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Monitoring for chloramphenicol (CAP) in aquaculture products is primarily performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which requires expensive equipment and specialized training. Many laboratories prefer to screen samples with facile and high-throughput enzyme-linked immunosorbent assay (ELISA) kits for CAP residues before submitting samples for LC-MS/MS quantification and confirmation. We evaluated the performance of a Ridascreen (R-Biopharm) ELISA kit for CAP in spiked and incurred crab and shrimp muscle at levels bracketing the minimum required performance level for analysis (0.3 ng/g). The Ridascreen ELISA kit incorporates antibody directed against CAP. Incurred CAP levels in crab and shrimp muscle were verified using LC-MS/MS. We found good repeatability (relative standard deviation) of the ELISA in spiked and incurred crab and shrimp muscle samples, with values ranging from 6.8 to 21.7%. Recoveries of CAP from tissues spiked at 0.15 to 0.60 ng/g ranged from 102 to 107%. Minimal cross-reactivity with blank crab and shrimp muscle matrix components was observed. ELISA data were highly correlated with those of LC-MS/MS for CAP in incurred muscle tissue. We believe this study to be the first evaluation of the performance and comparability of a CAP ELISA kit and LC-MS/MS for determination of CAP residues, as well as their elimination, in crab muscle. Our findings support the use of this ELISA kit for screening purposes and, when used in conjunction with validated instrumental methods, for regulatory monitoring of CAP in these species.

A headspace solid-phase microextraction gas chromatography-mass spectrometry (SPME GC-MS) method is described, to screen seafood for volatile organic compounds (VOCs) associated with petrochemical taint. VOCs are extracted from the headspace of heated sample homogenates by adsorption onto a SPME fiber and desorbed for analysis by GC-MS. Targeted compounds are determined semi-quantitatively using representative calibration standards for the various classes (alkanes, alkylbenzenes, indanes/tetralins, and naphthalenes) of VOCs analyzed. Sample preparation is minimal, and the analyses are rapid and automated with a capacity of 50 samples per day. The method was optimized in terms of headspace temperature, sample heating time, extraction time, and desorption time using oyster samples fortified with target compounds. Calibrations for hydrocarbon components were linear in the range of 8.3-167 ng/g; the limit of detection ranged between 0.05 and 0.21 ng/g, and the limit of quantitation between 0.16 and 0.69 ng/g. Good precision (RSD < 10 % at 16.7 ng/g for individual VOCs) and accuracy (recovery range 89-118 % at 25 ng/g) were obtained in oyster, crab, shrimp, and finfish matrices. The trueness of the method was demonstrated by quantifying VOCs at 1-2-ppb levels in oyster fortified with certified reference material NIST SRM 1491a. Following single laboratory validation, the method was employed for the determination of VOCs in seafood exposed to oil contaminated seawater and for the determination of background VOC levels in seafood species from the Gulf of Mexico and local food stores. The method as described can be used to supplement human sensory testing for petrochemical taint in seafood.

Brevetoxins in clams (Mercenaria sp.) exposed to recurring blooms of Karenia brevis in Sarasota Bay, FL, were studied over a three-year period. Brevetoxin profiles in toxic clams were generated by ELISA and LC-MS. Several brevetoxin metabolites, as identified by LC-MS, were major contributors to the composite brevetoxin response of ELISA. These were S-desoxyBTX-B2 (m/z 1018), BTX-B2 (m/z 1034), BTX-B5 (m/z 911), open A-ring BTX-B5 (m/z 929), and BTX-B1 (m/z 1018). Summed values of these metabolites were highly correlated (R(2) = 0.9) with composite B-type brevetoxin measurements by ELISA. S-desoxyBTX-B2, BTX-B2, and BTX-B1 were the most persistent and detectable in shellfish for several months after dissipation of blooms. These metabolites were selected as LC-MS biomarkers of brevetoxin exposure and reflective of composite B-type brevetoxins in hard clam. ELISA and LC-MS values were moderately correlated with toxicity of the shellfish by mouse bioassay. ELISA and LC-MS methods offer rapid screening and confirmatory determination of brevetoxins, respectively, as well as toxicity assessment in clams exposed to K. brevis blooms., (Published by Elsevier Ltd.)

Regulatory monitoring for nitrofuran drug residues in aquaculture products has largely focused on LC-MS/MS. In addition, there is a need for facile and high-throughput screening methods for monitoring programs. We evaluated the performance of Ridascreen (R-Biopharm) ELISA kits for nitrofuran drug residues in fish muscle, with verification by LC-MS/MS. Kits were available for 3-amino-2-oxazolidinone (AOZ) and 3-amino-5-morpholino-methyl-2-oxazolidinone (AMOZ) side-chains of furazolidone and furaltadone, respectively. We found good repeatability in fortified and incurred muscle samples, with RSDs ranging from 1.8% to 7.6%. Recoveries of AOZ and AMOZ from muscle fortified at levels of 0.5-2 ng/g ranged from 98% to 114%. Excellent selectivity was demonstrated. The minimum detection limits (MDLs) for AOZ and AMOZ in muscle were 0.05 and 0.2 ng/g, respectively. ELISA data were highly correlated with those of LC-MS/MS. Results of this study support the use of these kits as screening assays for nitrofuran residues in fish muscle., (Published by Elsevier Ltd.)

Invasive Indo-Pacific lionfish (Pterois volitans) have rapidly expanded in the Western Atlantic over the past decade and have had a significant negative impact on reef fish biodiversity, habitat, and community structure, with lionfish out-competing native predators for resources. In an effort to reduce this population explosion, lionfish have been promoted for human consumption in the greater Caribbean region. This study examined whether the geographical expansion of the lionfish into a known ciguatera-endemic region can pose a human health threat for ciguatera fish poisoning (CFP). More than 180 lionfish were collected from waters surrounding the US Virgin Islands throughout 2010 and 2011. Ciguatoxin testing included an in vitro neuroblastoma cytotoxicity assay for composite toxicity assessment of sodium-channel toxins combined with confirmatory liquid chromatography tandem mass spectrometry. A 12% prevalence rate of ciguatoxic lionfish exceeding the FDA guidance level of 0.1 µg/kg C-CTX-1 equivalents was identified in fish from the U.S. Virgin Islands, highlighting a potential consumption risk in this region. This study presents the first evidence that the invasive lionfish, pose a direct human health risk for CFP and highlights the need for awareness and research on this food safety hazard in known endemic areas.

Brevetoxin metabolites were identified and characterized in the hard clam (Mercenaria sp.) after natural exposure to Karenia brevis blooms by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Principal brevetoxins BTX-1 and BTX-2 produced by K. brevis were not detectable in clams. Metabolites of these brevetoxins found in clams included products of oxidation, reduction, hydrolysis and amino acid/fatty acid conjugation. Of highest abundance were cysteine and taurine conjugates. We also found glutathione, glycine-cysteine, and γ-glutamyl-cysteine conjugates. A series of fatty acid derivatives of cysteine-brevetoxin conjugates were also identified., (Published by Elsevier Ltd.)

Ciguatera fish poisoning (CFP) is endemic in certain tropical and subtropical regions of the world. CFP had not been described on the West Africa Coast until a 2004 outbreak in the Canary Islands. In 2008-2009, two additional outbreaks of ciguatera occurred. Individuals afflicted had consumed lesser amberjack (Seriola rivoliana) captured from nearby waters. Caribbean ciguatoxin-1 (C-CTX-1) was confirmed in fish samples by LC-MS/MS. Ciguatoxic fish in this region may pose a new health risk for the seafood consumer., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Ciguatera fish poisoning is a seafood-borne illness caused by consumption of fish that have accumulated lipid-soluble ciguatoxins. In the United States, ciguatera is responsible for the highest reported incidence of food-borne illness outbreaks attributed to finfish, and it is reported to hold this distinction globally. Ciguatoxins traverse the marine food web from primary producers, Gambierdiscus spp., to commonly consumed fish in tropical and subtropical regions of the world. Ciguatoxins comprise 12 known congeners among Caribbean and tropical Atlantic fish and 29 reported congeners among Pacific fish. Expanding trade in fisheries from ciguatera-endemic regions contributes to wider distribution and increasing frequency of disease among seafood consumers in non-endemic regions. Ciguatoxins produce a complex array of gastrointestinal, neurological and cardiological symptoms. Treatment options are very limited and supportive in nature. Information derived from the study of ciguatera outbreaks has improved clinical recognition, confirmation, and timely treatment. Such studies are equally important for the differentiation of ciguatoxin profiles in fish from one region to the next, the determination of toxicity thresholds in humans, and the formulation of safety limits. Analytical information from case and outbreak investigations was used to derive Pacific and Caribbean ciguatoxin threshold contamination rates for adverse effects in seafood consumers. To these threshold estimates 10-fold safety factors were applied to address individual human risk factors; uncertainty in the amount of fish consumed; and analytical accuracy. The studies may serve as the basis for industry and consumer advisory levels of 0.10ppb C-CTX-1 equivalent toxicity in fish from the tropical Atlantic, Gulf of Mexico, Caribbean, and 0.01ppb P-CTX-1 equivalent toxicity in fish from Pacific regions., (Published by Elsevier Ltd.)

Herein, we describe advancements in monitoring of brevetoxins in molluscan shellfish, with respect to exposure management and control of neurotoxic shellfish poisoning (NSP). Current knowledge of the fate of brevetoxins in molluscan shellfish, and the toxic potency of brevetoxin metabolites, is presented. We review rapid assays for measuring composite brevetoxins, and methodology for measuring constituent brevetoxins, in contaminated shellfish. The applicability of in vitro methods for estimating brevetoxin burden and composite toxicity in shellfish is assessed. Specific and measurable biomarkers of brevetoxin exposure and toxicity in shellfish, and of human intoxication, are described. Their utility in regulatory monitoring of toxic shellfish and in clinical diagnosis of NSP is evaluated., (Published by Elsevier Ltd.)

The use of nitrofuran drugs in food-producing animals continues to attract international concern as a food safety issue. Methods for monitoring nitrofuran residues have been directed to the intact side chain of tissue-bound metabolites. Semicarbazide, the side chain of nitrofurazone (NFZ), can enter food products from non-NFZ sources, suggesting the need for an alternative biomarker for confirmatory purposes. We characterized a cyano derivative as a major metabolite of NFZ in channel catfish (Ictalurus punctatus). The depletion of cyano metabolite was examined in the muscle of channel catfish after oral dosing (10 mg of NFZ/kg of body weight). Parent NFZ was rapidly eliminated in muscle, with a half-life of 6.3 h. The cyano metabolite was detected for up to 2 weeks, with an elimination half-life of 81 h. The cyano metabolite represents an alternative biomarker for confirming the use of NFZ in channel catfish.

The depletion of the nitrofuran drugs furazolidone, nitrofurazone, furaltadone, and nitrofurantoin and their tissue-bound metabolites [3-amino-2-oxazolidinone (AOZ), semicarbazide (SC), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AH), respectively] were examined in the muscle of channel catfish following oral dosing (1 mg/kg body weight). Parent drugs were measurable in muscle within 2 h. Peak levels were found at 4 h for furazolidone (30.4 ng/g) and at 12 h for nitrofurazone, furaltadone, and nitrofurantoin (104, 35.2, and 9.8 ng/g respectively). Parent drugs were rapidly eliminated from muscle, and tissue concentrations fell below the limit of detection (1 ng/g) at 96 h. Peak levels of tissue-bound AMOZ and AOZ (46.8 and 33.7 ng/g respectively) were measured at 12 h, and of SC and AH (31.1 and 9.1 ng/g, respectively) at 24 h. Tissue-bound metabolites were measurable for up to 56 days postdose. These results support the use of tissue-bound metabolites as target analytes for monitoring nitrofuran drugs in channel catfish.

Urine specimens from patients diagnosed with neurotoxic shellfish poisoning (NSP) were examined for biomarkers of brevetoxin intoxication. Brevetoxins were concentrated from urine by using solid-phase extraction (SPE), and analyzed by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine extracts were fractionated by LC, and fractions analyzed for brevetoxins by ELISA. In subsequent LC-MS/MS analyses, several brevetoxin metabolites of B-type backbone were identified, with elution profiles consistent with those of ELISA. The more abundant brevetoxin metabolites in urine were characterized structurally by LC-MS/MS. With the exception of BTX-3, brevetoxin metabolites in urine differed from those found in shellfish and in shellfish meal remnants. Proposed structures of these major urinary metabolites are methylsulfoxy BTX-3, 27-epoxy BTX-3, and reduced BTX-B5. BTX-3 was found in all specimens examined. BTX-3 concentrations in urine, as determined by LC-MS/MS, correlated well with composite toxin measurements by ELISA (r(2)=0.96). BTX-3 is a useful biomarker for confirmation of clinical diagnosis of NSP.

Brevetoxin uptake and elimination were examined in Eastern oyster (Crassostrea virginica) exposed to recurring blooms of the marine alga Karenia brevis in Sarasota Bay, FL, over a three-year period. Brevetoxins were monitored by in vitro assays (ELISA, cytotoxicity assay, and receptor binding assay) and LC-MS, with in vivo toxicity of shellfish extracts assessed by the traditional mouse bioassay. Measurements by all methods reflected well the progression and magnitude of the blooms. Highest levels recorded by mouse bioassay at bloom peak were 157 MU/100g. Oysters were toxic by mouse bioassay at levels >or=20 MU/100g for up to two weeks after bloom dissipation, whereas brevetoxins were measurable by in vitro assays and LC-MS for several months afterwards. For the structure-based methods, summed values for the principal brevetoxin metabolites of PbTx-2 (cysteine and cysteine sulfoxide conjugates), as determined by LC-MS, were highly correlated (r(2)=0.90) with composite toxin measurements by ELISA. ELISA and LC-MS values also correlated well (r(2)=0.74 and 0.73, respectively) with those of mouse bioassay. Pharmacology-based cytotoxicity and receptor binding assays did not correlate as well (r(2)=0.65), and were weakly correlated with mouse bioassay (r(2)=0.48 and 0.50, respectively). ELISA and LC-MS methods offer rapid screening and confirmation, respectively, of brevetoxin contamination in the oyster, and are excellent alternatives to mouse bioassay for assessing oyster toxicity following K. brevis blooms.

A new competitive electrochemiluminescence-based immunoassay for the type-2 brevetoxins in oyster extracts was developed. The assay was verified by spiking known amounts of PbTx-3 into 80% methanol extracts of Gulf Coast oysters. We also provide preliminary data demonstrating that 100% acetone extracts, aqueous homogenates, and the clinical matrixes urine and serum can also be analyzed without significant matrix interferences. The assay offers the advantages of speed ( 2 h analysis time); simplicity (only 2 additions, one incubation period, and no wash steps before analysis); low limit of quantitation (conservatively, 50 pg/mL = 1 ng/g tissue equivalents); and a stable, nonradioactive label. Due to the variety of brevetoxin metabolites present and the lack of certified reference standards for liquid chromatography-mass spectrometry confirmation, a true validation of brevetoxins in shellfish extracts is not possible at this time. However, our assay correlated well with another brevetoxin immunoassay currently in use in the United States. We believe this assay could be useful as a regulatory screening tool and could support pharmacokinetic studies in animals and clinical evaluation of neurotoxic shellfish poisoning victims.

Several novel brevetoxin derivatives were isolated and identified in Karenia brevis cultures and natural blooms by using solid phase extraction (SPE) and LC/MS(MS) techniques. These analogs were more polar compared with previously described brevetoxins, and were poorly extractable by conventional non-polar solvent (chloroform) partitioning. Brevetoxin analogs were structurally confirmed as hydrolyzed (open A-ring) forms of brevetoxins PbTx-1, PbTx-7, PbTx-2, and PbTx-3, and of oxidized PbTx-1 and PbTx-2. Some of these open A-ring derivatives were in greater abundance than their non-hydrolyzed counterparts. All were in much greater abundance in bloom water filtrate compared with cell-rich fractions. Open A-ring compounds were cytotoxic in mouse neuroblastoma (N2a) cell assay. In the K. brevis bloom-exposed Eastern oyster, brevetoxin metabolites with opened A rings were identified (e.g., open-ring cysteine-PbTx conjugates), contributing to their overall toxin burden.

The metabolism and elimination of brevetoxins were examined in the Eastern oyster (Crassostrea virginica) following controlled exposures to Karenia brevis cultures in the laboratory. After a 2-day exposure period ( approximately 62 million cells/oyster), elimination of brevetoxins and their metabolites was monitored by using liquid chromatography/mass spectrometry (LC/MS). Composite toxin in oyster extracts was measured by in vitro assay (i.e. cytotoxicity, receptor binding, and ELISA). Of the parent algal toxins, PbTx-1 and PbTx-2 were not detectable by LC/MS in K. brevis-exposed oysters. PbTx-3 and PbTx-9, which are accumulated directly from K. brevis and through metabolic reduction of PbTx-2 in the oyster, were at levels initially (after exposure) of 0.74 and 0.49 microg equiv./g, respectively, and were eliminated largely within 2 weeks after dosing. PbTx-7 and PbTx-10, the reduced forms of PbTx-1, were non-detectable. Conjugative brevetoxin metabolites identified previously in field-exposed oysters were confirmed in the laboratory-exposed oysters. Cysteine conjugates of PbTx-1 and PbTx-2, and their sulfoxides, were in the highest abundance, as apparent in LC/MS ion traces, and were detectable for up to 6 months after dosing. Composite toxin measurements by in vitro assay also reflected persistence (up to 6 months) of brevetoxin residues in the oyster. Levels of cysteine conjugates, as determined by LC/MS, were well correlated with those of composite toxin, as measured by ELISA, throughout depuration. Composite toxin levels by cytotoxicity assay were well correlated with those by receptor binding assay. Cysteine-PbTx conjugates are useful LC/MS determinants of brevetoxin exposure and potential markers for composite toxin in the Eastern oyster.

Brevetoxin (PbTx) metabolism was examined in the Eastern oyster (Crassostrea virginica) following exposure to a Karenia brevis red tide, by using LC/MS(/MS) and cytotoxicity assay. Metabolites observed in field-exposed oysters were confirmed in oysters exposed to K. brevis cultures in the laboratory. Previously, we identified a cysteine conjugate and its sulfoxide (MH(+): m/z 1018 and 1034) as metabolites of the brevetoxin congener PbTx-2. In the present study, we found a cysteine conjugate and its sulfoxide with A-type brevetoxin backbone structure (MH(+): m/z 990 and 1006), as probable derivatives of PbTx-1. We also found glycine-cysteine-PbTx (m/z 1047 and 1075), gamma-glutamyl-cysteine-PbTx (m/z 1147), and glutathione-PbTx (m/z 1176 and 1204) conjugates with A- and B-type backbone structures. Amino acid-PbTx conjugates react with fatty acids through amide linkage to form a series of fatty acid-amino acid-PbTx conjugates. These fatty acid conjugates are major contributors to the composite cytototoxic responses obtained in extracts of brevetoxin-contaminated oysters. Other brevetoxin derivatives found in oysters are consistent with hydrolytic ring-opening and oxidation/reduction reactions.

A thirteen-laboratory comparative study tested the performance of four methods as alternatives to mouse bioassay for the determination of brevetoxins in shellfish. The methods were N2a neuroblastoma cell assay, two variations of the sodium channel receptor binding assay, competitive ELISA, and LC/MS. Three to five laboratories independently performed each method using centrally prepared spiked and naturally incurred test samples. Competitive ELISA and receptor binding (96-well format) compared most favorably with mouse bioassay. Between-laboratory relative standard deviations (RSDR) ranged from 10 to 20% for ELISA and 14 to 31% for receptor binding. Within-laboratory (RSDr) ranged from 6 to 15% for ELISA, and 5 to 31% for receptor binding. Cell assay was extremely sensitive but data variation rendered it unsuitable for statistical treatment. LC/MS performed as well as ELISA on spiked test samples but was inordinately affected by lack of toxin-metabolite standards, uniform instrumental parameters, or both, on incurred test samples. The ELISA and receptor binding assay are good alternatives to mouse bioassay for the determination of brevetoxins in shellfish.

A functional pharmacologically-based assay for the brevetoxin group of sodium channel activators was developed using synaptoneurosomes isolated from the brains of CD1 mice. The assay can detect the depolarizing effect of brevetoxin congeners PbTx-2 and PbTx-3 as enhancements of the veratridine-dependent increase in fluorescence of the voltage-sensitive fluorescent probe rhodamine 6G. The assay is relatively rapid and can detect brevetoxin activity in the nanomolar range. The synaptoneurosomal assay has been used to analyse mussel tissue extracts spiked with PbTx-2, and composite toxicity, expressed as PbTx-3 equivalents in extracts of oysters naturally exposed to brevetoxins. In this latter context, the synaptoneurosomal technique was shown to compare favorably with the cytotoxicity assay, the receptor binding assay and HPLC/MS. Our results support the concept that this membrane potential assay detects brevetoxins based on their interaction with sodium channels.

Previously, we analyzed Eastern oysters (Crassostrea virginica) naturally exposed to a Karenia brevis red tide and found that brevetoxins (PbTx) are rapidly accumulated and metabolized. Several metabolites were isolated and later identified, including a cysteine-PbTx conjugate (MH(+): m/z 1018) and its sulfoxide product (m/z 1034). In the present study, we confirm and extend those findings by examining PbTx metabolism and elimination in oysters exposed to pure toxins (PbTx-2 and -3) under controlled conditions. Waterborne PbTx-3 was rapidly accumulated, but not metabolized, in the oyster and was largely eliminated within 2 weeks after exposure. In contrast, PbTx-2 was accumulated and rapidly metabolized. Metabolites of PbTx-2 included the reduction product PbTx-3 (m/z 897), and the cysteine conjugates (m/z 1018 and 1034) isolated previously from the field samples. Levels of the metabolite PbTx-3 in PbTx-2-exposed oysters were highest immediately after exposure and declined at a rate similar to parent PbTx-3 in PbTx-3-exposed oysters. Cysteine-PbTx persisted for 8 weeks after exposure. The same metabolites were confirmed in oysters exposed to laboratory cultures of K. brevis. PbTx metabolites contribute to neurotoxic shellfish poisoning (NSP) and should be included in analytical protocols for monitoring shellfish toxicity after a K. brevis red tide event., (Copright 2002 Elsevier Science Ltd.)

Rapid methods are described for determination of flumequine (FLU) residues in muscle and plasma of farm-raised channel catfish (Ictalurus punctatus). FLU residues were extracted from tissues with an acidified methanol solution, and extracts were cleaned up on C18 solid-phase extraction cartridges. FLU concentrations were determined by liquid chromatography (LC) using a C18 analytical column and fluorescence detection (excitation, 325 nm; emission, 360 nm). Mean recoveries of FLU from fortified muscle were 87-94% at 5 levels ranging from 10 to 160 ppb (5 replicates per level). FLU recoveries from fortified plasma were 92-97% at 5 levels ranging from 20 to 320 ppb. Limits of detection (signal-to-noise ratio, 3:1) for the method as described were 3 and 6 ppb for muscle and plasma, respectively. Relative standard deviations (RSDs) for recoveries were < or = 12%. Live catfish were dosed with 14C-labeled or unlabeled FLU to generate incurred residues. Recoveries of 14C residues throughout extraction and cleanup were 90 and 94% for muscle and plasma, respectively. RSDs for incurred FLU at 2 levels in muscle and plasma ranged from 2 to 6%. The identity of FLU in incurred tissues was confirmed by LC/mass spectrometry.

A peer-verified, liquid chromatographic (LC) method for simultaneous determination of residues of flumequine (FLU), nalidixic acid (NAL), oxolinic acid (OXO), and piromidic acid (PIR) in catfish muscle is presented. Sample workup involves homogenizing tissue with acetone, defatting with hexane, and extracting quinolones into chloroform. Sample is purified further by partitioning into base and then subsequently back-extracting into chloroform after acidifying the aqueous phase. After solvent is evaporated, the residue is diluted with mobile phase, and analytes are introduced into an LC system where separations are made with a 5 microns, reversed-phase polymer column and an isocratic, buffered acetonitrile-tetrahydrofuran mobile phase. Determinations are made by UV detection at 280 nm for PIR and by fluorescence detection (excitation at 325 excitation and emission at 365 nm) for the other 3 analytes. Each quinolone was used to fortify catfish muscle at 5, 10, 20, 40, and 80 ng/g. The following recoveries and relative standard deviation (RSD) values represent an average of the 5 levels for each analyte: FLU, 79.7% (RSD = 5.7%); OXO, 80.8% (RSD = 6.3%); PIR, 75.0% (RSD = 5.9%); and NAL, 87.1% (RSD = 10%). Assay of 5 levels (base incurred catfish, plus 4 dilutions with control catfish) of catfish muscle incurred with the 4 quinolones gave the following averages: FLU: base, 198 ng/g (RSD = 2.3%); dilutions, 98.0 ng/g (RSD = 4.2%), 61.6 ng/g (RSD = 4.4%), 21.6 ng/g (RSD = 2.8%), 9.24 ng/g (RSD = 8.7%); OXO, base, 257 ng/g (RSD = 6.9%); dilutions, 146 ng/g (RSD = 5.5%), 95.0 ng/g (RSD = 4.1%), 30.7 ng/g (RSD = 3.8%), 13.7 ng/g (RSD = 4.6%); PIR, base, 22.1 ng/g (RSD = 4.2%); dilutions, 13.7% ng/g (RSD = 6.7%), 6.49 ng/g (RSD = 15%), 2.65 ng/g (RSD = 15%); and NAL, base, 75.1 ng/g (RSD = 3.8%); dilutions, 42.3 ng/g (RSD = 5.1%), 24.1 ng/g (RSD = 6.3%), 8.59 ng/g (RSD = 4.8%). A second multiresidue analysis of the 4 quinolones was performed by an outside analyst. Average recoveries from catfish fortified at 5, 10, 20, and 40 ng/g were FLU, 75.9% (RSD = 4.0%); OXO, 84.0% (RSD = 5.5%); NAL, 85.6% (RSD = 8.9%); and PIR, 66.2% (RSD = 8.7%).

1. The disposition of proflavine (PRO) and acriflavine (ACR) were examined in channel catfish after intravascular (i.v.) dosing (1 mg/kg) or waterborne exposure (10 mg/l for 4 h). 2. After i.v. dosing, plasma concentration-time profiles of parent PRO and ACR were best described by two- and three-compartment pharmacokinetic models respectively. Terminal elimination half-lives of PRO and ACR in plasma were 8.7 and 11.4 h respectively. 3. In animals dosed with 14C-PRO or 14C-ACR, total drug equivalent concentrations were highest in the excretory organs and lowest in muscle, fat and plasma. In PRO-dosed animals, residues in the liver and trunk kidney were composed primarily of glucuronosyl and acetyl conjugates of PRO; residues in muscle were composed mostly (> 95%) of the parent drug. In ACR-dosed animals, the parent compound comprised > 90% of the total residues in all tissues examined. 4. PRO and ACR were poorly absorbed in catfish during waterborne exposure. At the end of a 4-h exposure, parent PRO and ACR concentrations in muscle were 0.064 and 0.020 microgram/g respectively. Levels in muscle declined below the limit of determination (0.005 microgram/g) within 1-2 weeks.

Malachite green (MG), a traditional agent used in aquaculture, is structurally related to other carcinogenic triphenylmethane dyes. Although MG is not approved for use in aquaculture, its low cost and high efficacy make illicit use likely. We developed sensitive and specific methods for determination of MG and its principal metabolite, leucoMG (LMG), in edible fish tissues using isotope dilution liquid chromatography atmosphere pressure chemical ionization mass spectrometry. MG and LMG concentrations were measured in filets from catfish treated with MG under putative use conditions (ca. 250 and 1000 ppb, respectively) and from commercial trout samples (0-3 and 0-96 ppb, respectively). Concentrations of LMG in edible fish tissues always exceeded those of MG. A rapid cone voltage switching acquisition procedure was used to simultaneously produce molecular ions for quantification and diagnostic fragment ions for confirmation of MG and metabolites. The accurate and precise agreement between diagnostic ion intensity ratios produced by LMG in authentic standards and incurred fish samples was used to unambiguously confirm the presence of LMG in edible fish tissue. This suggested the validity of using LMG as a marker residue for regulatory determination of MG misuse. Additional metabolites derived from oxidative metabolism of MG or LMG (demethylation and N-oxygenation) were identified in catfish and trout filets, including a primary arylamine which is structurally related to known carcinogens. The ability to simultaneously quantify residues of MG and LMG, and to confirm the chemical structure of a marker residue by using LC/MS, suggests that this procedure may be useful in monitoring the food supply for the unauthorized use of MG in aquaculture.

A liquid chromatographic (LC) method was developed for determination of acriflavine (ACR) and proflavine (PRO) residues in channel catfish muscle. Residues were extracted with acidified methanol solution, and extracts were cleaned up with C18 solid-phase extraction columns. Residue concentrations were determined on an LC cyano column, with spectrophotometric detection at 454 nm. Catfish muscle was individually fortified with ACR (purified from commercial product) and PRO at concentrations of 5, 10, 20, 40, and 80 ppb (5 replicates per level). Mean recoveries from fortified muscle at each level ranged from 86 to 95%, with relative standard deviations (RSDs) of 2.5 to 5.7%. The method was applied to incurred residues of ACR and PRO in muscle after waterborne exposure of channel catfish to commercial acriflavine (10 ppm total dye for 4 h). RSDs for incurred residues of ACR and PRO were in the same range as those for fortified muscle. Low residue concentrations (< 1% of exposure water concentration) suggested poor absorption of ACR and PRO in catfish.

Methylene blue (MB) is a thiazine dye that, although not regulated for use with edible fish, may sometimes be used as a chemotherapeutic agent in the aquaculture industry. A liquid chromatographic (LC) method was developed for the determination of MB in fish muscle. MB was extracted from catfish tissue with an acetonitrile-acetate buffer solution containing hydroxylamine and toluenesulfonic acid, partitioned into methylene chloride, and cleaned up on alumina and carboxylic acid solid-phase extraction cartridges. MB concentrations were determined by LC with visible detection at 660-665 nm. Recoveries of MB from catfish fortified at 10-50 ng/g were 75-90% (RSDs, 5-12%). Leucomethylene blue also can be recovered from catfish tissue as the MB colored form at low levels with similar recoveries. Analysis of catfish exposed to MB in a bath treatment at 5 ppm MB for 1 h recovered 10-20 ppb of the drug in the muscle tissue. The low tissue concentration suggests poor absorption of this drug compared with other antifungal dyes that tend to concentrate and remain in fish tissue at high levels.

A previously published liquid chromatographic (LC) method for determining residues of flumequine (FLU) and nalidixic (NAL), oxolinic (OXO), and piromidic (PIR) acids in catfish tissue was applied to salmon and shrimp muscle. Identities of all 4 residues in salmon and shrimp were confirmed by gas chromatography/mass spectrometry (GC/MS). The tissue is homogenized with acetone, the acetone extract is defatted with hexane, and the quinolones are extracted into chloroform. The extract is further purified by first partitioning into base and then back-extracting from a solution acidified to pH 6.0. Analytes are determined by LC with simultaneous UV and fluorescence detection. Muscle tissue was fortified with each quinolone at 5, 10, 20, 40, and 80 ng/g. Average recoveries and relative standard deviations (RSDs) for salmon, which represent an average of the 5 levels for each analyte, ranged from 75.9 to 90.8% and from 2.25 to 6.40%, respectively. Average recoveries and RSDs for shrimp ranged from 81.3 to 91.2% and from 7.34 to 10.7%, respectively. Identities of OXO, FLU, NAL, and PIR were confirmed in extracts of salmon and shrimp tissue fortified at 10 ng/g by determination of decarboxylated quinolones by GC/MS. Four diagnostic ions were monitored for OXO, FLU, and PIR, and 5 ions were monitored for NAL. All ion relative abundances were within 10% of those calculated for standard decarboxylated quinolones. Optimum conditions for decarboxylation and GC/MS confirmation are given.

A liquid chromatographic (LC) method was adapted and optimized for the determination of malachite green and its metabolites in fish plasma and muscle. Residues in plasma were extracted with acetonitrile, the extract was evaporated to dryness, and residues were resolubilized for LC analysis. Residues in muscle were extracted with an acetonitrile-acetate buffer mixture, reextracted with acetonitrile, and partitioned into methylene chloride with final cleanup on alumni and propylsulfonic acid solid-phase extraction columns. Residue levels were determined by using an LC cyano column with a PbO2 postcolumn and visible detection (618 nm). Overall mean recoveries of parent malachite green (MG-C) and its major metabolite, leuco-malachite green (MG-L), from plasma were 93 and 87%,respectively, at fortification levels ranging from 25 to 250 ppb. Overall mean recoveries of MG-C and MG-L from muscle were 85 and 95%, respectively, at fortification levels ranging from 5 to 100 ppb. Relative standard deviations (RSDs) of recoveries at all fortification levels ranged from 3.9 to 7.0% for plasma and from 2.1 to 5.2% for muscle. The method was applied to incurred residues in tissues sampled from catfish after waterborne exposure to [14C]MG-C. Mean recoveries of total radioactive residues in plasma and muscle throughout the extraction and cleanup process were 88 and 87%, respectively, and corresponding RSDs for MG-C and MG-L were in the same range as those for fortified tissues. MG-L was confirmed as the major metabolite of MG-C in catfish.

Chemical noise limits mass spectrometric detection of chloramphenicol (CAP) with electron capture ionization at low resolution, and makes CAP identification at concentrations of 5 parts per billion (ppb) difficult. Increasing the resolution from 1000 to 3500, however, was sufficient to separate the analyte signals from the noise signals, and resulted in a 100 times higher analytical sensitivity. The introduction of sweep gas in the ion source decreased the scattering of the quantitative results on average by a factor of 7, and thereby improved the precision of the analyses to an acceptable level (CV < 10%). Under such conditions, CAP residues of 1.5 and 2.1 ppb in shrimp as determined by electron capture gas chromatography/mass spectrometry can readily be identified by monitoring four diagnostic ions.

1. The pharmacokinetics, tissue distribution and excretion of the nitrofuran drug furazolidone have been examined in the channel catfish. [14C]Furazolidone was administered by intravascular or oral routes in a single dosage of 1 mg/kg body weight. 2. A two-compartment pharmacokinetic model best described parent furazolidone concentrations in the plasma after intravascular dosing. Elimination of parent compound was extremely rapid, with a terminal half-life of 0.27h and total body clearance of 1901 ml/h/kg. 3. After oral dosing, furazolidone concentrations in the plasma were highest at 1 h and were below the limit of determination (< 20 ng/ml) at 5 h. The oral bioavailability of parent furazolidone administered in solution was 58%, compared with 28% in a feed mixture. 4. Concentrations of furazolidone and its metabolites were highest in the excretory tissues and lowest in the muscle after oral dosing. Parent furazolidone comprised 10% of the total 14C in the muscle at 8 h and was not detectable (< 1 ng/g) at 24 h; total 14C concentrations declined from 274 to 59 ng furazolidone equiv./g between 8 and 168 h. Non-extractable (bound) residues comprised 18% of total 14C in muscle at 8 h and 33% at 168h. 5. Renal excretion was the primary route of elimination of 14C residues and accounted for nearly 55% of the oral dose.

A liquid chromatographic (LC) method was developed for the quantitation of furazolidone residues in shrimp muscle. The shrimp homogenate (1.0 g) is extracted with acetonitrile, and the extract is taken to dryness. The residue is dissolved in acetonitrile, and the solution is passed through alumina and C18 cleanup columns. The eluate is taken to dryness and reconstituted in a suitable solvent for reversed-phase (C18) LC with UV detection at 365 nm. Recoveries of furazolidone from shrimp homogenates spiked from 5 to 80 ng/g ranged from 74.3 to 79.7%, and relative standard deviations (RSDs) were 5.0-8.9%. RSDs for incurred furazolidone quantitated at 5.9 and 9.2 ng/g were 6.6 and 7.6%, respectively.

An interlaboratory study of a gas chromatographic method for determining chloramphenicol (CAP) residues in shrimp was conducted. An internal standard (Istd), the meta isomer of CAP, was added to the shrimp, and the treated shrimp were homogenized with ethyl acetate. The ethyl acetate extract was defatted with hexane, and the CAP was partitioned into ethyl acetate from an aqueous salt solution. The ethyl acetate was evaporated, and the dried residue was treated with Sylon, a trimethylsilyl derivatizing agent, to yield the trimethylsilyl derivative of CAP. A portion of the solution containing the derivative was injected into a gas chromatograph equipped with an electron capture detector. Levels of fortified and incurred CAP were calculated from the peak area ratio of standard CAP to Istd. Recoveries of CAP from tissue directly fortified at 5 ppb were 102% (within-laboratory relative standard deviation [RSDr] = 5.6%), 104% (RSDr = 5.5%), and 108% (RSDr = 6.3%) from Laboratories 1, 2, and 3, respectively. Incurred-CAP residues at 5 and 10 ppb levels were also determined, with the following results: Laboratory 1: composite A, 4.56 ppb (RSDr = 14.0%); composite B, 8.38 ppb (RSDr = 11.6%); Laboratory 2: composite A, 4.17 ppb (RSDr = 12.5%); composite B, 8.90 ppb (RSDr = 5.60%); Laboratory 3: composite A, 4.66 ppb (RSDr = 14.9%); composite B, 11.0 ppb (RSDr = 11.8%).

A liquid chromatographic (LC) method was developed for the simultaneous determination of nitrofurazone (NFZ), nitrofurantoin (NFT), and furazolidone (FZD) in catfish muscle tissue. The drugs were extracted from the tissue with acetonitrile, and the lipids were removed from the extract with hexane. The acetonitrile extract was evaporated by rotary evaporation, and the resultant drug residues were dissolved with LC mobile phase. The mixture was sonicated, centrifuged, and filtered. The drugs were determined by using LC with a C18 reversed-phase (ODS Hypersil) column, a mobile phase of acetonitrile-1% aqueous acetic acid (25 + 75), and a photodiode array ultraviolet detector at 375 nm. NFZ, NFT, and FZD were each determined in catfish tissue at 5 fortification levels (80, 40, 20, 10, and 5 ng drug/g tissue). Average recoveries of each of the 3 drugs at each level ranged from 70.7 to 101.5%, and relative standard deviations ranged from 2.2 to 18.6%. The limit of detection of each drug was approximately 1 ng drug/g tissue, and the limit of quantitation was 5 ng drug/g tissue. In the second part of the study, the method was used to determine nitrofuran residues incurred in catfish tissue. Live channel catfish were intravascularly doses (10 mg/kg body wt) with NFZ to generate drug-incurred fish muscle tissue. Incurred NFZ levels exceeded 400 ng drug/g tissue at 2 h after dosing but decreased rapidly to approximately 1 ng drug/g tissue by 8 h after dosing, as determined by this method.

The stress response to dorsal aorta cannulation and serial blood sampling was examined in the channel catfish Ictalurus punctatus. Channel catfish cannulated and repetitively sampled once a day for 7 days did not exhibit a change in response to surgery or to the 24h sample regime as measured by plasma cortisol, glucose, and chloride. In fish that were either serially bled (0, 1, 3, 6, 9, 12, 24, and 48h) immediately after surgery or allowed to recover 6 days before being serially bled, overall plasma cortisol levels were higher than those of fish bled every 24h. Catfish serially bled immediately after surgery had significantly higher plasma glucose levels compared with catfish allowed to recover from surgery 6 days before serial sampling. Although channel catfish recover from cannulation surgery in 24h, a longer recovery period may be needed prior to serial sampling if the samples are taken more frequently than every 24h.

1. Disposition of phenol red was examined in channel catfish (Ictalurus punctatus) after oral or intravascular (i.v.) dosing at 10 mg/kg body weight. 2. Phenol red was not detectable in plasma, urine, or bile after oral administration. 3. After i.v. dosing, plasma concentrations of phenol red were best described by a two-compartment pharmacokinetic model with distribution and elimination half-lives of 2.3 and 21 min, respectively. The apparent volume of distribution at steady state (Vss) was 225 ml/kg and total body clearance (Clb) was 658 ml/h per kg. Plasma protein binding was 19%. 4. Biliary excretion was the primary route of elimination of phenol red; in 24 h, 55% of the i.v. dose was excreted in bile compared with 24% in urine. No metabolites were detected in these fluids. 5. The use of anaesthesia during dosing had no effect on the quantitative excretion of phenol red by renal or biliary routes.

The pharmacokinetics, tissue distribution, and metabolism of 1-naphthol were examined in the channel catfish (Ictalurus punctatus). Catfish were administered [1-14C]1-naphthol intravascularly at 1, 5, or 25 mg/kg or orally at 1 mg/kg. Plasma data for 1-naphthol were fitted by a three-compartment pharmacokinetic model. There were dose-related changes in the area under the plasma concentration vs. time curve, apparent volume of distribution, and total body clearance after intravascular dosing. After oral dosing, peak plasma concentrations of 1-naphthol occurred at 1 hr; parent compound made up less than 15% of the total radioactivity, and the bioavailability was 32%. Plasma protein binding was 92% and was independent of concentration. 1-Naphthol and metabolites were rapidly eliminated from the tissues after oral dosing; less than 1% of the administered dose remained at 24 hr. Renal excretion was the primary route of elimination of total 14C. Approximately 60% of the oral dose was excreted in the urine within 48 hr. Parent 1-naphthol made up 1% of the urinary 14C. Major metabolites in the urine were sulfate and glucuronide conjugates, which composed 65 and 28% of the total 14C, respectively. Biliary excretion accounted for 7% of the oral dose. The glucuronide conjugate and an unidentified polar metabolite made up the majority of the biliary 14C. The high capacity of channel catfish for conjugative metabolism of 1-naphthol was demonstrated. The dose dependency of pharmacokinetic values could not be explained by saturable metabolism or plasma protein binding.

The pharmacokinetics, tissue distribution and excretion of 14C-labelled aflatoxin B1 (AFB1) were examined after oral administration (250 micrograms/kg body weight) in channel catfish (Ictalurus punctatus). Plasma concentrations of parent AFB1 were best described by a one-compartment pharmacokinetic model, in which peak plasma concentration (503 ppb) occurred at 4.1 hr after dosing. The absorption and elimination half-lives were 1.5 and 3.7 hr, respectively. AFB1 was highly bound (95%) to plasma proteins. Concentrations of 14C (in AFB1 equivalents) measured in the tissues were highest at 4 hr, ranging from 596 ppb in the plasma to 40 ppb in the muscle. AFB1 residues were rapidly depleted; at 24 hr the concentrations in the plasma and muscle were 32 and less than 5 ppb, respectively. Concentrations in the bile exceeded 2000 ppb (at 24 hr), whereas the highest concentration in the urine was 51 ppb (4-6-hr collection interval). Renal and biliary excretion accounted for less than 5% of the administered dose, indicating incomplete absorption. Pharmacokinetic modelling and tissue data demonstrate a very low potential for the accumulation of AFB1 and its metabolites in the edible flesh of channel catfish through the consumption of AFB1-contaminated feed.

The Bacillus stearothermophilus disk assay for penicillin in milk (AOAC official method) was adapted for the determination of ampicillin in fish muscle. The method was evaluated in 2 species of cultured fish: channel catfish and striped bass. Recoveries of ampicillin ranged from 99 to 104% when muscle specimens from both species were spiked at concentrations of 0.025-1.00 micrograms/g. The lower limit of determination (LOD) was 0.025 micrograms/g. The assay was applied to monitor the elimination of ampicillin from the muscle of striped bass after intravascular administration (dosage of 10 mg/kg body weight). The mean concentrations in the muscle declined from 1.160 micrograms/g at 2 h to 0.063 micrograms/g at 18 h. The half-life of ampicillin in the muscle was 3.6 h. Ampicillin concentrations were below LOD at 24 h. No inhibitory activity was observed in the muscle of control fish.

The pharmacokinetics and metabolism of a model organophosphorothioate, chlorpyrifos, was investigated in channel catfish (Ictalurus punctatus). Chlorpyrifos exhibited multicompartmental disposition after oral and intravascular administration, indicating slower distribution to peripheral storage tissues. The oral bioavailability of chlorpyrifos was 41%, substantially higher than in mammals. Muscle contained less than 5% of the oral dose, with an elimination half-life of about 3.3 days. Residues in the whole fish were greater than 95% chlorpyrifos, while excreta (bile and urine) primarily contained metabolites. The dephosphorylated metabolite, trichloropyridinol (TCP), was the major metabolite in the blood, while the glucuronide conjugate of TCP was the major metabolite in urine and bile. The toxic metabolite, chlorpyrifos oxon, was not detected in any samples of blood, tissues, or excreta. The metabolism of chlorpyrifos in catfish appeared similar to other species of fish and mammals. Extensive metabolism resulted in a low potential for chlorpyrifos to accumulate in catfish from dietary exposure.

The pharmacokinetics and metabolism of the model compound benzoic acid were examined after intravascular (iv) and po administration at 10 mg/kg in the channel catfish (Ictalurus punctatus). A two-compartment pharmacokinetic model best described the plasma disposition of parent benzoic acid after iv dosing. The following pharmacokinetic values were estimated: elimination half-life, 5.9 hr; total body clearance, 61 ml/hr/kg; and volume of distribution (steady-state), 369 ml/kg. Plasma protein binding of [14C]benzoic acid was 18%. Benzoic acid was rapidly and extensively absorbed after po administration; absorption half-life was 0.8 hr and bioavailability was 95%. Renal excretion was the primary route of elimination of benzoic acid and metabolites. More than 80% of the iv-administered 14C was recovered in the urine in 24 hr. Unchanged benzoic acid comprised more than 90% of the urinary radiolabel. The major urinary metabolite was benzoyltaurine, which comprised 6-7% of the excreted 14C. Channel catfish were qualitatively similar to other teleost fishes in the formation of the taurine conjugate of benzoic acid. In contrast, the primary mammalian metabolite is the glycine conjugate, hippuric acid.

The bioavailability of lysine in Maillard browned protein was investigated by plasma lysine response in rainbow trout (Salmo gairdneri). The concentrations of free lysine in the plasma were measured after feeding control and browned protein diets supplemented with graded levels of lysine. Bioavailability of lysine was estimated based on the amounts of supplemental lysine in the diets that resulted in rapid increases in plasma lysine. An approximately 80% loss in bioavailable lysine content was determined by this method in a fish protein isolate subjected to the Maillard browning reaction under mild conditions (40 d incubation at 37 degrees C). The nutritional damage to lysine determined by plasma lysine response was similar to that estimated in vitro by enzymatic hydrolysis and fluorodinitrobenzene reagent, but was underestimated by acid hydrolysis and trinitrobenzene sulfonic acid reagent. Rainbow trout are similar to other animals in their inability to utilize the deoxyketosyl (Amadori) compound of lysine formed in early Maillard reaction, and in their plasma response to dietary levels of essential amino acids.

An overview is presented on the absorption of drugs and other xenobiotics in mammals and fish. Species differences in gastrointestinal tract physiology as well as other factors that influence bioavailability of these compounds are considered. Results of recent studies on the bioavailability of drugs in aquatic animal species are discussed.

The toxicity of the plant flavonol, quercetin, to rainbow trout (Salmo gairdneri) was investigated. Quercetin, which had been confirmed to be mutagenic in the Ames Salmonella/mammalian microsome test, was fed to trout at levels of 1 or 5% in the diet for 8 months. Survival, growth and feed conversion efficiency, selected haematological parameters and the relative weights of heart, liver and spleen were unaffected by the ingestion of quercetin, and there were no histopathological changes in any of the tissues examined.

1. The tissue disposition and bioavailability of 3H-tetracycline were examined in the channel catfish (Ictalurus punctatus) after intravascular (4 mg/kg body weight) and per os (4 and 80 mg/kg) dosing. 2. The pharmacokinetics of the intravascularly administered drug using a two-compartment open model revealed plasma half-lives of 1.3 and 16.5 h for the distribution and elimination phases, respectively. 3. The drug was highly concentrated in both hepatobiliary and urinary compartments. The concentrations in the edible flesh were the lowest of any compartment examined. 4. The apparent bioavailability of the per os-administered drug was less than 5% and was influenced by the presence of feed material and the dosage. 5. The drug was 72% bound to plasma protein at both 4 and 24 h after intravascular administration.

The effect of the Maillard browning reaction in the diet of rainbow trout (Salmo gairdneri) on growth and amino acid availability was investigated. Chemical and enzymatic hydrolysis methods were applied for the detection of the losses of amino acids in a model protein browning system. Arginine and lysine exhibited the greatest losses in the mixture of fish protein isolate and glucose stored for 40 d at 37 degrees C. The apparent digestibility and absorption of individual amino acids, particularly lysine, was lower in trout fed browned protein than in those fed the control protein. Plasma lysine levels were significantly depressed, while the plasma levels of glucose and most other amino acids were elevated in relation to the loss in nutritive value of dietary protein after browning. The early Maillard reaction derivative of lysine, epsilon-deoxy-fructosyl-lysine, was recovered from browned protein (by using the in vitro enzymatic hydrolysis procedure) and from the plasma of trout fed browned protein. Analysis of plasma free amino acids provided an indication of lysine bioavailability and identified lysine as the first-limiting amino acid in the diets containing browned protein.