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HPLC analysis of Romet-30 in chinook salmon (Oncorhynchus tshawytscha): wash-out time, tissue distribution… Zheng, Ming 1993

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HPLC ANALYSIS OF ROMET-30® IN CHINOOK SALMON (Oncorhynchustshawytscha): WASH-OUT TIME, TISSUE DISTRIBUTION IN MUSCLEAND LIVER TISSUES, AND METABOLISM OF SULFADIMETHOXINEbyMING ZHENGB.Sc. (Pharm.), Shanghai Medical University, 1987M.Sc. (Pharm.), Ghent University, 1990A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Faculty of Pharmaceutical Sciences)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAApril 1993© Ming Zheng, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of  Pharmaceutical SciencesThe University of British ColumbiaVancouver, CanadaDate April 15, 1993DE-6 (2/88)iiABSTRACTAquaculture is a rapidly expanding industry that has contributedsubstantially to world-wide food production. Salmon culture constitutes animportant part of aquaculture industry. The production of farmed salmon inBritish Columbia reached 20,000 metric tons (mt) in 1991 and 1992 and ispredicted to grow to 26,700 mt by 1995 (Smith, 1993). However, due to thehigh stocking density in aquaculture and occasional poor husbandrypractices, the fish are predisposed to various diseases. Treatment protocolsfor bacterial infections include the use of antimicrobial agents such aspotentiated sulfonamides, oxytetracycline, macrolides, penicillins andquinolones.As antimicrobial residues in edible fish tissues after drugadministration is of utmost concern to the consumer's health, analyticalassay is required to monitor the antimicrobial residue levels in edible fishtissue. The focus of this research was to identify a "marker" tissue whichwould have a higher residue level than the muscle tissue, and that the ratiobetween this "marker" tissue and muscle tissue would be more or lessconsistent. An HPLC assay was developed for determination of Romet-30®residue levels in Chinook salmon muscle and liver tissues. By using ion-pairing reagent TBAH and granular sodium sulfate anhydrous duringextraction, the extraction recoveries averaged 66%, 78% and 83% for OMP,SDM and N4-Ac-SDM, respectively, in muscle tissue; 61% and 72% for SDMand N4-Ac-SDM, respectively, in liver tissue. OMP could not be quantified inliver tissue due to the presence of substantial amounts of co-extractedendogenous substances. The HPLC assay had a sensitivity of 0.05 ppm forOMP, SDM and N4-Ac-SDM in muscle tissue, and a sensitivity of 0.20 ppmiiifor SDM and N4-Ac-SDM in liver tissue.The HPLC assay was applied to the analysis of Romet-30 residues inChinook salmon muscle and liver tissues after administration of Romet-30®.Two intubation studies were carried out. SDM was found to be detectable upto day 20 in both studies, while OMP was detectable on day 20 in the secondstudy but was not detectable on day 20 in the first study. N4-Ac-SDM wasonly detectable on day 11 in both studies. However, in liver tissue, theconcentration of N4-Ac-SDM was not significantly different from that ofSDM. The presence of N4-Ac-SDM in liver tissue was confirmed by LC/MSand LC/MS/MS analyses.Both studies showed significant variations of Romet-30® residue levelsbetween individual fish. A consistent liver/muscle SDM residue ratio was notfound from the current study. However, it was suggested that the liver tissuebe used as a "monitoring" tissue since the overall concentration of SDM inliver was higher than that in muscle. Finally, since the analytical assay hasa sensitivity of 0.05 ppm, which is well below the tolerance level of 0.1 ppmfor Romet-30® (USFDA, 1984), the muscle tissue appears to be a good targettissue for Romet-308 residue analysis.ivTABLE OF CONTENTSAbstractTable of Contents^ ivList of Tables viiiList of Figures^ ixList of Schemes xiSymbols and Abbreviations^ xiiAcknowledgement^ xivDedication^ XV1^INTRODUCTION^ 11.1 Aquaculture 11.2 Salmon^ 21.2.1 Salmon 21.2.2 Salmon Culture^ 41.3 Bacterial Diseases in Salmon^ 51.3.1 Bacterial Kidney Disease 51.3.2 Furunculosis^ 71.3.3 Vibriosis 81.4 Major Antimicrobials for Treatment of Bacterial Fish Diseases^91.4.1 General Information^ 91.4.2 Sulfonamides^ 101.4.3 Oxytetracycline 121.4.4 Quinolones 131.5 Fish Vaccination^ 151.6 Problems in Chemotherapy in Aquaculture^ 161.6.1 Drug Residues in Edible Tissues 161.6.2 Development of Antimicrobial ResistanceBacterial Community^ 171.7 Background Information: Romet-30®^ 181.7.1 Chemistry^ 181.7.2 Mechanism of Action^ 191.7.3 Toxicity in Fin Fish 221.7.4 Pharmacokinetics of SDM in Aquatic Species^22V^1.7.4.1^Pharmacokinetics of SDM in Lobster^221.7.4.2^Pharmacokinetics of SDM in ChannelCatfish^ 231.7.4.3^Pharmacokinetics of SDM in Rainbow Trout 241.7.4.4 Pharmacokinetics of OMP in Rainbow Troutand Channel Catfish^ 261.7.4.5^Effect of OMP on the Bioavailability,Distribution, and Pharmacokinetics ofSDM in Rainbow Trout(Oncorhynchus mykiss)^ 281.7.5 Metabolism of SDM and OMP in Aquatic Species^281.7.6 Palatability of Medicated Feed 301.8 Analytical Methods for SDM and OMP in Fish Tissue^301.8.1 Analytical Methods for SDM^ 301.8.2 Analytical Methods for OMP 341.9 Research Hypothesis^ 361.10 Research Goals 372 EXPERIMENTAL^382.1 Materials and Supplies 382.1.1 Drugs^ 382.1.2 Chemicals and Reagents^ 382.1.3 Solvents 392.2 Instrumentation^ 392.2.1 HPLC System 392.2.2 LC/MS and LC/MS/MS Experiments^ 392.2.3 Miscellaneous^ 402.3 Stock Solutions 402.3.1 Siilfadimethoxine and Ormetoprim Standard Solutions^402.3.2 N4-acetyl-sulfadimethoxine Standard Solution^402.3.3 Sulfisoxazole Internal Standard Solution 412.3.4 TBAH (0.5 M)^ 412.3.5 Sodium Hydroxide (1 M)^ 412.3.6 Sodium Carbonate/Sodium Bicarbonate Buffer, pH 10^412.3.7 Phosphate Buffer (0.1 M), pH 4.0^ 412.4 Synthesis of N4-Ac-SDM^ 422.5 Intubation Studies 422.6 Extraction Procedures in Muscle and Liver Tissues^43vi2.7 Calibration Curves^ 462.7.1 Calibration Curves in Muscle Tissue^ 462.7.2 Calibration Curves in Liver Tissue 462.8 Extraction Recovery Studies^ 472.8.1 Extraclion Recoveries of SDM, OMPand N4-Ac-SDM in Muscle Tissue^ 472.8.2 Extraction Recoveries of SDM and N4-Ac-SDMin Liver Tissue^ 472.9 Intra-assay Variability Studies^ 482.9.1 Intra-assay Variability in Muscle Tissue^482.9.2 Intra-assay Variability in Liver Tissue 482.10 Inter-assay Variability Studies^ 492.10.1^Inter-assay Variability in Muscle Tissue^492.10.2 Inter-assay Variability in Liver Tissue 492.11 Confirmation of the Presence of N4-Ac-SDM in Liver Tissueby LC/MS and LC/MS/MS^ 492.12 Effect of the Addition of Salts During the Extraction^502.13 Inyestigation. of Possible Hydrolysis ofN4-Ac-SDM during Extraction^ 502.14 Statistics^ 503^RESULTS AND DISCUSSION^ 513.1 Development of Chromatographic Conditions fqr theSeparation of SDM, OMP, Sulfisoxazole and N4-Ac-SDM^513.1.1 Initial Development of HPLC Mobile Phasefor the Separation of SDM, OMP andSulfisoxazole in Muscle Tissue^ 513.1.2 Dqtection and Confirmation of the Presence ofN4-Ac-SDM in Liver and Muscle Tissues^533.1.2.1^Detection of N4-Ac-SDM in Liverand Muscle Tispue^ 533.1.2.2^Synthesis of N4-Ac-SDM 553.1.2.3 Confirmation of the Presence of N4-Ac-SDMin Liver and Muscle Tissues^553.1.3 Summary of the Developed HPLC Mobile PhasesUsed in the Current Assay in Chinook SalmonMuscle and Liver Tissues^ 62vii3.2 Extraction Protocol^ 673.2.1 Formation of Ion-Pair Between SDM and TBAH^673.2.2 Addition of Inorganic Salts to IncreaseExtraction Recovery^ 683.3 Investigation of Possibility of the Hydrolysis of N4-Ac-SDMduring Extraction^ 703.4 Determination of Extraction Recoveries^ 713.4.1 Extraction Recoveries from Muscle Tissue^713.4.2 Extraction Recoveries from Liver Tissue 713.5 Linearity of the Assay^ 733.5.1 Linearity of the Assay in Muscle Tissue^733.5.2 Linearity of the Assay in Liver Tissue 733.6 Determination of Intra-assay Variabilities 763.6.1 Intra-assay Variabilities of OMP, SDM andN4-Ac-SDM in Muscle Tissue^ 763.6.2 Intra-assay Variabilities of SDM and N4-Ac-SDMin Liver Tissue^ 763.7 Determination of Inter-assay Variabilities^ 783.7.1 Inter-assay Variabilities of OMP, SDM andN4-Ac-SDM in Muscle Tissue 783.7.2 Inter-assay Variabilities of SDM and N4-Ac-SDMin Liver Tissue^ 783.8 Intubation Studies 803.9 Romet-30® Residues in Muscle and Liver Tissues from theIntubation Studies^ 803.9.1 Romet-30,, Residue Data from Muscle Tissue^803.9.2 Romet-30Q9 Residue Data from Liver Tissue 923.9.3 Liver/Muscle Concentration Ratios of SDM 1054^CONCLUSION^ 113REFERENCES 115V•111List of Tables1. List of Scientific, Common and Market Names of Salmon 32. Pharmacokinetic parameters of SDM in aquatic species 253. Pharmacokinetic parameters of OMP in rainbow troutand channel catfish 274. Extraction recoveries of OMP, SDM and N4-Ac-SDMfrom muscle tissue with or without addition ofdifferent inorganic salts 695. Extraction recoveries of OMP, SDM and N4-Ac-SDMfrom muscle tissue 726. Extraction recoveries of SDM and N4-Ac-SDM from liver tissue 727. Calibration curves of OMP, SDM and N4-Ac-SDM inmuscle tissue 748. Calibration curves of SDM and N4-Ac-SDM in liver tissue 759. Results of intra-aasay variability study in muscle tissue 7710. Results of intra-assay variability study in liver tissue 7711. Results of inter-assay variability study in muscle tissue 7912. Results of inter-assay variability study in liver tissue 7913. OMP/SDM/N4-Ac-5DM concentrations in Chinook salmonmuscle tissue from the first study 8214. OMP/SDM/N4-Ac-SDM concentrations in Chinook salmonmuscle tissue from the second study 8615. SDM/N4-Ac-SDM concentrations in Chinook salmon liver tissuefrom the first study 9416. SD1VI/N4-Ac-SDM concentrations in Chinook salmon liver tissuefrom the second study 9817. Liver/muscle SDM concentration ratios from the first study 10718. Liver/muscle SDM concentration ratios from the second study 108ixList of Figures1. Structures of sulfadimethoxine and ormetoprim^112. Structure of oxytetracycline^ 123. Structures of quinolones used in aquaculture^ 144. Structure of para-aminobenzoic acid^ 195. Biosynthesis of folic acid and THF 216. Structure of DHF analog formed by sulfonamideand dihydropteridine derivative^ 217. Structure of N4-acetyl-sulfadimethoxine^ 298. Structure of 14C-ormetoprim^ 359. Chromatograms of a standard solution containing ormetoprim,sulfadimethoxine and the internal standard, and amuscle sample spiked with OMP/SDM/I.S.^ 5210. Structure of sulfisoxazole^ 5311. Chromatograms of a liver sample from the secondintubation study^ 5412. A chromatogram of a blank Chinook salmon liver extractand a representative chromatogram of a Chinook salmonliver extract^ 5713. A chromatogram of a Chinook salmon liver extract froman intubated fist and a chromatogram of an admixtureof OMP/SDM/N4-Ac-SDM/I.S. with the above liver extract^5914. Flow injection LC/MS analysis of N4-Ac-SDM15. LC/MS/MS Koduct ion spectra of the protonated molecular ion(M+H)+ of N4-Ac-SDM obtained by flow injection16. Chromatograms of a standard solution and a blankmuscle sample17. Chromatograms of a standard solution and a blankmuscle sample18. OMP, SDM and N4-Ac-SDM residue profile in Chinook salmonmuscle tissue from the first study19. OMP, SDM and N4-Ac-SDM residue profile in Chinook salmonmuscle tissue from the second study606163659091x20. SDM and acetylated SDM residue profile in Chinook salmonliver tissue from the first study^ 10221. SDM and acetylated SDM residue profile in Chinook salmonliver tissue from the second study 10322. Liver/muscle SDM concentration ratios from the firstintubation study^ 10923. Liver/muscle SDM concentration ratios from the secondintubation study 11024. SDM residue profile in Chinook salmon liver and muscletissues from the first study^ 11125. SDM residue profile in Chinook salmon liver and muscletissues from the second study 112xiList of Schemes1. Extraction protocol in Chinook salmon muscle or liver tissues^452. Formation of tetrabutylammonium ion-pair withsulfadimethoxine^ 673diSymbols and AbbreviationsBKD^bacterial kidney diseaseBV bioavailabilityC.V.%^coefficient of variationCI chemical ionizationClb^ total body clearanceDAD diode array detectorDHF^dihydrofolic acidECD electron-capture detectorg^ gramGC/MS gas chromatography/mass spectrometryGC/MS/MS^gas chromatography/tandem mass spectrometryh^ hourHPLC high performance liquid chromatographykg^ kilogramLC/MS liquid chromatography/mass spectrometryLC/MS/MS^liquid chromatography/tandem massspectrometrym/z^ mass-to-charge ratiomin minuteml^ millilitermint million metric tonsmt^ metric tonN4-Ac-SDM^N4-acetyl-sulfadimethoxineng^ nanogramnm nanometerOMP^ormetoprimPABA^para-aminobenzoic acidppm parts per millionS.D.^standard deviationS.E.M. standard error of the meanSDM^sulfadimethoxinet112a half life for distribution phaset1120^half life for elimination phaset1127 half life for elimination phase in athree-compartment modelTBAH^tetrabutylammonium hydroxideTHF tetrahydrofolic acidTLC^thin-layer chromatographyTmax time for the drug to reach its maximumconcentrationVc^apparent volume of distribution of centralcompartmentVd apparent volume of distributionVP^apparent volume of distribution of peripheralcompartmentVp d apparent volume of distribution of deepperipheral compartmentV^ apparent volume of distribution of shallowpsperipheral compartmentVss apparent volume of distribution at steady statexivAcknowledgementI would like to express my very sincere appreciation to my supervisor,Dr. Keith McErlane, for his enthusiasm, encouragement and guidancethroughout my research and studies. I appreciate the helpful suggestionsand discussions from the other members of my research committee: Drs.Frank Abbott, Gail Bellward (chairperson), Helen Burt and David Kitts. Iwould like to extend my appreciation to Dr. David Kitts for his criticismduring the thesis preparation.Many thanks to Mr. Ron Aoyama, Mr. Michael Gentleman, Ms. Hei-yiLiu and Ms. Jacqueline Walisser for their assistance in the intubationstudies. Special thanks to Ms. Hei-yi Liu for her contribution to the assaydevelopment.I would like to express my deep gratitude to Dr. Frank Abbott for hiscoordination, and to Mr. Anthony Borel for performing the LC/MSexperiments. Also thanks Ms. Sue Panesar and Mr. Roland Burton for theirassistance and patience in computer consultation.Finally, the financial support from the Science Council of BritishColumbia, the Department of Fisheries and Oceans, and NSERC to thisresearch project is gratefully acknowledged.XVDEDICATIONThis thesis is dedicated to my parents, Hui-qin Louand Zhan-pei Zheng11. INTRODUCTION1.1 AquacultureAquaculture is the animal husbandry practice of aquatic organisms.According to the Food and Agriculture Organization (FAO, 1990) of theUnited Nations, in 1988 the total world aquatic food production was 98million metric tons (mmt), in which 14 mmt was from aquaculture and 84mmt from commercial harvesting. Aquaculture production consists of finfish,crustaceans, molluscs and seaweeds. Of the 14 mmt aquaculture productionin 1988, 7 mmt was from finfish (FAO, 1990). With the human populationexpected to reach 6 billion by the year 2000, an annual aquatic foodproduction of 138 rru-nt would be needed (NRC, 1992). However, thecommercial harvesting production of 100 mmt is generally accepted to be thenatural limits (NRC, 1992). Therefore, a continuous growth in aquacultureproduction would be an effective supplement to the anticipated demand.Presently, there are some problems and limitations existing in thedevelopmental potential of the aquacultural industry. Since aquaculture isgenerally conducted in inland and at coastal waters, the competition forspace has been a primary problem. Aquatic farm sites may obstructnavigational channels, compete for shorelines with other commercialinterests such as logging and may interfere with recreational interests.Water pollution is another particular concern. Such pollution is the result ofthe excretory wastes produced in aquaculture system and the excess feedwhich may contain antibiotics used to control diseases or hormones used tostimulate growth. In addition, the water quality itself in the rearing regionis a limiting factor for aquaculture.2Another factor that will affect aquaculture is the availability of animalproteins for fish and crustacean feeds. Predatory aquatic species requirerelatively high levels of total animal proteins. These proteins are mostlyobtained by the inclusion of dietary fish meal. However, the supply of fishmeal fluctuates due to events that may be global in scope, such as majorchanges in ocean currents caused by El Nino (Stickney, 1990). In addition,the development of surimi industry has had an enormous impact on theavailability of fish proteins destined for aquacultural use. Enormousquantities of fish that were once only used as sources of fish meal are nowtaken by the surimi industry and are turned into analogue products, such asartificial crab legs, shrimp or scallops, that are used directly for humanconsumption (Stickney, 1990).1.2 Salmon Culture1.2.1 SalmonSalmon consists of one Atlantic species and six pacific species (Table 1;Dore, 1990a). All the species have a similar life cycle. The alevins emergefrom the fertilized eggs and develop up to the smolt stage in freshwater. Thesmolts move downstream to the ocean and live in seawater for the next one tothree years. When sexually mature, adult salmon swim upstream to theriver of their origin to spawn. Most of the adult fish die after spawning.3Table 1List of Scientific, Common and Market Names of Salmon (Dore, 1990a)Latin Name^Common Name Market NameOncorhynchus tshawytscha^King Salmon^Chinook, SpringOncorhynchus keta^Chum Salmon^KetaOncorhynchus kisutch^Coho Salmon^Silver, Medium RedOncorhynchus gorbuscha^Pink Salmon^HumpbackOncorhynchus nerka^Sockeye Salmon Red, BluebackOncorhynchus masou^Cherry Salmon CherrySalmo salar^Atlantic Salmon Atlantic Salmon41.2.2 Salmon CultureThe world production of salmon reached almost 1 mmt in 1989 (FAO,1991), of which 185,000 mt were from aquaculture production (OECD, 1991).Norway alone produced 115,000 mt of farmed salmon (OECD, 1991). Thefarmed salmon production in British Columbia (B.C.) in 1989 was 12,385 mtwith a landed value of Can $82.1 million, in which 75% was Chinook (Eganand Kenney, 1990). The production in B.C. is forecasted to increase to26,700 mt with a value of $187 million and may become B.C.'s second largestfood commodity by 1995 (Smith, 1993).Salmon culture is primarily practiced in anchored, floating net penswhich are approximately 15 meters on the side and 10 to 15 meters deep,with nets also forming the bottom. Floating sea cages, which are located inoffshore seawaters, have also been introduced which may resolve thecompetition for space and offer better water quality to the fish. The mostrecent technique utilized in aquaculture is salmon ranching. Ranching takesadvantage of the instinct of salmon that it will return to the river of its birth.The salmon farmers plant salmon smolts in a particular stream, and let themswim out to sea, feed and grow. As the salmon mature, they return to theoriginal stream. The advantages of salmon ranching are that the salmon arecaught at their maximum size and the fish are raised at low cost. Althoughthis technique has been successfully practiced in China, Iceland, the formerSoviet Union and in parts of Japan, its application is not widespread due touncontrolled commercial fishing while the salmon are returning (Dore,1990b).51.3 Bacterial Diseases in SalmonFish are infected by various pathogens in nature. However, intensiveaquaculture and poor fish husbandry practice predispose fish to bacterial,parasitic and viral diseases due to a variety of stress factors such as lowoxygen, crowding and elevated ammonia (Herman and Bullock, 1986). Theseconditions enhance the opportunity of introduction, transfer and rapiddissemination of infectious diseases (Rohovec, 1990).Prevention and control of fish diseases can be accomplished by goodfish husbandry practices, use of antimicrobials and vaccination. Good fishhusbandry practices may include environmental management such asminimization of stress factors, good fish nutrition (Cho, 1990) and selection ofincoming fish eggs and smolts (Stuart, 1983). The use of antimicrobials andvaccination will be discussed in detail in the following sections.1.3.1 Bacterial Kidney Disease (BKD)BKD is the most commonly diagnosed disease in pen-reared salmon.The British Columbia Salmon Farmers Association (BCSFA) ranks BKD asthe industry's number one problem (Hicks and Pennell, 1987). It is primarilyseen in Pacific salmon after salt water entry. Coho and Chinook salmon aremore susceptible than Atlantic salmon (Hicks, 1989a). The disease is causedby gram-positive Renibacterium salmoninarum which has been found only insalmonid species. The optimum temperature for the growth of the organismis from 15 to 18°C (Sindermann, 1988a). The transmission of the organismcan occur vertically (parent to offspring), as well as horizontally (fish to fish).The transmission is normally by the oral or cutaneous routes (Post, 1983a),however, R. salmoninarum can also be transmitted inside the egg (Post,1983b).6BKD is a chronic to subacute disease which develops slowly insusceptible salmon. The infected fish may look apparently normal and insome cases feed vigorously. However, healthy-appearing fish may diesuddenly. The obviously sick fish may have large abscesses under the skin,hemorrhages at the base of fins and in the muscle. The internal organs mayhave white to grey-white granulomas in the heart, spleen or kidney.The best method to prevent BKD is the application of good fishhusbandry practice. Stress factors must be kept at a minimum and carefulscreening of diseased smolts can also reduce the risk. Injection ofprespawning female Chinook salmon with erythromycin at 11 mg/kg of bodyweight results in concentrations up to 0.6 part per million (ppm) in the eggs,which persists for 30 to 60 days after the injection (Bullock and Leek, 1986).This allows prolonged contact of drug with the causative pathogen, R.salmoninarum, and reduces vertical transmission of BKD from parent tooffspring. More recently, it has been found that injection of penicillin G,oxytetracycline, cephradine and rifampicin into maturing female coho salmoncan also reduce the vertical transmission of BKD (Brown et al., 1990).Dietary modification may also help to reduce the prevalence of BKD.Lall et al. (1985) found that Atlantic salmon fed with a diet containing highlevels of iodine (4.5 mg/kg of feed) and fluorine (4.5 mg/kg of feed) hadsignificantly lower BKD infection rates: 3% and 5%, respectively, for twoconsecutive studies. In two control studies in fish fed with normalcommercial feed, the infection rates were 95% and 38%, respectively.Early diagnosis of the pathogen and early initiation of the treatmentwill control the disease more effectively. There is no effective therapy for theterminal stages of BKD. The drug of choice is erythromycin at a dosage of100 mg/kg fish/day for 21 days (Herman and Bullock, 1986). Treatment with7sulfamerazine at 200 mg/kg fish/day for 14 days can temporarily arrest theprogression of the disease, but will not cure the disease (Sindermann, 1988a).1.3.2 FurunculosisFurunculosis is a highly communicable septicemia disease which iscaused by gram-negative Aeromonas salmonicida (Hicks, 1989b), which hasan optimum growth temperature from 20 to 22°C (Post, 1983c). The diseasemay occur in both salmonids and non-salmonids. Furunculosis infectionsmay be acute or chronic. In the acute disease state, symptoms may not beobserved or a slight reddening at the base of the fins may be seen. However,acute outbreaks of furunculosis may occur with sudden onset and mortalitiesmay be high (Austin, 1983). In subacute or chronic disease states, the onsetis normally gradual and the mortalities are low. Extensive focal musclelesions such as furuncles, erythema at the bases of the fins, andhemorrhaging throughout the peritoneal cavity are characteristic symptoms(Austin, 1983). Since the symptoms of this disease are quite similar to otherfish diseases such as vibriosis, laboratory diagnosis is required to identify thepathogen.Screening of smolts helps to prevent the vertical transmission by thebacterium. The diseased fish are usually treated with antimicrobials such asRomet-30 which is a potentiated sulfonamide containing a 5:1 mixture ofsulfadimethoxine (SDM) and ormetoprim (OMP), oxytetracycline and oxolinicacid. Oxolinic acid is the most extensively used antimicrobial in Europe andJapan, however, it has not been approved for use in fish in Canada nor in theUnited States and is thus used only on an experimental basis (Rohovec,1991). Oxolinic acid is administered at a dose of 10 mg/kg of fish/day for 10days (Sindermann, 1988b). Oxytetracycline is administered at a dose of 888mg/kg of fish/day for 10 days (Sindermann, 1988b), and Romet-30® at adosage of 50 mg/kg of fish/day for 5 days (Bullock et al., 1983a).1.3.3 VibriosisVibriosis is a hemorrhagic septicemia which is very similar tofurunculosis. It is one of the most serious diseases of salmonids reared insalt water and accounts for 5% to 10% of the mortalities in pen-reared salmon(Hicks, 1989c). The annual losses in Japan alone reached $22 million (Smith,1988). The first proven outbreak of vibriosis in Canada was recorded on July22, 1968, in Nanaimo and West Vancouver (British Columbia), in four speciesof Pacific salmon that were cultured in sea water (Evelyn, 1969).Vibriosis is caused by gram-negative Vibrio anguillarum or Vibrioordalli, however, the majority of isolates are found to be V. anguillarum(Hicks, 1989c). The optimum growth temperature for V. anguillarum isbetween 18 to 20°C (Post, 1983d). It is believed that several stress factors,such as physical transfer, rapid changes in water temperature and salinity,overcrowding, rough handling, low oxygen level, and high suspended solids inthe water, may precipitate the disease outbreak, particularly when the watertemperature exceeds 10°C (Sindermann, 1988c).The symptoms of chronic infections are similar to those observed withfurunculosis and include external hemorrhagic and necrotic lesions and wide-spread hemorrhaging throughout the peritoneal cavity (Sindermann, 1988d).In acute outbreaks of vibriosis, a swollen liquified spleen may be detected(Hicks, 1989c). Laboratory evaluations are required to diagnose thecausative organism.A vibriosis vaccine has been developed and is available commercially.Vaccination is thought to be the best method to prevent the disease (Hicks,91989c). Outbreaks are usually treated with a potentiated sulfonamide suchas Romet-30 or with oxytetracycline.1.4 Major Antimicrobials for Treatment of Bacterial Fish Diseases1.4.1 General InformationAntimicrobial compounds are used both therapeutically for thetreatment of disease outbreaks and prophylactically where the fish might beat risk of disease infection or when the fish are transferred to a totally newenvironment. Romet-30® and oxytetracycline are currently the approvedantimicrobials for the treatment of bacterial fish diseases in Canada and theUnited States (Meyer, 1989; Bunn, 1992). Sulfamerazine is still approved foruse in the United States, however, the production of sulfamerazine in fishfeed formulations has been discontinued.Antimicrobials are usually administered orally as additives in fishfood. Oral administration is the most appropriate route for treating largenumbers of infected fish. However, as diseased fish frequently lose theirappetite, the desired therapeutic concentrations of the drugs may not beachieved. In addition, the taste of the antimicrobials may further reduce thepalatability of the feed. An alternative method of drug administrationinvolves immersion of fish in a bath containing the drug in solution.Penetration of the antimicrobial agent takes place via the gills (Michel,1986). The major drawback of this approach is the disposal of the drugsolution.Parenteral injection, while labour intensive, provides rapid onset andcontrolled drug administration. It is thus generally reserved for breedingstock and certain valuable fish, however, the handling process itself subjects10the fish to stress.1.4.2 SulfonamidesAs mentioned earlier, Romet-30 is a potentiated sulfonamideconsisting of a 5:1 mixture of SDM and OMP (Fig. 1). It was approved by theFood and Drug Administration (FDA) in the United States for controllingfurunculosis in salmonids on November 11, 1984 (U.S. FDA, 1984) and forcontrolling enteric septicemia in channel catfish on May 23, 1986 (U.S. FDA,1986). It was approved by the Health Protection Branch (HPB) in Canada forthe above purposes on October 7, 1990 (Bunn, 1992). The drug isadministered in the diet at a dosage of 50 mg/kg of fish/day for 5 consecutivedays followed by a 42-day withdrawal period (U.S. FDA, 1984). The drug hasalso been reported as effective in controlling enteric redmouth disease insalmonids (Bullock et al., 1983b) and vibriosis (Austin, 1988) in salmonids.Tribrissen® is another potentiated sulfonamide which is a 5:1 mixtureof sulfadiazine and trimethoprim. While not currently registered in NorthAmerica for aquaculture use, Tribrissen® is effective against many gram-negative bacterial fish diseases (Austin, 1988; Bose and Post, 1983).OC H3C H2NH2^OC H3C H311 OC H3SO2NH^\(NN-=(OC H3NH2SulfadimethoxineOrmetoprimFig. 1 Structures of Sulfadimethoxine and Ormetoprim.121.4.3 Oxytetracycline (Terramycin())Oxytetracycline (Fig. 2) has been successfully used as a feed additivefor the control of furunculosis, coldwater disease, columnaris, entericredmouth disease, gill disease and vibriosis (Austin, 1988).As with other tetracyclines, oxytetracycline is deposited in the bones offish after the administration of the drug (Herman, 1969), and could bedetected in Atlantic salmon bones for more than two years followingadministration (Odense and Logan, 1974). Due to the chelating ability oftetracyclines, the absorption of oxytetracycline might be reduced if the co-administered feed contains high levels of calcium, magnesium, zinc or iron.A study by Salte and Leist91 (1983) proposed a withdrawal time of60 days at 10°C and 100 days at 7°C to 10°C for rainbow trout following oraloxytetracycline administration of 75 mg/kg for no more than 10 consecutivedays. More recently, Aoyama et al. (1991) reported that oxytetracycline couldnot be detected (detection limit=0.05 ppm) in Chinook salmon muscle tissue35 days following 10 days of medicated feed administration at a dose of80 mg/kg fish. The water temperature varied from 7.8 to 10.3°C during the52 days study period.Fig. 2 Structure of Oxytetracycline.131.4.4 QuinolonesQuinolones represent the latest generation of antimicrobials. Fourquinolones: nalidixic acid, oxolinic acid, piromidic acid and flumequine(Fig. 3), have been investigated for potential application in fisherieschemotherapy. Oxolinic acid has now been widely accepted in Europe andJapan (Austin, 1988). The use of oxolinic acid in fisheries was banned by theDepartment of Agriculture in the United States and by the Bureau ofVeterinary Drugs in Canada in 1992 due to its carcinogenic effect (Sheppard,1993).Quinolones are effective against systemic bacterial infections causedby gram-negative pathogens. Oxolinic acid has been used in the treatment offurunculosis, vibriosis, enteric redmouth disease and columnaris (Austin etal., 1983; Austin, 1988; Rodgers and Austin, 1983). Although at high levelssome quinolones can suppress infections of gram-positive R. salmoninarum,they are generally ineffective for this application at practical dosageregimens (Austin, 1985a).Oxolinic acid is used at a dosage of 10 mg/kg of fishiday for 10 days.Oxolinic acid is absorbed rapidly both by oral and bath techniques, and iseliminated rapidly (Alderman, 1988). A study by Ishida (1992) found thatafter a single oral administration (40 mg/kg) and a single intravascularinjection (20 mg/kg), tissue concentrations of oxolinic acid in rainbow troutkept in sea water decreased to undetectable levels by 72 hours, whereas theconcentrations in rainbow trout kept in fresh water were detectable up to atleast 244 hours. The author concluded that the elimination rate of oxolinicacid was faster in seawater than in freshwater, i.e., the elimination ofoxolinic acid was a function of the salinity of the fish's environment.Therefore, the above differences should be kept in mind when determiningC-,I-LI 4 'H3 C^N^N,,11COOH(a)(d)14dosage regimen or withdrawal times for the fish reared at differentconditions. A withdrawal time of 21 days is recommendeded for a 10-daytreatment of oxolinic acid at a dosage of 10 mg/kg of fish/day in rainbow troutand Atlantic salmon (Austin, 1988).HOOC (b)I^IHOOCN0(c)Fig. 3 Structures of Quinolones Used in Aquaculture: (a) Nalidixic acid ;(b) Oxolinic acid; (c) Piromidic Acid; (d) Flumequine.151.5 Fish VaccinationIn both human and veterinary medicine, vaccination constitutes theprimary means of disease prevention. Three fish vaccines are commerciallyavailable for vibriosis, enteric redmouth disease and furunculosis. Amongthese three, only vaccines against vibriosis and enteric redmouth diseasehave proved successful (Home et al., 1984).These vaccines are usually prepared from formalin-inactivated crudebroth cultures (Austin, 1985b). Vaccination may be performed byintraperitoneal injection, oral vaccination, immersion or spray. Each methodhas its advantages and disadvantages (Ellis, 1988; Horne, 1984). Theinjection method gives rapid and high level protection and is economical, butit is also labor intensive, slow and impractical when fish are below 15 g. Theminimum size for a large scale operation is usually between 30 g and 50 g.The immersion technique is the most practical method for fry and small fishand is the most common method used. However, it is costly, less protectiveand causes a similar degree of stress to the fish as does the injection method.The spray technique is less efficacious than the immersion method, sinceequal exposure of all the fish to the vaccine cannot be ensured. A study byJohnson et al. (1982) showed the fish size of 1 g was the minimum size atwhich salmonids could be effectively immunized by the direct immersionmethod using V. anguillarum and Y. ruckeri bacterins, and betterimmunization occurred in 2.5 g or larger sizes. The development of immunitywas also found to be temperature dependent. With an immersion time of5 seconds, protective immunity was developed within 5 days at 18°C andwithin 10 days at 10°C.Fish vaccination is still a relatively new technique in aquaculture.Rigorous and thorough studies of the immunologic capacity of the fish, and a16more in depth understanding of the antigenic components of the bacteria arenecessary for the development of reliable and consistently effective vaccinesagainst major fish diseases such as BKD and furunculosis. However,vaccines will not kill the pathogen in the host, therefore, although thevaccinated fish are resistant to the disease, they may become carriers of thepathogens and these fish could become a potential hazard by spreading thecarried pathogen to susceptible populations. To avoid this potential hazard,the vaccinated fish should not be transferred to non-vaccinated healthygroups.1.6 Problems in Chemotherapy in Aquaculture1.6.1 Drug Residues in Edible TissuesDrug residues, including metabolites derived from parent drugs, inedible fish tissue are of primary concern to consumers because of possibleadverse effects induced by these drug residues. Therefore, a withdrawal timeis necessary to allow the drug levels in edible tissue to decline to tolerancelevels established for each drug used in aquaculture practice.There are two possible adverse effects which could be induced by thedrug residues: hypersensitivity and development of drug resistant bacteria inhumans. Cases of hypensensitivity due to consumption of or skin contactwith milk containing residual penicillin have been reported (Erskine, 1958;Vickers et al., 1958; and Wicher et al., 1969). However, the individuals in theabove cases had previously received penicillin therapeutically before theincidences. It is thought that although minute amounts of a drug such aspenicillin can elicit a hypersensitivity reaction, the drug residues in edibletissue are too low to trigger allergic reactions (Bevill, 1989; Woodward, 1990).17However, hypersensitivity reactions may occur in patients pre-sensitized bytherapeutic administration of the drug (Woodward, 1990).The other issue is the development of antimicrobial resistant bacteriain the consumer due to the digestion of drug residues in the fish or otheranimal tissues. However, the residue levels allowed in edible animal tissuesat the time of marketing are considered to be sufficiently low that bacterialresistance to the antimicrobial would not develop (Bevill, 1989).1.6.2 Development of Antimicrobial Resistant Bacterial CommunityThe development of antimicrobial resistant bacteria afterchemotherapy has long been noticed in veterinary medical practice as well asin human medical practice. Bacteria develop resistance to antimicrobialsthrough chromosomal changes, or through the exchange of genetic materialsfrom resistant strains via plasmids or transposons (Neu, 1992). The latterroute is more serious because the developed resistance can be transferredfrom one bacterium to another (Michel, 1986).Antimicrobial resistant pathogens have been developed to virtually allantimicrobial agents commonly used in aquaculture (Austin, 1988; Meyerand Schnick, 1989; Michel, 1986). The antimicrobial resistant bacteria ofanimal origin can cause serious diseases in humans. In a case report byHolmberg et al. (1984), 18 patients were infected by Salmonella newport thatwas resistant to ampicillin, carbenicillin and tetracycline, and 11 of themwere hospitalized for salmonellosis. Further investigation found that thesepatients consumed hamburgers originating from cattle fed subtherapeuticchlortetracycline and the beef was believed to be contaminated by S. newportwhich had already developed drug resistance. Although this case happenedin cattle, it could also occur in fish.18The other risk to public health is the release of antimicrobial resistantbacteria from fish farms via the effluent (Austin, 1988). To reduce the risk ofresistance developing, the antimicrobials must not be abused. A drug isselected to which the pathogen is most sensitive, and the drug should bedistributed by authorized personnel with appropriate instructions.Rotational use or combination use of two antimicrobials may help to reducethe risk of resistance developing. In addition, it is suggested that certainmedically important drugs such as doxycycline, isoniazid and rifampicinwhich are used to control tuberculosis should be banned from use on fishfarms (Austin, 1985).1.7 Background Information: Romet-30®1.7.1 ChemistryRomet-308 (Hoffmann-La Roche Inc., Nutley, New Jersey) is apotentiated sulfonamide with a combination of 5 parts SDM [N1-(2,6-dimethoxy-4-pyrimidiny1)-sulfanilamide] and 1 part OMP [2,4-diamino-5-(4,5-dimethoxy-2-methylbenzy1)-pyrimidine] (Fig. 1).SDM has the following physicochemical properties: white, odorlesscrystalline powder; molecular formula, C12H14N404S; molecular weight,310.33; pH of saturated aqueous solution, 5.5 - 6.5; maximum UV absorption,272 ± 2 nm; melting range, 197°C-202°C (Roche, 1980a). OMP possesses thefollowing physicochemical properties: white to off-white crystalline powder;molecular formula, C14H18N402; molecular weight, 274.4; maximum UVabsorption, 279 ± 2 nm; melting range, 231°C-235°C (Roche, 1980b).191.7.2 Mechanism of ActionSulfonamides are structural analogs of para-aminobenzoic acid (PABA,Fig. 4), which is an essential growth ingredient for a variety ofmicroorganisms involving in the biosynthesis of one-carbon units (Sammes,1990). One-carbon units are required for many enzyme-catalyzed reactions,in which the one-carbon units are transferred to various substrates(Lehninger, 1982; Stryer, 1988).COOHFig. 4 Structure of Para-aminobenzoic Acid20During the biosynthetic process (Fig. 5), the dihydropteridinederivative, 2-amino-4-hydroxy-6-hydroxymethyl-dihydropteridine (1), iseither coupled with PABA, with the involvement of dihydrofolic acid (DHF)synthetase, leading to the dihydropteric acid (2) which is then conjugatedwith glutamic acid to form dihydrofolic acid (DHF, 3). Compound (1) mayalso react directly with the PABA-glutamic acid conjugate (4), again with theinvolvement of DHF synthetase, to give DHF (3). DHF can then undergoeither reversible oxidation to folic acid (5), or reversible reduction totetrahydrofolic acid (THF) (6), which acts as the one-carbon carrier. Theenzyme involved in the latter step, dihydrofolate reductase, can be inhibitedby sulfonamide potentiators, such as trimethoprim and ormetoprim.The sulfonamides are competitive antagonists of PABA, bindingstrongly to DHF synthetase and thus inhibiting the formation of DHF (3).Thus by using a potentiated sulfonamide, the biosynthesis of one-carbonunits is synergistically inhibited by the sulfonamide and the sulfonamidepotentiator.Sulfonamides were also found to be able to substitute for PABA to formDHF analogs (7) (Fig. 6), which are inactive one-carbon carriers (Brown,1962). Sulfonamides do not inhibit the growth of mammalian cells by thismechanism because mammalian cells cannot synthesize folic acid as theyacquire folic acid from dietary sources.OHSO2NHRII-1f\Lre1H (7)21Fig. 5 Biosynthesis of folic acid (5) and THF (6) (Sammes, 1990).Fig. 6 Structure of DHF analog formed by sulfonamide and dihydropteridinederivative (1). R = substituent on N1 in the sulfonamide molucule.221.7.3 Toxicity in Fin FishRomet-30® has not been reported to elicit toxic effects in fish atpractical dosage regimens. A study by Bullock et al. (1973) showed noevidence of gross or histological toxic effects in viscera, kidney or musclewhen Romet-30® was fed at a rate of 500 mg/kg/day to trout for 14 days. Thecurrent practical dosage is 50 mg/kg of fish/day for 5 days (U.S. FDA, 1984).1.7.4 Pharmacokineties of SDM in Aquatic SpeciesPharmacokinetic studies of SDM in aquatic species have been reportedin lobster (Homarus americanus), channel catfish (Ictalurus punctatus) andtwo species of rainbow trout (Salmo gairdneri and Oncorhynchus mykiss),whereas the studies on OMP have only been reported in channel catfish(Ictalurus pun,ctatus) and rainbow trout (Salmo gairdneri).1.7.4.1 Pharmacokinetics of SDM in LobsterBarron et al. (1988) reported that a two compartment pharmacokineticmodel best described the disposition of 14C-SDM in lobster after singleintrapericardial drug administration. The pharmacokinetic behavior wasindependent of dose up to 55 mg/kg and was similar in both sexes. The totalbody clearance (Clb), apparent volume of distribution (Vd) and half life forelimination phase (t11213) were 13.8 ml/h/kg, 1369 ml/kg and 76.7 h,respectively. Binding to haemolymph proteins was linear with 53.5% bound.The majority of SDM distributes into muscle, shell and haemolymph duringthe early phases (up to 4 hours), while the hepatopancreas and the digestivetract held the largest fraction of the residual drug two weeks after dosing.231.7.4.2 Pharmacokinetics of SDM in Channel CatfishTwo pharmacokinetic studies of SDM were reported in channel catfish(Michel et al., 1990; Squibb et al., 1988). A two-compartment model wasrequired for both studies to best describe the results. In the study (Squibb etal., 1988), the fish were administered 14C-SDM (sodium salt) or 35S-SDM(free base form) at a dose of 40 mg/kg both intravenously and orally. Similarresults were obtained for both isotopically labelled compounds. Theestimated half-lives for SDM in blood were 0.06 h and 12.8 h for distributionand elimination phases, respectively. SDM was rapidly absorbed after oraladministration. The bioavailabilities for the sodium salt form of SDM andfree base form were 34.1% and 31.4%, respectively, and were not significantlydifferent. The elimination half-life of SDM in muscle tissue was 12.1 h. SDMwas mainly eliminated via the hepatobiliary pathway with a half-life of107.3 h in bile. The plasma protein binding was about 18%, and was non-specific and dose-independent. Multiple dosing with 14C-SDM(40 mg/kg/day for 5 days) did not significantly alter the tissue elimination ofSDM in muscle, skin and liver. However, the elimination half-life in bile wasreduced from 107 to 29 h.In the study by Michel et al. (1990), the fish were administered 14C-SDM at a dose of 40 mg/kg intravenously and orally. The results weresimilar to those reported by Squibb et al. (1988). The disposition andelimination were best described by a two-compartment model. The half-livesfor SDM in blood were 0.09 h and 12.6 h for the distribution and eliminationphases, respectively. The bioavailability was 34%. The elimination half-lifein muscle was 13.1 h. SDM became concentrated in the bile with time andhad a half-life of 115.5 h. The plasma protein binding was 18% and was non-specific and dose-independent.241.7.4.3 Pharmacokinetics of SDM in Rainbow TroutPharmacokinetic studies of SDM in rainbow trout have been reportedby Kleinow and Lech (1988) and Kleinow et al. (1992). The data were foundto best fit a two-compartment model. Following a single intravenousadministration of 35S-SDM (42 mg/kg) the pharmacokinetic parameters oft1/2a, t11213, Vss and Clb were, respectively, estimated to be 0.38 h, 15.9 h,421.6 ml/kg and 21.8 ml/kg/h as determined by an HPLC method, and 0.63 h,17.0 h, 500.8 ml/kg and 22.7 ml/kg/h as determined by a liquid scintillationcounting method. In contrast to the study in channel catfish (Squibb et al.,1988), multiple-dose administration of SDM (42 mg/kg at 24-h intervals up to96 h) to rainbow trout lengthened t11213 to 35.2 h from 17.0 h. In addition,the bioavailabilities of the sodium salt SDM (42 and 126 mg/kg) and free baseSDM (42 mg/kg) following single oral administrations were 63%, 50% and34%, respectively. The bioavailabilities between the two doses of sodium saltSDM were not significantly different. Plasma protein binding was 16% andwas non-saturable and non-specific. A tissue distribution study showed thatSDM attained the highest level in bile, followed by the intestine, liver, blood,skin, kidney, spleen, gill, muscle, and fat.A summary of some of the pharmacokinetic parameters in the abovethree aquatic species is presented in Table 2.25Table 2Pharmacoldnetic parameters of SDM in aquatic species.Channel catfish(Michel et al., 1990)^34^18^0.09(Squibb et al., 1988)^31/34 18.4 0.06Rainbow trout^34-64^15.8^0.63/(Kleinow et al., 1992) 0.38t11213 d(h)Vde^Clbf^Tmaxg(ml/kg) (ml/kg/h)^(h)76.7 1369 13.812.6 402 22.112.8 662 3-617.0/ 500.8/ 23.7/ 10-2415.9 421.6 21.8Species^BVa^Percentageb^tin c(%)^bound (h)Lobster^ 53.5^1.97(Barron et al., 1988)a. Oral bioavailability;b. Percentage of SDM bound to plasma protein;c. Half-life for SDM in blood for the distribution phase;d. Half-life for SDM in blood for the elimination phase;e. Apparent volume of distribution;f. Total body clearance of SDM;g. Time for SDM to reach its maximum concentration in plasma.261.7.4.4 Pharmacokinetics of OMP in Rainbow Trout and Channel CatfishThe first pharmacokinetic study of OMP was reported in rainbow troutby Droy et al. (1990). Single doses of 14C-OMP (8 mg/kg) were administeredintravascularly and orally to two groups of rainbow trout. The distributionand elimination half-lives were 0.54 h and 17.5 h, respectively, after a singleintravascular dose, and were increased to 0.67 h and 36.7 h, respectively,after multiple doses (4 consecutive doses at 8 mg/kg at every 25 h). Thebioavailability was estimated to be 87%. The plasma protein binding was 31-33% and was non-specific and non-saturable. The disposition of OMP washighest in bile, kidney and liver. Significant OMP residues were detected inskin (0.90 ppm) and muscle (0.15 ppm) at 38 days.In a pharmacokinetic study in channel catfish (Plaska et al., 1990),14C-OMP was given as single intravascular and oral doses at 4 mg/kg. Thebioavailability was estimated at 52% and the pharmacokinetics were bestdescribed by a triexponential compartment model, with half-lives of 0.39 h,4.9 h and 49 h for a, B and y phases, respectively. OMP was widelydistributed in the tissues with the highest concentrations in liver, kidney andspleen. The muscle tissue contained about 50% of the intravascularlyadministered dose at 2 h, however, 0.6% of the dose remained in the muscleat 72 h with a concentration of 0.04 ppm. The skin contained 1.2% of theintravascularly administered dose at 72 h with a concentration of 0.71 ppm.OMP was extensively metabolized in channel catfish, however, themetabolites were not identified.A summary of some of the pharmacokinetic parameters of OMP fromthe above studies is presented in Table 3.Table 3Pharmacokinetic parameters of OMP in rainbow trout and channel catfish.Species Dose^BV Percentage t /2 a^ti/2t3^tia y Vssa^Clb^vb^vc^v d^vde T f(mg/kg)^(%)^bound^(h)^(h)^(h)^(ml/kg) (ml/kg/h) (ml/kg) (my/kg) (ms/kg) (n-Pdkg) 11(lhaxr)Rainbow trout^8^87^31-33^0.54^17.5^4986^228^1042^3944^ 12(single)8^ 0.67^36.7(multiple)Channel catfish^4^52^0.39^4.9^49^192^537^1061^3905^6a. Vss = Apparent volume of distribution at steady state;b. Vc = Apparent volume of distribution of central compartment;c. VP = Apparent volume of distribution of peripheral compartment;d. V = Apparent volume of distribution of shallow peripheral compartment;pse. Vpd = Apparent volume of distribution of deep peripheral compartment;f. Tmax = Time for OMP to reach its maximum plasma concentration after oral administration.281.7.4.5 Effect of OMP on the Bioavailability, Distribution, andPharmacokinetics of SDM in Rainbow Trout (Oncorhynchus mykiss)To determine the effects of combination therapy, a study in rainbowtrout was carried out by Droy et al. (1989). A combination of SDM/OMP(SDM/OMP, 42/8 mg/kg) was administered by the intravascular and oralroutes to two groups of rainbow trout (3 fish/group). The mainpharmacokinetic parameters for SDM were: t112a 0.40 h; t11213 16.08 h;Vd 503.85 ml/kg; Clb 23.04 ml/kg/h. The oral bioavailability of SDM was 38%and peak plasma concentration was reached at 20 h. The plasma binding ofthe two drugs was not altered by each other, and with approximate values of16% for SDM and 31% for OMP. The distribution study revealed the highestconcentrations of SDM were found in bile, intestine, and liver. These resultswere similar to those reported by Kleinow and Lech (1988) and Kleinow et al.(1992). Hence, the pharmacokinetics of SDM were not influenced by thecombination usage of SDM and OMP.1.7.5 Metabolism of SDM and OMP in Aquatic SpeciesThe metabolism of SDM differs from species to species (Bridges et al.,1968). In human and monkey, the major metabolite was N1-glucuronide,while in rabbit and guinea pig, it was N4-acetyl-SDM. SDM was excretedunchanged in the dog. In the rat, equal amounts of the unchanged drug andN4-acetyl-SDM were the main elimination products.A study in channel catfish following a single oral dose at 40 mg/kg(Squibb et al., 1988) showed that in plasma and muscle tissues, SDM waspresent primarily (>95%) as the parent drug, however, in the liver, N4-acetyl-SDM (Fig. 7) and SDM were both present. In bile, more than 90% of SDMwas excreted as N4-acetyl-SDM. In rainbow trout (Kleinow et al., 1992), it29was found that N4-acetyl-SDM predominated in the bile (86%) 20 h after asingle oral dose of 42 mg/kg, while the liver contained slightly greateramounts of N4-acetyl-SDM (55%) than the parent drug (35%). In plasma,SDM was present primarily in the =changed form (84%). In lobster,however, N4-acetyl-SDM was only a minor metabolite present in hemolymphand hepatopancreas (Barron and James, 1988).The metabolism of OMP has not been well investigated in aquaticspecies. A single study was reported in channel catfish by Plaska et al.(1990) who suggested that OMP was extensively metabolized to nonpolar andpolar metabolites and that the nonpolar metabolite might be N-acetylatedOMP. However, the identities of these metabolites were not established. OC H3SO2NHNOC4CH3CONHFig. 7 Structure of N4-Acetyl-Sulfadimethoxine301.7.6 Palatability of Medicated FeedFish feed with the addition of antimicrobials usually has decreasedpalatability and affects the intake of the medicated feed. As a result, thedesired therapeutic concentration might not be achieved in the fish. A studyshowed that channel catfish feed containing Romet-30® had decreasedpalatability due to OMP (Poe and Wilson, 1989). This could be improved byinclusion of at least 16% fish meal in the feed at a rate of 1.65% Romet-30®in catfish feeds (Robinson et al., 1990).1.8 Analytical Methods for SDM and OMP in Fish Tissues1.8.1 Analytical Methods for SDMThe Tishler method (Tishler et al., 1968) is typical of non-chromatographic methods for determination of sulfonamides in animaltissues. In this method, 50 g of animal tissue is homogenized with 90 to100 ml of a chloroform and acetone mixture (1:1). The filtered extracts areflash-evaporated and the residue is taken up in hexane and acetone andtransferred to 0.5 N hydrochloric acid. The solution is centrifuged and theaqueous layer filtered. The color is developed by utilizing the Bratton-Marshall color reaction (Bratton and Marshall, 1939), in which sulfonamidesare diazotized by adding 0.1% NaNO2 solution at acidic condition (pH 1-2),followed by adding 0.5% ammonium sulfamate solution to destroy the excessnitrous acid. The final color is developed by the coupling of the diazocompound with 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride (NED).The absorbance is measured at 545 nm. The recoveries from swine, poultry,and calf tissues ranged from 76 to 100% at the 0.1 ppm level. The methodcan detect 0.05 ppm of sulfonamides. This method can be used to quantify31sulfonamides or other compounds containing a free primary aromatic aminogroup and hence lacks specificity in complex biological systems.A thin-layer chromatographic (TLC) method (Reimer and Suarez,1991) was developed for screening sulfonamide residues, including SDM, insalmon muscle tissue. In this assay, a matrix solid phase dispersion methodwas employed in which the tissue sample was ground with C18-derivatizedsilica gel, firmly packed into a column and washed with 10% toluene inhexane and eluted by dichloromethane. The dichloromethane eluant wasevaporated, the residues were constituted in 50 Ill methanol and eluted onsilica high-performance thin-liquid chromatographic plates. Thesulfonamides were detected after spraying the plate with fluorescaminesolution. The method had recoveries ranging from 57 to 63% for 5 differentsulfonamides. The lowest-detectable level was 0.04 ppm for SDM. Therecovery of SDM was 63%.Analyses by gas-chromatographic (GC) methods require methylation atthe N1-position on the sulfonamide molecules by diazomethane (CH2N2) toprovide derivatives with sufficient volatility. A GC method with electron-capture detection (ECD) was developed for determining sulfonamides invarious animal tissues (Goodspeed et al., 1978). Tissue samples wereextracted with acetone, partitioned with ethyl ether and 1 N hydrochloricacid, and the acid layer was adjusted to pH 6.5 and extracted withdichloromethane. The residue was methylated with diazomethane andacetylated with pentafluoropropionic anhydride [PFPA, (CF3CF2C0)20],followed by GC/ECD. The assay had an average recovery of 81.5% at the0.1 ppm level, 79.1% at the 0.5% ppm level, and 76.0% at the 1.0 ppm level.The sensitivity of the assay was not presented.32Garland et al. (1980) reported a gas chromatographic-chemicalionization mass spectrometry (GC/CIMS) method for determining SDM inporcine and bovine livers and kidneys. A deuterated analogue of SDM, SDM-d6, was used as an internal standard. The tissue were extracted withdichloromethane and methylated with diazomethane followed by acetylationwith PFPA and analysis by GC/CIMS, with isobutane as reagent gas. Theassay was able to measure SDM at the 0.1 ppm level.Takatsuki and Kikuchi (1990) reported a GC/MS method usingselected ion monitoring (SIM) mode for the simultaneous determination of sixsulfonamides in egg and animal tissues. The samples were extracted withacetonitrile and were passed through a silica cartridge column for analyteisolation. The resulting sulfonamides were methylated with diazomethane inether. After evaporation, the residue was dissolved in methylene chlorideand further purified by silica gel column chromatography. The analytes werefinally analyzed by GC/MS in SIM mode. SDM was monitored at m/z 260.Methylated sulfonamides were found to be more stable than non-methylatedones and had a much better recovery (85 to 87%) than non-methylated ones(15%) from silica columns. The detection limit was 0.03-0.05 ppm.A gas chromatographic/tandem mass spectrometric (GC/MS/MS)method was described by Matusik et al. (1990). The quantitation of foursulfonamide residues, including SDM, in bovine and porcine liver was carriedout by using GC/ECD, while the identities of these sulfonamides wereconfirmed by MS/MS daughter ion scan. Sulfonamides were methylatedprior to GC analysis. The recoveries were 80% for sulfamethazine,sulfachloropyridazine and sulfadimethoxine and 46% for sulfathiazole. Aresidual level of 0.1 ppm could be successfully confirmed.33High-performance liquid chromatographic (HPLC) assays are themainstream in analysis of sulfonamides in animal tissues (Bevill et al., 1978;Hone et al., 1991; Horii et al., 1990; Kleinow et al., 1992; Klimowicz, 1989;Long et al., 1990; Mengelers et al., 1989; Nose et al., 1987; Pleasance et al.,1991; Vree et al., 1990; Walisser et al., 1990; Weiss et al., 1987). Nose et al.(1987) used an alumina column to isolate antimicrobial agents, including foursulfonamides from cultured fish muscle samples, prior to HPLC analysis.The extract was transferred onto an alumina column, eluted successivelywith acetonitrile and 1% NaOH solution, and fractions corresponding toindividual antimicrobials (including SDM) were collected, combined andanalyzed on a Nudeosil C18 HPLC column. The recoveries of theantimicrobial agents were 80%. The lower limit of detection of the drugs was1-2 ng for 10 ill injection.Long et al. (1990) described a matrix solid phase dispersion method forthe isolation of SDM from catfish muscle tissue before analysis of SDM onHPLC. Half a gram of tissue was blended with octadecylsilyl (ODS)derivatized silica and packed into a column. The column was first washedwith 8 nil of hexane, followed by 8 ml of dichloromethane. Thedichloromethane eluant was evaporated, reconstituted in 0.5 ml HPLCmobile phase and analyzed. An average relative recovery of 101.1±4.2%(peak area ratios of SDM/internal standard from extracted fortified sampleswere compared to those of pure standards which had undergone identicalextraction procedures) was achieved. The minimum detectable limit was0.05 ppm.Pleasance et al. (1991) reported an HPLC method with diode arraydetection (DAD) and an LC/MS/MS method with ion-spray technique fordetermining 21 sulfonamides in cultured salmon flesh. By using DAD,34complete UV spectra of the individual sulfonamide were obtained to help theconfirmation of peak identities. In addition LC/MS/MS technique providedfurther structural information about each drug. A sensitivity of 0.025 ppmwas achieved for SDM.Some researchers have employed the use of 14C or 35S labelled SDMin pharmacokinetic studies (Squibb et al., 1988; Michel et al., 1990; Kleinowand Lech, 1988; Kleinow et al., 1992). Aniline labelled 14C-SDM was utilizedfor all studies. Generally, a tissue sample was digested in tissue solubilizers(e.g., Protosol) and the radioactivity was quantified by liquid scintillationcounting. The major drawback in these studies is that it was the totalradioactivity, representing the parent drug and metabolites, that wasassayed. A study by Kleinow et al. (1992) showed that some pharmacokineticparameters determined by 35S-SDM counting were quite different from theones by HPLC method, e.g., t112oc and Vss were 0.63 h and 500.8 mUkg,respectively, by 35S-SDM counting, and were 0.38 h and 421.6 mUkg,respectively, by HPLC method. Thus, the lack of specificity of radioisotopeassays make such techniques unsuitable for pharmacokinetic studies if theparent drug is not separated from its metabolites.1.8.2 Analytical Methods for OMPSpectroflurometric methods have been reported for the determinationof OMP in feeds and animal tissues (Fellig et al., 1971; Osadca and De Ritter,1970; Osadca et al., 1974). In all assays, OMP was oxidized withpermanganate to yield the fluorescent product, 4,5-dimethoxy-o-toluic acid.Radioisotope techniques have also been reported for pharmacokinetic studiesin rainbow trout (Droy et al., 1990) and channel catfish (Plakas et al., 1990).14c_omp (Fig. 8) was employed for both studies.H2N OC H3NH2^OC H3H335The simultaneous determination of SDM and OMP was reported byWalisser et al. (1990) and Weiss et al. (1987). In the former method, SDMand OMP were extracted with acetonitrile from salmon muscle tissue and theanalytes were isolated using Sep-Pak C18 cartridges. The drugs weredetermined by HPLC on an Ultrasphere ion-pair column. The minimumdetectable quantity of SDM and OMP was 0.2 ppm at a signal-to-noise ratioof 5:1. The mean recoveries for SDM and OMP were 55% and 67%,respectively. In the latter method, tetrabutylammonium hydroxide was usedto form the tetrabutylammonium ion pair of SDM which allowed bothcompounds to be extracted into dichloromethane at pH 10 and analyzed byHPLC. The average recovery exceeded 90% for both SDM and OMP invarious tissues from cattle, chickens and catfish. The detection limit was0.05 ppm for both compounds.14C-OrmetoprimFig. 8 Structure of 14C-Ormetoprim.361.9 Research HypothesisPrevious studies conducted by other investigators indicated that liverand kidney tissues usually contain higher levels of drug residues than muscletissue in rainbow trout and channel catfish (Herman and Bullock, 1986;Kleinow and Lech, 1988; Kleinow et al., 1992; Michel et al., 1990). Thus it isproposed that a tissue could be identified which will accumulate a higherlevel of Romet-30 than muscle tissue, and that the ratio of the residuesbetween this tissue and muscle tissue will be consistent. Therefore, theconcentration of Romet-30® in this "marker" tissue can be used to indicatethe concentration of Romet-30® in muscle tissue. In this way, when theRomet-30® level has fallen near or below the lower detection limit of theassay, the level in the "marker" tissue would still be detectable and greaterconfidence in the results could be achieved.371.10 Research Goals1) To intubate Chinook salmon with Romet-30® at a dosage regimenof 40 mg/kg of fish/day for 10 days;2) To improve the sensitivity of the previous assay (Walisser et al.,1990) for Romet-30® in muscle tissue to a more sensitive assay;3) To adopt this assay for analysis of Romet-30® in Chinook salmonliver tissue and to assay Romet-30 concentrations in liver tissue;4) To determine if a consistent concentration relationship existsbetween muscle and liver tissues;5) To determine wash-out times of Romet-30® in muscle tissue.382. EXPERIMENTAL2.1 Materials and Supplies2.1.1 DrugsTwo lots of Romet-30® were obtained from Hoffmann-La Roche(Nutley, New Jersey; Lot# D125H4) and Syndel Laboratories (Vancouver,British Columbia; Lot# 4106), and were used in two intubation studiesconducted in September, 1990 and January, 1992, respectively.2.1.2 Chemicals and ReagentsAll chemicals and reagents were analytical grade and were usedwithout further purification. Ormetoprim and sulfadimethoxine wereobtained from Hoffmann-La Roche. Tricaine methanesulfonate (MS-222) wasobtained from Syndel Laboratories. Sulfisoxazole was obtained from SigmaChemical Company (St. Louis, Missouri). Tetrabutylammonium hydroxide(TBAH, 40%) and sodium carbonate were obtained from Aldrich ChemicalCompany, Inc. (Milwaukee, Wisconsin). Phosphoric acid (85%) was obtainedfrom Fisher Scientific Company (Fair Lawn, New Jersey). Sodium sulfateanhydrous granular, sodium sulfate anhydrous powder, sodium chloride,ammonium acetate, sodium hydroxide, sodium bicarbonate, disodiumhydrogen orthophosphate heptahydrate (Na2HPO4.7H20) and aceticanhydride were obtained from BDH (Toronto, Ontario). Zinc sulfate wasobtained from BDH Chemicals Ltd. (Poole, England).392.1.3 SolventsHPLC grade methanol, acetonitrile and dichloromethane wereobtained from BDH. Purified water was produced using a Milli-Q waterpurification system (Millipore, Mississauga, Ontario).2.2 Instrumentation2.2.1 HPLC SystemThe HPLC system consisted of a Beckman Model 100A pump(Fullerton, California), a Shimadzu SIL-9A auto injector, Shimadzu SPD-6AUV spectrophotometric detector and Shimadzu C-R6A Chromatopac dataprocessor (Kyoto, Japan), a Beckman Ultrasphere ion-pair column (5p,m, 250x 4.6 mm I.D.) (San Ramon, California), and a guard column with a BrownleeRP-18 cartridge (15 x 3.2 mm I D ) (Santa Clara, California).2.2.2 LC/MS and LC/MS/MS ExperimentsLC/MS and LC/MS/MS experiments were performed on a Sciex API Illtriple quadrupole mass spectrometer (Thornhill, Ontario), equipped with anIonSpray interface and an APCI source. The samples were delivered by flowinjection with 10 glimin aqueous 50% methanol. A dwell time of 6 ms/daltonwas used for full-scan LC-MS analyses. APCI mass spectra were acquired inthe first quadrupole Ql, and selected precursor ions were fragmented in theradio frequency only quadrupole Q2 at a collision energy of 36 eV. Argon wasused as the target gas at an indicated thickness of 5.48 x 1014molecules/cm2. Product ions were examined in the third quadrupole Q3.402.2.3 Miscellaneous SuppliesOther equipment included: a Brinlunann Polytron Model PT 10/35homogenizer (Brinkmann instruments, Rexdale, Canada), a Beckman GPcentrifuge (Palo Alto, California), a Fisher Model 220 pH meter, an AS290automatic SpeedVac concentrator (Savant Instruments, Inc., Farmingdale,New York), a Thermolyne vortex mixer and Thermolyne Dry-bath (Dubuque,Iowa), a capillary melting point apparatus (Arthur H. Thomas Co.,Philadelphia, Pennsylvania), a Kontes filter assembly (Vineland, NewJersey), 0.45 gm Nylaflo® membrane filters (13 mm and 47 mm diameter,Gelman Sciences, Ann Arbor, Michigan), and blood collection tubes (10 ml,Becton Dickinson VACUTAINER Systems, Rutherford, New Jersey).2.3 Stock SolutionsQuantities given are representative of average values.2.3.1 Sulfadimethoxine and Ormetoprim Standard SolutionsA mixture of SDM and OMP was prepared by dissolving SDM andOMP in 50 ml of acetonitrile to give a concentration of 200 gg/ml for bothstandards. This stock solution was further diluted to give a series ofstandard solutions with concentrations of 100, 20, and 2 gghnl.2.3.2 N4-Acetyl-Sulfadimethoxine Standard SolutionA stock solution of synthetic N4-Ac-SDM was prepared in 25 ml ofacetonitrile to give a concentration of 400 pg/ml. This stock solution wasfurther diluted to give concentrations of 200, 100, 20, and 2 jig/mi.412.3.3 Sulfisoxazole Internal Standard SolutionSulfisoxazole internal standard solution was prepared in 100 ml ofacetonitrile to give a final concentration of approximately 60 gg/ml.2.3.4 TBAH (0.5 M)TBAH working solution (0.5 M) was prepared by diluting 6 ml of 40%TBAH (approximately 1.5 M) with 12 ml of purified water.2.3.5 Sodium Hydroxide (1 M)Sodium hydroxide (1 M) was prepared by dissolving 8 g of sodiumhydroxide pellets in 200 ml of purified water.2.3.6 Sodium Carbonate/Sodium Bicarbonate Buffer, pH 10 (Delory and King,1945)Sodium carbonate stock solution (0.2 M) was prepared by dissolving21.2 g of sodium carbonate in 1000 ml of purified water. Sodium bicarbonatestock solution (0.2 M) was prepared by dissolving 16.8 g of sodiumbicarbonate in 1000 ml of purified water. The buffer was prepared by mixing27.5 ml of the above sodium carbonate solution and 22.5 ml of the abovesodium bicarbonate solution and further dilution to 200 ml with purifiedwater.2.3.7 Phosphate Buffer (0.1 M), pH 4.0Na2HPO4-7H20 (26.8 g) was dissolved in 900 ml of purified water.The pH of the solution was adjusted to 4.0 with phosphoric acid. The solutionwas transferred to a 1000 ml volumetric flask and was diluted up to volumewith purified water.422.4 Synthesis of N4-Ac-SDMAn excess of 5 ml of acetic anhydride (approximately 53 mmole) wasadded to 2 g of SDM (approximately 6 mmole) and the mixture was heated ona Thermolyne Dry-bath at 70°C for 20 min with occasional manual shaking.The excess acetic anhydride was evaporated under nitrogen in a 40°C waterbath. The resulting crystals were filtered and washed with water. Theproduct was recrystallized from acetonitrile. Its melting point wasdetermined to be 219-222°C on a capillary melting point apparatus, while theMerck Index value is 220-223°C (The Merck Index, 1976). The purity wasdetermined on the HPLC system which was used for the analysis of SDM andN4-Ac-SDM in liver samples.2.5 Intubation StudiesTwo intubation studies were conducted in September, 1990 andFebruary, 1992. In the first study, twenty-eight Chinook salmon weighingbetween 520 and 2475 g (1255±101; mean±S.E.M.) were obtained from HardySea Farms (Hardy Island, British Columbia). During the study, three of thefish died. The water temperatures varied between 10.8 and 11.6°C duringthe 20-day study period.In the second study, forty-one Chinook salmon were obtained from SaltSpring Aquafarms (Salt Spring Island, British Columbia). Due to therelatively high mortality rate, only 26 fish were available for final analyses.The fish weighed between 615 and 1800 g (1094±57; mean_±S.E.M.). Thewater temperatures ranged from 8.0 to 9.0°C throughout the 20-day study.In both studies, the fish were maintained in flowing seawater tanks atthe West Vancouver Laboratory of the Department of Fisheries and Oceans.A suspension of Romet-30® in water (25 mg/nil) was freshly prepared prior to43each intubation. To administer Romet-30 , salmon were removed from thetank individually by net and immersed in a tricaine methanesulfonate (MS-222) bath at a concentration of 43 mg/ml. Sodium bicarbonate (43 mg/ml)was also added to the anesthetic solution in order to buffer the change in pHcaused by the addition of MS-222 which otherwise would be irritating to thefish.After the fish were anesthetized, they were weighed and held forintubation. In the second study, all fish were tagged a specific number andweighed on the first day of the intubation. The fish were re-weighed on thefifth day to check if there were any weight change. The Romet-30® solutionwas administered daily for 10 days at a dosage of 40 mg/kg. Followingintubation, each fish was put into another tank and held by hand or net untilthe fish recovered from the anesthetic.In the first study, 6 fish were sampled each time on each of days 11(first day after the cessation of the intubation), 14 and 17, and 7 fish weresampled on day 20. In the second study, 6 fish were sampled on each of days11 and 14, and 7 fish were sampled on days 17 and 20. The fish weresacrificed by a blow to the cranium. Blood samples were immediatelycollected in heparinized tubes by venipuncture of the caudal vein orcardiopuncture. The fish were transported to the laboratory, samples of skin,muscle, liver and kidney were removed, rinsed briefly with tap water, andfrozen at -20°C until required for analysis.2.6 Extraction Procedures in Muscle and Liver TissuesA schematic diagram of extraction procedures in muscle and livertissues is shown in Scheme 1.44A 5 g sample of muscle or liver tissue was dissected and placed in a50 ml centrifuge tube. An aliquot of 200 ill of sulfisoxazole internal standardsolution (approximately 12 i.tg sulfisoxazole) was added along with 300 gl of0.5 M TBAH, 1 ml of pH 10 Na2CO3/NaHCO3 buffer and 1 ml of 1 N NaOH.After brief manual mixing with a pasteur pipette, 15 ml of dichloromethanewas added and the sample was homogenized at medium speed (at 4 to 5 onthe control panel) for 20-30 seconds. Granular sodium sulfate anhydrous(2 g) was added to the homogenate and the sample was vortex-mixed for2 min. The mixture was then centrifuged at 3000 rpm (2000 x g) for 15 min.After centrifugation, the top aqueous layer was removed to a test tube, theinterfacial solid tissue plug was pushed aside, and the dichloromethane layerwas removed. The aqueous layer was returned to the tube containing theresidual tissue plug and an additional 10 ml of dichloromethane was added.The sample was vortex-mixed for 2 min and centrifuged. Thedichloromethane layer was removed, combined with the initialdichloromethane layer and evaporated under a nitrogen stream in a 40°Cwater bath. The residue was reconstituted in 1 ml of HPLC mobile phase(0.1 M pH 4.0 phosphate buffer:acetonitrile:methanol, 75:12:13). Aftercentrifugation for 5 min at 2500 rpm (1430 x g), the bottom clear layer wasremoved and filtered through a 0.45 pin membrane filter. An aliquot of 20 illof the filtrate was injected onto the HPLC system.45Scheme 1 Extraction Protocol in Chinook SalmonMuscle or Liver TissuesFive grams of muscle or liver sample12 pg Internal standard sulfisoxazole addedStored at 4 °C for 1 hour300 pl of 0.5 M TBAH1 ml of pH 10 Na2CO2/1NaHCO3 buffer1 ml of 1M-NaOH15 ml of dichloromethane addedHomogenized at medium speed for 20-30 sec.2 g of Na2SO4 anhydrous added and vortex mixed for 2 min.Centrifuged at 2000 x g for 10 min.Bottom dichloromethane layer drawn and tissue plugre-extracted with 10 ml of dichloromethane byvortex mixing for 2 min.Dichloromethane extracts combined andevaporated under nitrogen stream at 40 °CResidues reconstituted in 1 ml of HPLC mobile phaseFiltered through 0.45 pm membrane20 pl injected onto the HPLC for analysis462.7 Calibration Curves2.7.1 Calibration Curves in Muscle TissueThe calibration curves were determined in a series of 5 g muscle tissuesamples to which were added 200 IA of internal standard solution (60 [tg/m1)and appropriate volumes of SDM/OMP and N4-Ac-SDM standard solutions togive final concentrations of 0.05, 0.10, 0.20, 0.50, 2.00, 5.00 and 10.0 ppm foreach of the standards. The samples were stored at 4°C for 1 hour, followed byextraction as described in section 2.6. The calibration curve was constructedby plotting the peak area ratios of SDM, OMP and N4-Ac-SDM to theinternal standard against the concentrations of SDM, OMP and N4-Ac-SDMadded.2.7.2 Calibration Curves in Liver TissueThe calibration curves in liver tissue were determined in a similarfashion to section 2.7.1. To a series of liver tissue samples (5 g each) wereadded 200 1.4,1 of internal standard solution and appropriate volumes ofSDM/OMP and N4-Ac-SDM standard solutions to give final concentrations of0.20, 0.50, 2.00, 5.00, 15.0 and 25.0 ppm for each of the standards. Thesamples were then stored at 4°C for 1 hour, followed by extraction asdescribed in section 2.6. The calibration curves were constructed by plottingthe peak area ratios of SDM and N4-Ac-SDM to internal standard againstthe concentrations of SDM and N4-Ac-SDM added in the liver samples.472.8 Extraction Recovery Studies2.8.1 Extraction Recoveries of SDM, OMP and N4-Ac-SDM in Muscle TissueThe extraction recoveries were determined at 0.50 ppm and 5.00 ppmconcentration levels. To six muscle tissue samples (5 g each) were added125 pi of SDM/OMP and 125 ill of N4-Ac-SDM standard solutions (20 gg/m1)to give final concentrations of 0.50 ppm (equivalent to 0.50 1.1g/g tissue) foreach of the three standards. To a second series of six samples (5 g each) wereadded 125 pl of SDM/OMP and 125 Ill of N4-Ac-SDM standard solutions(200 gg/m1) to give final concentrations of 5.00 ppm for each of thesecompounds. The samples were stored at 4°C for 1 hour, followed byextraction as described in section 2.6, except the internal standard was addedjust before the combined dichloromethane extract was evaporated to dryness.Two solutions containing SDM/OMP and N4-Ac-SDM at 0.50 and 5.00 ppmalong with the internal standard were prepared in parallel, but withoutundergoing extraction.2.8.2 Extraction Recoveries of SDM and N4-Ac-SDM in Liver TissueThe extraction recoveries of SDM and N4-Ac-SDM in liver tissue weredetermined at 0.50, 5.00 and 20.0 ppm, respectively. Two liver tissuesamples (5 g each) were analyzed at each concentration level. To the first twosamples were added 125 gl of SDM/OMP and 125 p1 of N4-Ac-SDM standardsolutions (20 mg,/m1) to give final concentrations of 0.50 ppm for each of thestandards. To the second two samples were added 125 gl of SDM/OMP and125 IA of N4-Ac-SDM standard solutions (200 p.g/m1) to give finalconcentrations of 5.00 ppm for each of the standards. To the last two sampleswere added 500[1.1 of SDM/OMP and 500111 of N4-Ac-SDM standard solutions48(200 gg/m1) to give final concentrations of 20.0 ppm for each of the standards.The samples were stored at 4°C for 1 hour, followed by extraction asdescribed in section 2.6, except the internal standard was added just beforethe combined dichloromethane extract was evaporated to dryness. Standardsolutions of SDM/OMP and N4-Ac-SDM at 0.50, 5.00 and 20.0 ppm alongwith the internal standard were prepared in parallel without undergoingextraction.2.9 Intra-assay Variability StudiesAll studies for the determination of intra-assay variabilities atspecified concentrations were completed in one day for each tissue type.2.9.1 Intra-assay Variability in Muscle TissueThe intra-assay variability was determined by the analysis of sixmuscle samples to which SDM/OMP and N4-Ac-SDM were added at0.50 ppm, and six muscle samples to which SDM/OMP and N4-Ac-SDM wereadded at 5.00 ppm. An aliquot of 200 ill of the internal standard solution(60 [tg/m1) was added to each sample. After storage at 4°C for 1 hour, thesamples were extracted as described before (section 2.6) and assayed.2.9.2 Intra-assay Variability in Liver TissueThe study on intra-assay variability in liver tissue was conducted at0.50, 5.00 and 15.0 ppm concentration levels. Six liver samples wereanalyzed at each of the above three concentrations following the sameprocedures as outlined in section 2.9.1.492.10 Inter-assay Variability Studies2.10.1 Inter-assay Variability in Muscle TissueIn a similar fashion to the intra-assay variability study, the inter-assay variability study was conducted at 0.50 and 5.00 ppm concentrationlevels. Six samples were assayed at each concentration level. However, theassay of the six samples at the same concentration level was not completedon the same day; rather one sample was assayed per day and the six sampleswere assayed over a 6-day period.2.10.2 Inter-assay Variability in Liver TissueThe inter-assay variability in liver tissue was determined in a similarmanner described above (2.10.1). Six samples were assayed at eachconcentration level of 0.50, 5.00 and 15.0 ppm, respectively. The analysiswas completed over a 6-day period.2.11 Confirmation of the Presence of N4-Ac-SDM in Liver Tissue by LC/MSand LC/MS/MSTen grams of liver sample from the second intubation study (fish Nos.3 and 5 on day 11) were extracted as previously described (section 2.6). Afterreconstitution, the samples were injected onto the same HPLC system asdescribed before. The fraction of the effluent corresponding to the syntheticN4-Ac-SDM on the standard chromatogram was collected. The collectedfraction was evaporated to dryness at 40 °C in a SpeedVac concentrator. Theresidues were re-extracted with dichloromethane and evaporated to dryness.The residues were reconstituted with 100 [1,1 of acetonitrile and an aliquot of1 ill was injected onto the Sciex API /// mass system for LC/MS and50LC/MS/MS analyses by flow injection.2.12 Effect of the Addition of Salts During the ExtractionTo investigate the possible role of sodium sulfate anhydrous granularduring the extraction, several other inorganic salts were added to the spikedmuscle samples during the extraction. A total of 18 muscle samples werespiked with SDM/OMP and N4-Ac-SDM at 0.50 ppm. In these samples,inorganic salt was not added to three samples; 2 g of sodium sulfateanhydrous granular was added to three samples; 2 g of sodium sulfateanhydrous powder was added to three samples; 2 g of sodium chloride wasadded to three samples; 2 g of ammonium acetate was added to threesamples; 2 g of zinc sulfate was added to the last three samples. The sampleswere extracted as described before (section 2.5) and the internal standardsolution (200 ptl) was added just before the final evaporation.2.13 Investigation of Possible Hydrolysis of N4-Ac-SDM during ExtractionThree muscle tissue samples were spiked with N4-Ac-SDM at5.00 ppm and extracted as described in section 2.5 followed by HPLCanalysis. A N4-Ac-SDM standard solution at 5.00 ppm was prepared inparallel without undergoing extraction.2.14 StatisticsAll statistical comparisons were performed with Analysis of Variance(ANOVA) at P = 0.05 using software NCSS (by Dr. Jerry L. Hintze, Kaysville,Utah).513. RESULTS AND DISCUSSION3.1 Development of Chromatographic Conditions for the Separation of SDM,OMP, Sulfisoxazole and N4-Ac-SDM3.1.1 Initial Development of HPLC Mobile Phase for the Separation of SDM,OMP and Sulfisoxazole in Muscle TissueAn HPLC mobile phase was initially developed for the separation ofSDM, OMP and the internal standard, sulfisoxazole, in Chinook salmonmuscle tissue, based on previous research in this laboratory (Walisser et al.,1990). The mobile phase consisted of 0.1 M pH 4.0 phosphate buffer-acetonitrile-methanol at 75:12:13 (v/v/v). The chromatograms for the threestandards and a muscle sample spiked with the standards are shown inFigure 9. Sulfisoxazole (Fig. 10) was chosen as an internal standard for theanalysis because it has a similar chemical structure to SDM and elutesbetween OMP and SDM with a retention time of approximately 13 minutes(Fig. 9).52.;^A0^l'o^SO MIN0^ib^20^30 Kul^0^10^15^20^25^30Figure 9Chromatograms of a standard solution (panel A) containing ormetoprim(OMP), sulfadimethoxine (SDM) and the internal standard (I.S.), a musclesample spiked with OMP/SDM/I.S. (panel B), and a blank muscle sample(panel C).Chromatographic conditions:Column: Ultrasphere I.P. 5 i.tm (250 x 4.6 mm I.D.);Mobile phase: acetonitrile-methanol-0.1 M pH 4.0 phosphate buffer (12:13:75,v/v/v);Flow rate : 1.0 ml/min;UV detection wavelength: 280 nm;Attenuation: 3 (throughout the thesis, unless specified).H NOHII IS—N53H3C^C H3Fig. 10 Structure of Sulfisoxazole.3.1.2 Detection and Confirmation of the Presence of N4-Ac-SDM in Liver andMuscle Tissues3.1.2.1 Detection of N4-Ac-SDM in Liver and Muscle TissuesWhen the above HPLC mobile phase was used for the analysis in liversamples from the intubation studies, a peak was found to elute after SDM(Fig. 11). As SDM has been reported to be extensively metabolized to N4-Ac-SDM in channel catfish and rainbow trout (Kleinow et al., 1992; Squibb et al.,1988), this peak was speculated to be N4-Ac-SDM. A similar peak was alsopresent in muscle samples, however, it was a minor peak and was onlypresent in the samples on day 11 from the first study. Thus it was initiallythought to be due to an endogenous substance in muscle tissue.30 miN011 'o^2b54CaH^ -\--/\I^t.../. \ Figure 11Chromatogran of a liver sample from the second intubation study. N4-Ac-SDM is the N -acetylated metabolite of SDM. Refer to Fig.9 for other peakidentities.Chromatographic conditions:Column: Ultrasphere I.P. 5 gm (250 x 4.6 mm I D);Mobile phase: acetonitrile-methanol-0.1 M pH 4.0 phosphate buffer (12:13:75,v/v/v);Flow rate : 1.0 ml/min;UV detection wavelength: 280 nm;Attenuation: 1.55Preliminary extraction studies with liver samples indicated that therewere substantial amounts of co-extractable endogenous substances present.These were particularly evident in the early portion of the chromatogramswhere OMP eluted (Fig. 11). Despite numerous alternations of the extractionprotocol and HPLC mobile phases, it was not possible to resolve OMP fromco-eluting endogenous substances and hence this compound could not bedetermined accurately in liver tissue. Furthermore, based on thepreliminary analytical results in muscle samples from the first intubationstudy, SDM had been shown to exhibit higher residue levels and longerwash-out times than OMP. Thus, SDM represented a more importantxenobiotic for the purpose of drug residue detection.3.1.2.2 Synthesis of N4-Ac-SDMTo confirm the identity of the peak observed in liver samples, N4-Ac-SDM was synthesized from SDM. N4-Ac-SDM was synthesized by additionof acetic anhydride to SDM. The purity of this synthetic material wasdetermined on HPLC by using a modified mobile phase which had acomposition of acetonitrile-methanol-0.1 M pH 2.5 phosphate buffer at11:23.5:75 (v/v/v) and was found to be greater than 99.5% by comparing thepeak area of N4-Ac-SDM with the peak area of SDM.3.1.2.3 Confirmation of the Presence of N4-Ac-SDM in Liver and MuscleTissuesTo compare the retention time of the synthetic N4-Ac-SDM with thenewly resolved peak in the liver sample, a standard solution consisting ofOMP/SDM/N4-Ac-SDM/internal standard and a blank liver sample spikedwith OMP/SDM/N4-Ac-SDM/internal standard were prepared and analyzed56by HPLC. Due to the presence of N4-Ac-SDM, the HPLC mobile phase wasmodified to a composition of acetonitrile-methanol-0.1 M pH 2.5 phosphatebuffer at 11:23.5:75 (v/v/v) to obtain better resolution of SDM and N4-Ac-SDM from the endogenous substances. The results (Fig. 12) show that thesynthetic N4-Ac-SDM had the same retention time as the newly resolvedpeak. Analysis of an admixture of the synthetic N4-Ac-SDM and a liverextract from an intubated salmon did not show any evidence of skewing ofthis peak (Fig. 13).The presence of N4-Ac-SDM was further confirmed by LC/MS andLC/MS/MS analyses. The dominant protonated molecular ion [M+H]E(m/z 353) of N4-Ac-SDM was observed by LC/MS analysis from both thesynthetic standard and the collected fraction from liver sample (Fig. 14).Although some background ions were present in the collected fraction (Fig.14B), they were apparently caused by the co-collected endogenouscompounds. Further structural information was acquired by the use ofLC/MS/MS technique. The precursor ion m/z 353 was fragmented in thesecond quadrupole Q2, and the product ions were analyzed in the thirdquadrupole Q3. The resulted product ion spectra from both samples showedvery similar fragmentation patterns (Fig. 15).The LC/MS and LC/MS/MS analyses could not be performed for muscletissue because the concentration of N4-Ac-SDM in muscle tissue was not highenough to meet the sensitivity requirement of the instrument. However, theabove HPLC, LC/MS and LC/MS/MS results confirmed the presence of N4-Ac-SDM in liver and muscle tissues.57 Alb^2b^3b MIN 0^10^20^310 MINFigure 12A chromatogram of a blank liver extract (panel A) and a representativechromatogram of a liver extract (panel B) from fish No. 4 on day 11 in thesecond intubation study. Refer to Fig. 11 for peak identities.Chromatographic conditions:Column: Ultrasphere I.P. 5 gm (250 x 4.6 mm I.D.);Mobile phase: acetonitrile-methanol-0.1 M pH 2.5 phosphate buffer(11:23.4:75, v/v/v);Flow rate : 1.0 ml/min;UV detection wavelength: 280 nm.58continued from Figure 12Chromatogravi of a blank Chinook salmon liver sample spiked with 0.50 ppmOMP/SDM/M*-Ac-SDM.Figure 13Panel A: chromatogram of a liver extract from an intubated salmon No. 2 onday 14 in the second study;Panel B: chromatogram of an admixture of OMP/SDM/N4-Ac-SDM/I.S. withthe above liver extract. Refer to Fig. 11 for peak identities.Chromatographic conditions:Column: Ultrasphere I.P. 5 gm (250 x 4.6 mmMobile phase: acetonitrile-methanol-0.1 M pH 2.5 phosphate buffer(11:23.4:75, v/v/v);Flow rate : 1.0 ml/min;UV detection wavelength: 280 nm.01 .10'353ISO34125. 475391364B24275•_332360sal250^313219 347279295so6041+H).Figure 14Flow injection LC/MS analysis of N4-Ac-SDM. (A), from N4-Ac-SDMsynthetic standard; (B) from HPLC fraction containing N4-Ac-SDM from liversamples.164164350131 —CH3C0' +211..931981141-11+412502t487218^ 287,111^J 200^250^300^360enh10013475.so12510817392550^TOO^ISO10218J 20013476100A12593.550.25,50198 154134soi218aisco^061ssFigure 15LC/MS/MS product ion spectra of the protonated molecular ion (M+H)± of/V-Ac-SDM obtained by flow injection. (A), from IV-Ac-SDM syntheticstandard; (B) from HPLC fraction containing N4-Ac-SDM from liver samples.The formation of [M+H-H2S02]+ was confirmed by Pleasance et al. (1991).623.1.3 Summary of the Developed HPLC Mobile Phases Used in the CurrentAssay in Chinook Salmon Muscle and Liver TissuesFor the analysis of OMP, SDM and N4-Ac-SDM in muscle tissue, twodifferent mobile phases were necessary in order to obtain separation of theanalytes from the endogenous substances. One mobile phase consisted ofacetonitrile-methanol-0.1 M pH 4.0 phosphate buffer at 12:13:75 (v/v/v) andwas used for the analysis of OMP. This mobile phase allowed the separationof OMP from the co-extracted early eluting endogenous substances on thechromatograms (Fig. 16). However, N4-Ac-SDM could not be separated fromthe late co-eluting endogenous substances.The other mobile phase consisted of acetonitrile-methanol-0.1 M pH2.5 phosphate buffer at 11:23.4:75 (v/v/v) and was used for the analysis ofSDM and N4-Ac-SDM. This mobile phase enabled resolution of SDM andN4-Ac-SDM from the endogenous substances (Fig. 17). Since only SDM andN4-Ac-SDM were to be analyzed in the liver tissue, this mobile phase wasalso used for the assay in liver samples.CI;H63 ^FA Bt Ct;HI6^lb^2'0^3b0^0^1020^310 laxFigure 16Chromatograms of a standard solution (panel A) and a blank muscle sample(panel B). Refer to Fig. 11 for peak identities.Chromatographic conditions:Column: Ultrasphere I.P. 5 iim (250 x 4.6 mm I D.);Mobile phase: acetonitrile-methanol-0.1 M pH 4.0 phosphate buffer (12:13:75,v/v/v);Flow rate : 1.0 ml/min;UV detection wavelength: 280 nm.6 lb^20^30 ram^6^lb^20^30 xxxgcontinued from Figure 16Chromatograms of a spiked muscle sample (panel C) and a muscle sample(fish No. 5 on day 11) from the second intubation study (panel D).64DIvzt\ \ P-■___..--...,65Figure 17Chromatograms of a standard solution (panel A) and a blank muscle sample(panel B). Refer to Fig. 11 for peak identities.Chromatographic conditions:Column: Ultrasphere I.P. 5 gm (250 x 4.6 mm I.D.);Mobile phase: acetonitrile-methanol-0.1 M pH 2.5 phosphate buffer(11:23.4:75, v/v/v);Flow rate : 1.0 ml/min;UV detection wavelength: 280 nm.DZacnio4ivZCx0ratg x0tn9H66^?)41'\,---0^10^20mix^0^102  KINcontinued from Figure 17Chromatograms of a spiked muscle sample (panel C) and a muscle sample(fish No. 5 on day 11) from the second intubation study (panel D).7 -s-N0OCH3H2NOCH3673.2 Extraction Protocol3.2.1 Formation of Ion-Pair Between SDM and TBAHSDM is an amphoteric compound and it is best extracted at pH 6.0 and6.5, while OMP is best extracted at pH 10. To extract both compoundssimultaneously, TBAH was added under basic conditions during theextraction to form a tetrabutylammonium ion-pair with SDM (Weiss et al.,1987) (Scheme 2). Similar ion-pairs were also formed with sulfisoxazole andN4-Ac-SDM. The ion pairs and OMP were then readily extracted intodichloromethane at pH 10.H3COYNIX°N CH3[OH3(CH2)314N+ Do.^ 0(TBAH)^ H2N^S—N IICH3(OH2)34N -Scheme 2 Formation of Tetrabutylammonium Ion -pairwith Sulfadimethoxine (SDM).683.2.2 Addition of Inorganic Salts to Increase Extraction RecoveryIn order to increase the recoveries of the analytes, 2 g of sodium sulfateanhydrous granular was added after the tissue was homogenized (Scheme 1)and was blended with the homogenate by vortex-mixing. Sodium sulfateanhydrous has been reported to dehydrate tissue samples to facilitate betterexposure of the tissue matrix to the organic solvent (Horwitz, 1981).However, in the present study, the quantity of sodium sulfate added was notsufficient to absorb all the water added (1 ml of sodium hydroxide solutionand 1 ml of Na2CO3/NaHCO3 buffer).To further investigate the role of the addition of inorganic salts duringextraction, sodium sulfate anhydrous granular, sodium sulfate anhydrouspowder, sodium chloride, ammonium acetate and zinc sulfate were addedindividually to the homogenates.The data presented in Table 4 summarizes the results of this study.As the results show, sodium sulfate anhydrous granular provided the highestrecoveries for all three compounds among the salts used. Only sodiumsulfate anhydrous powder produced similar results. Although the resultsobtained by the addition of sodium sulfate anhydrous powder were notsignificantly different from the ones obtained by the addition of sodiumsulfate anhydrous granular, there was a trend that the former results wereconsistently lower than the latter results. The reason for this is thought to bedue to adsorption of small quantities of the drug.69Table 4Extraction recoveries of OMP, SDM and N4-Ac-SDMa from muscle tissuewith or without addition of different inorganic saltsSalts AddedRecovery (%)bOMP SDM N4-Ac-SDMNo salt 54±1.7c 68±6.9c 72±0.2cNa2SO4granular66±0.6 83±1.7 79±1.1Na2SO4powder61±1.7 79±1.3c 77±1.7NaC1 59±2.2 55±2.4c 61+1.4cNH4Ac 52+1.3c 79±2.5c 82±3.4ZnSO4 32±3.1c 75±6.9c 65±0.4ca. All three compounds were added at 0.50 ppm;b. Results are mean values of three measurements (n=3);c. Values which are significantly different from the ones obtained by theaddition of Na2SO4 anhydrous granular (P=0.05).70It is unlikely that "salting-out" effects contributed to the improvedrecoveries observed with sodium sulfate since the addition of NaC1 andNH4Ac did not improve the recoveries. Although ZnSO4 has certain proteinprecipitation effects under alkaline conditions (Horwitz, 1981; Somogyi,1945), the recoveries obtained were significantly lower than those withsodium sulfate anhydrous. Hence, protein precipitation was not consideredto be a factor which contributed to the improved recoveries with sodiumsulfate anhydrous either. Considering the fact that there were more than2 ml of aqueous solution present during the extraction, the dehydration effectof sodium sulfate anhydrous was also not considered to be responsible for theimproved recoveries. It is speculated that the improved recoveries by theaddition of sodium sulfate anhydrous granular were, at least in part, becauseof some physical actions of the crystals of sodium sulfate during vortexmixing. It was also found that the samples containing sodium sulfateanhydrous granular were easier to process since the interfacial tissue plugswere more tightly packed after centrifugation. As a result, thedichloromethane extract was much easier to remove. Such factors mighthave also reduced the loss of the drugs during extraction.3.3 Investigation of Possibility of the Hydrolysis of N4-Ac-SDM duringExtractionSince there was the potential for deacetylation of N4-Ac-SDM,especially in acidic conditions (Horwitz, 1981), three muscle samples werespiked with N4-Ac-SDM (5.00 ppm) and extracted following the protocoldescribed earlier. After HPLC analysis, there was no evidence of degradationof N4-Ac-SDM during extraction as determined by HPLC analysis.713.4 Determination of Extraction Recoveries3.4.1 Extraction Recoveries from Muscle TissueThe results of the recovery study in muscle tissue are shown inTable 5. N4-Ac-SDM had the highest recovery among the three. Therecoveries at 0.50 ppm were not significantly different from those at 5.00 ppm(P=0.05).3.4.2 Extraction Recoveries from the Liver TissueThe results of the recovery study in liver tissue are shown in Table 6.The recoveries of N4-Ac-SDM were significantly higher than the recoveries ofSDM at all concentrations tested (P=0.05). The recoveries determined atdifferent concentrations for each individual compound were not significantlydifferent from each other (P=0.05). The recoveries of both compounds werelower in liver tissue than in muscle tissue.72Table 5Extraction recoveries of OMP, SDM and N4-Ac-SDM from muscle tissueConcentration^Recoverya^Recovery^Recoveryof each standard^of OMP of SDM of N4-Ac-SDM(Ppm)^(%) (%) (%)^0.50^65±2.4^76±0.70^82±1.35.00^68±1.5^79±0.70^84±0.60a. Presented as mean ± S.E.M. (n=6).Table 6Extraction recoveries of SDM and N4-Ac-SDM from liver tissueConcentration of^Recoverya^14coveryeach standard of SDM of N4-Ac-SDM(Ppm) (%) (%)0.50^58±2.0^69±5.05.00 64±2.0 74±4.020.0^62±2.0^74±0.50a. Presented as mean ± S.E.M. (n=2).733.5 Linearity of the Assay3.5.1 Linearity of the Assay in Muscle TissueThe calibration curves for OMP, SDM and N 4-Ac-SDM were linearover the concentration range of 0.05 to 10.0 ppm (Table 7). At the same time,the lower detection limit of the assay was determined to be 0.05 ppm for allthree analytes at a signal-to-noise ratio of 5:1.3.5.2 Linearity of the Assay in Liver TissueThe calibration curves for SDM and N 4-Ac-SDM were linear over theconcentration range of 0.20 to 25.0 ppm (Table 8). The lower detection limitof the assay was determined to be approximately 0.20 ppm for both analytesat a signal-to-noise ratio of 5:1.74Table 7Calibration curves for OMP, SDM and N4-Ac-SDM in muscle tissueConcentration(ppm)Area Ratio (analyte/I.S.)OMP SDM N4-Ac-SDM0.05 0.01531 0.01676 0.019160.10 0.01820 0.04258 0.037620.20 0.03196 0.08526 0.072170.50 0.08141 0.1928 0.17822.00 0.3354 0.8045 0.77625.00 0.9005 2.164 1.99710.0 1.644 3.993 3.830OMP:Y = 4.638 x 10-3 + 0.1669X;SDM:Y = 1.201 x 10-2 + 0.4042X;N4-Ac-SDM:Y = 3.403 x 10-3 + 0.3858X;r2 = 0.9989;r2 = 0.9992;F2 = 0.9998.75Table 8Calibration curves of SDM and N4-Ac-SDM in liver tissueConcentration(Ppm)Area ratio(SDM/I.S.)Area ratio(N4-Ac-SD1V1/I.S.)0.20 0.08861 0.093320.50 0.2380 0.18862.00 0.7184 0.69885.00 1.800 1.79115.0 5.743 5.63425.0 8.944 8.678SDM:Y = 4.549 x 10-2 + 0.3619X;^r2 = 0.9993;N4-Ac-SDM:Y = 4.755 x 10-2 + 0.3523X;^r2 = 0.9990.763.6 Determination of Intra-assay Variabilities3.6.1 Intra-assay Variabilities of OMP, SDM and N4-Ac-SDM in MuscleTissueIntra-assay variability, also known as within-day variation, is thevariation of the analytical process observed among the samples which areanalyzed on the same day. The results of intra-assay variability study at0.50 and 5.00 ppm levels in muscle tissue are shown in Table 9. The intra-assay variabilities were lower than 5% for all three compounds.3.6.2 Intra-assay Variabilities of SDM and N4-Ac-SDM in Liver TissueThe results of intra-assay variability study in liver tissue at 0.50, 5.00and 15.0 ppm are presented in Table 10. The intra-assay variabilities arelower than 10% for both compounds at all concentrations.77Table 9Result of intra-assay variability study in muscle tissueConcentration(Ppm)Variabilityof OMP(%)Variabilityof SDM(%)Variabilityof IV-Ac-SDM(%)0.50*5.00*3.64.82.80.891.20.19*• Six samples were analyzed at each concentration.Table 10Results of intra-assay variability study in liver tissueConcentration(ppm)Variabilityof SDM(%)Variabilityof IV-Ac-SDM(%)0.50* 4.8 7.25.00* 4.6 3.515.0* 5.1 7.2* Six samples were analyzed at each concentration.783.7 Determination of Inter-assay Variabilities3.7.1 Inter-assay Variabilities of OMP, SDM and N4-Ac-SDM in MuscleTissueInter-assay variability, also defined as day-to-day variation, is thevariation of the overall analytical process observed among the samples whichare analyzed during a certain period of time. The results of inter-assayvariability study in muscle tissue at 0.50 and 5.00 ppm are shown inTable 11. The inter-assay variabilities were lower than 10% at bothconcentrations.3.7.2 Inter-assay Variabilities of SDM and N4-Ac-SDM in Liver TissueThe results of inter-assay variability study in liver tissue at 0.50, 5.00and 15.0 ppm are shown in Table 12. The inter-assay variabilities werelower than 10% at all three concentrations for both compounds.79Table 11Results of inter-assay variability study in muscle tissueConcentration(Ppm)Variabilityof OMP(%)Variabilityof SDM(%)Variabilityof 1\14-Ac-SDM(%)0.50*5.00*7.96.75.45.83.53.0* Six samples were analyzed at each concentration.Table 12Results of inter-assay variability study in liver tissueConcentration(Ppm)Variabilityof SDM(%)Variabilityof IV-Ac-SDM(%)0.50* 9.7 7.85.00* 7.5 5.615.0* 6.7 5.9* Six samples were analyzed at each concentration.803.8 Intubation StudiesA previous study in this laboratory (Walisser et al., 1990) showed thatthere was a large variation of SDM and OMP concentrations in muscle tissueamong the fish sampled on a given day after a ten-day period ofadministration of medicated feed. The variation could have been due to "sizehierarchy effect" which resulted in larger (in terms of size) fish feeding moreaggressively than others (Peter, 1979).To minimize the feeding variations between individual fish,Romet-30® was administered by means of gastric gavage in the presentstudy. Two such studies were undertaken. In the first study, the fish wereweighed on a daily base and then dosed at a rate of 50 mg/kg/day for 10 days.Since the handling during the weighing process itself is a stress factor for thefish, the second study was undertaken in which the fish were initiallyweighed and tagged for identification and weight. The fish were re-weighedon the fifth day to check if there was any change in weight. If there was, thedosage was then adjusted.3.9 Romet-30® Residues in Muscle and Liver Tissues from the IntubationStudies3.9.1 Romet-30® Residue Data from Muscle TissueThe analytical results of Romet-30® residues in muscle tissues afterthe intubation studies are given in Tables 13 to 14. Figures 18 and 19present the concentration-time curves of Romet-30® residues in muscletissue from the two intubation studies.Although OMP and SDM existed at 1:5 ratio in the drug substancesadministered, this ratio was not seen from OMP and SDM residue levels in81muscle tissue which suggests a difference in the rate of absorption and/orelimination between the two compounds.In both intubation studies, SDM was detectable up to day 20, whileOMP was detectable on day 20 in the second study but was not detectable onday 20 in the first study. Sulfonamides have been found to be rapidlyeliminated initially, but a small persistent residue may remain for a longerperiod (Alderman, 1988). This finding is also true for the present study,particularly for the second study as the residue levels of SDM on days 14, 17and 20 were not significantly different from one another (P=0.05) (Fig. 19).The metabolite of SDM, N4-Ac-SDM, was found to have the lowestresidue level among the three analytes and was not detectable after day 11.This indicates that the distribution of N4-Ac-SDM to the muscle tissue waslimited and/or the elimination from the muscle tissue was rapid. This is inagreement with results reported by other investigators (Kleinow and Lech,1988; Kleinow et al., 1992; Squibb et al., 1988).82Table 13.1OMP/SDM/N4-Ac-SDM concentrations in Chinook salmon muscle tissuecollected on day 11 (the first day after the cessation of the ten-day drugadministration) from the first studyFish No. OMP SDM N4-Ac-SDM(ppm) (143m) (Ppm)1 2.43 3.21 0.302 1.68 2.78 0.133 1.09 2.72 N.D.a4 1.88 2.81 0.445 2.02 6.67 0.146 1.80 2.79 0.12Mean: 1.82 3.50 0.18S.D.: 0.43 1.58 0.14C.V.%: 22.7 44.8 77.1S.E.M.: 0.17 0.64 0.05a) Below the detection limit which was 0.05 ppm.83Table 13.2OMP/SD1VI/N4-Ac-SDM concentrations in Chinook salmon muscle tissuecollected on day 14 (the fourth day after the cessation of the ten-day drugadministration) from the first studyFish No. OMP(Ppm)SDM(Ppm)N4-Ac-SDM(PPIn)1 0.67 0.24 N.D.a2 1.38 13.0 0.753 0.41 0.60 N.D.4 0.38 0.49 N.D.5 0.26 0.30 N.D.6 0.71 0.15 N.D.Mean: 0.49 2.47 N.D.S.D.: 0.40 5.18C.V.%: 40.3 210S.E.M.: 0.16 2.12a) Below the detection limit which was 0.05 ppm.84Table 13.3OMP/SDM/N4-Ac-SDM concentrations in Chinook salmon muscle tissuecollected on day 17 (the seventh day after the cessation of the ten-day drugadministration) from the first studyFish No. OMP(Ppm)SDM(Ppm)N4-Ac-SDM(Ppm)1 0.31 1.52 N.D.a2 0.12 1.47 N.D.3 0.25 1.26 N.D.4 0.12 N.D. N.D.5 0.10 0.61 N.D.6 0.16 0.22 N.D.Mean: 0.18 0.85 N.D.S.D.: 0.08 0.6648.2 78.0S.E.M.: 0.03 0.27a) Below the detection limit which was 0.05 ppm.85Table 13.4OMP/SDM/N4-Ac-SDM concentrations in Chinook salmon muscle tissuecollected on day 20 (the tenth day after the cessation of the ten-day drugadministration) from the first studyFish No. OMP SDM Nil-Ac-SDM(PPIn) (ppm) (Ppm)1 N.D.a 0.40 N.D.2 N.D. 0.11 N.D.3 N.D. N.D. N.D.4 0.08 1.72 N.D.5 N.D. N.D. N.D.6 N.D. 0.10 N.D.7 N.D. 0.21 N.D.Mean: N.D. 0.36 N.D.S.D.: 0.61C.V.%: 170S.E.M.: 0.23a) Below the detection limit which was 0.05 ppm.86Table 14.1OMP/SDM/N4-Ac-SDM concentrations in Chinook salmon muscle tissuecollected on day 11 (the first day after the cessation of the ten-day drugadministration) from the second studyFish No. OMP(PPIn)SDM(ppm)N4-Ac-SDM(Ppm)1 3.60 14.7 1.792 3.22 7.58 0.223 4.00 14.0 1.624 3.85 12.0 0.685 3.88 7.08 0.946 2.28 0.64 N.D.aMean: 3.47 9.33 0.88S.D.: 0.65 5.32 0.72C.V.%: 19.1 56.8 83.2S.E.M.: 0.26 2.17 0.30a) Below the detection limit which was 0.05 ppm.87Table 14.2OMP/SDM/N4-Ac-SDM concentrations in Chinook salmon muscle tissuecollected on day 14 (the fourth day after the cessation of the ten-day drugadministration) from the second studyFish No. OMP(PPIn)SDM(ppm)N4-Ac-SDM(ppm)1 2.06 0.40 N.D.a2 2.46 0.69 0.053 1.50 0.56 N.D.4 0.46 0.16 N.D.5 2.26 2.66 0.056 0.90 0.39 N.D.Mean: 1.61 0.81 N.D.S.D.: 0.80 0.9250.3 114S.E.M.: 0.32 0.38a) Below the detection limit which was 0.05 ppm.88Table 14.3OMP/SDM/N4-Ac-SDM concentrations in Chinook salmon muscle tissuecollected on day 17 (the seventh day after the cessation of the ten-day drugadministration) from the second studyFish No. OMP(PPIn)SDM(Ppm)N4-Ac-SDM(Plmn)1 0.22 0.34 N.D.a2 0.65 0.79 N.D.3 0.47 0.56 0.054 0.12 0.44 N.D.5 0.14 0.08 N.D.6 0.25 0.13 N.D.7 0.12 0.05 N.D.Mean: 0.28 0.34 N.D.S.D.: 0.20 0.28C.V.%: 72.0 80.7S.E.M.: 0.08 0.10a) Below the detection limit which was 0.05 ppm.89Table 14.4OMP/SDM/N4-Ac-SDM concentrations in Chinook salmon muscle tissuecollected on day 20 (the tenth day after the cessation of the ten-day drugadministration) from the second studyFish No. OMP(Ppm)SDM(Prom)N4-Ac-SDM(PPrn)1 0.05 0.70 N.D.a2 N.D. 0.18 N.D.3 0.05 0.10 N.D.4 0.05 0.58 N.D.5 0.06 3.58 0.076 0.13 0.70 N.D.7 N.D. 0.14 N.D.Mean: 0.05 0.85 N.D.S.D.: 0.04 1.23C.V.%: 90.2 144S.E.M.: 0.02 0.46a) Below the detection limit which was 0.05 ppm.T•--„,,.i.-„;?-----■,...„2--0-0 OMP•—• SDMA Acetyl SDMI-ii-Figure 18OMP, SDM and Acetylated SDM Residue Profile inChinook Salmon Muscle Tissue from the First Study,--:.LiV5^10.000-HEa_a^1 .000coa)m(T)a)^0.100rxcr)=Ci0.01010 12^14^16^18^20Days after the Initiation of Drug Administrationvi^10.000-FI• 1.000cncT)0.100L.z0-0 OMP• —• SDMA Acetyl SDM12^14^16^18^200.010 ^10Figure 19OMP, SDM and Acetylated SDM Residue Profile inChinook Salmon Muscle Tissue from the Second StudyDays after the Initiation of Drug Administration92The rates of absorption and elimination of antimicrobials used in.^.^-aquaculture have been reported to be temperature dependent (Alderman,1988). Although the fish in both studies were given the same dose ofRomet-30® at the same intervals, the residue levels of OMP, SDM andN4-Ac-SDM on day 11 in the second study were significantly higher (P=0.05)than those in the first study. Since the first intubation study was carried outin September with water temperatures ranging from 10.8 to 11.6°C and thesecond study was carried out in February with water temperatures rangingfrom 8.0 to 9.0°C, the temperature difference was likely responsible for theinitial residue level differences between the two studies.The above initial residue level differences might also be attributed toother factors. As mentioned earlier, the second group of fish had a highermortality rate (36%) than the first group's (11%). The dead fish usually hadlarge scale skin lesions. Hence, the larger percentage of the diseased fish inthe second group was thought to be responsible for the residue leveldifferences as well. In addition, although Chinook salmon were used for bothstudies, they were purchased from different fish farms. It is possible that thefish at different fish farms were raised under different environments (e.g.,water conditions, disease history, feed differences, stock density, etc.). Allthese factors might contribute to the variations in absorption, distribution orelimination of the drug between these two groups of fish, suggesting that theexperimental data should not be extrapolated to other fish species orexperimental conditions for which there is no experimental evidence.3.9.2 Romet-30® Residue Data from Liver TissueTables 15 and 16 present the analytical results of the residue levels ofSDM and N4-Ac-SDM in liver tissues from the two intubation studies.93Figures 20 and 21 show the concentration-time curves of SDM and N4-Ac-SDM in liver tissues from the two studies.The residue levels of SDM and N4-Ac-SDM are not significantlydifferent from each other (P=0.05) on each individual sampling day. Thisindicates that the liver is a major metabolism site for SDM; which was alsofound in channel catfish (Squibb et al., 1988) and rainbow trout (Kleinow andLech, 1988; Kleinow et al., 1992). It has also been reported that N4-Ac-SDMpredominated in the bile in channel catfish and rainbow trout (Kleinow andLech, 1988; Kleinow et al., 1992; Squibb et al., 1988) which suggested hepaticmetabolism and biliary excretion were important elimination pathways forSDM in fish. In addition, Kleinow et al. (1992) found that SDM, andparticularly N4-Ac-SDM, underwent extensive enterohepatic recirculation.This may explain the longer duration of N4-Ac-SDM residue in liver tissue(detectable up to day 20) than in muscle tissue (not detectable after day 11).94Table 15.1SDM/N4-Ac-SDM concentrations in Chinook salmon liver tissue collected onday 11 (the first day after the cessation of the ten-day drug administration)from the first studyFish No. SDM (ppm) N4-Ac-SDM (ppm)1 3.58 2.722 2.38 3.243 1.92 0.744 6.40 13.15 6.56 12.66 3.62 3.12Mean: 4.08 5.92S.D.: 2.14 6.36C.V.%: 52.4 107S.E.M.: 0.62 1.8395Table 15.2SDM/N4-Ac-SDM concentrations in Chinook salmon liver tissue collected onday 14 (the fourth day after the cessation of the ten-day drug administration)from the first studyFish No. SDM (PPIn) N4-Ac-SDM (PPIn)1 0.71 N.D.a2 16.8 6.463 2.32 1.624 0.78 0.545 N.A.b N.A.6 1.00 0.72Mean: 4.69 2.00S.D.: 6.90 2.65C.V.%: 147 133S.E.M.: 2.30 0.88a) Below the detection limit which was 0.20 ppm;b) Unable to quantify due to interference from endogenous substances.96Table 15.3SDM/N4-Ac-SDM concentrations in Chinook salmon liver tissue collected onday 17 (the seventh day after the cessation of the ten-day drugadministration) from the first studyFish No. SDM (prom) N4-Ac-SDM (ppm)1 1.79 1.782 2.10 1.143 2.56 5.604 N.A.a N.A.5 1.50 1.466 0.46 0.56Mean: 1.68 2.11S.D.: 0.79 2.00C.V.%: 47.1 95.0S.E.M.: 0.25 0.63a) Unable to quantify due to interference from endogenous substances.97Table 15.4SDM/N4-Ac-SDM concentrations in Chinook salmon liver tissue collected onday 20 (the tenth day after the cessation of the ten-day drug administration)from the first studyFish No. SDM (ppm) N4-Ac-SDM (ppm)1 1.82 1.892 N.D.a N.D.3 N.A.b N.A.4 4.84 6.625 2.16 1.806 N.D. N.D.7 0.38 N.D.Mean: 1.65 1.87S.D.: 1.90 2.64C.V.%: 115 141S.E.M.: 0.60 0.84a) Below the detection limit which was 0.20 ppm;b) Unable to quantify due to interference from endogenous substances.98Table 16.1SDM/N4-Ac-SDM concentrations in Chinook salmon liver tissue collected onday 11 (the first day after the cessation of the ten-day drug administration)from the second studyFish No. SDM (ppm) N4-Ac-SDM (ppm)1 7.82 5.362 10.6 3.033 17.4 21.64 17.2 3.625 7.88 7.296 1.85 0.83Mean: 10.5 6.95S.D.: 5.81 7.6255.6 110S.E.M.: 1.68 2.2099Table 16.2SDM/N4-Ac-SDM concentrations in Chinook salmon liver tissue collected onday 14 (the fourth day after the cessation of the ten-day drug administration)from the second studyFish No. SDM (Prom) N4-Ac-SDM (ppm)1 1.42 0.872 2.46 2.563 2.12 3.514 0.66 0.835 3.73 1.976 0.94 1.56Mean: 1.89 1.88S.D.: 1.10 1.04C.V.%: 58.2 55.1S.E.M.: 0.32 0.30100Table 16.3SDM/N4-Ac-SDM concentrations in Chinook salmon liver tissue collected onday 17 (the seventh day after the cessation of the ten-day drugadministration) from the second studyFish No. SDM (ppm) N4-Ac-SDM (ppm)1 1.56 0.602 2.56 3.263 1.94 3.454 1.62 0.935 0.49 0.636 0.94 0.507 0.26 N.D.aMean: 1.34 1.34S.D.: 0.85 1.50C.V.%: 63.9 112S.E.M.: 0.23 0.40a) Below the detection limit which was 0.20 ppm.101Table 16.4SDM/N4-Ac-SDM concentrations in Chinook salmon liver tissue collected onday 20 (the tenth day after the cessation of the ten-day drug administration)from the second studyFish No. SDM (ppm) N4-Ac-SDM (PPIn)1 2.94 3.332 2.05 1.243 2.39 1.964 0.22 N.D.a5 9.07 5.586 0.49 0.247 0.56 0.48Mean: 2.68 1.80S.D.: 2.89 2.03C.V.%: 108 113S.E.M.: 0.77 0.54a) Below the detection limit which was 0.20 ppm.Figure 20SDM and Acetylated SDM Residue Profile in ChinookSalmon Liver Tissue from the First StudyDays after the Initiation of Drug Administration6i1.000 -0-0 SDM•—• Acetyl SDM1 0.000 -i0.100 ^10114^ 18Figure 21SDM and Acetylated SDM Residues Profile in ChinookSalmon Liver Tissue from the Second StudyDays after the Initiation of Drug Administration104In the liver tissue, SDM and N4-Ac-SDM were also eliminated rapidlyat the initial stage but persisted in the liver at lower concentrations in thelatter sampling periods. This was evident again in the second study (Fig. 21).As in the muscle tissue, the residue levels of SDM and N4-Ac-SDM in theliver on day 11 in the second study were significantly higher (P=0.05) thanthose in the first study, a result which might be due to the temperaturedifference at which these two studies were conducted.Both studies showed variations in drug residue levels betweenindividual fish although gastric gavage was used to minimize the variationsin drug administration. This is particularly evident with one fish taken onday 14 in the first study (Table 13.2 and Table 15.2). SDM concentration wasunexpectedly high (13.0 ppm in muscle and 16.8 ppm in liver) and waspossibly due to kidney or liver malfunction (BED could not be diagnosed, butis endemic to salmon species) which might have resulted in higher levels ofSDM due to compromised kidney or liver function. In order to obtainadditional data that would (it was anticipated) provide greater agreementbetween expected tissue concentrations over time, the second intubationstudy was undertaken.If the above assumption that the fish had compromised kidney or liverfunction were correct, then the metabolite, N4-Ac-SDM, might have beenexpected in relatively lower quantity than that would be found in other fish.While the ratio of SDM to N4-Ac-SDM in liver was high (2.6:1) in that fish,ratios of this magnitude were also evident in some of the fish in the secondstudy and the concentrations of SDM in those fish were in a reasonable rangecomparing to other fish on the same sampling day. Although the data do notallow for the conclusion that biological variations might be in the extreme inthis fish, the biological variations were most likely responsible for the105variations of Romet-30 residues between individual fish.Due to the significant data variations, the wash-out time forRomet-30® could not be reliably determined from the present study. Whatthe data do show is that the absorption and the elimination of antimicrobialsin salmon are highly variable processes and depend on a number of factorsbeyond the control of the producer. This supports the need for monitoringprograms for farmed fish which have received antimicrobials and againemphasizes that the experimental data should not be extrapolated to otherfish species or conditions for which there is no experimental evidence.3.9.3 Liver/Muscle Concentration Ratios of SDMThe primary goal of the present research was to identify a "marker"tissue which would have a higher residue level than the muscle tissue, andthat the ratio between this "marker" tissue and muscle tissue would be moreor less consistent.Because only SDM and N4-Ac-SDM could be determined in liver tissueand N4-Ac-SDM was not detectable after day 11 in muscle tissue, it waspossible only to calculate the concentration ratios of SDM as shown in Tables17 and 18, and Figures 22 and 23. The results indicate that a consistentconcentration ratio of SDM did not exist between the liver and the muscletissues and, therefore, the liver is not a suitable "marker" tissue for themuscle tissue in the current study. However, as also shown in Figures 24and 25, the overall concentration of SDM in the liver was higher than that inthe muscle. Hence, the liver tissue could be used as a "monitoring" tissuewhich serves as an indicator to the extent of SDM residue content in themuscle, i.e., as the SDM residue level in the liver declines to 0.1 ppm, theSDM residue level in the muscle has most likely decreased below the above106level.The inconsistent liver/muscle concentration ratios could be attributedto highly variable biological processes, e.g., absorption, distribution orelimination, among the individual fish. Although a "marker" tissue has notbeen identified in the current study, the analytical assay developed for theanalysis of Romet-30 in Chinook salmon muscle tissue possesses satisfyingsensitivity (0.05 ppm) to meet the current residue regulation for Romet-30®in edible fish tissue (0.1 ppm; USFDA, 1984). Due to this analyticalsensitivity, the muscle tissue appears to be a good target tissue for SDM andOMP residue analysis.107Table 17Liver/muscle SDM concentration ratios from the first study1 3.01.33.91.6N.A.a6.723456141 4.6N.D./0.11N.A.2.82.16/N.D.N.D./0.101.923456720a) Data in liversubstances;tissue not available due to interference from endogenousFish No Liver/Muscle Ratio of SDM1234561.10.850.712.30.981.3171234561.21.42.0N.A.2.42.1Day11b) Concentration in muscle or liver was below the detection limit of the HPLCassay.108Table 18Liver/muscle SDM concentration ratios from the second studyDay^Fish No^Liver/Muscle Ratio of SDM111^0.532 1.43 1.24 1.45^1.16 2.9141^3.62 3.63 3.84 4.15^1.46 2.4171^4.62 3.23 3.54 3.75^6.16 7.27 4.8201^4.22 113 244 0.385^2.56 0.707 4.07 —5 —0003 — 0 00 o0 0oo1 8- o01Figure 22Liver/Muscle SDM Concentration Ratios from theFirst Intubation Study10^12^14^16^18^20Days after the Initiation of Drug Administration^11—^ o9 —^7—^ o05—gi §3— o1—^8^oo 8188Figure 23Liver/Muscle SDM Concentration Ratios from the Secondlntubation Study (Fish No.3 on Day 20 not included dueto its high value)10^12^14^16^18^20^22Days after the Initiation of Drug Administrationa.] 10.000 —vi-H1.000 —a)-oCl)0.100 —0.050 ^0-0 LiverA MuscleFigure 24SDM Residue Profile in Chinook Salmon Liver andMuscle Tissues from the First Study10 12^14^16^18^20^22Days after the Initiation of Drug Administration0-0 Muscle•--e LiverFigure 25SDM Residue Profile in Chinook Salmon Liver andMuscle Tissues from the Second Study0.010 ^10 12^14^16^18^20Days after the Initiation of Drug Administration1134. CONCLUSIONAn HPLC assay was developed for the analysis of Romet-30® residuesin Chinook salmon muscle and liver tissues. An ion-pairing reagent TBAHwas utilized to form ion-pairs with SDM, N4-Ac-SDM and the internalstandard, sulfisoxazole, under basic condition which facilitated simultaneousextraction with OMP. Granular sodium sulfate anhydrous was found to beable to increase the extraction recoveries of the analytes. To resolve theanalytes from the endogenous substances, an HPLC mobile phase consistingof acetonitrile-methanol-0.1 M pH 4.0 phosphate buffer at 12:13:75 (v/v/v)was required to quantify OMP, while an HPLC mobile phase consisting ofacetonitrile-methanol-0.1 M pH 2.5 phosphate buffer at 11:23.4:75 (v/v/v) wasrequired to quantify SDM and N4-Ac-SDM. The assay provided goodlinearity, reasonable precision and recoveries. The assay also had asensitivity of 0.05 ppm in muscle which satisfies the current regulation onRomet-30® residues in edible fish tissue.The assay was found to be less sensitive in liver tissue (0.20 ppm) dueto the presence of substantial amounts of co-extractable endogenoussubstances, especially in the early portion of the chromatograms where OMPeluted. Thus, only SDM and N4-Ac-SDM could be quantified in liver tissue.The assay was applied to the analysis of Romet-30 residues inChinook salmon muscle and liver tissues following administration of Romet-30® by gastric gavage. Two intubation studies were performed. In muscletissue, a 5:1 ratio of SDM to OMP which exists in Romet-308 was not seenwhich suggests a difference in the rate of absorption and/or elimination of thetwo compounds. In both studies, SDM was detectable up to day 20, whileOMP was detectable on day 20 in the second study but not detectable on day20 in the first study. N4-Ac-SDM had the lowest residue level and was only114detectable on day 11 in both studies which reveals very limited distribution ofN4-Ac-SDM to muscle tissue and/or rapid elimination from muscle tissue.The initial concentrations of OMP and SDM in the second study weresignificantly higher (P=0.05) than those in the first study, a result which isspeculated to be due to the differences in water temperatures at which thetwo studies were conducted, the disease state of the fish between the twogroups, and/or the different sources from which these fish were obtained.The difference in water temperatures might attributed to the differences inabsorption and/or elimination rate of the drug.In liver tissue, the residue level of N4-Ac-SDM was not significantlydifferent from the residue level of SDM (P=0.05) which indicates that theliver is a major metabolic site for SDM in Chinook salmon. In both liver andmuscle tissues, small amounts of SDM were found to persist for a long time.Both studies showed variations in drug residue levels betweenindividual fish. The biological variations between individual fish werethought to be most likely responsible for these results. A consistentliver/muscle SDM residue ratio was not found in the present study whichcould be a result of the biological variations between individual fish. Hence,the liver tissue is not a suitable "marker" tissue. However, since the overallSDM concentration in liver was higher than that in muscle, it was suggestedthat the liver tissue could be used as a "monitoring" tissue for the SDMconcentration in muscle. In addition, since the developed analytical assay inmuscle tissue had a higher sensitivity (0.05 ppm) than the toleranceregulation for Romet-30® (0.1 ppm), the muscle tissue appears to be a goodtarget tissue for SDM and OMP residue analysis.115REFERENCESAlderman, D.J. 1988. 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