Rapid procedure for determination of nicarbazin residues in chicken tissues

A procedure is described for the quantitation of nicarbazin residues in chicken tissues. The method includes extraction of tissue with chloroform-ethyl acetate-dimethyl sulfoxide (50 + 50 + 0.8), adsorption on neutral alumina, and subsequent elution of the residues with methanol-pH 6.0 phosphate buffer (1 + 1). Extracts are separated on a 15 cm, 5 micron C18 column with methanol-pH 6.0 phosphate buffer (6.5 + 3.5) as the mobile phase. The dinitrocarbanilide portion of the complex is detected and quantitated with an electrochemical detector in the reductive mode. Recoveries, based on dinitrocarbanilide, were greater than 95% in liver, breast, and thigh muscle tissues fortified with 0.25-8.0 ppm nicarbazin.     

Liquid chromatographic determination of zoalene and its metabolites in chicken tissues with electrochemical detection

A procedure is described for the quantitation of Zoalene (3,5-dinitro-o-toluamide) and its 2 major monoamino metabolites in chicken tissues. The method includes blender extraction of tissue with chloroformethyl acetate (1 + 1), adsorption of the drug and metabolites on neutral alumina, and subsequent elution of the residues with pH 3.5 formate buffer-methanol (6.5 + 3.5). Recovered residues were separated on a 5 micron C18 column with the alumina eluting solvent as the LC mobile phase. The parent drug and metabolites were detected and quantitated with an electrochemical detector in the reductive mode with a minimum level of reliable measurement of 0.1 ppm. Overall mean recoveries greater than 85% were obtained with Zoalene and its 2 monoamino metabolites in breast, thigh, and liver tissues fortified with 0.25-2.00 ppm. The results on tissues from chickens fed a diet containing 0.0125% Zoalene are presented.     

Liquid chromatographic determination of amprolium in chicken tissues, using post-column reaction and fluorometric detection

A method is presented for determination of amprolium residues in chicken muscles by a liquid chromatographic post-column reaction system. The drug is extracted from muscles with methanol, and the extract is concentrated to 3-4 mL. This aqueous solution is rinsed with n-hexane and cleaned up by alumina column chromatography. The drug is separated from the interferences on a LiChrosorb RP-8 column, reacted with ferricyanide in alkaline solution, and quantitated by fluorometric detection at 367 nm (excitation) and 470 nm (emission). Recoveries of amprolium added to chicken muscles at levels of 0.1 and 0.2 ppm were 74.9 and 80.9%, respectively. The detection limit was 1 ng for amprolium standard and 0.01 ppm in chicken muscles.     

Liquid chromatographic determination of spiramycin residues in chicken tissues

A liquid chromatographic (LC) method is described for determination of spiramycin residues in chicken muscles. The drug is extracted from muscles with acetonitrile, the extract is concentrated to 3-4 mL and rinsed with n-hexane followed by ethyl ether, and the drug is extracted with chloroform. LC analysis is carried out on a Zorbax BP-C8 column, and spiramycin is detected spectrophotometrically at 231 nm. Recoveries of spiramycin added to chicken muscles at 0.2 and 0.1 ppm were 93.9 and 89.0%, respectively. The detection limit was 5 ng for spiramycin standard, and 0.05 ppm in chicken muscles.     

Liquid chromatographic determination of monensin in chicken tissues with fluorometric detection and confirmation by gas chromatography-mass spectrometry

An accurate, sensitive method is described for the determination of monensin residue in chicken tissues by liquid chromatography (LC), in which monensin is derivatized with a fluorescent labeling reagent, 9-anthryldiazomethane (ADAM), to enable fluorometric detection. Samples are extracted with methanol-water (8 + 2), the extract is partitioned between CHCl3 and water, and the CHCl3 layer is cleaned up by silica gel column chromatography. Free monensin, obtained by treatment with phosphate buffer solution (pH 3) at 0 degrees C, is derivatized with ADAM and passed through a disposable silica cartridge. Monensin-ADAM is identified and quantitated by normal phase LC using fluorometric detection. The detection limit is 1 ppb in chicken tissues. Recoveries were 77.6 +/- 1.8% at 1 ppm, 56.7 +/- 7.1% at 100 ppb, and 46.5 +/- 3.7% at 10 ppb fortification levels in chicken. Gas chromatography-mass spectrometry is capable of confirming monensin methyl ester tris trimethylsilyl ether in samples containing residues greater than 5 ppm.     

Liquid chromatographic determination of six sympathomimetic drugs in dosage forms

A simple and rapid stability-indicating liquid chromatographic method is described for quantitative determination of 6 sympathomimetic drugs in various liquid and solid formulations. Analyses were carried out on a C18 reverse phase column using 0.01M 1-octanesulfonic acid, sodium salt in 0.2% acetic acid-methanol (70 + 30) as the mobile phase with photometric detection at 220 nm. Coefficients of variation for 5 consecutive injections of a mixed standards solution ranged from 0.62% for metaraminol to 1.40% for epinephrine. Standard recoveries ranged from 98.8% for metaraminol to 100.8% for epinephrine. The method was linear between 0.2 and 10 micrograms of drug injected and was used successfully to analyze 17 commercial products in a variety of dosage forms.     

Modified fast procedure for the detection and screening of antiglycative phytochemicals

In an attempt to elevate temperature to facilitate glycation, a nonenzymatic reaction by incubation of bovine serum albumin (BSA) and fructose at 50 °C for 24 h has been developed. As conducted and compared to a routine procedure by incubation of BSA and fructose at 37 °C for 168 h, the reactant fluorescence intensities and SDS-PAGE-detected glycated BSA quantities produced by both test temperatures increased with time of incubation. As the Amadori products and α-dicarbonyl compounds during incubation were quantified, both quantities produced at each temperature also increased with an increase of time of incubation, and their trends of changes at both temperatures were similar. In practical application for the detection and screening of the antiglycative phytochemicals, each of 20 peanut root extracts was introduced to a series of BSA-fructose solutions and incubated at 37 and 50 °C for 168 and 24 h, correspondingly. All extracts exhibited notable activities and varied depending on peanut origins. Pair comparison of the resultant antiglycative activities determined at 37 and 50 °C showed that both determined activities for each peanut root extract deviated limitedly. As further analyzed, SDS-PAGE-detected glycated BSA quantities formed at 50 °C were closely proportional to the antiglycative activities determined on the basis of their fluorescence intensities. It is of merit to demonstrate that fluorescence-based determination of BSA-fructose reactant after incubation at 50 °C for 24 h is practical and time-saving in the detection and screening of antiglycative phytochemicals.     

Rapid, low cost thin-layer chromatographic screening method for the detection of ochratoxin A in green coffee at a control level of 10 microg/kg.

A thin-layer chromatographic (TLC) screening method was developed for the detection of ochratoxin A (OTA) in green coffee at a control level of 10 microg/kg. The method is based on extraction of OTA with a mixture of phosphoric acid and dichloromethane, purification by liquid-liquid partition into sodium hydrogen carbonate, separation by normal-phase TLC, and detection by visual estimation of fluorescence intensity under a UV lamp at 366 nm. The method was validated by performing replicate analyses of uncontaminated green coffee material spiked at 3 different levels of OTA (5, 10, and 20 microg/kg), and also by comparing results obtained on a series of test trial green coffees naturally contaminated with OTA (range 0.2 to 136.7 microg/kg) with those measured by a quantitative immunoaffinity/HPLC method. The agreement between the two methods was excellent, and neither false positive nor false negative results were recorded. This screening method is rapid, simple, robust, and very cheap, which makes it particularly well adapted for implementation in coffee-producing countries.     

Practical screening procedure for sulfathiazole in honey

A simple, inexpensive procedure is described for rapidly screening small samples of honey for sulfathiazole (ST), a drug formerly used but not approved in the United States for the prophylactic treatment of American foulbrood disease of bees. The method uses 2 plastic tubes arranged in tandem. The upper tube contains a bed of alumina, which removes some interfering pigments. The lower tube contains a very small bed of anion exchange resin in the HSO4- form, which traps the ST. The drug is eventually eluted and detected using the Bratton-Marshall diazotization-coupling reagents. Honey containing 0.1 ppm ST can be readily detected. An optional dye concentration step permits the detection of as little as 25 ppb ST.     

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.     

A rapid spectrofluorometric screening method for enrofloxacin in chicken muscle

A simple spectrofluorometric method was developed for screening enrofloxacin (ENRO) in chicken muscle. A single-step extraction with acidic acetonitrile gave the best results without further cleanup. Following centrifugation the supernatants were excited at 324 nm and the emission was measured at 442 nm. Using this procedure, 18 chicken breast samples from 3 producers were tested. The results showed background signal levels significantly lower than those corresponding to 300 microg/kg ENRO, the FDA approved tolerance level. Statistical treatment of these data established a threshold which can be used in subsequent screening of ENRO at the tolerance level. The calibration curve revealed a satisfactory linear relationship (R(2) = 0.9991) in a range of 0-700 microg/kg ENRO in fortified chicken breast. ENRO-incurred samples were examined using this approach, and the results agreed with those obtained from more extensive separation followed by high-performance liquid chromatography. Because the threshold can be set at the 3 sigma limit, reliable screening can be accomplished with an error rate of less than 0.26%. Based on this investigation, a high-throughput screening method for ENRO in chicken tissue is proposed.     

Residues in broiler chick tissues from low level feedings of seven chlorinated hydrocarbon insecticides.

Two chlorinated insecticide feeding studies from 1967-1968, using broiler chicks, have been completed. In Study I, lindane, heptachlor epoxide, dieldrin, endrin, methoxychlor, and DDT were fed in combination at 3 levels: 0.05, 0.15, and 0.45 ppm. Data show that, in fat tissues, heptachlor epoxide attained a level approximately 20 times the respective levels in the feed; dieldrin 15 times; endrin 10 times; p,p'-DDT 9 times; lindane 3 times; and o.p'-DDT less than the feeding levels. Of the DDT metabolites, p.p'-DDE and p,p'-DDD, only p,p'-DDE was significant at 3 times the 0.45 ppm feeding level. Endrin ketone, a metabolite of endrin, reached plateau levels approximately equal to feeding levels. All residue levels in liver tissues were less than 0.02 ppm. In Study II, technical chlordane was fed singly at the 0.05, 0.15, and 0.45 ppm levels. Results are tabulated for both total chlordane and for 6 identifiable isomers. Total chlordane in fat tissues attained plateau levels 3-5 times the respective feeding levels. Total chlordane levels in liver and breast tissues were all less than 0.01 ppm.     

Rapid fluorescence screening assay for enrofloxacin and tetracyclines in chicken muscle

A simple, rapid fluorescence assay was developed for screening both enrofloxacin (ENRO) and tetracyclines in chicken muscle at the U.S. tolerance levels (300 ng/g and 2 microg/g, respectively). Screening for both classes of antibiotics is accomplished using one extraction, thus simplifying and expediting the process. The method requires an initial extraction of chicken muscle with 1% acetic acid in acetonitrile, centrifugation, and analysis of the supernatant for ENRO fluorescence. After addition of ammonium hydroxide, magnesium chloride, and methanol, followed by centrifugation and filtration, the supernatant can be measured for tetracycline fluorescence. Chlortetracycline (CTC) was chosen as a representative tetracycline to demonstrate the method, as it displays intermediate sensitivity among the three tetracyclines approved in the U.S. Comparison of the fluorescence of control and tolerance-level-fortified samples of both ENRO and CTC shows no overlap. Setting a threshold as the average fortified fluorescence minus 3sigma allows for successful screening, as illustrated with blind samples as controls or fortified with ENRO and/or CTC over a range of concentrations. This method can provide an alternative or supplemental approach to currently used microbial screening assays.     

Rapid quantitative thin layer chromatographic screening procedure for sulfathiazole residues in honey

A procedure is presented for the quantitative determination of sulfathiazole residues in honey. Induced fluorescence of sulfathiazole is measured by fluorescent scanning densitometry; sulfaquinoxaline is added as an internal standard for quantitation. Recovery is greater than 98% and results are linear over the range 0.05-0.60 mg/kg. The detection limit, CL (k = 3), is 0.02. The procedure allows a single analyst to process 50-60 samples/day.     

A screening method for determining nitrofuran drug residues in animal tissues.

A method was developed for measuring low levels of total nitrofurans in animal tissues and milk. The antimicrobial nitrofurans (5 or more products) used in agriculture are extracted from tissue with aqueous acid in the presence of ethyl acetate. After centrifugation and evaporation, the organic residue is washed with hexane and the nitrofurans are hydrolyzed to 5-nitrofuraldehyde in aqueous acid at 70 degrees C. The hydrolysis product is extracted with benzene and measured by gas-liquid chromatography with electron capture detection. Recoveries of nitrofurazone and furazolidone from fortified poultry and swine tissues at the levels of 0.5 and 0.1 ppm are 75 and 65%, respectively. This procedure can be used to detect the total nitrofuran content of as little as 10 ppb muscle tissues and milk, 100 ppb liver, and 50 ppb fat with no interference from related veterinary nitrodrugs.     

A convenient thin layer chromatographic cleanup procedure for screening several mycotoxins in oils.

A thin layer chromatographic cleanup development with benzene-hexane (3+1) effectively removed lipids and some contaminants from mixtures of mycotoxins in corn oil, olive oil, peanut oil, soybean oil, and seed extracts. A second development in the same direction as the first, using toluene-ethyl acetate-formic acid (6+3+1) or benzene-acetic acid (9+1), separated the mycotoxins. Satisfactory separation was achieved for commercial oils spiked with sterigmatocystin, zearalenone, ochratoxins A, B, and C, and aflatoxins B1, B2, G1, and G2. This technique permits detection of 5 ppb aflatoxin B1 in corn.     

Development of an enzyme-linked immunosorbent assay for the determination of maduramicin in broiler chicken tissues.

Maduramicin is one of the most widely used coccidiostats in the world. A rapid and accurate analytical method for this drug should provide producers and users with an effective management tool. The current chromatographic methods are sensitive but labor-intensive. This paper reports the development of an enzyme-linked immunosorbent assay (ELISA) based on an immunoaffinity chromatography cleanup procedure for the analysis of maduramicin in broiler chicken tissues (including muscle, liver, and fat). Recoveries from fortified tissue homogenates at levels of 30.0-120.0 microg kg(-)(1) ranged from 76.4 to 107.5% with coefficients of variation of 3.8-16.4%. The limits of detection were 1.0 ng g(-)(1) in muscle, 2.8 ng g(-)(1) in liver, and 1.5 ng g(-)(1) in fat. The ELISA results from the analysis of incurred residue in tissue samples showed the cleanup procedure is viable.     

Thin-layer chromatography/bioautography method for detection of monensin in poultry tissues

Monensin is an effective anticoccidial agent widely used in the poultry industry. Because of concerns over its toxicity, a sensitive, reliable, fast, and simple method for residue detection in poultry tissues is desirable from both a diagnostic and a regulatory view. Many methods of detection are excluded due to this ionophore's complex chemical and biological nature. A common method used for its detection is thin-layer chromatography/bioautography (TLC/B), although this is usually only semiquantitative. A new TLC/B method for monensin detection in poultry tissues was developed and is reported here. Recovery from cardiac muscle, skeletal muscle, and liver and kidney tissues is in the range of 93-97%. A detection limit of 250 micrograms/kg with 99% reliability was achieved with this method. Lower limits (to 10 micrograms/kg) were detectable, but with lower reliability (60%). Quantitative analysis is not possible on samples from fatty tissue.     

Engineering of a broad specificity antibody for simultaneous detection of 13 sulfonamides at the maximum residue level

Sulfa antibiotics (sulfonamides) are a group of molecules sharing the p-aminobenzenesulfonamide moiety. Sulfonamides are used in veterinary and human medicine. Sometimes, the meat or milk of medicated animals is contaminated with residual sulfonamides. Current analytical methods for sulfonamides are unfit for screening of food, because they are either too laborious, insensitive, or specific for a few sulfa compounds only. A rapid immunoassay for detection of all sulfas in a single reaction would thus be useful. Previously, we used protein engineering to improve the broad specificity of sulfa antibody 27G3. In this study, we improved the best mutant of the previous studies with site-directed mutagenesis. The new mutants recognized different sulfonamides with affinities sufficient for detection of all 13 tested sulfonamides below the MRL level. We furthermore demonstrated the functionality of one mutant in some real sample matrices.