Liquid chromatographic determination of para-toluenesulfonamide in edible fillet tissues from three species of fish.

Chloramine-T (N-sodium-N-chloro-p-toluene-sulfonamide) is a candidate therapeutic drug for treating bacterial gill disease, a predominant disease of a variety of fish species. Research has been initiated to obtain the U.S. Food and Drug Administration's (FDA) approval for the use of chloramine-T on a variety of fish species. An attribute of a therapeutic aquaculture drug that must be characterized before the FDA approves its use is depletion of the drug's marker residue (the drug's parent compound or metabolite of highest concentration in an edible tissue). para-Toluenesulfonamide (p-TSA) is the primary degradation product and marker residue for chloramine-T in rainbow trout. To conduct residue depletion studies for chloramine-T in fish, a robust analytical method sensitive and specific for p-TSA residues in edible fillet tissue from a variety of fish was required. Homogenized fillet tissues from rainbow trout (Oncorhynchus mykiss), walleye (Stizostedion vitreum), and channel catfish (Ictalurus punctatus) were fortified at nominal p-TSA concentrations of 17, 67, 200, 333, and 1000 ng/g. Samples were analyzed by isocratic reversed-phase liquid chromatography (LC) with absorbance detection at 226 nm. Mean recoveries of p-TSA ranged from 77 to 93.17%; relative standard deviations ranged from 1.5 to 14%; method quantitation limits ranged from 13 to 18 ng/g; and method detection limits ranged from 3.8 to 5.2 ng/g. The LC parameters produced p-TSA peaks without coelution of endogenous compounds and excluded chromatographic interference from at least 20 chemicals and drugs of potential use in aquaculture.     

Performance of a proposed determinative method for p-TSA in rainbow trout fillet tissue and bridging the proposed method with a method for total chloramine-T residues in rainbow trout fillet tissue

Chloramine-T is an effective drug for controlling fish mortality caused by bacterial gill disease. As part of the data required for approval of chloramine-T use in aquaculture, depletion of the chloramine-T marker residue (para-toluenesulfonamide; p-TSA) from edible fillet tissue of fish must be characterized. Declaration of p-TSA as the marker residue for chloramine-T in rainbow trout was based on total residue depletion studies using a method that used time consuming and cumbersome techniques. A simple and robust method recently developed is being proposed as a determinative method for p-TSA in fish fillet tissue. The proposed determinative method was evaluated by comparing accuracy and precision data with U.S. Food and Drug Administration criteria and by bridging the method to the former method for chloramine-T residues. The method accuracy and precision fulfilled the criteria for determinative methods; accuracy was 92.6, 93.4, and 94.6% with samples fortified at 0.5X, 1X, and 2X the expected 1000 ng/g tolerance limit for p-TSA, respectively. Method precision with tissue containing incurred p-TSA at a nominal concentration of 1000 ng/g ranged from 0.80 to 8.4%. The proposed determinative method was successfully bridged with the former method. The concentrations of p-TSA developed with the proposed method were not statistically different at p < 0.05 from p-TSA concentrations developed with the former method.     

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.     

High-performance liquid chromatographic analysis of oxytetracycline in chinook salmon following administration of medicated feed.

A high-performance liquid chromatographic assay was developed to detect oxytetracycline (OTC) in chinook salmon muscle tissue. A solid-phase extraction protocol was used to recover OTC and the internal standard, epitetracycline hydrochloride, from the salmon tissue samples. OTC was analyzed using a mobile phase of methanol-0.02 M phosphate buffer, pH 2.25 (60:190), an ultraviolet detection wavelength of 365 nm and 250 mm x 4.6 mm I.D. Ultrasphere ODS column. A linear calibration curve (r2 = 0.999) of OTC in salmon muscle tissue from 0.05 to 3.0 ppm was obtained. Using a signal-to-noise ratio of 5:1, the OTC detection limit was 0.5 ppm in salmon muscle tissue. OTC recovery (74.4%) and intra-assay variability (2.3%) were optimized for salmon muscle tissue. An in vivo feeding study was performed by administrating OTC-medicated feed for a period of 10 days, followed by a 42-day sampling period. The half-life for the elimination of OTC in chinook salmon muscle tissue was found to be 5.4 days.     

Rapid and simple determination of oxytetracycline in chicken products.

A rapid and simple method for determining residual oxytetracycline (OTC) in chicken products (muscle, liver, and eggs) by high-performance liquid chromatography (HPLC) was developed. Samples were prepared by homogenization with acetonitrile-n-hexane (5 + 4, v/v) followed by centrifugation to minimize fat and protein contents. OTC in the acetonitrile layer was free from interfering compounds when examined by HPLC using a LiChrospher 100 RP-8 end-capped column, a mobile phase of acetonitrile-acetic acid-0.01 M disodium EDTA (28 + 2 + 70, v/v/v), and a photodiode array detector. Average recoveries from samples spiked with OTC (0.1, 0.2, and 1.0 ppm) were > 88%, with coefficients of variation between 2.3 and 5.1%. The limit of detection was 0.05 ppm.     

Liquid chromatographic determination of benzocaine and N-acetylbenzocaine in the edible fillet tissue from rainbow trout.

A method was developed for determining benzocaine and N-acetylbenzocaine concentrations in fillet tissue of rainbow trout. The method involves extracting the analytes with acetonitrile, removing lipids or hydrophobic compounds from the extract with hexane, and providing additional clean-up with solid-phase extraction techniques. Analyte concentrations are determined using reversed-phase high-performance liquid chromatographic techniques with an isocratic mobile phase and UV detection. The accuracy (range, 92 to 121%), precision (R.S.D., < 14%), and sensitivity (method quantitation limit, < 24 ng/g) for each analyte indicate the usefulness of this method for studies characterizing the depletion of benzocaine residues from fish exposed to benzocaine.     

Immunochemical determination of oxytetracycline in fish: Comparison between enzymatic and time-resolved fluorometric assays

An indirect competitive enzyme-linked immunosorbent assay (ELISA) with photometric detection of horseradish peroxidase (HRP) activity, was developed in plate to detect oxytetracycline (OTC) in Gilthead sea bream (Sparus aurata) samples. The results were compared to those obtained by time-resolved fluoroimmunoassay (TR-FIA) using a secondary antibody with coproporphyrin of platinum (II) (PtCP) as marker. The limits of detection obtained in fish extract were 16 and 0.08 μg kg⁻¹ for photometric and fluorometric detections, respectively; therefore, they were suitable for fish quality control according to the maximum residue level established by the European Union.An extraction procedure using methanol:water 70:30 (v/v) + 1 mL EDTA 0.1 M, and different clean-up procedures based on solid-phase extraction (C₁₈, polymeric reversed phase, SCX, Si) was assayed. The matrix effects were overcome by means of an average tetracycline-free fish extract calibration curve used for quantification.The OTC optimized ELISA can also be applied to determine tetracycline and chlortetracycline residues with good results. Thus, the developed immunoassay could be considered as a generic assay for the most used tetracyclines in aquaculture antibiotic treatments.In order to confirm the utility of the developed immunoassay as a semi-quantitative methodology, fish samples obtained from different supermarkets were analyzed. Results correlate well with those obtained with a reference HPLC method.     

Determination of oxytetracycline in salmon by liquid chromatography with metal-chelate fluorescence detection

A liquid chromatography (LC) method is described for the determination of oxytetracycline (OTC) in farmed Atlantic salmon muscle tissue. The method involves homogenization of salmon tissue, extraction of OTC into Mcllvaine-EDTA buffer, acid precipitation of proteins, cleanup through tandem solid-phase extraction cartridges (Strata-X and aminopropyl), elution with mobile phase containing slightly alkaline buffer and Mg2+, and LC separation with metal-chelate induced fluorescence detection. Salmon tissue was fortified with 0.10, 0.25, 0.50, 0.75, and 1.0 microg/g (ppm) oxytetracycline. Average absolute recoveries were 84, 76, 70, 76, and 85%, respectively, with relative standard deviation (RSD) values all less than 9%. The interassay average recovery was 78%, with a 4.2% RSD. Determination was based on a standard graph using peak areas with standard solutions equivalent to 0.0625, 0.125, 0.25, 0.50, and 1.0 ppm in tissue. A set of 5 matrix controls (unfortified salmon tissue) were also analyzed, in which no OTC was detected. The lowest standard was used as the limit of quantitation.     

Simultaneous screening and confirmation of multiple classes of drug residues in fish by liquid chromatography-ion trap mass spectrometry

LC-ion trap mass spectrometry was used to screen and confirm 38 compounds from a variety of drug classes in four species of fish: trout, salmon, catfish, and tilapia. Samples were extracted with acetonitrile and hexane. The acetonitrile phase was evaporated, redissolved in water and acetonitrile, and analyzed by gradient chromatography on a phenyl column. MS² or MS³ spectra were monitored for each compound. Qualitative method performance was evaluated by the analysis over several days of replicate samples of control fish, fish fortified with a drug mixture at 1 ppm, 0.1 ppm and 0.01 ppm, and fish dosed with a representative from each drug class. Half of the 38 drugs were confirmed at 0.01 ppm, the lowest fortification level. This included all of the quinolones and fluoroquinolones, the macrolides, malachite green, and most of the imidazoles. Florfenicol amine, metronidazole, sulfonamides, tetracyclines, and most of the betalactams were confirmed at 0.1 ppm. Ivermectin and penicillin G were only detectable in the 1 ppm fortified samples. With the exception of amoxicillin, emamectin, metronidazole, and tylosin, residue presence was confirmed in all the dosed fish.     

Determination of perfluorinated compounds in fish fillet homogenates: method validation and application to fillet homogenates from the Mississippi River

We report herein a simple protein precipitation extraction-liquid chromatography tandem mass spectrometry (LC/MS/MS) method, validation, and application for the analysis of perfluorinated carboxylic acids (C7–C12), perfluorinated sulfonic acids (C4, C6, and C8), and perfluorooctane sulfonamide (FOSA) in fish fillet tissue. The method combines a rapid homogenization and protein precipitation tissue extraction procedure using stable-isotope internal standard (IS) calibration. Method validation in bluegill (Lepomis macrochirus) fillet tissue evaluated the following: (1) method accuracy and precision in both extracted matrix-matched calibration and solvent (unextracted) calibration, (2) quantitation of mixed branched and linear isomers of perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) with linear isomer calibration, (3) quantitation of low level (ppb) perfluorinated compounds (PFCs) in the presence of high level (ppm) PFOS, and (4) specificity from matrix interferences. Both calibration techniques produced method accuracy of at least 100 ± 13% with a precision (%RSD) ≤18% for all target analytes. Method accuracy and precision results for fillet samples from nine different fish species taken from the Mississippi River in 2008 and 2009 are also presented.     

High-performance liquid chromatographic analysis of Romet-30 in Chinook salmon (Oncorhynchus tshawytscha): wash-out time, tissue distribution in muscle, liver and skin, and metabolism of sulfadimethoxine

A high-performance liquid chromatographic (HPLC) assay was developed for the determination of Romet-30 residue levels in Chinook salmon muscle and liver tissues. The extraction recoveries averaged 66, 78 and 83% for ormetoprim (OMP), sulfadimethoxine (SDM) and N⁴-acetyl-SDM (N⁴-Ac-SDM), respectively, in muscle tissue; 61 and 72% for SDM and N⁴-Ac-SDM, respectively, in liver tissue. The HPLC assay had a lower detection limit of 0.05 μg/g for OMP, SDM and N⁴-Ac-SDM in muscle tissue, and a lower detection limit of 0.20 μg/g for SDM and N⁴-Ac-SDM in liver tissue. OMP could not be quantified in liver due to the presence of substantial amounts of co-extracted endogenous substances. The HPLC assay was applied to the analysis of Romet-30 residues in Chinook salmon after gastric intubation with Romet-30 at water temperatures between 8.0 and 9.0°C. Different disposition characteristics were found in muscle and liver tissues. The presence of N⁴-Ac-SDM in liver tissue was confirmed by MS and MS-MS analyses. An estimation of SDM and N⁴- Ac-SDM residue levels in skin tissue was obtained and the disposition of these two compounds was similar to the disposition in muscle tissue.     

Determination of putrescine and cadaverine in seafood (finfish and shellfish) by liquid chromatography using pyrene excimer fluorescence

A liquid chromatography (LC) method is described for the easy determination of the biogenic diamines putrescine (PUT) and cadaverine (CAD) in canned tuna, frozen tuna loin, fresh mahimahi fillet, frozen raw shrimp, cooked lump crabmeat, and fresh and cold-smoked salmon. The method is also a useful screen for histamine (HTA). The method involves homogenization of fish tissue, extraction of biogenic amines into borate-trichloroacetic acid solution, centrifugation, and derivatization of supernatant with 1-pyrenebutanoic acid succinimidyl ester. The derivatized diamine species allow for the intramolecular excimer fluorescence of the pyrene moiety at a higher emission wavelength than is possible for the endogenous tissue monoamines, thus providing visual specificity of detection. All seafood species were fortified with 0.5, 1.0, 5.0, 10.0, and 15.0 microg/g (ppm) of PUT and CAD. Determination was based on standard graphs for PUT and CAD using peak areas with standard solutions equivalent to 0.375, 1.0, 5.0, 10.0, and 20.0 ppm in tissue. A set of five matrix controls (unfortified seafood tissue) were also analyzed; endogenous PUT was found in all samples except the canned tuna, and CAD found only in the shrimp, crab, and cold-smoked salmon. The background amines were thus subtracted prior to determining spike recovery. The intra-assay average recoveries ranged from 71 to 94% across species and spike levels.     

Development of a Monoclonal Antibody-Based ELISA for the Detection of Oxytetracycline and 4-Epi-Oxytetracycline Residues in Chicken Tissues

In this study, a specific monoclonal antibody (Mab) against oxytetracycline (OTC) and its metabolite 4-epi-Oxytetracycline (4-epi-OTC) was produced. Based on this Mab, a sensitive and reliable method indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the detection of OTC and 4-epi-OTC from chicken meats. The ic-ELISA showed a 50% inhibition (IC₅₀) value of 2.01 ± 0.16 ng/ml and a detection limit of 0.13 ± 0.03 ng/ml. The recoveries from chicken muscles and livers spiked with OTC of 50–600 ng/g were 83.33–88.25% and 84.62–86.12%, respectively. The intra-assay coefficients of variation (CVs) were 4.73–9.31%, and the inter-assay CVs were 6.44–11.01%. The method showed a positive correlation with the traditional method HPLC (R² = 0.997) within a certain concentration of OTC used in this assay. The method developed in this study was simple and independent of specific expensive equipment. Thus, it could be useful as a convenient method to detect OTC residues.     

Simultaneous multiresidue determination of tetracyclines and fluoroquinolones in catfish muscle using high performance liquid chromatography with fluorescence detection

Efficient methods are needed for analysis of veterinary drug residues in food. A number of methods are available for single analytes. Multiresidue methods are now increasingly available. It is still rare, however, to find methods not involving mass spectrometry which allow for analysis of more than one class of drug residue. An efficient multiresidue method for the simultaneous determination of fluoroquinolones (FQs) and tetracyclines (TCs) in catfish muscle has now been developed. This method involves an extraction of the analytes with a mixture of acetonitrile and citrate buffer containing magnesium chloride. After centrifugation and evaporation of the supernatants, the residues are determined using high performance liquid chromatography with fluorescence detection. With this method, five fluoroquinolones and three tetracyclines were determined in fortified catfish muscle at levels of 20, 50, and 100 ng g(-1). Average recoveries for ciprofloxacin (CIP), sarafloxacin (SAR), danofloxacin (DANO), enrofloxacin (ENRO), difloxacin (DIF), oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) were in the range of 60-92% with good relative standard deviations. The limits of quantitation ranged from 0.15 to 1.5 ng g(-1). Utilization of the method to successfully analyze catfish muscle samples incurred with enrofloxacin and with oxytetracycline is described.     

Europium-sensitized luminescence determination of oxytetracycline in catfish muscle

An europium-sensitized time-resolved luminescence (TRL) method was developed to determine oxytetracycline (OTC) in cultivated catfish muscle. Extraction of OTC from fish muscle was performed with pH 4.0 ethylenediaminetetraacetic acid (EDTA)-McIlvaine buffer and clean up with hydrophilic-lipophilic balanced copolymer solid phase extraction (SPE) cartridges. The eluate was used without further concentration for TRL measurement in pH 9.0 micellar tris(hydroxylmethyl)aminomethane (TRIS) buffer. Cetyltrimethylammonium chloride (CTACl) was used as surfactant and EDTA as a co-ligand. The excitation and emission wavelengths were set at 388 and 615 nm, respectively. The linear dynamic range was 0-1000 ng g⁻¹ (R²=0.9995). The recovery was 92-112% in the fortification range of 50-200 ng g⁻¹ and the limits of detection (LOD) ranged from 3 to 7 ng g⁻¹. Incurred catfish samples were used to demonstrate the performance of the method around 100 ng g⁻¹, the European Union maximum residue level.     

Improvement of chemical analysis of antibiotics. XVII. Application of an amino cartridge to the determination of residual sulphonamide antibacterials in meat, fish and egg

A simple, rapid and reliable method for the determination of residual sulphonamide antibacterials (SAs) (sulphathiazole, sulphisozole, sulphamethoxazole, sulphadiazine, sulphamerazine, sulphadimidine, sulphamonomethoxine, sulphadimethoxine, sulphamethoxypyridazine and sulphaquinoxaline) in meat, fish and egg was developed using a combination of high-performance liquid chromatography (HPLC) and clean-up with an amino-type prepacked cartridge. SAs were extracted with ethyl acetate and applied to a Baker 10 amino cartridge. After elution from the cartridge, SAs were determined by HPLC. The recoveries at the level of 0.5 ppm were 73.7-99.1% and the detection limits were 0.05 ppm. The analysis time per sample was about 45 min.     

Improvement of chemical analysis of antibiotics. IX. A simple method for residual tetracyclines analysis in honey using a tandem cartridge clean-up system

A simple, rapid and precise analytical method for the residual tetracyclines in honey has been established using a tandem cartridge clean-up system (prepacked reversed-phase and ion-exchange cartridges) followed by high-performance liquid chromatography. The recoveries of oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DC) from honey spiked at a level of 1.0 ppm are 87.1, 85.3, 98.0 and 99.0%, respectively, with coefficients of variation of 1.1-3.9%. The detection limits in honey are 0.02 ppm for OTC and TC, and 0.05 ppm for CTC and DC, respectively. The time required for the analysis of four samples is only 1 h.     

A new liquid chromatographic method for routine determination of oxytetracycline marker residue in the edible tissues of farm animals

A simple, rapid, and specific ion-pair liquid chromatographic method for routine determination of the marker residue of oxytetracycline (OTC), namely OTC and 4-epi-oxytetracycline (4-epiOTC), in edible animal tissues (muscle, liver, kidney, and fat) has been developed. Minced tissue samples were acidified at pH 2.7 with 2 mol L(-1) sulfuric acid and extracted with acetonitrile. The extracts were purified by treatment with ammonium sulfate solution and concentrated into 0.1 mol L(-1) phosphoric acid. Baseline separation was carried out isocratically on a Nucleosil 100-5 C(18), 5-microm column using an acetonitrile-0.01 mol L(-1) disodium hydrogen phosphate (20:80, v/v) mobile phase that contained both positively (tetrabutylammonium) and negatively (octanesulfonate) charged pairing ions and EDTA, and was adjusted to pH 3.8. Detection was by UV at 370 nm. The method was fully validated according to Commission Decision 2002/657/EC. Overall recoveries were better than 82.6% and overall relative standard deviation was better than 6% for all the tissues examined. The good analytical characteristics of the method allowed limits of quantification as low as 30 ng g(-1) for muscle and fat and 50 ng g(-1) for liver and kidney, for both OTC and 4-epiOTC, to be realized. The method was successfully used to determine the OTC marker residue in tissues of two sheep intramuscularly administered a commercial OTC formulation.     

Determination of 3-trifluoromethyl-4-nitrophenol and 3-trifluoromethyl-4-nitrophenol glucuronide in edible fillet tissue of rainbow trout and channel catfish by solid-phase extraction and liquid chromatography

3-Trifluoromethyl-4-nitrophenol (TFM) is a pesticide used for the selective control of sea lampreys (Petromyzon marinus) in stream and river tributaries of the Great Lakes. To determine concentrations of TFM and TFM glucuronide in the edible fillet tissue of fish during sea lamprey control treatments, an analytical method was developed to determine the concentrations of these residues in rainbow trout (Oncorhynchus mykiss; RBT) and channel catfish (Ictalurus punctatis; CCF). Homogenized fillets were extracted with methanol-water (80 + 20). TFM and TFM glucuronide were isolated from coextractives by C18 solid-phase extraction. TFM glucuronide was hydrolyzed to TFM by the addition of beta-glucuronidase to the TFM glucuronide extract. The extracts were analyzed separately by liquid chromatography with UV-visible detection. Recoveries from TFM-fortified CCF and RBT tissues were 84.1 and 96.1%, respectively. The method detection limits (MDLs) are 2.4 ng/g for TFM-fortified tissues of CCF and 3 ng/g for those of RBT. Recoveries were 78.8 and 77% from TFM glucuronide-fortified CCF and RBT tissues, respectively. The MDLs for TFM glucuronide-fortified tissues are 3.5 and 6.9 ng/g for CCF and RBT, respectively.     

Hyaluronate depolymerization following thermal decomposition of oxytetracycline

Depolymerization of sodium hyaluronate (HA) by tetracyclines was investigated. Reduction in HA molecular weight was followed by size exclusion chromatography with a low angle laser light scattering detector. On mixing with oxytetracycline hydrochloride (OTC) solution and incubating at 37 degrees C, HA was gradually depolymerized. OTC, a representative antibiotic, is known as a photosensitizer, and phototoxic side effects relevant to radicals have been reported. However, HA depolymerization required no irradiation. As time passed, OTC solution incubated at 37 degrees C got colored reddish brown, even in the dark. With reversed-phase HPLC separation, several peaks derived from decomposed OTC appeared. One of the peaks had an absorbance in the visible range. A quantitative correlation between the discoloration and the HA depolymerization rate was obtained. On the other hand, when samples were incubated below 25 degrees C, change of color was slight, and practically no HA depolymerization was observed after up to 4 h. Oxygen depletion by nitrogen saturation or addition of mannitol also prevented the depolymerization. Under anaerobic conditions, the color of the solution did not change, whereas it turned red under aerobic conditions in the presence of mannitol. The mannitol did not inhibit the OTC decomposition, but it preserved HA from damage. On the basis of the known decomposition of OTC and the results of HPLC separation, anhydrooxytetracycline can be proposed as the derivative causing HA depolymerization.