Assay of oxolinic acid residues in salmon muscle by liquid chromatography with fluorescence detection: interlaboratory study.

A previously developed method that uses a simplified sample preparation and fluorometric detection of liquid chromatographic eluates for the determination of oxolinic acid in salmon muscle has been collaboratively studied. Five laboratories participated in the study to analyze, in quintuplicate, blank salmon muscle fortified at 10, 20, 50, and 100 micrograms/kg (ppb), and 2 incurred samples from salmon given feed with medicated oxolinic acid. The tissue, 2 g mixed with 2 g Na2SO4, is extracted with ethyl acetate and centrifuged, and the solvent is evaporated. The residue is partitioned in a mixture of hexane and 0.01 M oxalic acid, and the aqueous phase is chromatographed using fluorescence detection at 327 nm excitation and 369 nm emission. Mean recoveries ranged from 77.2 to 84.5% in spiked samples with reproducibility relative standard deviation (RSDR) ranging from 11.5 to 18.3%. Treated salmon were found to contain 8.71 and 53.8 micrograms/kg with RSDR of 18.6 and 16.7%, respectively. The corresponding repeatability relative standard deviations (RSDR) were 5.8-12.2%, and 7.7 and 6.2%. The method is recommended for regulatory purposes in Canada.     

Quantitative and confirmatory analyses of malachite green and leucomalachite green residues in fish and shrimp

Liquid chromatographic methods are presented for the quantitative and confirmatory determination of malachite green (MG) and leucomalachite green (LMG) for channel catfish, rainbow trout, tilapia, basa, Atlantic salmon, and tiger shrimp. Residues were extracted from tissues with ammonium acetate buffer and acetonitrile and isolated by partitioning into dichloromethane. LMG was quantitatively oxidized to the chromic MG with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Extracts were analyzed for total MG by liquid chromatography with both visible detection (LC-VIS) at 618 nm for routine screening and ion trap mass spectrometry (LC-MSn) with no discharge-atmospheric pressure chemical ionization for residue confirmation. The method was validated in each species fortified with LMG at 1, 2, 4, and 10 ng/g (ppb), and average recoveries ranged from 85.9 to 93.9%. Quantitative data were consistent for the two detection methods, with measured method detection limits of 1.0 ng/g for LC-VIS and 0.25 ng/g for LC-MSn. Incurred tissues from catfish, trout, tilapia, and salmon that had been treated with MG were also extracted and analyzed as part of this study.     

Determination of albendazole and its major metabolites in the muscle tissues of Atlantic salmon,tilapia, and rainbow trout by high performance liquid chromatography with fluorometric detection

A liquid chromatographic procedure for the determination of albendazole ([5-(propylthio)-1H-benzimidazol-2yl]carbamic acid methyl ester) and its major metabolites, albendazole sulfoxide, albendazole sulfone, and albendazole-2- aminosulfone in rainbow trout, tilapia, and salmon muscle with adhering skin tissue is described. The muscle tissue samples are made alkaline with potassium carbonate and extracted with ethyl acetate. The extracts are further subjected to cleanup by utilizing a number of liquid-liquid extraction steps. After solvent evaporation, the residue is reconstituted in mobile phase and chromatographed. The chromatography is carried out on a reversed phase Luna C(18) column, using acetonitrile/methanol/buffer as a mobile phase and a fluorescence detector. The average recoveries from the fortified muscle tissue of the three fish species for albendazole (25-100 ppb), albendazole sulfoxide (15.5-62 ppb), albendazole sulfone (1-10 ppb), and albendazole-2- aminosulfone (10-100 ppb) were 94, 77, 82, and 67%, respectively. The average CV for each compound was < or =10%. The procedure was validated and then applied to the determination of albendazole and its three major metabolites in the muscle tissue of the three fish species obtained after orally dosing with albendazole.     

Gas-liquid chromatographic determination of 3,6-dichloropicolinic acid in soils.

A gas-liquid chromatographic (GLC) method has been developed for the determination of the experimental herbicide 3,6-dichloropicolinic acid (Dowco 290) in soils. The method involves extraction of 1 g soil samples with 1N NaCl at approximately pH 7, methylation with diazomethane utilizing a microgenerator, and detection by electron capture GLC. Interferences are small, so that a cleanup step is not necessary even at the 6 ppb level. The procedure is rapid, requiring only 45 min/sample. Recoveries range from 84 to 94% at the 6-1000 ppb level with a minimum detectable limit of 6 ppb. Standard deviations for the percentage recovery values vary from 10.9 to 2.3 for the tested range of 6.7-670 ppb, respectively.     

Liquid chromatographic method for determination of citreoviridin in corn and rice

Citreoviridin, a neurotoxic mycotoxin, has been found as a natural contaminant in corn left unharvested in the southeastern United States and in rice of several Asian countries, including Japan. A reliable analytical method for the quantitative determination of citreoviridin in corn and rice is described. Corn or rice is extracted with dichloromethane, and the extract is partially purified on silica and amino solid-phase extraction (SPE) columns. The extract is analyzed for citreoviridin by normal-phase liquid chromatography, using a mobile phase of ethyl acetate-hexane (75 + 25) at 1.5 mL/min and a fluorescence detector to measure the yellow fluorescence (388 nm excitation, 480 nm emission). With a 100 microL injection loop, the relationship between concentration and injection volume is linear for 20-60 microL injections. Recoveries of citreoviridin added to yellow corn at 10-50 ng/g were 91.0-96.9%; recoveries from white corn (10-50 ng/g added) were 96.8-102.8%. Recoveries of 5000 ng/g added to white corn were 89.0%, indicating that heavily contaminated samples can be assayed by the method. Minimum detection limits were 10 ng for citreoviridin standard and 2 ng/g for citreoviridin added to corn. White rice fermented with Penicillium citreo-viride (1524 ppm) was mixed with and serially diluted with uncontaminated ground corn to obtain citreoviridin-contaminated corn (ca 25 ppb). When the samples were assayed by the method, a mean level of 24.4 +/- 1.65 ppb (6.5% coefficient of variation) was obtained. Four fermented rice food samples and 3 commercial rice samples were investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Residue level determination of the aquatic herbicide fluridone and a potential photoproduct (N-methylformamide) in water

Methods are presented for the determination of the aquatic herbicide fluridone and N-methylformamide (NMF), a potential photoproduct of fluridone which has thus far been observed only in a laboratory aqueous photolysis study. Water samples are passed through a Sep-Pak C18 cartridge to extract fluridone while NMF passes unretained through the cartridge with the water. Fluridone is eluted from the cartridge with methanol, which is then concentrated for analysis by liquid chromatography with UV detection at 313 nm. The water eluate containing NMF is concentrated for analysis by rotary vacuum evaporation at 40-46 degrees C. Methanol is added to help evaporate the water, and glycerol is added as a keeper solvent to retain NMF during the evaporation. The residual NMF is dissolved in methanol for analysis by gas chromatography with a Hall electrolytic conductivity detector operated in the nitrogen mode. The assay limits of detection are 1 and 2 ppb for fluridone and NMF, respectively. Recoveries averaged 98% for fluridone at 1-400 ppb and 87% for NMF at 2-50 ppb.     

Analysis of 2-alkylcyclobutanones with accelerated solvent extraction to detect irradiated meat and fish

A new analytical procedure has been developed to analyze 2-alkylcyclobutanones to detect gamma-ray-irradiated fat-containing foodstuffs. Samples were extracted with an accelerated solvent extraction system via hot and pressurized ethyl acetate in cells. A large amount of fat in the extract was precipitated and removed with filtration by standing at -20 degrees C after the addition of acetonitrile. The extract was further cleaned with a 1 g silica gel mini column, and the radiolytic compounds of 2-docecylcyclobutanone (2-DCB) and 2-tetradecylcyclobutanone (2-TCB) were determined with gas chromatography with mass spectrometry (GC/MS). Sample preparation time before GC/MS was 7-8 h. At first, the procedure was evaluated with a recovery test in eight samples spiked with 2-DCB and 2-TCB at 20 ng/g, resulting in 70-105% recoveries with mostly less than 10% relative standard deviations. The procedure was further evaluated with beef, pork, chicken, and salmon samples irradiated with gamma-rays from 0.7 to 7.0 kGy at -19 degrees C. Both 2-DCB and 2-TCB in most samples were detected with good dose-response relations at all doses, while salmon was detected more than 2 kGy irradiation. The amounts of 2-alkylcyclobutanones produced reflected precursor fatty acids levels in samples, especially for the combination of 2-TCB and stearic acid. The results indicated that the production rate of 2-TCB to stearic acid was more obvious than that of 2-DCB to palmitic acid in frozen samples with gamma-ray irradiation.     

High pressure liquid chromatographic determination of furazolidone in turkey tissue.

A high pressure liquid chromatographic method for determining furazolidone in turkey tissue has been developed. Tissues are ground with methanol and centrifuged. For lower levels of furazolidone, 2--40 ppb, the supernate is evaporated to dryness and redissolved before it is injected onto the liquid chromatographic column. Using a reverse phase column and an ultraviolet absorption detector set at 365 nm, the assay is linear over the concentration range 2--400 ppb with a coefficient of variation of less than 4%. Average recovery from fortified tissues was 96% with a coefficient of variation of 6% at the 50--400 ppb level, and 105% with a coefficient of variation of 11% at the 2--40 ppb level.     

Development of a fluorescent latex immunoassay for detection of a spectinomycin antibiotic

There is a need to develop a rapid and sensitive method to detect spectinomycin residues in animal tissues. A latex fluorescent immunoassay was designed using reagents developed for this assay. The spectinomycin antibody was produced in sheep, and the immunoglobulin (IgG) was purified through a Protein G affinity column and was immobilized onto latex particles. Spectinomycin was labeled with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein (DTAF). The optimum assay conditions consisted of preincubating the latex-IgG with spectinomycin in buffer solutions or in bovine kidney extracts. DTAF-spectinomycin was added and was further incubated. The bound spectinomycin-DTAF/IgG-latex complex was separated by centrifugation at 4000 g for 10 min. The fluorescence signals of the unbound spectinomycin-DTAF in the supernatant were measured at 485/535 nm excitation/emission. The measured signals were directly proportional to the concentration of spectinomycin in the samples, and spectinomycin was detected at 0-100 ppb with minimum detectability of 5 ppb. The mean regression correlation of four trials in buffer was 0.936 when the % bound complex vs spectinomycin concentration was plotted. Analysis of the kidney extract spiked with 0-100 ppb spectinomycin had a regression correlation of 0.959. This assay provides a rapid screening method for low ppb detection of spectinomycin.     

Method for determination of pentobarbital in dry dog food by gas chromatography/mass spectrometry

A procedure has been developed and validated for measuring the concentration of pentobarbital residues in dry, extruded animal feed in the range of 3-200 ng/g (ppb) with an estimated limit of quantitation of 2 ppb. The method was developed for surveillance purposes: to measure the concentration of euthanizing agent which might be present in feeds incorporating rendered products which themselves might include some fraction of euthanized animals. A previously published qualitative procedure was modified by adding isotopically labelled pentobarbital as an internal standard. Dry feed was ground and extracted with methanol. The extract was loaded on a mixed-mode (C-18, anion exchange) solid-phase extraction cartridge designed for barbiturate residues. Pentobarbital was eluted and derivatized for gas chromatography/mass spectrometry in positive ion chemical ionization mode. Quantitation was based on the ratio of dimethyl-pentobarbital MH+ (m/z 255) vs dimethyl-pentobarbital-d(5) (m/z 260) in standards and extracts. Accuracy ranged from 112% at 3 ppb to 96% at 200 ppb, with relative standard deviations ranging from 4% at 3 ppb to 2% at 200 ppb.     

Multiresidue assay for benzimidazole anthelmintics by liquid chromatography and confirmation by gas chromatography/selected-ion monitoring electron impact mass spectrometry

A multiresidue method utilizing all-disposable labware has been developed for 8 benzimidazole anthelmintics from ovine, bovine, and swine muscle and liver tissues. After an initial extraction with ethyl acetate and subsequent evaporation, a 3-component extraction using hexane, ethanol, and 0.2N HCl was used for final cleanup. Clean extracts were produced for separation and determination by reverse-phase liquid chromatography at 298 nm, using methanol and aqueous buffer as mobile phase. A synthesized internal standard, 2-(n-butylmercapto)benzimidazole, was used for quantitation of all drugs. Results are included along with statistical information verifying the performance of the method. Spiked control tissues and incurred drug tissues were used for an intralaboratory study with a concentration range of 50-1470 ppb. A series of standard curves at 0, 50, 100, and 200 ppb were analyzed. Overall recovery at the 100 ppb level averaged 92% (CV 8%) in liver tissues, across all 3 species and 88% (CV 5%) in muscle tissues across all 3 species. Results were confirmed by gas chromatography/mass spectrometry with acid hydrolysis of the remaining extract in 2N HCl followed by re-extraction of the amine and derivatization to the tert-butyldimethylsilyl derivative. The anthelmintics were identified by gas chromatography/selected ion monitoring electron-impact mass spectrometry. Ion ratio measurements were taken and compared to standard material. CVs averaged 10% or less for all drugs tested.     

Liquid chromatography analysis of erythromycin A in salmon tissue by electrochemical detection with confirmation by electrospray ionization mass spectrometry

A rapid and sensitive method is described for the quantitation of erythromycin A (EA) in edible salmon tissue by liquid chromatography (LC) analysis using either electrochemical detection (ED) or electrospray ionization mass spectrometric (ESI/MS) detection. The salmon tissue is extracted with 10 mM ammonium formate. The extract is then purified by solid phase extraction using a hydrophilic-lipophilic balanced (HLB) polymeric-based C18 packing, followed by partitioning of EA into methylene chloride at alkaline pH, evaporation, and final dilution. The mean recoveries of EA at 50, 100, 200, and 400 ppb levels in fortified salmon tissue were 63.8 +/- 6.0 and 75.5 +/- 5.4% by LC-ED and LC-ESI/MS, respectively. There was no evidence of formation of the anhydro-EA (m/z 716) decomposition product of EA (m/z 734) that was reported to occur by other published methods.     

Determination of volatile N-nitrosamines in frankfurters containing minced fish and surimi

A method was developed to quantitatively measure volatile N-nitrosamines, particularly N-nitrosodimethylamine (NDMA), in cured minced fish or surimi-meat frankfurters. This method is free from artifact formation. First, 2 dry solid-phase extraction columns are prepared. Solvent is passed through the top column containing the fish-meat into a second column containing acid Celite. The eluate from the Celite column is then passed through a third column containing silica gel. Nitrosamines are eluted from the acid Celite column and then from the silica gel column into the same receiver. Recovery of the internal standard, N-nitrosoazetidine, added at the 10 ppb level, was 86.5%. In addition, a few samples of nitrite-treated salmon (lox) were also tested for N-nitrosamines. The results show that the method is applicable to samples containing nitrite-treated fish and fish-derived products.     

Determination of benzo(a)pyrene in foods

An analytical method was developed for determining benzo(a)pyrene in foods, suitable for routine use. The method consists of 4 cleanup steps: (1) alkali cleavage of sample, (2) preliminary silica gel column chromatography, (3) selective extraction with concentrated sulfuric acid, and (4) further silica gel column chromatography. Recoveries of benzo(a)pyrene added to 50 g (or 10 g) food at levels of 0.4 ppb (or 2 ppb) ranged from 70% for short-necked clam and mackeral to 85% for chicken meat. The sulfuric acid extraction step affords a simple method for isolating benzo(a)pyrene from various kinds of interfering substances which could not be separated by existing methods.     

Liquid chromatographic determination of multiple sulfonamide residues in bovine milk

A liquid chromatographic method has been developed for simultaneous determination of residues of 10 sulfonamide drugs at 10 ppb and above in raw bovine milk. The method is based on a chloroform-acetone extraction, evaporation of organic phase, dissolution of residues in an aqueous potassium phosphate solution, and extraction of fatty residue into hexane. The aqueous layer is collected, filtered, injected onto an LC system, and detected by ultraviolet absorption at 265 nm. To elute all 10 sulfonamides isocratically, 2 chromatographic conditions are required. Seven sulfonamides can be quantitated with 12% methanol in the mobile phase; 4 sulfonamides can be quantitated with 30% methanol. Sulfamethazine, the most widely used sulfonamide, is detected on both systems. Recoveries are 44-87% for individual sulfonamides, with only 2 below 60%. Coefficients of variation are 3-13% at 10 ppb.     

Determination of gibberellic acid residues on fruits by synchronous scanning derivative spectrofluorometry

A synchronous derivative spectrofluorometric method is described for the determination of the plant growth regulator, gibberellic acid (GA3). The method is based on the formation of a fluorogen in concentrated sulfuric acid. The reaction is carried out at 85% sulfuric acid and in aqueous medium. The common fluorometric method with a linear dynamic range of 137-400 ppb, and a detection limit of 48 ppb is described. The synchronous first and second derivative method has linear dynamic ranges between 7.6-40 ppb and 12-40 ppb, with detection limits of 3.5 and 6.7 ppb, respectively. The influence of reaction variables and of other plant growth regulators present, and the application to residues on oranges, lemons, and grapes, are also described.     

Sensitive determination of ethopabate residues in chicken tissues by liquid chromatography with fluorometric detection

A liquid chromatographic (LC) method is described for determination of ethopabate residues in chicken tissues. The drug is extracted from tissues with acetonitrile, and the extract is concentrated to 2-3 mL. This aqueous solution is rinsed with ethyl acetate and cleaned up by Florisil column chromatography. LC analysis is carried out on a Zorbax ODS column, and ethopabate is quantitated by using a fluorometric detector set at 306 nm (excitation) and 350 nm (emission). Recoveries of ethopabate added to chicken tissues at levels of 0.01 and 0.05 ppm were 87.8 and 92.7%, respectively. The detection limit was 100 pg for ethopabate standard, and 0.5 ppb in chicken tissues.     

Free polyunsaturated fatty acids cause taste deterioration of salmon during frozen storage

Increased intensity of train oil taste, bitterness, and metal taste are the most pronounced sensory changes during frozen storage of salmon (Refsgaard, H. H. F.; Brockhoff, P. B.; Jensen, B. Sensory and Chemical Changes in Farmed Atlantic Salmon (Salmo salar) during Frozen Storage. J. Agric. Food Chem. 1998a, 46, 3473-3479). Addition of each of the unsaturated fatty acids: palmitoleic acid (16:1, n - 7), linoleic acid (C18:2, n - 6), eicosapentaenoic acid (EPA; C20:5, n - 3) and docosahexaenoic acid (DHA; C22:6, n - 3) to fresh minced salmon changed the sensory perception and increased the intensity of train oil taste, bitterness, and metal taste. The added level of each fatty acid ( approximately 1 mg/g salmon meat) was equivalent to the concentration of the fatty acids determined in salmon stored as fillet at -10 degrees C for 6 months. The effect of addition of the fatty acids on the intensity of train oil taste, bitterness and metal taste was in the order: DHA > palmitoleic acid > linoleic acid > EPA. Formation of free fatty acids was inhibited by cooking the salmon meat before storage. Furthermore, no changes in phospholipid level were observed during frozen storage. The results suggest that enzymatic hydrolysis of neutral lipids plays a major role in the sensory deterioration of salmon during frozen storage.     

Liquid chromatographic determination of basic nitrogen-containing polynuclear aromatic hydrocarbons in smoked foods

Several smoked foods were analyzed for basic nitrogen-containing polynuclear aromatic hydrocarbon (NPAH) content by a relatively rapid liquid chromatographic (LC) technique. The analyzed products included both domestic and imported market basket commodities. Nanogram quantities of NPAH standards were detected by UV and fluorescence detectors connected in series. The NPAHs were extracted from basic aqueous ethanolic solution into cyclohexane, extracted from cyclohexane into 6N HCl, and extracted back into cyclohexane after neutralization of the acid. The NPAHs were then purified by filtering the extract through deactivated basic alumina. The eluate from this step was concentrated to dryness, and the residue was dissolved in 95% ethanol and analyzed by LC, using a Vydac C-18 column and acetonitrile-water (9 + 1) as the mobile phase. Recoveries of 3 NPAHs, 5,7-dimethylbenz(a)acridine, dibenz(a,j)acridine, and dibenz(a,h)acridine, each added to salmon and sausage at the 5 ppb level, ranged from 62 to 101% by fluorescence measurement and from 64 to 106% by UV measurement. None of the NPAHs used as standards were found by either fluorescence or UV detection at levels greater than or equal to 5 ppb in any of the foods analyzed.     

Metabolism of a new herbicide, [(14)c]pyribenzoxim, in rice

The in vivo metabolism of a new herbicide pyribenzoxim (benzophenone Ο-[2,6-bis(4,6-dimethoxypyrimidin-2-yloxy)benzoyl]oxime) in rice was carried out using container trials. Two radiolabeled forms of [carbonyl-(14)C]pyribenzoxim (P1) and [ring-(14)C(U)]pyribenzoxim (P2) were treated separately as formulations for foliar treatment by single applications of 50 g of active ingredient (ai)/ha at the 4-6 leaves stage. At 0, 7, 30, and 60 days after treatment (DAT), samples of panicle, foliage/rest of plant, and roots were taken for analysis. Upon harvest (120 DAT), rice plants were separated into grain, husk, straw, and root parts. Total radioactive residues (TRRs) at each sampling date were determined to show that the final radioactive residues at harvest were low in grain, husk, straw, and roots, accounting for <17 ppb. The concentration of final residues in the rice plant decreased rapidly, and less than 0.1% of initial TRRs remained at harvest. At 7 DAT, metabolite 1 [M1, 2,6-bis(4,6-dimethoxypyrimidin-2-yloxy)benzoic acid] and two unknown compounds (other-1 and other-2) were detected in foliage extract, accounting for 3.5% TRRs (21.0 ppb), 3.1% TRRs (19.0 ppb), and 9.0% TRRs (54.3 ppb), respectively, while 26.1% of M1 was observed in solvent wash. Any other metabolites were not detected in the plant, including expected metabolite M3 (benzophenone oxime). On the basis of the results obtained, a metabolic pathway of pyribenzoxim in a rice plant was proposed.