 injections) (triplicate TABLE 4.1 VARIATION INTRA-ASSAY (CV)

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using the mean peak height ratio values for duplicate

injections. The overall daily variation among samples

containing 50-1000 PPB SDM was 13% for muscle and 14% for plasma extracts, and 11% and 10% for N-acetyl SDM in muscle and plasma samples (Table 4.2).

RECOVERY OF METHOD

Absolute recovery of tritiated SDM at a tissue concentration of 150 PPB was 79% ± 4% for muscle and 67% ±

5% for plasma. The mean relative recoveries of SDM and N- acetyl SDM based on a comparison of the peak height ratios of standard solutions and extracted spiked tissues were 97% ± 4% and 91% ± 3% for SDM in muscle and plasma and

126% ± 5% and 112% ± 6% for N-acetyl SDM in muscle and plasma. The high relative recovery of N-acetyl SDM was consistent across all concentrations examined and may

result from an increased extraction efficiency of the acetylated compound compared to the two non-acetylated compounds. The 4-N-acetylated sulfonamides (except

sulfapyrimidines) have decreased aqueous solubility

compared to the parent compounds [4.25] and would be

expected to have different solubility in extracting solvents than the parent compounds. The use of standard curves developed with peak height ratio values for

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. H 00 7.9 ± ± 3.5 14.3 10.1 21.0 16.0 SDM N-acetyl SDM ± ± 4.7 MSPD Extracts+ 8.8 9.1 7.6 11.4 N-acetyl SDM Muscle Plasma 6.96.6 5.0 11.0 8.3 SDM 12.8 12.8 5.9 16.6 ± ± 7.3 ± 8.3 3.8 3.3 4.4 13.2 11.9 13.9 10.5 11.8 24.5 25.5 ± ± 4.0 N-acetyl N-acetyl SDM Standards* 2.5 1.7 5.8 5.1 5.0 9.0 SDM ± ± 2.5 50 100 200 4.4 400 3.9 SDM 1000 (ng/g) Overall CV (mean ± ± SD) (mean concentration t t N = extractions/conc. 6 mean of duplicate injections) (using * * N mean = duplicate6 of injections) (using TABLE 4.2 TABLE 4.2 VARIATION INTER-ASSAY (CV)

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fortified tissues eliminated the extraction inequity

problem and resulted in near 100% agreement between known

tissue concentration and calculated tissue concentration for SDM and N-acetyl SDM. Therefore, quantitation of N-

acetyl SDM should be performed using standard curves developed with fortified samples.

The modified MSPD method for plasma extraction had a

lower absolute recovery than the MSPD method for muscle. However, the plasma method offers several advantages. The

technique of vortex-mixing a biological fluid, such as

plasma, with in the extraction column eliminates the

need for manual blending of the sample and C18 and transfer

of the blended material from mortar to column that is

required with the MSPD method for solid tissues. For

tissues not requiring mechanical disruption of tissue

architecture, this adaptation greatly speeds the

extraction process and makes it more amenable to

automation. The need for mortar and pestle cleanup and concerns about possible carryover are also eliminated with this modification. Additionally, the small sample size required (100 /xl) is advantageous in experimental studies

involving small animals such as fish and rodents. The

modified MSPD method has also been used with milk for the extraction of several /3-lactam antibiotics (unpublished

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data) and the method potentially can be applied to other

biological fluids and compounds.

For comparison of the plasma MSPD method with solid phase extraction (SPE), fortified catfish plasma was extracted using a packing of 400 mg of CI8 in a Poly-Prep

column (n=3). SPE yielded recovery and chromatography

similar to that obtained with the plasma MSPD method.

However, SPE required an additional sample dilution step

and more importantly, flow of the diluted sample and

solvents through the SPE column required either positive

column head pressure or aspiration. While chromatography and recovery were similar with both methods, the ability to utilize gravity flow of solvents is a distinct advantage in some laboratories and essential for field

applications.

A tiered approach to residue monitoring utilizes

rapid screening assays for examining large numbers of

samples and subsequent quantitation and/or confirmation of identified suspect samples. We previously presented an

evaluation of commercially available enzyme immunoassays for screening channel catfish muscle for SDM residues

using MSPD derived extracts [4.10]. A number of other techniques have been used for detection and quantitation of sulfonamides in animal tissues. A 1981 review of analytical methods for sulfonamides in animal tissues is

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available [4.11,4.12]. More recently, several methods for single or multiresidue analysis of sulfonamide residues in

fish tissues have been published [4.13-4.21]. Several classical multi-residue methods for the

isolation of SDM from fish tissues have been reported. An extraction method reported by Horie et al. yielded

recoveries of 73-80% for SDM fortified at 1 /xg/g muscle in several fish species. The limit of detection for SDM was 0.05 /xg/g [4.13]. Nose et al. reported a multi-residue

method for three fish species that provided SDM recoveries of 88-92% from muscle tissue fortified at 0.5 /xg/g. The method of recovery determination was not provided. The

lower limit of detection for SDM was 0.06 /xg/g [4.14]. Horie et al. published a multi-residue method for the

determination of eight antibacterials in the muscle of five species of cultured fish. Recovery of SDM varied among species from 82-87% at a tissue concentration of 1

/xg/g. The detection limit of the method was 0.05 /xg/g [4.15]. An extraction and LC method reported by Ueno et al. for sulfamonomethoxine, SDM, and their N-acetylated metabolites provided SDM recoveries of 88 and 81% from

rainbow trout plasma and muscle fortified at 2 PPM,

respectively. This study was the only method found in the

literature for extraction and analysis of N-acetyl SDM in fish tissues. N-acetyl SDM recoveries were 83 and 89%

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 191 from muscle and plasma. The method of recovery

determination was not provided. The detection limits were

0.05 /xg/g and limits of quantitation were 0.5 /xg/g for both compounds [4.16].

Two methods are reported for the simultaneous extraction and LC analysis of SDM and OMP in fish tissue.

Weiss et al. reported a mean recovery of 97 % for SDM in catfish muscle. The limit of quantitation was 0.05 /xg/g [4.17], Walisser et al. reported a mean relative

recovery, using peak height ratio comparisons, of 55% for SDM from chinook salmon muscle fortified at 0.5-6 /xg/g. The minimum detectable concentration was 0.2 /xg/g [4.18],

Several MSPD methods have been reported for the

isolation of SDM from fish tissues. Long et al. reported a relative recovery of 101% based on peak height ratios

for SDM in catfish muscle using a single residue MSPD

extraction procedure. The minimum detectable limit was

0.05 /xg/g [4.19]. In two studies, Reimer & Suarez used

MSPD for the simultaneous extraction of five sulfonamides from salmon muscle with quantitation by thin layer

chromatography (TLC) or LC. For TLC, the method detection limit (MDL) of SDM was 0.13 /xg/g and recovery was 63% at

concentrations of 0.5 and 2 /xg/g. In a second study, they reported 75% recovery for SDM fortified at 0.4-5 fig/g and a MDL of 0.15 /xg/g using LC analysis [4.20,4.21].

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Tolerances for animal drugs as set by the United States Food and Drug Administration (FDA) are often based

on total residue present, including parent drug and all

metabolites, in edible tissues (4.24]. The FDA tolerance

for total SDM residues in channel catfish muscle is 100

nU/5 [4.26]. The primary metabolite of SDM in channel

catfish is 4-N-acetylsulfadimethoxine [4.27]. The relative contribution of this metabolite to total SDM residue varies among tissues and species. In channel

catfish, N-acetyl SDM accounts for over 90% of total SDM residue in bile but in muscle and plasma SDM is present mainly (>95%) as parent compound [4.27]. However, in

rainbow trout muscle the N-acetyl metabolite accounts for

over half of total SDM residue [4.28]. Additionally, the ratio of parent SDM:N-acetyl SDM at tissue concentrations approaching tolerance is not known. Under the FDAs general food safety requirements, individual metabolite

identification, concentration, and persistence should be

obtained for metabolites comprising 100 ppb or >10% of the

total residue (whichever is lower) at zero withdrawal [4.29], Extraction, detection, quantitation, and

confirmatory methods should, therefore, be capable of simultaneous extraction or analysis of the parent drug and it's important metabolites at tolerance for such use and

in situations where marker residues are not determined.

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The USDA has indicated that a method of residue

analysis should take no more than 48 hours to perform

[4.24]. The MSPD procedure and LC method as used in this study can easily be run in less than a half-workday. The

sensitivity and recovery of SDM with this method is comparable to recoveries reported with published methods

for SDM in fish tissues [4.13-4.21]. However, most of

these studies used classical methods of analyte isolation

involving homogenization, liquid-liquid partitioning, and multiple sample cleanup steps. With improvements in

determinative methods a major limiting factor in a residue monitoring program becomes the isolation of the analyte(s)

from tissues. Classical methods of extraction are the backbone of present residue monitoring programs and have

withstood the test of time but are time, labor, and

solvent use intensive and limit the number of samples that can be practically analyzed in a day. Additionally,

several of the methods lack the sensitivity required for detection of SDM residues at present tolerance. Newer

methods of analyte isolation that eliminate many of the disadvantages of traditional extraction methods are needed

if we are to fully benefit from recent advances in determinative aspects of residue analysis.

One such isolation method is the MSPD procedure. The MSPD theory and process have been the subject of recent

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reviews [4.30,4.31]. In general, its main advantages are that it provides a more rapid and solvent sparing

isolation method than do classical methods, provides

adequate recovery at tolerance, and is amenable to

automation. Therefore, the use of MSPD allows one to examine a larger number of samples per day and reduces

labor and solvent costs. Solvent purchase and disposal

costs and potential environmental contamination problems increasingly dictate that solvent use in routine analytical methods be reduced. The combined use of MSPD

and a microbore analytical column often yields a greater than 50% reduction in solvent use compared to classical extraction and analysis techniques.

The extraction and LC methods presented in this study

provide procedures to simultaneously analyze SDM and its major metabolite in channel catfish muscle and plasma. These methods provide rapid and simple extraction with

adequate recovery of analytes, quantitation at tolerance

with acceptable accuracy and precision, and minimal solvent use. The applicability of these procedures to

channel catfish muscle and plasma samples containing

incurred SDM residues are presented in Chapter 5.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 9 5 REFERENCES

4.1 Consumer Reports, "Is our fish fit to eat". February 1992, pp. 103-114. 4.2 Broussard, M.C., Jr. (1991) Aquaculture: Opportunities for the nineties. Journal of Animal Science, 69:4221-4228. 4.3 Meyer, F.P. (1991) Aquaculture disease and health management. Journal of Animal Science, 69:4201-4208. 4.4 Johnsen, P.B. (1991) Aquaculture Product quality issues: Market position opportunities under mandatory seafood inspection regulations. Journal of Animal Science, 69:4209-4215.

4.5 Ahmed, F.E. (Editor) (1991) Seafood Safety,National Academy Press, Washington, DC 4.6 Mitchell, G.A. (1991) Compliance issues and proper animal drug use in aquaculture. Veterinary and Human Toxicology, 33(Sup 1):9-10. 4.7 Van Dresser, W.R., and Wilcke, J.R. (1989) Drug residues in food animals. Journal of the American Veterinary Medical Association, 194(12):1700-1710. 4.8 Paige, J.C. (1993) Analysis of illegal residues in meat and poultry. FDA Veterinarian, 8(2):5-6.

4.9 Miller, R.W. (1991) Get hooked on seafood safety. FDA Consumer, 25(5):6-11.

4.10 Walker, C.C. and Barker, S.A. (1993) Extraction and enzyme immunoassay of sulfadimethoxine residues in channel catfish (Ictalurus punctatus) muscle. Journal of the Association of Official Analytical Chemists, accepted for publication. 4.11 Horwitz, W. (1981) Analytical methods for sulfonamides in foods and feeds. I. Review of methodology. Journal of the Association of Official Analytical Chemists, 64(1):104-130. 4.12 Horwitz, W. (1981) Analytical methods for sulfonamides in foods and feeds. II. Performance characteristics of sulfonamide methods. Journal of

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the Association of Official Analytical Chemists, 64(4):814-824.

4.13 Horie, M., Hoshino, Y., Nose, N., Iwasaki, H. & Nakazawa, H. (1985) Simultaneous determination of antibiotics and synthetic antibacterials in fish by high performance liquid chromatography. Eisei Kagaku 31(6):371-376. 4.14 Nose, N. Hoshino, Y., Kikuchi, Y., Horie, M., Saitoh, K., Kawachi, T., and Nakazawa, H. (1987) Simultaneous liquid chromatographic determination of residual synthetic antibacterials in cultured fish. Journal of the Association of Official Analytical Chemists, 70(4):714-717.

4.15 Horie, M., Saito, K., Hoshino, Y., Nose, N., Nakazawa, H., and Yamane, Y. (1991) Simultaneous determination of residual synthetic antibacterials in fish by high-performance liquid chromatography. Journal of Chromatography, 538:484-491. 4.16 Ueno, R., Uno, K., Kato, M., Kubota, S.S., and Aoki, T. (1991) Simultaneous determination of sulfamonomethoxine, sulfadimethoxine and their N4- acetylated forms in fish tissues by high performance liquid chromatography. Nippon Suisan Gakkaishi, 57(3):549-554.

4.17 Weiss, G., Duke, P.D., and Gonzales, L. (1987) HPLC method for the simultaneous analysis of sulfadimethoxine and ormetoprim in tissues and blood of cattle, chickens, and catfish. Journal of Agricultural and Food Chemistry, 35(6):905-909.

4.18 Walisser, J.A., Burt, H.M., Valg, T.A., Kitts, D.D., and McErlane, K.M. (1990) High-performance liquid chromatographic analysis of Romet-30* in salmon following administration of medicated feed. Journal of Chromatography, 518:179-188. 4.19 Long, A.R., Hsieh, L.C., Malbrough, M.S., Short, C.R. and Barker, S.A. (1990) Matrix solid phase dispersion isolation and liquid chromatographic determination of sulfadimethoxine in catfish (Ictalurus punctatus) muscle tissue. Journal of the Association of Official Analytical Chemists, 73(6):868-871.

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4.20 Reimer, G.J., and Suarez, A. (1991) Development of a screening method for five sulfonamides in salmon muscle tissue using thin-layer chromatography. Journal of Chromatography, 555:315-320

4.21 Reimer, G.J. and Suarez, A. (1992) Liquid chromatographic confirmatory method for five sulfonamides in salmon muscle tissue by matrix solid phase dispersion. Journal of the Association of Official Analytical Chemists International, 75(6)1979—981.

4.22 Nielsen, P. (1973) The metabolism of four sulphonamides in cows. Biochemical Journal (London), 136:1039-1045.

4.23 Glaser, J.A., Foerst, D.L., McKee, G.D., Quave, S.A., and Budde, W.L. (1981) Trace analyses for wastewater. Environmental Science & Technology, 15(12):1426-1435. 4.24 Livingston, R.C. (1985) Antibiotic residues in animal-derived food. Journal of the Association of Official Analytical Chemists, 68(5):966-967. 4.25 Vree, T.B., and Hekster, Y.A. (1987) Clinical pharmacokinetics of sulfonamides and their metabolites - sulfadimethoxine. Antibiotics and Chemotherapy, 37:25-30.

4.26 United States Food and Drug Administration, Code of Federal Regulations (1988) Title 21, Part 556:640, Washington, D.C.

4.27 Squibb, K.S., Michel, C.M.F., Zelikoff, J.T., O'Connor, J.M. (1988) Sulfadimethoxine pharmacokinetics and metabolism in the channel catfish (Ictalurus punctatus). Veterinary and Human Toxicology, 3 0(Supplement 1):31-35. 4.28 Hara, T. and Inoue, S. (1967) Concentrations of various sulfonamides in the tissues of rainbow trout after oral dosing. Bulletin of the Japanese Society of Scientific Fisheries 33 (7):618-623. 4.29 Weber, N.E. (1983) Metabolism and kinetics in the regulation of animal drugs. Journal of Animal Science, 56(1):244-251.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 9 8 4.30 Barker, S.A., Long, A.R., and Short, C.R. (1989) Isolation of drug residues from tissues by solid phase dispersion. Journal of Chromatography 475:353-361.

4.31 Barker, S.A., and Long, A.R. (1992) Tissue drug residue extraction and monitoring by matrix solid phase dispersion (MSPD)-HPLC analysis. Journal of Liquid Chromatography, 15(12):2071-2089.

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PLASMA/MUSCLE RATIOS OF SULFADIMETHOXINE RESIDUES IN CHANNEL CATFISH (Ictalurus punctatus)

INTRODUCTION

Pending federal legislation concerning mandatory seafood inspection will require a variety of analytical

methods for the detection, quantitation, and confirmation of chemical residues in food fish [5.1]. A tiered

approach to residue monitoring [5.2] uses rapid screening

assays for an initial examination of large numbers of

samples and more definitive methods of analysis for

quantitation and identification of chemical residues in samples identified with screening assays. Thus,

regulatory agencies will need practical methods that are rapid, accurate, precise, and specific for these purposes. Traditional screening methods, such as microbial

inhibition tests and chromatographic methods, as are

presently used in poultry and red meat inspection, lack

one or more of these characteristics. Additionally, there

is a lack of screening methods available for use by the producer in a field setting for the pre-harvest detection

of illegal drug residues. Screening methods compatible

with field use could impact in a positive manner the function of an aquatic animal drug residue monitoring

program by reducing the marketing of fish containing

199

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 200 violative drug residues, would help prevent economic loss

to producers resulting from the condemnation of large lots

of harvested fish, and as part of a quality assurance

program, help allay consumer concerns regarding aquaculture product safety.

One technique with potential field use application as

a residue screening method is the enzyme-linked

immunosorbent assay (ELISA). Enzyme-linked immunosorbent assays for detection of chemical residues are relatively

inexpensive, easy to use, require no special equipment,

and are presently commercially available for a range of compounds [5.3,5.4]. They also provide the sensitivity and specificity required for residue monitoring at

tolerance [5.5]. Nonetheless, a major limiting factor in

the practical use of these tests for residue detection in tissues is the need for analyte extraction from tissue samples prior to testing.

To facilitate the implementation of ELISAs for chemical residue monitoring several strategies for analyte

extraction from target tissues have been used. These vary from simple tissue maceration [5.6] to matrix solid phase

dispersion [5.5,5.7]. However, the simplest residue screening method is one that requires no tissue

extraction, but rather uses the presence of the drug and/or its metabolite(s) in a readily accessible reference

biological fluid to indicate the presence of violative

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residues in a target tissue(s). However, before such a

technique can be implemented, a relatively constant

relationship between the drug concentration in the target tissue and the reference biological fluid must be

demonstrated. This is necessary because a relatively constant relationship between marker residue and target

tissue residue concentration among animals is needed to provide the sensitivity and specificity required of a

residue screening assay.

Biological fluids have been proposed as specimens for pre- or postmortem residue monitoring in several food

animal species and the tissue concentration relationships for several sulfonamides in domestic food animal species

have been evaluated [5.8-5.13]. However, little

information regarding such drug concentration relationships in fish species is available in the

literature. Several studies have examined the

pharmacokinetic and tissue depletion behavior of

sulfonamides in a limited number of fish species [5.14- 5.18], but none have examined the concentration ratio of

total sulfadimethoxine residues in biological fluids and

target tissues over the range of tissue concentrations expected in channel catfish.

The determination of such a drug concentration ratio would permit the use of a readily obtainable biological

fluid such as plasma to predict the presence of violative

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drug residues in edible tissues. This could enhance the

application of an on-site test and increase throughput in

a regulatory laboratory. The determination of such ratios must include data on total residue including the relative

contribution by the parent compound and its important metabolites. Such information is relevant in relating

concentration of the chemical species detected by the screening assay (marker compounds) to drug residue

concentration in the tissue of interest.

We previously evaluated the performance of several commercially available ELISAs for detection of

sulfadimethoxine in channel catfish muscle about the US Food and Drug Administration tolerance of 100 ng/g

[Chapter 3,5.5]. All of the ELISAs examined cross-reacted

equally well with the parent sulfonamide and its primary metabolite in channel catfish, 4-N-acetylsulfadimethoxine (N-acetyl SDM). Therefore, to use such ELISAs with plasma

to identify fish containing violative SDM tissue levels one must first know the ratio of total SDM residue in

plasma, including parent and cross-reacting metabolites, to total SDM residue in muscle.

Romet-30®, a potentiated sulfonamide containing

sulfadimethoxine (SDM) and ormetoprim (OMP) in a 5:1 ratio, is one of only two systemic antibacterial agents

currently marketed for use in aquaculture in the United

States. Romet-30® is frequently used in channel catfish

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. aquaculture for the control of enteric septicemia and other infectious bacterial diseases of catfish. The

channel catfish is the major aquaculture species in the US

and accounted for 56% of the total US aquacultural production in 1988 [5.19] with 360 million pounds produced in 1990 [5.20]. Drug residue violations in the United

States' red meat and poultry industries most frequently result from a failure by the producer to observe the

stated withdrawal time for an approved drug [5.21,5.22].

If a similar trend holds for aquaculture, the greatest number of residue violations would be expected for

approved drugs that have been used by the producer in a manner not in accordance with label directions.

Therefore, practical methods for detection, quantitation,

and confirmation of SDM and OMP residues in channel catfish are urgently needed.

The purpose of the present study was to determine the

concentration ratio of total SDM residues, including the parent compound and its N-acetyl metabolite, between channel catfish plasma and muscle to enable the use of

plasma drug concentration as an indicator of muscle drug

concentration. This paper presents the first report of such tissue concentration ratios over the range of SDM tissue concentrations expected when Romet®-30 is used

according to label instructions. Additionally, information regarding sulfadimethoxine depletion in

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channel catfish administered Romet®-30 at a water

temperature of 27°C is provided.

EXPERIMENTAL

REAGENTS AND EXPENDABLE MATERIALS

(a) SOLVENTS — Commercially obtainable liquid

chromatography grade solvents were used without further

purification. (b) WATER — Triple-distilled and passed through a Modulab™ Polisher I water purification system (Continental Water Systems Corp., San Antonio, TX) used for LC

analyses. (c) CHEMICALS — Note: Wear rubber gloves and

protective clothing when handling. Sulfadimethoxine (SDM) and Sulfamethoxazole (SMX) were obtained from Sigma

Chemical Co., (St. Louis, MO).

(d) 4-N-ACETYLSULFADIMETHOXINE — The procedure of Nielsen [5.23] was used for synthesis. Identity confirmed

with high performance liquid chromatography and direct

probe, Cl, positive ion mass spectrometry. (e) STANDARD SOLUTIONS — Stock solutions of SDM, N- acetyl SDM, and SMX were prepared and serially diluted in

methanol to provide separate fortification solutions for

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each compound. Fortification solutions of 5, 10, 20, 40,

100, 200, 500, 1000, & 2000 /ig/ml were prepared for SDM;

4, 10, 20, 50, 100, & 200 for N-acetyl SDM; and 100 Mg/ml for the internal standard SMX. All solutions were

stored in the dark at 4°C.

(e) FORTIFIED MUSCLE MID PLASMA EXTRACTS — Extracts of fortified muscle and plasma for daily standard

calibration curves were obtained as follows:

MUSCLE — 0.5 g of catfish muscle was injected with 5 nl each of separate SDM and N-acetyl SDM fortification

solutions appropriate to give tissue concentrations (SDM:N-acetyl SDM) of 50:0, 100:0, 200:0, 400:40,

1000:100, 2000:200, 5000:500, and 10000:1000 ng/g. SMX was added as an internal standard by the injection of 5 fil

of a 100 ng SMX/ml methanol fortification solution to give a tissue concentration of 1000 ng/g. The samples were processed and extracted by MSPD as described in chapter 4.

PLASMA — 0.5 ml aliquots of catfish plasma were placed in 1.5 ml polypropylene test tubes and 5 nl each of separate

SDM and N-acetyl SDM fortification solutions were added to achieve plasma drug concentrations of 50:0, 100:0, 200:0,

400:40, 1000:100, 2000:200, 5000:500, 10000:1000, and

20000:2000 ng/g (SDM:N-acetyl SDM). Five ^1 of a 100 ng SMX/ml methanol fortification solution was added to each tube as an internal standard, vortex mixed, and extracted

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using a modification of the MSPD procedure as given in chapter 4.

EXPERIMENTAL ANIMALS, DRUG ADMINISTRATION, AND SAMPLING

Eighty-four laboratory-reared male and female channel

catfish (Ictalurus punctatus) with a mean body weight of 59 ± 15 g were obtained from an indoor recirculating water

culture system and assigned to seven circular fiberglass tanks, each containing 200 liters of water. The fish were

maintained under flow-through conditions with a 13.5 hour light : 10.5 hour dark photoperiod. Water temperature was maintained at 27 ± 1°C, pH of 8.41 ± .04, and total

hardness of 20 mg/1. The fish were allowed to acclimate

for 4 weeks prior to drug administration. Experimental fish were fed commercial non-medicated catfish pellets (Grow Big Floater, SF Services, Inc.,

North Little Rock, AR) at a rate of 2% of body weight once daily before and after the drug treatment period. Romet®-

30 (Hoffman-La Roche, Inc., Nutley, NJ) medicated channel catfish feed (Grow Big Floater R-30 Medicated, SF

Services, Inc., North Little Rock, AR) containing SDM and ormetoprim at concentrations of 0.42% and 0.084% respectively was fed free-choice at a rate of 1% of body

weight once a day for five consecutive days to six tanks

of fish. This feeding rate provided a daily dose of 50 mg

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Romet®-30/kg fish body weight. Control fish were fed the commercial non-medicated feed throughout the experiment.

Groups of 12 fish (1 tank) plus 2 control fish were sacrificed at sampling times of 6, 12, 24, 48, 72, and 96

hours following the fifth day of medicated feed administration. Blood was collected from the caudal vein

with a heparinized syringe and the plasma separated and placed in 1.5 ml polypropylene test tubes (Micro Tube,

cat. no. 72.690.475, Sarstedt, Inc., Newton, NC). Skinless muscle fillets were obtained and stored in Whirl-

Pak® sample bags (Nasco). All samples were stored frozen at -20°C and analyzed within 2 months following

collection.

EXTRACTION PROCEDURE

(a) MUSCLE — 0.5 g of catfish fillet was placed in a

glass mortar containing 2 g of CI8. Sulfamethoxazole (SMX)

was added as an internal standard by the injection of 5 /il

of a 100 jug SMX/ml methanol fortification solution to give a SMX tissue concentration of 1000 ng/g. The muscle

samples were allowed to stand 2 minutes and extracted

using the matrix solid phase dispersion (MSPD) technique reported in Chapter 4.

(b) PLASMA — 0.5 ml aliquots of catfish plasma were

placed in 1.5 ml polypropylene test tubes and 5 pi of a

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100 ng SMX/ml methanol fortification solution added to

each tube as an internal standard and the sample was

vortex mixed. Plasma samples were extracted using a modification of the MSPD procedure as given in Chapter 4.

LIQUID CHROMATOGRAPHIC ANALYSIS

(a) APPARATUS — All analyses were performed using a

Hewlett Packard 1090 liquid chromatograph equipped with a variable volume auto-injector and a photodiode array

detector set at 265 nm (20 nm bandwidth), reference spectrum of 450 nm (100 nm bandwidth), and spectrum range of 210-320 nm. LC control, data acquisition, and peak

integration were performed using Hewlett-Packard HPLC30

ChemStation Software (DOS Series)(Part No. G1300-900006). A reversed phase narrow-bore column packed with

octadecylsilyl (ODS) derivatized silica (200 X 2.1 mm, 0DS Hypersil, 5 /im, Part No. 799160D-572, Hewlett-Packard,

Wilmington, DE) maintained at ambient temperature was used for all analyses.

(b) SOLVENT — The mobile phase was a mixture of 0.017

M aqueous H3P04 (pH 2.4)(PB) and acetonitrile (ACN)

delivered at an isocratic flow rate of 0.4 ml/min. Solvent ratio's of 70:30 (PB:ACN) for muscle analysis and

73:27 for plasma analysis were used. The mobile phase was

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filtered over a 0.45 pm. membrane filter (FP Vericel™,

Prod. No. 66480, Gelman Sciences, Inc., Ann Arbor, MI) and

degassed with helium before use. A 10 pi aliquot of muscle extract or a 25 pi aliquot of plasma extract was

injected for LC analysis.

DATA ANALYSIS

(a) DAILY CALIBRATION CURVES — Daily calibration

curves for SDM and N-acetyl SDM for both standards and

extracted fortified muscle and plasma samples were constructed by plotting the peak height ratio of analyte to internal standard (SMX) using data derived from single sample injections.

(b) DETERMINATION OF TISSUE CONCENTRATION — Tissue concentrations of experimental plasma and muscle samples

were determined by the internal standard method using

calibration curves obtained with extracts of fortified

plasma or muscle. The concentrations of samples

containing <1000 PPB analyte were determined using five point daily calibration curves derived from fortified

tissue extracts containing 50-1000 PPB. The concentrations of samples containing >1000 PPB analyte

were determined using daily calibration curves of concentrations of 50-20000 PPB.

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Experimental tissue samples containing drug concentrations less than the method detection limit (MDL)

were reported as zero. Samples containing drug

concentrations greater than the MDL but less than the

practical limit of quantitation (LOQ) (i.e. trace quantities) were reported in Tables 5.1-5.6 as the

estimated concentration value. However, samples in the above two categories were not used for calculation of plasma:muscle concentration ratios. For samples

containing trace drug quantities, the estimated

concentration was used for calculation of mean drug concentration values at each sampling period.

RESULTS AND DISCUSSION

Considerable variation in total SDM tissue

concentration among individual fish within a sampling

period was observed (Tables 5.1-5.6). Total SDM concentrations in individual experimental fish ranged from

1.4-24.8 fig/ml and 0.6-12.6 ;xg/g for plasma and muscle, respectively, for the twelve fish sampled at the six hour

post-dose sampling period. Mean total SDM residue concentrations for plasma and muscle samples for this same

period were 9.1 fig/ml and 5.3 fig/g and relative standard

deviations of 82.3% and 80.7% respectively. Similar large

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ro H H 1.7 0.3 1.5 1.8 2.5 81% 17% 4.24 1.22 0.1 0.56 6.90 2.0 5.11 1.8 1.50 1.6 9.82 7.97 9.07 1.3 12.63 2.0 Total Total Residue SDM 82% 2.16 1.71 0.90 1.9 13.66 • ** • 19% 0.1 1.6 1.61.5 9.04 15.13 1.51.5 1.62 2.47 0.90 1.8 1.2 11.72 4 ______83% SDM 1.16 4.01 0.3 7.50 6.57 1.9 6.28 1.5 11.70 6.96 4.84 1.7 9.11 5.25 1.8 9.10 4.64 0.79 0.59 2.0 1.54 0.70 2.2 8.54 11.89 1.9 24.86 87% 6.78 1.23 0.51 2.4 1.43 9.35 10.29 22.34 16% 3.7 1.20 3.43.7 1.16 3.7 7.30 2.9 12.51 7.31 1.7 14.46 ______63% 0.26 0.5 0.08 0.1 1.96 0.41 3.2 7.83 0.33 3.5 12.51 0.72 2.6 13.24 0.160.74 3.2 3.4 1.19 0.74 0.11 0.39 2.0 1.67 1.11 0.11 0.06 0.69 3.4 0.53 2.7 0.47 0.66 N-acetyl N-acetyl SDM 64% 1.43 0.81 1.14 0.24 1.89 0.42 1.74 1.95 2.52 0.79 2.35 0.38 ------______1 3 7 2 0.52 4 6 5 8 9 1112 0.20 SD 10 SEM RSD Mean 1.28 Fish # # Fish Plasma Muscle P:M Plasma Muscle P:M Plasma Muscle P:M * * concentration parts million per in TABLE 5.1 SULFADIMETHOXINE AND RESIDUES* PLASMA:MUSCLE RATIOS - HOURS6 POST-DOSE I

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to H to 1.8 *** 0.4 0.2 *** 1.1 *** *** *** *** P:M 4.40 112%1.27 22% 0.42 0.27 3.49 2.1 11.78 1.9

Muscle 0.58 0.26 116% 8.41 0.38 0.18 0.33 0.71 0.29 7.80 6.95 Plasma of quantitation of quantitation

1.5 1.7 7.23 3.93 0.1 2.43 1.9 21.92 0.4 1.1 0.9 2.1 19.58 8.92 2.2 2.4 0.57 0.18 1.5 7.42 4.43 1.7 P:M SDM TotalResidue SDM 1.16 0.26 0.24 3.75 10.64 Muscle 0.38 122% 113% 26% 6.19 3.55 0.21 7.52 4.02 2.17 0.24 0.180.60 1.3 0.38 1.6 0.82 6.096.46 3.06 6.08 0.42 2.0 0.18 7.48 0.48 0.25 2.0 5.62 19.98 16.53 9.19 1.8 19.13 9.95 1.9 Plasma *** 1.2 0.5 *** 1.5 3.0 *** *** 3.2 *** 4.1 17.28 8.36 40% 3.4 4.6 2.6 P:M ------1.13 1.7 0.38 0.12 0.43 (0.04) (0.04) Muscle N--acetyl SDM N--acetyl 93% 108% 0.12 0.96 0.41 0.28 0.22 1.40 Plasma 1516 17 0.231819 0.05 1.34 0.14 0.88 24 0.20 14 2.60 0.77 22 1.94 23 2.29 0.55 13 1.80 0.68 SD 2021 0.14 SEM RSD Mean 1.04 Fish Fish # () () method trace level £ detection of limit and < limit of *** *** unable to accurately determine due to value(s) < limit concentration method the < of detection limit TABLE 5.2 SULFADIMETHOXINE AND RESIDUES* PLASMAsMUSCLE RATIOS - 12 HOURS POST-DOSE

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to 10 H *** 1.8 0.1 *** *** 2.0 *** *** 1.6 P:M ------148% 16% 4.182.02 2.2 0.90 2.99 0.3 1.34 1.6 0.10 0.08 1.56 1.7 3.82 1.6 9.81 0.09+

Muscle --- 138% 3.57 4.91 6.15 9.40 0.07 0.25 0.18 2.09 3.26+ 15.39 of quantitation of quantitation 1.7 0.2 13% *** 1.7 1.6 *** 0.1 1.48 1.4 1.6 2.63 P:M Plasma ------3.57 2.2 0.82 2.73 1.17 9.04 1.5 Muscle --- 1.29 141% 149% 3.02 1.83 0.13 0.08 1.67 0.17 0.10 2.17 1.34 3.22* 0.09+ 35.4+ (0.03) 0.5 4.27 *** *** *** 20% 1.9 5.54 3.50 1.6 2.6 *** *** 3.2 0.48 0.26 1.8 0.69 0.33 2.1 2.5 13.44 2.5 2.1 ------126% 0.61 2.7 7.75 0.26 0.32 0.21 0.08 0.2 0.14 3.4 1.92 1.04 1.8 2.39 1.18 0.22 0.77 --- Muscle P;M Plasma -acetyl SDM-acetyl SDM TotalResidue SDM N- --- 120% 0.55 0.66 0.46 0.47 0.21 0.07 (0.04)+ Plasma 30 28 (0.04) 31 0.08 35 1.66 25 0.43 0.17 36 26 0.62 SD 33 0.05 29 27 1.95 34 32 SEM 0.20 RSD Mean Fish # () () of trace methodlevel £ detection limit of < and limit t t Outlier - excluded from calculations *** *** limit < unable to accurately determine due to value(s) — - concentration method< the of detection limit TABLE 5.3 SULFADIMETHOXINE AND RESIDUES4 PLASMA:MUSCLE RATIOS - 24 HOURS POST-DOSE

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. w H 4^ a a a *** *** AAA AAA AAA *** *** AAA *** a a a *** a a a *** *** *** P:M ------0. 02 0. 0.03 192% 0.01 0.05 SDM Residue SDM

------—- 0.03 0.20 0.09 0.05 0.07 149% 0.02 0.10 0.09 0.13 0.05 Plasma Muscle of quantitation of quantitation

a a a a a a a a a a a a *** a a a *** *** a a a AAA 1.4 a a a *** a a a a a a P:M ------SDM Total —- 0.05 Muscle ------0.12 0.090.04173% 1.4 0.03 192% 0.02 0.02 0.01 0.01 0.07 (0.04) 0.05 (0.04) Plasma a a

a a a *** a a a *** *** a a a a a a a *** a a a *** a a a *** a a a a a a *** P?M ------—— ------Muscle acetvl SDM acetvl method detectionof limit < and of limit t N------0. 02 0. 134% Plasma 38 39 37 41 46 0.03 40 42 0.06 44 4748 0.08 SD 0.03 4345 0.06 0.05 SEM 0.01 RSD Mean Fish # () () trace level *** *** unable to accurately determine to value(s) due < limit concentration methodthe < detection of limit TABLE 5.4 SULFADIMETHOXINE AND RESIDUES* PLASMA:MUSCLE RATIOS - 48 HOURS POST-DOSE

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o i *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** P:M Ratio 0 0 0 0 0 0 0 0 0 0 0 .01 0.05 0.00 343% 0.00 Muscle -- -— ------Plasma Total Residue SDM *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** Ratio ______-- —— SDM ------0.00 0.05 0.00 0.01 343% Muscle P:M ------Plasma *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** Ratio ______—- -- -— ------N-acetyl N-acetyl SDM «•* ------Plasma Muscle P:M 50 53 49 55 57 59 51 58 60 52 54 56 SD SEM RSD Mean Fish # () () trace method level £ of detectionlimit < and limit of quantitation *** *** unable to accurately determine due to value(s) < limit quantitation of £ * * concentration parts per million in concentration methodthe < detection of limit TABLE 5.5 SULFADIMETHOXINE AND RESIDUES* PLASMA;MUSCLE RATIOS - 72 HOURS POST-DOSE

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to 0> H *** *** *** *** *** *** *** *** *** *** *** *** *** P : M P : ------Total Total Residue SDM ------______-— ------9 6 HOURS POST-DOSE *** *** *** *** *** *** *** *** *** *** *** *** *** *** RATIOS ------—— ------SDM Muscle P:M Plasma Muscle —— ------—_ — ~ ------* AMD PLASMA:MUSCLE * *** ****** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** Ratio Ratio Ratio ------Muscle P:M Plasma N-acetyl N-acetyl SDM ------SULFADIMETHOXINE SULFADIMETHOXINE RESIDUES Plasma 61 68 62 63 64 65 66 69 67 70 SD 71 SEM RSD Mean Fish # () () trace method level £ detection limit of and < limit quantitation of *** *** unable to determineaccurately to due value(s) < quantitation of limit * * concentration parts per million in concentration method< the detection of limit TABLE 5.6

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variations in tissue antibacterial concentrations have

been reported in other studies with fish fed free-choice

medicated feed containing a potentiated sulphonamide

[5.14,5.24,5.25]. A number of factors can influence the consumption,

metabolism, distribution, and elimination of xenobiotics in the body [5.26-5.30]. These include individual animal

factors and systemic variation within an experimental unit

attributable to age, weight, sex, and reproductive status.

Variability due to the analytical technique employed may

also contribute to scattering of data, but does not appear to be the case here.

Additionally, palatability may affect the consumption of medicated feed and hence actual dose administered per

fish. Variable medicated feed intake may be partially

responsible for the range of tissue drug concentrations seen among fish within a single group in this study. Fish

feeds containing potentiated sulfonamides, such as Romet®-

30 or Tribrissen® (sulfadiazine-trimethoprim), reportedly have decreased palatability compared to non-medicated

feeds [5.25,5.30,5.31]. For Romet®-30 medicated feed the

ormetoprim component has been identified as the factor responsible for decreased palatability [5.31].

In a related study, Walisser et aI. suggested the variation in drug tissue concentration among individual

Chinook salmon within a group treated with Romet®-30 may

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have been the consequence of hierarchial feeding within a

tank [5.24]. Hierarchial feeding, however, appears not to

be the primary cause of variability of tissue drug

concentration in this study. In the present study all experimental fish actively fed on non-medicated feed but

were observed to have reduced feeding activity when given

the Romet®-30 medicated feed. Further, feeding activity

with the medicated feed was not uniform among all fish

within a tank. Individual fish fed more actively than

others on the medicated feed although sufficient

quantities of feed were freely available to all fish. The low palatability of Romet®-30 medicated feeds also has implications in regards to drug delivery and

efficacy in diseased fish in addition to its effect on drug residue concentration. In general, sick fish consume

less medicated feed than healthy fish. It is conceivable that the decrease in feed intake by sick fish would be

exacerbated by administration of an unpalatable feed.

Sick fish may, therefore, exhibit greater variability in medicated feed intake and attain lower and more variable

tissue drug concentrations than the fish in this experiment.

Nevertheless, despite the large variation in tissue drug concentrations a mean plasma:muscle drug

concentration ratio of 1.8:1 ± 0.3:1 (SD) was obtained for

fish over all concentration ranges and sampling periods.

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The 95% Cl for the mean plasma:muscle drug concentration ratio is (1.7:1, 1.8:1). The plasma:muscle total SDM

concentration ratio in individual fish ranged from 1.3:1 to 2.5:1 with a 95% confidence interval for individual

fish of (1.2:1, 2.4:1). The general correlation between plasma and muscle concentrations attained in this study is

illustrated in Figure 5.1. A correlation coefficient of 0.967 and visual examination of the plot indicate a strong correlation between plasma and muscle total SDM

concentrations over a large concentration range. The

points on the graph represent individual fish with plasma and muscle SDM concentrations above the practical limits of quantitation of the analytical method. The plotted

line and its slope were determined by linear regression. Figure 5.2 illustrates similar results for samples

containing muscle drug concentrations less than 2000 ng/g.

Although samples containing concentrations below the LOQ

were not included in this plot, an examination of total

plasma and muscle concentrations of such samples in Tables 5.1-5.3 indicates the relationship between total SDM residues in plasma and muscle is maintained at

concentrations about tolerance. This relatively stable plasma:muscle ratio should permit the use of plasma drug concentration to predict violative muscle drug concentration.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 220

15 >1.8 slop* r -r 0.967 (Thousands) 12 9 6 Muscle Total Sulfadimethoxine (ng/g) 3 0 CATFISH FOLLOWING ADMINISTRATION OFROMET® MEDICATED FEED. LIQUID CHROMATOGRAPHIC ANALYSIS WITH DIODEARRAY DETECTION. 25 20

~ 0 « £ t o 3 rt = rt o> m C C £ » S X}

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2000 Slop* -1.4 Slop* 1600 1200 800 Muscle Total Sulfadimethoxine (ng/g) 400 0 CATFISHFOLLOWING ADMINISTRATION OFROMET® CONCENTRATION MEDICATED <2000 FEED. ng/g. MU8CLE 600 1200 1800 3000 2400 3 O E e © O © E © X c cn c (O 15 w sz '• o t- FIGURE 5.2 PLASMA:MU8CLECORRELATION OF TOTAL SDMRESIDUES FROM INDIVIDUAL CHANNEL

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 222 Such a drug concentration ratio should enable one to

use channel catfish plasma as the test material in an

ELISA to determine qualitatively the presence of violative

muscle residues of SDM. The number of fish from a Romet®-

30 treated pond required to ensure detection of a residue violation problem is dependent upon the percentage

violative in the sampled population (pond) and the desired percent probability of detection [5.2]. Additional field studies to determine the expected percent violative fish

in a catfish pond treated with Romet®-30 medicated feed

are needed. Sulfadimethoxine depletion from plasma and muscle

approximates first-order kinetics (Figures 5.3, 5.4) based on the sampling intervals used in this study. This is in

agreement with data from Squibb et al. [5.16]. The calculated half-lives for total SDM residues at a water

temperature of 27°C were approximately 11 hours for muscle

and 13 hours for plasma. The label withdrawal period for

Romet®-30 following administration to channel catfish is

three days [5.33]. The estimate, based on the total SDM residue depletion curve using mean tank values, of the

time required for the mean concentration to reach the tolerance of 100 ng/g muscle is 49 hours. In this study however, no fish contained total SDM residues of >0.1 PPM in muscle by the 48 hour post-dose sampling period. This

indicates the present three day drug withdrawal period for

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 223

r -1.0r r - 1.0 Muscle Plasma 60 48 36 Time (hours) 24 12 0 SAMPLING PERIOD. POLYNOMIAL 2° CURVE FIT. ROMET®MEDICATED FEED. WATER TEMPERATURE 27°C. MEANVALUES (n=12) AT EACH 0 4 2 6 8 10 (0 3 w o c (0 ■a o c c © O o c W O S Q 75 H £ co CL 0. FIGURE 5.3 TOTAL SDMRESIDUE DEPLETION IN CHANNEL CATFISH FOLLOWING ADMINISTRATIONOF

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 224

R - .999 - R R - .996 - R Plasma Muscle 96 72 48 Time (hours) 24 0 SAMPLING PERIOD. SEMI-LOG PLOT. LINEAR CURVE FIT. ROMET®MEDICATED FEED. WATER TEMPERATURE 27°C. MEANVALUES AT (n=12) EACH 100 1000 10000 r c o L- o rt e c C o o CL O 2 CO GQ (L Q 75 FIGURE 5.4 TOTAL SOMRE8IDUE DEPLETION INCHANNEL CATFISH FOLLOWINGADMINISTRATION OF

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Romet®-30 is adequate for the SDM component for channel

catfish at a water temperature of 27°C. This is in

agreement with Brown's estimate of an -48 hour mean

depletion time at 28°C [5.17]. All control fish (n=12) in

the study were negative for SDM and the N-acetyl

metabolite at the LODs for the analytical methods

employed. An evaluation of the performance of commercial ELISAs when using plasma as a means to identify fish

containing violative SDM residues is planned but was

outside the scope of present research goals.

REFERENCES

5.1 Mitchell, G.A. (1991) Compliance issues and proper animal drug use in aquaculture. Veterinary and Human Toxicology, 33(Supplement 1):9-10. 5.2 Compound Evaluation and Analytical Capability, National Residue Program Plan, J. Brown (Editor), United States Department of Agriculture, Food Safety and Inspection Service, Science and Technology Program, Washington, D.C., 1991.

5.3 Newsome, H. (1986) Potential and advantages of immunochemical methods for analysis of foods. Journal of the Association of Official Analytical Chemists, 69(6):919-923.

5.4 Golden, C.A. (1991) Overview of the state of the art of immunoassay screening tests. Journal of the American Veterinary Medical Association, 198(5):827-830.

5.5 Walker, C.C. and Barker, S.A. (1993) Extraction and enzyme immunoassay of sulfadimethoxine residues in channel catfish (Ictalurus punctatus) muscle. Journal of AOAC International, Accepted for publication, July, 1993.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 2 6

5.6 Wu, C.C., Lin, T.L., Heitz, J.R., and Brake, N.P. "On site rapid screening test for antibiotic residue in channel catfish", in Proceedings of the 24th Annual International Association for Aquatic Animal Medicine Conference, B.F. Andrews (Editor), Chicago, IL, 1993, volume 24, p. 101. 5.7 Renson, C., Degand, G., Maghuin-Rogister, G., Delahaut, P. (1993) Determination of sulphamethazine in animal tissues by enzyme immunoassay. Analytica Chimica Acta, 275:323-328.

5.8 Epstein, R.L., and Ashworth, R.B. (1989) Tissue sulfonamide concentration and correlation in turkeys. Journal of the American Veterinary Medical Association, 50(6):926-928.

5.9 Randecker, V.W., Reagan, J.A., Engel, R.E., Soderberg, D.L., and McNeal, J.E. (1987) Serum and urine as predictors of sulfamethazine levels in swine muscle, liver, and kidney. Journal of Food Protection, 50(2):115-122. 5.10 Ashworth, R.B., Epstein, R.L., Thomas, M.H., and Frobish, L.T. (1989) Sulfamethazine blood/tissue correlation study in swine. American Journal of Veterinary Research, 47(12):2596-2603. 5.11 Bevill, R.F., Sharma, R.M., Meachum, S.H., Wozniak, S.C., Bourne, D.W,A., and Dittert, L.W. (1977) Disposition of sulfonamides in food-producing animals: Concentrations of sulfamethazine and its metabolites in plasma, urine, and tissues of lambs following intravenous administration. American Journal of Veterinary Research, 38(7):967-972. 5.12 Bourne, D.W,A., Bevill, R.F., Sharma, R.M., Gural, R.P., and Dittert, L.W. (1977) Disposition of sulfonamides in food-producing animals: Pharmacokinetics of sulfamethazine in lambs. American Journal of Veterinary Research, 38(7):967-972.

5.13 Bourne, D.W.A., Dittert, L.W., Koritz, G.D., and Bevill, R.F. (1978) Disposition of sulfonamides in food-producing animals: VII. Disposition of sulfathiazole in tissue, urine, and plasma of swine following intravenous administration. Journal of Pharmacokinetics and Biopharmaceutics, 6(2):123-134.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 2 7

5.14 Salte, R., and Liestrol, K. (1983) Drug withdrawal from farmed fish: Depletion of oxytetracycline, sulfadiazine, and trimethoprim from muscular tissue of rainbow trout (Salmo gairdneri). Acta Veterinaria Scandinavica, 24:418-430.

5.15 Kleinow, K.M., and Lech, J.J. (1988) A review of the pharmacokinetics and metabolism of sulfadimethoxine in the rainbow trout (Salmo gairdneri). Veterinary and Human Toxicology, 30(Supplement 1):26-30.

5.16 Squibb, K.S., Michel, C.M.F., Zelikoff, J.T., O'Connor, J.M. (1988) Sulfadimethoxine pharmacokinetics and metabolism in the channel catfish (Ictalurus punctatus). Veterinary and Human Toxicology, 3 0(Supplement 1):31-3 5. 5.17 Brown, L.A. (1989) Effect of water temperature on withdrawal periods for a potentiated sulphonamide used in Channel Catfish, in Current trends in fish therapy, Proceedings of a joint WAVSD and DVG meeting. Deutsche Veterinarmedizinische Gesellschaft, pp. 116-128. 5.18 Alavi, F.K., Rolf, L.L., and Clarke, C.R. (1993) The pharmacokinetics of sulfachlorpyridazine in channel catfish, Ictalurus punctatus. Journal of Veterinary Pharmacology and Therapeutics, 16:232-236.

5.19 Broussard, M.C., Jr. (1991) Aquaculture: Opportunities for the nineties. Journal of Animal Science, 69:4221-4228. 5.20 Miller, R.W. (1991) Get hooked on seafood safety. FDA Consumer, 25(5):6-11. 5.21 Van Dresser, W.R., and Wilcke, J.R. (1989) Drug residues in food animals. Journal of the American Veterinary Medical Association, 194(12):1700-1710.

5.22 Paige, J.C. (1993) Analysis of illegal residues in meat and poultry. FDA Veterinarian, 8(2):5-6. 5.23 Nielsen, P. (1973) The metabolism of four sulphonamides in cows. Biochemical Journal (London), 136:1039-1045.

5.24 Walisser, J.A., Burt, H.M., Valg, T.A., Kitts, D.D., and McErlane, K.M. (1990) High-performance liquid chromatographic analysis of Romet-30* in salmon

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following administration of medicated feed. Journal of Chromatography, 518:179-188. 5.25 Hormazabal, V., and Rogstad, A. (1992) Simultaneous determination of sulphadiazine and trimethoprim in plasma and tissues of cultured fish for residual and pharmacokinetic studies. Journal of Chromatography, 583:201-207. 5.26 Baggot, J.D. Principles of drug disposition in domestic animals: the basis of veterinary clinical pharmacology. W.B. Saunders, Co., Philadelphia, PA 1977.

5.27 Dittert, L.W. (1977) Pharmacokinetic prediction of tissue residues. Journal of Toxicology and Environmental Health, 2:735-756. 5.28 Mercer, H.D., Baggot, J.D., and Sams, R.A. (1977) Application of pharmacokinetic methods to the drug residue profile. Journal of Toxicology and Environmental Health, 2:787-801. 5.29 Braun, W.H., and Waechter, J.M., Jr., (1983) Sources of uncertainty in pharmacokinetic prediction. Journal of Animal Science, 56(1):235-243. 5.30 Juskevich, J.C. (1987) Potential applications of pharmacokinetics toward minor species-use approvals: human food safety. Veterinary and Human Toxicology, 29(Supplement 1):89-92. 5.31 Poe, W.E., and Wilson, R.P. (1989) Palatability of diets containing sulfadimethoxine, ormetoprim, and Romet 30 to channel catfish fingerlings. The Progressive Fish Culturist, 51:226-228. 5.32 Robinson, E.H., Brent, J.R., Crabtree, J.T., and Tucker, C.S. (1990) Improved palatability of channel catfish feeds containing Romet-30®. Journal of Aquatic Animal Health, 2:43-48.

5.33 United States Food and Drug Administration, New Animal Drugs for use in Animal Feed, Code of Federal Regulations (1988) Title 21, Part 558:575, Washington, D.C.

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SUMMARY AND CONCLUSIONS

The goal of the research presented in preceding

chapters was to develop the analytical methods and basic

pharmacokinetic relationships needed for establishing a drug residue monitoring and surveillance program for

sulfadimethoxine residues in channel catfish. The process applied and the results obtained may serve as a model for monitoring programs for other veterinary drugs used in

aquaculture. Screening and quantitative analytical

methods were developed and the relationships between drug concentrations in tissue compartments were examined.

An effective residue monitoring program should have analytical methods available for use on two levels - in

regulatory agency laboratories and at the producer level, i.e. pond-side tests. One of the major weaknesses of present residue monitoring programs in general is the

relative lack of qualitative assays available for use on­

site by the producer or veterinarian. Further, there is a

lack of rapid and practical drug residue extraction and determinative methods available for the analysis of veterinary drug residues by regulatory agency laboratories.

229

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On-site qualitative residue detection tests will

permit pre-marketing identification of individual food

animals or treatment groups of animals containing violative drug residues. Early identification of such

animals would have a positive impact on ensuring a safe food supply and help prevent producer losses resulting

from carcass condemnation. The US Department of

Agriculture Food Safety Inspection Service has indicated its commitment to developing rapid and practical tests for

such purposes for use in several food animal species. However, initial methods development efforts have involved

microbial inhibition tests (MIT). Although MITs may be suitable in some cases, they are generally not amenable to field use.

Because the effectiveness of a residue monitoring

program begins at the producer level, education of aguaculturists in proper drug use, maintenance of

treatment records, and observation of recommended drug withdrawal periods may help prevent the marketing of fish

with violative drug residues. A problem unique to

aquaculture, however, is the poikilothermic nature of fish and their temperature-dependent elimination rate for drugs. In general, for each 1°C decrease in temperature

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there is a corresponding 10% decrease in drug elimination

rate in fish. Therefore, at low water temperatures, even

when medications are administered according to label instructions and the recommended withdrawal time observed,

violative drug residues in harvested fish may result. As a result, on-site qualitative tests for detection of such

residues are more urgently needed in aquaculture than in

some other forms of animal agriculture.

An ideal drug residue screening test for field use is one that is rapid, inexpensive, and simple to run;

requires minimal training and no complex or costly equipment; and provides adequate sensitivity, specificity,

and ruggedness. Such an ideal test would require minimal

or no sample extraction. Although a variety of methods

are available for field use, commercially available ELISA kits, using a biological fluid as a marker tissue, most nearly meet the above needs.

One of the research goals for this dissertation was the determination of an appropriate marker residue in a

biological fluid to indicate drug residue concentration in channel catfish muscle. However, a relatively constant

relationship between drug concentration in the target tissue and the reference biological fluid need first be demonstrated. Total sulfadimethoxine (SDH) residue in plasma was chosen as the marker residue because of the

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relative ease of obtaining plasma samples and the fact that all commercially available ELISAs for detection of

SDM residues were demonstrated to detect parent SDM and

its primary metabolite, 4-N-acetylsulfadimethoxine (N- acetyl SDM), equally well. To determine total SDM

residues in plasma and muscle, high performance liquid

chromatographic (LC) methods were developed for both

matrices for the simultaneous analysis of parent SDM and N-acetyl SDM. These LC methods were used to determine plasmarmuscle total drug concentration ratios for

individual fish. From these data, plasma:muscle

correlations and a 95% confidence interval for plasma:muscle ratios of individual fish were calculated.

The excellent correlation obtained over a range of

concentrations demonstrates that total plasma SDM concentration accurately reflects total muscle SDM residue in the channel catfish. Thus, the results indicate that,

with an appropriate dilution factor, plasma may be used in an on-site ELISA or other screening assay to identify catfish with violative SDM residues. At present, all

commercially available qualitative ELISAs for SDM have a

detection cut-off of 10 ng/ml. Therefore, if one uses the lower end of the 95% confidence interval for plasma:muscle

ratios of individual fish (1.2:1 or 120 ng/ml plasma:100 ng/g muscle) and an assay detection cut-off level of 10

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ng/ml, then plasma diluted by a factor of 1:11

(plasma:buffer) and analyzed by ELISA should correctly

identify >95% of fish containing SDM muscle residues at tolerance (100 ng/g). Further, an even greater percent of

fish containing SDM at concentrations well above tolerance should be accurately identified using this protocol. This

could be determined pond-side or plasma analysis used in a regulatory laboratory to greatly increase throughput for

initial screening of samples. The application of this

protocol to samples containing incurred SDM residues for

the identification of fish containing violative SDM residue by ELISA analysis of plasma is part of the continuing research.

LABORATORY ANALYSIS OF RESIDUES

The second part of an effective monitoring program

involves regulatory laboratory function. The analytical needs of a regulatory agency are somewhat different than

those of the producer. The tiered approach to residue monitoring requires rapid screening tests for an initial

\ examination of large numbers of samples and more

definitive analytical methods for quantitation and identification of residues in samples identified by

screening assays. A regulatory laboratory is typically

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presented with animals of unknown treatment history and

must therefore examine samples for a number of unknown

compounds. To effectively examine such samples for a range of compounds requires the use of rapid screening tests.

Animals presented for slaughter and inspection may

have been treated with approved or non-approved compounds. For detection of approved compounds, marker residues are

often available. As previously discussed, marker residues

permit the use of a biological fluid for drug residue

screening. However, for detection of non-approved compounds, marker residues are often not well defined. Identification of animals containing such residues requires an analysis of the target tissue itself.

Screening methods for such tissues are limited, in part,

by the drug extraction process. Thus, the need for rapid

and efficient extraction of the drug from the target tissue.

The combined use of matrix solid phase dispersion for extraction and ELISA for detection allows one to rapidly and effectively screen large numbers of muscle samples for

violative residues in a laboratory setting. Further, the

use of an ELISA employing the microtiter well format and

optical density determination allows one to examine a number of samples simultaneously rather than serially and

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may provide significant time savings over individual

testing. Chapter 3 presents the results of efforts to

develop a rapid muscle extraction method that provides an

extract compatible with commercially available ELISAs for

the detection of total SDM residues in channel catfish muscle. The results presented offer several rapid

screening protocols with excellent sensitivity and specificity that will permit a regulatory laboratory to examine large numbers of catfish muscle samples.

For regulatory enforcement, samples identified by screening tests as suspect require more definitive

quantitation and identification of any drug residue

present. Quantitation and presumptive identification of

veterinary drug residues is most commonly accomplished using LC with UV or diode array detection. An analytical

protocol employing matrix solid phase dispersion extraction and LC with diode array detection was developed

here for the simultaneous extraction and determination of SDM and N-acetyl SDM as residues in channel catfish

muscle. The results of this method development effort are presented in Chapter 4. This rapid procedure provides

adequate recovery of the two analytes and accuracy and precision acceptable for drug residue analysis about tolerance. Demonstration of the applicability of this

procedure to samples containing incurred SDM residues is

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provided in Chapter 5. This protocol should, therefore,

be suitable for use by regulatory agencies for

quantitation and presumptive identification of total SDM residues in channel catfish muscle. Further, this LC

method is potentially suitable for use in other fish

species, where information on marker residues is lacking, for the determination of total SDM residues.

CONCLUSIONS

Protocols were developed and are presented in this dissertation for the rapid extraction, screening, and

determination of sulfadimethoxine residues in channel catfish. Protocols are included for field application or regulatory laboratory use. From these data it is possible to construct a model for a comprehensive tissue residue

monitoring program for aquaculture but with general applicability to any species of food animal. Such a model

begins with prevention of violative drug residues by producer education concerning avoidance of drug residues

in fish and provides the producer with simple analytical

methods for detection of violative drug residues under field conditions. Included in this model are rapid and efficient methods of drug extraction and analysis for use

by regulatory laboratories. These methods should decrease

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'turnaround tine and increase the number of samples

analyzed by a regulatory agency laboratory as a result of

greater efficiency of residue screening and determinative protocols. For these reasons, the use of such a model

should improve the effectiveness of residue monitoring in channel catfish and in food animals in general.

With additional study to determine possible species-

specific matrix effects, these protocols may be extended to other cultured fish species in which Romet-30®-

medicated feeds may be used. Such protocols could

facilitate compliance with the Extra-Label Drug Use policy of the FDA and improve and expand implementation of the IR-4 initiative for drug approval in minor species such as

fish. A major concern with these two programs is the possibility of illegal residues resulting from limited

data relating to drug depletion times and metabolite

profiles in non-approved species and with non-approved

uses. As a result, the IR-4 initiative generally requires a procedure for assuring that animals marketed following

experimental drug use contain no residues. A testing

protocol such as the one provided here offers a reasonable

assurance that such fish are free of unwanted residues and

may facilitate attainment of the IR-4 goal of increasing the availability of medications for use in minor species. Further, the process and results offered may serve as a

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new approach for the development of a national aquaculture

products drug residue monitoring program.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX A OPTICAL DENSITY VALUES OBTAINED WITH THE IDS SULFADIMETHOXINE ONE-STEP ELISA*

Sample # OD Well A OD Well B Mean OD Mean ______Blank/Sample

0 nq SDM/q 1 0.817 0.822 0.820 1.000 6 1.211 1.162 1.187 1.000 11 1.401 1.352 1.377 1.000 16 0.962 0.952 0.957 1.000 21 1.304 1.150 1.227 1.000 26 0.928 0.913 0.921 1.000 31 1.197 1.164 1.181 1.000 36 0.887 0.993 0.940 1.000 41 1.084 0.956 1.020 1.000 46 1.025 1.210 1.118 1.000 51 1.096 1.019 1.058 1.000 56 0.966 0.964 0.965 1.000

Mean 1.064 1.000 Standard Deviation 0.158 0.000 CV f%)f=inter-assav variation) 14.9% 0.0%

25 na SDM/a 2 0.664 0.613 0.639 0.779 7 0.920 0.948 0.934 0.787 12 0.937 1.010 0.974 0.707 17 0.652 0.705 0.679 0.709 22 0.703 0.687 0.695 0.566 27 0.617 0.757 0.687 0.746 32 0.735 0.796 0.766 0.648 37 0.595 0.650 0.623 0.662 42 0.540 0.655 0.598 0.586 47 0.693 0.814 0.754 0.674 52 0.737 0.740 0.739 0.698 57 0.683 0.640 0.662 0.685

Mean 0.729 0.687 Standard Deviation 0.117 0.067 CV (%)f=inter-assav variation) 16.1% 9.8% * using matrix solid phase dispersion extracts of fortified catfish muscle; 10 minute incubation OD Optical Density IDS International Diagnostics Systems, Inc. SDM sulfadimethoxine

appendix con'd. 239

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Sample # OD Hell A OD Hell B Mean OD Mean Blank/Sample

50 nq SDM/q 3 0.516 0.452 0.484 0.591 8 0.528 0.588 0.558 0.470 13 0.688 0.762 0.725 0.527 18 0.571 0.584 0.578 0.603 23 0.588 0.546 0.567 0.462 28 0.525 0.517 0.521 0.566 33 0.555 0.586 0.571 0.483 38 0.549 0.538 0.544 0.578 43 0.394 0.450 0.422 0.414 48 0.510 0.610 0.560 0.501 53 0.594 0.612 0.603 0.570 58 0.516 0.455 0.486 0.503

Mean 0.551 0.522 Standard Deviation 0.074 0.060 CV f%)(=inter-assav variation) 13.5% 11.4%

100 ncr SDM/a 4 0.356 0.236 0.296 0.361 9 0.504 0.534 0.519 0.437 14 0.358 0.418 0.388 0.282 19 0.309 0.379 0.344 0.359 24 0.224 0.188 0.206 0.168 29 0.307 0.289 0.298 0.324 34 0.375 0.366 0.371 0.314 39 0.346 0.370 0.358 0.381 44 0.240 0.253 0.247 0.242 49 0.330 0.344 0.337 0.302 54 0.344 0.317 0.331 0.313 59 0.319 0.272 0.296 0.306

Mean 0.332 0.316 Standard Deviation 0.078 0.069 CV f%)(=inter-assav variation) 23.5% 21.8% * 10 minute incubation time OD Optical Density IDS International Diagnostics Systems, Inc. SDM sulfadimethoxine

appendix con'd.

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Sample # OD Well A OD Well B Mean OD Mean Blank/Sample

250 nq SDM/q 5 .142 .146 .144 .176 10 .288 .317 .303 .255 15 .252 .275 .264 .191 20 .164 .184 .174 .182 25 .318 .315 .317 .258 30 .159 .153 .156 .169 35 .190 .210 .200 .169 40 .201 .258 .230 .244 45 .133 .124 .129 .126 50 .176 .175 .176 .157 55 .160 .160 .160 .151 60 .143 .152 .148 .153

Mean 0.200 0.186 Standard Deviation 0.064 0.043 CV f%W=inter-assav variation) 31.9% 23.4% * 10 minute incubation time OD Optical Density IDS International Diagnostics Systems, Inc. SDH sulfadimethoxine

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX B OPTICAL DENSITY VALUES OBTAINED WITH THE SIGNAL SULFAMETHAZINE ELISA*

Sample t OD Well A OD Well B Mean OD Mean Blank/Sample

0 na SDM/a 1 1.635 1.538 1.587 1.000 6 1.329 1.296 1.313 1.000 11 1.210 1.217 1.214 1.000 16 1.340 1.442 1.391 1.000 21 1.921 1.629 1.775 1.000 26 1.876 1.771 1.824 1.000 31 1.777 1.904 1.841 1.000 36 1.744 1.821 1.783 1.000 41 1.956 1.872 1.914 1.000 46 1.584 1.718 1.651 1.000 51 1.612 1.532 1.572 1.000 56 1.709 1.692 1.701 1.000

Mean 1.630 1.000 Standard Deviation 0.223 0.000 CV (%)(=inter-assay variation) 13.7% 0.0%

25 na SDM /a 2 1.483 1.176 1.330 0.838 7 1.114 1.144 1.129 0.860 12 1.305 1.141 1.223 1.008 17 1.187 1.186 1.187 0.853 22 1.696 1.638 1.667 0.939 27 1.630 1.579 1.605 0.880 32 1.670 1.602 1.636 0.889 37 1.573 1.548 1.561 0.875 42 1.682 1.T27 1.705 0.891 47 1.446 1.499 1.473 0.892 52 1.286 1.293 1.290 0.820 57 1.373 1.380 1.377 0.809

Mean 1.432 0.880 Standard Deviation 0.202 0.054 CV (%)(=inter-assav variation) 14.1% 6.1% * using matrix solid phase dispersion extracts fortified catfish muscle; 30 minute incubation OD Optical Density SDH sulfadimethoxine

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Sample # OD Well A OD Well B Mean OD Mean Blank/Sample

50 nq SDM/q 3 1.218 1.119 1.169 0.737 8 1.003 1.067 1.035 0.789 13 1.100 1.043 1.072 0.883 18 1.293 1.190 1.242 0.893 23 1.553 1.515 1.534 0.864 28 1.592 1.517 1.555 0.852 33 1.473 1.477 1.475 0.801 38 1.475 1.442 1.459 0.818 43 1.534 1.506 1.520 0.794 48 1.359 1.350 1.355 0.820 53 1.211 1.282 1.247 0.793 58 1.275 1.397 1.336 0.786

Mean 1.333 0.819 Standard Deviation 0.181 0.046 CV (%)(=inter-assav variation) 13.6% 5.6%

100 nq SPM/q 4 1.296 0.896 1.096 0.691 9 0.885 0.926 0.906 0.690 14 0.953 0.885 0.919 0.757 19 0.971 0.900 0.936 0.673 24 1.247 1.193 1.220 0.687 29 1.206 1.209 1.208 0.662 34 1.035 1.013 1.024 0.556 39 1.268 0.997 1.133 0.635 44 1.263 1.288 1.276 0.666 49 1.183 1.138 1.161 0.703 54 0.825 0.887 0.856 0.545 59 1.001 1.068 1.035 0.608

Mean 1.064 0.656 Standard Deviation 0.139 0.061 CV (%)f=inter-assav variation) 13.1% 9.3% * 30 minute incubation time OD Optical Density SDM sulfadimethoxine

appendix con'd.

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Sample # OD Well A OD Well B Mean OD Mean Blank/Sample

250 ppb 5 0.742 0.665 0.704 0.443 10 0.748 0.761 0.755 0.575 15 0.794 0.749 0.772 0.636 20 0.682 0.698 0.690 0.496 25 0.968 1.016 0.992 0.559 30 1.010 1.052 1.031 0.565 35 1.200 1.248 1.224 0.665 40 1.007 0.960 0.984 0.552 45 1.011 1.038 1.025 0.535 50 0.888 0.921 0.905 0.548 55 0.839 0.820 0.830 0.528 60 0.844 0.943 0.894 0.525

Mean 0.900 0.552 Standard Deviation 0.159 0.058 CV f%>f=inter-assav variation) 17.7% 10.5% * 30 minute incubation time OD Optical Density SDM sulfadimethoxine

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VITA

Calvin Charles Walker was born November 9, 1951 in

Moss Point, Mississippi. He attended Our Lady of

Victories Central High School in Pascagoula, Mississippi, and graduated in 1969.

He attended Mississippi State University and

graduated in 1973 with a Bachelor of Science degree in Animal Science. He was admitted to the Auburn University School of Veterinary Medicine in 1974 and was awarded the degree of Doctor of Veterinary Medicine in 1978.

Following graduation from Auburn University, he entered private veterinary practice in Gulfport, Mississippi and

established veterinary practices in Ocean Springs and

Gautier, Mississippi. He holds licenses to practice

Veterinary Medicine in Alabama and Mississippi. In the fall of 1989 he accepted the Ethyl Corporation

Graduate Fellowship in Toxicology at Louisiana State University and began graduate studies for the Doctor of

Philosophy degree in Veterinary Medical Sciences (Toxicology Option). He completed all requirements for this degree in the fall of 1993.

245

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candidate: Calvin Charles Walker

Major Field: Veterinary Medical Sciences

Title of Dissertation: Sulfadimethoxine Analysis in Channel Catfish: A Model Drug Residue Monitoring Program For Aquaculture

Approved:

and f!hai rman

Dean of the Graduate School

EXAMINING COMMITTEE:

Date of Kxamination:

11 - 2-93

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