Determination of quinolones and fluoroquinolones in fish tissue and seafood by high-performance liquid chromatography with electrospray ionisation tandem mass spectrometric detection

A reversed-phase high-performance liquid chromatographic method with tandem mass-spectrometric detection was developed and validated for the simultaneous analysis of eight quinolones and fluoroquinolones (oxolinic acid, flumequine, piromidic acid, enrofloxacin, ciprofloxacin, danofloxacin, sarafloxacin and orbifloxacin) in trout tissue, prawns and abalone. The analytes were extracted from homogenised tissue using acetonitrile and the extracts subjected to an automated two-stage solid-phase extraction process involving polymeric reversed-phase and anion-exchange cartridges. Good recoveries were obtained for all analytes and the limit of quantification was 5 microg/kg (10 microg/kg for ciprofloxacin). The limit of detection was 1-3 microg/kg, depending on the analyte and matrix. Confirmation of the identity of a residue was achieved by further tandem mass-spectrometric analysis. A procedure for estimating the uncertainty associated with the measurement is presented.     

Determination of nifursol metabolites in poultry muscle and liver tissue. Development and validation of a confirmatory method

A method is described for the identification and quantitative determination of 3,5-dinitrosalicylic acid hydrazide (DSH), the marker residue of nifursol metabolites in poultry (turkey, broiler) muscle and liver tissue. The method is based on the acid-catalysed hydrolysis of tissue-bound metabolites to free DSH and in situ derivatisation with 2-nitrobenzaldehyde to the corresponding nitrophenyl derivative NPDSH. A structural analogue of DSH, 4-hydroxy-3,5-dinitrobenzoic acid hydrazide (HBH) was synthesised to serve as an internal standard. The analytes were isolated from the matrix by liquid-liquid extraction with ethyl acetate. Determination was performed by LC-MS/MS with negative electrospray ionisation. The [M - H](+) ions of NPDSH and NPHBH at m/z 374 were fragmented by collision induced dissociation (CID) producing transition ions at m/z 182, 183 and 226. The transition ions at m/z 182 and 226 were selected for monitoring of NPDSH while the transition ion at m/z 183 was selected for NPHBH. The method has been validated according to the EU criteria of Commission Decision 2002/657/EC at 0.5, 1.0 and 1.5 microg kg(-1) in muscle and liver tissue. A decision limit (CC(alpha)) was obtained of 0.04 and 0.025 microg kg(-1) in muscle and liver, respectively. Similarly a detection capability (CC(beta)) was obtained of 0.10 and 0.05 microg kg(-1) in muscle and liver, respectively. The introduction of HBH as an internal standard did not lead to a significant improvement of the quantitative performance of the method. In fact for liver better performance characteristics were obtained when the IS was not taken into account. Nevertheless, as a qualitative marker for recovery, HBH could still be very useful in the analysis of unknown samples.     

Validation of a high-performance liquid chromatography method for the determination of oxytetracycline,tetracycline, chlortetracycline and doxycycline in bovine milk and muscle

High-performance liquid chromatography with diode-array detection (HPLC-DAD) was optimised and validated for the determination of tetracyclines in bovine milk and tissues. Milk and tissue samples were extracted and purified using a solid-phase extraction HLB Oasis cartridge and analysed using HPLC-DAD set at 365 nm. The analyses were carried out using the mobile phase of 0.01 M oxalic acid-acetonitrile-methanol (60:25:15, v/v/v) on a C8 column (250 x 4.6 mm I.D., 5 microm). Recoveries of tetracyclines from spiked samples at the three concentrations (0.5, 1 and 1.5) of the maximum residues limits (corresponding to 100 microg/kg for milk and the muscle) were higher than 81.1% in milk and 83.2% in muscle. The method was successfully validated for bovine milk and muscle in compliance with requirements set by draft SANCO/ 1805/ 2000 European Decision. The decision limit (CCalpha) was in the range 113.2-127.2 microg/kg and 107.7-129.9 micro/kg for all compounds in milk and muscle, respectively. The detection capability (CCbeta) was in the range 117.2-131.3 microg/kg and 114.9-133.1 microg/kg for all compounds in milk and muscle, respectively.     

Development and validation of an HPLC confirmatory method for residue analysis of ten quinolones in tissues of various food-producing animals, according to the European Union Decision 2002/657/EC

The aim of this work was to develop an HPLC method for the simultaneous determination of ten quinolones: enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid, and flumequine, in various tissues of food-producing animals. Separation was achieved on a PerfectSil Target column (250 mm x 4 mm, ODS-3, 5 microm), by MZ-Analysentechnik (Germany), at room temperature. The mobile phase consisted of 0.1% TFA-CH(3)OH-CH(3)CN and was delivered by a gradient program of 35 min. The detection and quantitation was performed on a photodiode array detector at 275 and 255 nm. Caffeine (7.5 ng/microL) was used as the internal standard (IS). Analytes were isolated from tissue samples by 0.1% methanolic TFA solution. SPE, using LiChrolut RP-18 cartridges, was applied for further purification. The extraction protocol was optimized and the final recoveries varied between 92.0 and 107.4%. The method was fully validated according to Commission Decision 2002/657/EC. Limits of quantitation for the examined quinolones extracted from each tissue were much lower than the respective Maximum Residue Levels, ranging between 30 and 50 microg/kg for bovine tissue, between 30 and 55 microg/kg for ovine tissue, and between 40 and 50 microg/kg for porcine tissue.     

Determination of residues of enrofloxacin and its metabolite ciprofloxacin in chicken muscle by capillary electrophoresis using laser-induced fluorescence detection

A method for the residue analysis of the veterinary antimicrobial agent enrofloxacin and its active desethyl metabolite ciprofloxacin in chicken muscle tissue has been developed and validated. The detection of the analytes was performed by laser-induced fluorescence (LIF) detection using a HeCd laser (lambda(ex) = 325 nm) providing an enhancement in sensitivity and selectivity compared to conventional UV detection. The assay has been validated with satisfying results. The limits of quantification for enrofloxacin and ciprofloxacin were 5 microg/kg and 20 microg/kg, respectively, with a fivefold preconcentration yielded by a sample clean-up with a simple liquid-liquid extraction procedure. Calibration graphs were linear from 5 to 1000 microg/kg for enrofloxacin and from 20 to 1000 microg/kg for ciprofloxacin. The assay allows the detection of contaminated muscle samples at the required maximum residue limit of the European Union, which is 100 microg/kg for the sum of enrofloxacin and ciprofloxacin.     

Rapid method for the determination of non-steroidal anti-inflammatory drugs in animal tissue by liquid chromatography-mass spectrometry with ion-trap detector

A rapid and new liquid chromatography-mass spectrometry with ion-trap detection method for the determination of meloxicam (MLX), flunixin meglumine (FLU), carprofen (CPF), and tolfenamic acid (TOLF) in animal tissue is described. MRLs between 10 and 500 microg kg(-1) in muscle and between 65 and 1000 microg kg(-1) in liver, from different animal species have been established in the EU for these compounds. After chemical hydrolysis, an organic extraction from homogenised tissue was performed. Final extract was injected in a liquid chromatograph with an ion-trap mass spectrometer with electrospray interface. Four identification points (one precursor and two product ions) and a minimum of one ion ratio was monitored for each compound. For quantitative purposes flunixin-D3 (FLU-D3) was used as internal standard. The method was validated using fortified blank muscle and liver from different animal species according to the 2002/657/EC European decision criteria. The decision limits (CCalpha) and detection capabilities (CCbeta) were determined and their values were at concentrations near the MRL for each substance.     

Determination of quinolones in chicken tissues by liquid chromatography with ultraviolet absorbance detection

This paper presents an analytical method for the determination of quinolones in chicken tissues. The procedure involves pre-treatment by solid-phase extraction (SPE) and subsequent liquid chromatography (LC) with UV absorbance detection. Different SPE disposable cartridges and extractants of the tissue samples were tested, and various columns were systematically tested. The mobile phase was composed of acetonitrile and citric buffer at pH 4.5, with an initial composition of acetonitrile-water (12:88, v/v) and using linear gradient elution. Recoveries were 66-91% in the concentration range 30-300 microg kg(-1). The detector response was linear in this range. The limits of detection were 16-30 microg kg(-1). These values were lower than the maximum residue limits established by the European Union.     

Multiresidue method for simultaneous determination of ten quinolone antibacterial residues in multimatrix/multispecies animal tissues by liquid chromatography with fluorescence detection: single laboratory validation study

Quinolone antibacterials are veterinary drugs authorized for use in food animal production. The analysis of residual amounts of drugs in food from animal origin is important for quality control of products for consumers. For this purpose, Maximum Residue Limits (MRLs) have been set up by a European Union Council Regulation on Veterinary Drug Residues (No. 90/2377/EEC and subsequent), and 8 quinolones received MRLs at concentration levels depending on both the matrix and the animal species of interest. A method was developed for screening and confirming 10 quinolone residues (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, oxolinic acid, sarafloxacin) in a wide variety of matrixes of different animal species. It involves extraction of the residues from the biological tissues/fluids by acidic aqueous solution, centrifugation and filtration prior to injection on a C18 narrow-bore column, and detection through a 3-step-mode fluorescence detector. The method was validated during a 2-week study for a set of 8 species-matrixes (i.e., bovine raw milk, bovine muscle, porcine muscle, porcine kidney, porcine liver, fish flesh and skin, poultry muscle, whole egg). Residues were quantified down to 15 microg/kg with limits of detection and quantitation ranging from 4 to 11 and 13 to 36 microg/kg, respectively, which are sufficient compared to the wide range of MRLs set for these substances (from 30 microg/kg for danofloxacin in milk to 1900 microg/kg for difloxacin in poultry liver). The limit of performance of the method in terms of CCalpha and CCbeta, the critical concentrations stated in the Decision No. 2002/657/EC and the ISO Standard No. 11843, has been calculated for the authorized (MRL) substances but only estimated in the case of the nonauthorized (non-MRL) substances.     

Detection of streptomycin and dihydrostreptomycin residues in milk,honey and meat samples using an optical biosensor

Immuno-biosensor inhibition assays for the detection of streptomycin and dihydrostreptomycin residues in whole cows' milk, honey, pig kidney and pig muscle are reported. The antibody showed high cross-reactivity with dihydrostreptomycin in various foodstuffs (buffer 103%, milk 96%, honey 84%, kidney extract 129% and muscle extract 98%). There was no significant cross-reaction with other aminoglycosides or commonly used antibiotics. A streptomycin derivative was used to prepare a stable, reusable sensor chip surface. The assay allowed the direct analysis of bovine whole milk (fat content approximately 3.5%). Honey samples required dilution with buffer, while kidney and muscle samples from pigs were homogenized in an aqueous extraction buffer and clarified by centrifugation. The limit of detection for each assay was determined from known streptomycin-free samples (n = 20; mean - (3 x standard deviation)) and the results were as follows: milk 30 microg kg(-1), honey 15 microg kg(-1), kidney 50 microg kg(-1) and muscle 70 microg kg(-1). Repeatability (or relative standard deviation) between runs were calculated (n = 3) at the respective Community maximum residue limits (MRL) and 0.5 x MRL with the exception of honey since no European MRL exists at present. Results were determined as 4.3% (200 microg kg(-1)) and 2.8% (100 microg kg(-1)) in milk, 13.3% (40 microg kg(-1)) and 9.5% (20 microg kg(-1)) in honey, 7.1% (1000 microg kg(-1)) and 7.6% (500 microg kg(-1)) in kidney and 7.1% (500 microg kg(-1)) and 11% (250 microg kg(-1)) in muscle.     

Development of an HPLC multi-residue method for the determination of ten quinolones in bovine liver and porcine kidney according to the European Union Decision 2002/657/EC

A sensitive multi-residue analytical method was developed for the determination of ten quinolones: enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid, and flumequine in bovine liver and porcine kidney. A simple liquid extraction step followed by a solid phase extraction clean up procedure was applied for the extraction of quinolones from liver and kidney tissues. Recoveries of the extraction varied between 82 and 88% for bovine liver and 92 and 95% for porcine kidney. Separation was performed on an ODS-3 PerfectSil Target (250 x 4 mm) 5 microm analytical column at 25 degrees C. The mobile phase consisted of a mixture of TFA 0.1%-CH(3)CN-CH(3)OH, delivered at a flow rate of 1.2 mL/min according to a gradient program. Elution of quinolones and the internal standard (caffeine, 7.5 ng/microL) was complete within 27 min. Photodiode array detection was used for monitoring the eluants at 275 and 255 nm. The method was fully validated according to the European Union Decision 2002/657/EC, determining linearity, selectivity, decision limit, detection capability, accuracy, and precision. The LODs of the specific method of quinolone determination in bovine liver varied between 3 and 7 microg/kg and in porcine kidney between 3 and 4 microg/kg.     

Determination of multiresidue quinolones regulated by the European Union in pig liver samples. High-resolution time-of-flight mass spectrometry versus tandem mass spectrometry detection

The use of liquid chromatography coupled to orthogonal acceleration time-of-flight mass spectrometry (LC-ToF-MS) provides an attractive alternative to liquid chromatography coupled to quadrupole (LC-MS) or triple quadrupole mass spectrometry (LC-MS/MS) in multiresidue analysis. ToF-MS provides accurate mass information and a significantly higher mass resolution than quadrupole analyzers. In this work, the influential parameters in time-of-flight detection using an electrospray ionization (ESI) source were studied using a central composite design to obtain the main effects and their two-factor interactions. The method developed uses LC-ESI-ToF-MS to determine and characterize quinolones regulated by the EU in pig liver samples below the maximum residue limits (MRLs). Linearity, decision limit, detection capability, detection and quantification limits, precision and recoveries were determined and adequate results were obtained, with quantification limits between 1.5 and 6 microg kg(-1) and recoveries higher than 60% for all quinolones. Limits of detection are lower than 2 microg kg(-1). Results obtained using LC-ESI-ToF-MS were compared with those obtained using LC coupled to a quadrupole and to triple quadrupole mass spectrometer. The work described in this paper illustrates the suitability and excellent confirmatory potential of LC-ToF-MS for multiresidue analysis in food samples.     

Determination of tetracycline antibiotics in salmon muscle by liquid chromatography using post-column derivatization with fluorescence detection

A simple and accurate cleanup procedure using polymeric sorbent was developed for the determination of oxytetracycline (OTC) and tetracycline (TC) residues in salmon muscle. It was applied to the analysis of 20 salmon samples during a month period. The OTC and TC residues were extracted with ethylenediaminetetracetic acid (EDTA)-McIlvaine buffer acidified at pH 4.0 and cleaned up by solid-phase extraction with a polymeric sorbent. The advantages of the polymeric sorbent over the silica-based sorbent in the cleanup of salmon muscle samples are described. A liquid chromatographic method with post-column derivatization and fluorescence detection is proposed because of its sensitivity and specificity. The average recoveries of OTC and TC from muscle salmon tissue fortified at 50, 100, and 200 microg/kg levels, ranged from 83.9 to 93.4% with a coefficient of variation between 4.09 and 5.80%. The limit of quantitation for OTC and TC in salmon muscle was 50 microg/kg.     

Development of a high-performance liquid chromatography method for the simultaneous quantification of quinoxaline-2-carboxylic acid and methyl-3-quinoxaline-2-carboxylic acid in animal tissues

A method of high-performance liquid chromatography with UV detection has been established for simultaneous quantitative determination of quinoxaline-2-carboxylic acid (QCA) and methyl-3-quinoxaline-2-carboxylic acid (MQCA), the marker residues for carbadox (CBX) and olaquindox (OLA), respectively, in the muscles and livers of porcine and chicken and in the muscle of fish. Tissue samples were subject to acid hydrolysis followed by liquid-liquid extraction and Oasis MAX solid-phase extraction clean-up. The method was validated according to the EU Commission Decision 2002/657/EC. The decision limits (CCalpha) were 0.7-2.6microg/kg and the detection capabilities (CCbeta) were 1.3-5.6microg/kg for QCA and MQCA in tissues. The recoveries of QCA and MQCA, spiked at levels of 2-100microg/kg, were from 70 to 110%; the relative standard deviation values were <20%. This simple, fast and economic method could be applied to the monitoring for the possible misuse of CBX and OLA in animal edible tissue samples.     

Development of HPLC methods for the determination of cyadox and its main metabolites in goat tissues.

Rapid and sensitive high-performance liquid-chromatographic methods were developed for the determination of cyadox, an antimicrobial growth-promoter, and its main metabolites (1,4-bisdesoxycyadox, quinoxaline-2-carboxylic acid) in goat muscle, liver, kidney and fat. Cyadox (CYX) and 1,4-bisdesoxycyadox (BDCYX) in fat were extracted with acetonitrile, and in other tissues with ethyl acetate. Quinoxaline-2-carboxylic acid (QCA) was isolated from tissue hydrolysates by solvent extraction, cleaned up with ion-exchange chromatography, followed by a final liquid-liquid extraction step. UV detections of CYX, BDCYX and QCA were performed at 305, 280 and 320 nm, respectively. The average recoveries of CYX, BDCYX and QCA in spiked tissues at levels of 25, 50, 100 microg/kg were 65 - 92%. The inter-day relative standard deviation for three compounds in different tissues was 5 - 16%. The quantitation limit was 25 microg/kg, and the detection limit was 15 microg/kg for three compounds in various tissues. Incurred goat tissues were analyzed to demonstrate the validity of the described methodologies. The present methods were highly selective and could be used in the metabolism and residue studies of cyadox.     

Multiresidue determination of eleven quinolones in milk by liquid chromatography with fluorescence detection

A liquid chromatographic method with fluorescence detection was developed for simultaneous determination of norfloxacin, ofloxacin, ciprofloxacin, pefloxacin, lomefloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, and flumequine in milk The samples were extracted with 10% trichloroacetic acid/acetonitrile (9 + 1, v/v) and cleaned by Strata-X reversed-phase solid-phase extraction cartridges. The 11 quinolones were separated on a reversed-phase C18 column (Hypersil BDS-C18) with mobile phase gradient elution and detected with fluorescence by means of a wavelength program. The recoveries for milk fortified with the 11 quinolones at 3 levels were 69-88% with acceptable relative standard deviations of <9% (intraday) and <14% (interday). The limits of detection were 23 microg/L for enrofloxacin, and 1-9 microg/L for the other 10 quinolones.     

Liquid chromatographic determination of ampicillin residues in porcine muscle tissue by a multipenicillin analytical method: European Collaborative Study

A collaborative study involving 14 laboratories was conducted to determine residues of ampicillin in porcine muscle tissue by using a liquid chromatographic method developed for multipenicillin analysis that can quantitate 8 penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, ampicillin, amoxicillin, nafcillin, oxacillin, cloxacillin, and dicloxacillin) at trace levels in muscle tissue. This method involves extraction of the penicillins with phosphate buffer, pH 9, followed by cleanup and concentration on a C18 solid-phase extraction column and reaction with benzoic anhydride at 50 degrees C and with 1,2,4-triazole and mercury(II) chloride solution, pH 9.0, at 65 degrees C. The derivatized compounds are eluted isocratically on a C8 column with a mobile phase of acetonitrile and phosphate buffer (pH 6; 0.1 M) containing sodium thiosulfate and the ion-pair reagent tetrabutylammonium hydrogen sulfate. The penicillins are detected by UV absorption at 325 nm. The limit of detection and the limit of determination (quantitation) of the method were calculated to be approximately 3-5 and 25 microg/kg, respectively, in accordance with the criteria of European Union (EU) Decision No. 93/256/EEC. In this first interlaboratory study, collaborators were instructed to monitor 4 different penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, ampicillin, and amoxicillin) by analyzing 8 blind samples of muscle tissue in triplicate. These samples were prepared from 2 materials containing different concentrations of incurred ampicillin (63.5 microg/kg for material No. 1 and 358.1 microg/kg for material No. 2) and 1 blank material. The repeatability relative standard deviation and the reproducibility relative standard deviation were 10.2 and 17.4%, respectively, for material No. 1 and 7.0 and 16.0%, respectively, for material No. 2. These results demonstrate that the method is suitable for the determination of ampicillin residues in muscle tissue at the EU maximum residue limit (50 microg/kg) and above. However, the identification of positives by this procedure may need additional confirmation by techniques with greater specificity, such as liquid chromatography combined with mass spectrometry, or tandem mass spectrometry. Investigations regarding the basis of interlaboratory testing studies will further demonstrate the suitability of multiresidue methodology for detecting and quantitating other compounds in the family of penicillin antibiotics.     

Development and validation of an HPLC confirmatory method for the determination of tetracycline antibiotics residues in bovine muscle according to the European Union regulation 2002/657/EC

A high-performance liquid chromatographic method with diode-array detection, at 351 nm, was developed and validated for the determination of five tetracyclines (TCs): minocycline, tetracycline, oxytetracycline, chlortetracycline, and doxycycline in bovine muscle. Samples were macerated with a buffer solution, centrifuged, and purified using Abselut Nexus SPE cartridges. The separation of the examined TCs was achieved on an Inertsil ODS-3 5 microm, 250 x 4 mm analytical column, at ambient temperature. A multistep gradient elution was followed using 0.05 M oxalic acid and CH3CN, at a flow rate of 1.65 mL/min. The procedure was validated according to the European Union regulation 2002/657/EC determining selectivity, stability, decision limit, detection capability, accuracy, and precision. The results of the validation process demonstrate that the method can be readily applied to European Union statutory veterinary drug residue surveillance programmes. Mean recoveries of TCs from bovine muscle samples spiked at three concentrations (100, 250, and 400 ng/g) were in the range of 98.7-103.3%. Method's LOQ values achieved were 40 microg/kg for MNC, CTC, and DC and 25 microg/kg for OTC and TC. The decision limits (CCalpha) were in the range of 104.7-109.8 microg/kg, while the detection capability (CCbeta) was in the range of 108.4-116.7 microg/kg for all compounds.     

Multi-residue liquid chromatography/tandem mass spectrometry method for the detection of non-steroidal anti-inflammatory drugs in bovine muscle: optimisation of ion trap parameters

A multi-residue liquid chromatography/tandem mass spectrometry method (LC/MS2) was developed for the detection of the non-steroidal anti-inflammatory drugs acetylsalicylic acid (via the marker residue salicylic acid), flunixin, phenylbutazone, tolfenamic acid, meloxicam and ketoprofen, in bovine muscle. After extraction of the bovine muscle with acetonitrile, the cleanup was performed using a Oasis HLB column. The evaporated eluate was reconstituted and analysed by LC/MS2. To obtain optimal detection of salicylic acid and phenylbutazone, the ion trap mass spectrometric parameters activation q and maximum ion injection time, respectively, were optimised. The activation q for salicylic acid was increased to obtain reliable detection of both salicylic acid and its product ion. The maximum ion injection time for the time segment containing phenylbutazone was decreased since there were not enough scans across the chromatographic peak of this compound. The multi-residue method was able to detect the different analytes below or at the maximum residue limit (MRL) or minimum required performance limit (MRPL) or, in the case of phenylbutazone and ketoprofen, at 100 and 20 microg kg(-1), respectively.     

Natural occurrence of ochratoxin A in dried figs

Ochratoxin A (OTA) contamination in dried figs was investigated using high performance liquid chromatography (HPLC) with fluorescence detection after extraction with methanol and orthophosphoric acid and clean up by an immunoaffinity column. The limit of detection for OTA was 0.12 microg kg(-1). One hundred and fifteen samples were taken during the drying stage from 7 different districts in the Aegean Region in 2003 and 2004. Fifty-five (47.2%) of the 115 samples were found to contain detectable levels of ochratoxin A, ranging from 0.12 to 15.31 microg kg(-1). However, the OTA level for a majority of the samples was low, with only 4 samples containing OTA exceeding 1 microg kg(-1). The calculated overall median for the OTA level was below the limit of detection and the overall mean was estimated as 0.52 microg kg(-1). Frequency of ochratoxin A contamination in dried figs harvested in 2003 and 2004 are 47 and 50%, respectively. Highest contamination ratio was determined in dried figs from Erbeyli (60%), followed by Selcuk (56%), and Ortaklar (50%).     

Determination of florfenicol amine in channel catfish muscle by liquid chromatography

A method for quantifying florfenicol amine (FFA) in channel catfish muscle was validated according to U.S. Food and Drug Administration guidelines. FFA is the proposed marker residue for the veterinary antibiotic florfenicol in catfish muscle for regulatory surveillance purposes. The method includes acid hydrolysis followed by sample cleanup with ethyl acetate extraction, basification, solid-phase extraction, and quantitation by liquid chromatography with UV detection. The assay was validated at 5 concentrations in the range of 0.075-35 microg/g muscle. The overall mean recovery of FFA from fish tissues fortified at these concentrations ranged from 85.7 to 92.3%, 4.8-17.2% relative standard deviation (RSD). The assay limit of detection was 0.044 microg/g muscle based on analysis of control muscle. Catfish muscle samples containing incurred florfenicol residues were analyzed in quintuplicate with RSD < 5%. Acid hydrolysis has previously been demonstrated to convert florfenicol and its known metabolites to FFA and to release a significant amount of FFA from nonextractable florfenicol residues in tissues containing incurred residues in other species. By using acid hydrolysis, this method should yield a more accurate estimate of the total florfenicol-related residue level in muscle tissue from florfenicol-treated catfish than could be achieved by solvent extraction alone.