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.     

Detection of sulfonamides in animal feeds.

The animal feed sample is extracted with ethanol or acetone and the extract is evaporated to dryness. Another portion of the same sample, spiked at the 3 ppm level with 8 sulfonamides, is similarly extracted and the extract is evaporated to dryness. The residue from each solution is dispersed with 5 ml 0.1N NaOH and, following the addition of 1 ml 1N HCl and mixing, the solution is filtered. The filtrate is mixed with Celite, transferred to a column, and eluted with ammoniacal ether. Aliquots of the concentrated sample and control eluates are spotted on a neutral Adsorbosil-1 thin layer chromatographic (TLC) plate. Following development in chloroform-methanol (95+5) and drying, the plate is sprayed with an alcoholic solution of p-dimethylaminobenzaldehyde until the control chromatogram shows 6 yellow spots which are, from top to bottom; sulfadimethoxine (SO), the combined sulfamerazine (SM)-sulfamethazine (SH)-sulfaquinoxoline (SQ) spot, sulfadiazine (SD), sulfapyridine (SP), sulfathiazole (SZ), and sulfaguanidine (SG). A spot on the sample chromatogram can be identified if the Rf is identical to one of the 5 sulfonamides not overlapping with another compound. If the Rf of the sample spot is approximately the same as that of the combined SM-SH-SQ spot, more definite identification can be obtained by using basic Adsorbosil-1 TLC plates, with chloroform-methanol (92+8) as the developing solvent.     

Atomic absorption spectroscopic determination of lead extracted from acid-solubilized tissues.

A method is presented for determining lead in a variety of tissues. Lyophilized samples are solubilized with nitric acid at room temperature in glass screw-cap culture tubes. Following neutralization with sodium hydroxide and sodium bicarbonate, the lead is extracted into methyl isobutyl ketone as the pyrrolidine dithiocarbamate complex and analyzed by flame atomic absorption spectrophotometry. Brain, heart, liver, lung, and spleen gave recoveries ranging from 92 to 102% with standard deviations of less than 8%. Aorta, kidney, and rib were unsuitable for analysis by this method. A large number of samples can be analyzed without specialized equipment or intricate experimental steps. The detection limit is 35 ng/g tissue (wet weight) and sensitivity is approximately 140 ng/g tissue (wet weight).     

A method for extraction of pentachlorophenol from plant tissues

Pentachlorophenol (PCP) was extracted from well homogenized and acidified plant samples with diethyl ether. Partial purification was carried out by the partition of PCP between ether extract and aqueous sodium hydroxide (5%), then between the acidified sodium hydroxide solution and benzene. The final purification of PCP was achieved by its good gas chromatographic separation during the quantitative determination. Techniques for optimizing the efficiency of the method were described in detail. The efficiency of the method was found to increase with the increase of the starting quantities of PCP, where it reached 89.2% for amounts greater than 400 ng. The loss of PCP due to its presence in the plant tissues was 29.3 and 21.6% for concentrations of PCP lower than 35.5 and higher than 46.2 ppb in the plant respectively. The overall recovery of PCP from plant tissues was found to be about 60% and 70% at the mentioned low and high concentrations in the plant.     

On-line HPLC-DPPH screening method for evaluation of radical scavenging phenols extracted from apples (Malus domestica L.)

An on-line HPLC-DPPH screening method for phenolic antioxidants in apple methanol/water (80:20, v/v) extracts was applied. The determination of antioxidants was based on a decrease in absorbance at 515 nm after postcolumn reaction of HPLC-separated antioxidants with the 2,2'-diphenyl-1-picrylhydrazyl radicals (DPPH*). Each of the antioxidants separated by the HPLC column was observed as a negative peak corresponding to its antioxidative activity. The on-line method was applied for quantitative analysis of the antioxidants. A linear dependence of negative peak area on concentration of the reference antioxidants was observed. For validation of the on-line method the limit of detection, LOD (microg/mL), and the limit of quantification, LOQ (microg/mL), of the phenolic compounds were determined. Comparison of the UV and DPPH radical quenching chromatograms with authentic compounds identified catechin, chlorogenic acid, caffeic acid, epicatechin, and phloridzin in the apple cultivars (Lobo, Golden Delicious, and Boskoop), and the distribution of total antioxidant activity was calculated.     

A screening method for protein characterization and differentiation.

A circular paper chromatographic method was developed for the separation of the amino acids in proteins into 7 subgroups. Butanol-acetic acid water (4+1+1) was used as the developing solvent. Eluted ninhydrin-stained aminograms gave rise to graphic profiles or numerical indexes based on absorbance percentages. The profiles can be used to compare protein-containing samples. Twenty different samples were studied through 190 comparisons of graphic profiles and coefficients of correlation, with only 4% misleading results. The method showed excellent reproducibility for the identification or differentiation of proteins and has the advantage of being performed with low-priced apparatus and reagents.     

ELISA for sulfonamides and its application for screening in water contamination

Two enzyme-linked immunosorbent assays (ELISAs) were tested for their suitability for detecting sulfonamides in wastewater from various stages in wastewater treatment plants (WWTPs), the river into which the wastewater is discharged, and two swine-rearing facilities. The sulfamethoxazole ELISA cross-reacts with several compounds, achieving detection limits of <0.04 microg/L for sulfamethoxazole (SMX), sulfamethoxypyridine, sulfachloropyridine, and sulfamethoxine, whereas the sulfamethazine (SMZ) ELISA is more compound specific, with a detection limit of <0.03 microg/L. Samples from various stages of wastewater purifications gave 0.6-3.1 microg/L by SMX-ELISA, whereas river samples were approximately 10-fold lower, ranging from below detection to 0.09 microg/L. Swine wastewater samples analyzed by the SMX-ELISA were either at or near detectable limits from one facility, whereas the other facility had concentrations of approximately 0.5 microg/L, although LC-MS/MS did not confirm the presence of SMX. Sulfamethazine ELISA detected no SMZ in either WWTP or river samples. In contrast, wastewater samples from swine facilities analyzed by SMZ-ELISA were found to contain approximately 30 microg/L [piglet (50-100 lb) wastewater] and approximately 7 microg/L (market-weight hog wastewater). Sulfamethazine ELISA analyses of wastewater from another swine facility found concentrations to be near or below detection limits. A solid phase extraction method was used to isolate and concentrate sulfonamides from water samples prior to LC-MS/MS multiresidue confirmatory analysis. The recoveries at 1 microg/L fortification ranged from 42 +/- 4% for SMZ to 88 +/- 4% for SMX ( n = 6). The ELISA results in the WWTPs were confirmed by LC-MS/MS, as sulfonamide multiresidue confirmatory analysis identified SMX, sulfapyridine, and sulfasalazine to be present in the wastewater. Sulfamethazine presence at one swine-rearing facility was also confirmed by LC-MS/MS, demonstrating the usefulness of the ELISA technique as a rapid and high-throughput screening method.     

Rapid immunochemical screening method for aflatoxin B1 in human and animal urine

A method has been developed to determine the presence of aflatoxin B1 in the urine of animals (including humans) by utilizing commercial immunochemical kits that can be used in the field. Urine is treated with diatomaceous earth and filtered to clarify the sample; 2-3 ppb aflatoxin B1, corresponding to about 300 ppb in the ingested feed/food, can be detected in the filtered urine without further purification. To improve sensitivity, the urine filtrate is passed through a C18 solid phase column to extract the aflatoxin. The column is washed with acetonitrile-water (15 + 85) and water, aflatoxin B1 is eluted with methanol-water (7 + 3), and water is added to the eluate, which is then tested for aflatoxin with the test kit. The limit of detection is 0.2 ppb, reflecting consumption of 40 ppb or more aflatoxin in the feed/food. When the initial sample volume is adequate, purification through the C18 column step is usually sufficient. For limited sample volumes, the eluate from the C18 column is mixed with water, added to an immunosorbent affinity column, and washed with water to remove excess sample matrix and impurities. Aflatoxin B1 is eluted with acetonitrile. The extract is evaporated under nitrogen and the residue is redissolved in methanol-water (25 + 75). At this purification stage, the limit of detection is reduced to 0.05 ppb.     

Thin layer chromatographic/bioautographic method for identification of antibiotic residues in animal tissues

An analytical method for the identification of the residues from 14 commonly used antibiotics is presented. The technique is based on selective tissue extraction followed by thin layer chromatography (TLC)/bioautography. Antibiotic residues are extracted from the tissues with methanol and methanol-HCl (98 + 2). The methanol extract is further extracted with chloroform to isolate groups of antibiotics. The extracts are spotted onto TLC plates and developed in suitable solvent systems. Developed plates are placed on set medium seeded with Bacillus subtilis and a bioautograph is produced. The locations of zones of inhibition are used to identify antibiotic residues. Recoveries of antibiotics were quantitative, while the effect of naturally inhibiting components of the matrix was minimized. The sensitivity of the method can be adjusted through minor modifications, which allows its use in routine regulatory analysis.     

Thin layer chromatographic screening method for sulfadiazine residues in calf tissues, plasma, and urine

Because of the lack of specificity of the Bratton-Marshall procedure for assaying sulfonamides, a sensitive, specific tissue residue assay for sulfadiazine (SDZ) was developed. The methodology has been extended to provide a highly sensitive screen for sulfonamide residues, which employs 2-dimensional thin layer chromatography in conjunction with fluorescamine derivatization. The procedure described, which has been developed for SDZ in calf tissues, involves direct ethyl acetate extraction of tissue homogenates. Following evaporation of the organic phase, a portion of the residue is spotted on a 20 X 20 cm silica gel 60 plate, which is then developed in 2 dimensions with solvent systems devised to separate SDZ from endogenous substances as well as from 12 other sulfonamides that might be present in calf tissues. The presence of SDZ at a concentration of 0.1 ppm or its absence is easily demonstrated in calf kidney, liver, muscle, plasma, and urine. The basic method can be modified for a particular sulfonamide in a target tissue and can be used as a quantitative assay for sulfonamide residues.     

High-performance liquid chromatographic method for determination of biogenic amines in feedstuffs, complete feeds, and animal tissues

Numerous methods to analyze biogenic amines in biological materials have been described. A versatile and rapid methodology to analyze these compounds in feedstuffs, complete feeds, and animal tissues, however, has not been reported. The current method was developed to address this need. Biogenic amines in feedstuffs, complete animal feeds, and animal tissues were extracted with 10% trichloroacetic acid, reacted with O-phthaladehyde using high-performance liquid chromatographic employing a cation exchange column. Detection limits were 50 pmol/mL for tyramine, histamine, putrescine, and spermine; 40 pmol/mL for cadaverine; and 25 pmol/mL for spermidine. Extraction efficiency of biogenic amines in feedstuffs, duodenum, liver, ileum + jejunum, and whole shrimp and shrimp hepatopancreas ranged between 99-105, 93-135, 80-85, 65-102, 88-98, and 88-97%, respectively. It can be concluded that the current method can be applied to individual feedstuffs, complete feeds, and animal tissues for the rapid and accurate determination of concentration of biogenic amines.     

Methods for determination of ionophore-type antibiotic residues in animal tissues

Methods for the analysis of polyether antibiotics in animal tissues and fluids are described. For monensin and nigericin, only methods based on bioautography are available. For lasalocid, in addition to TLC bioautography for quantitation in chicken skin and fat, LC methods based on fluorescence detection have been developed for quantitation in animal blood, milk, liver, skin, and fat. In addition, a confirmatory method for lasalocid is described; this is based on purification by LC followed by silylation and pyrolysis gas chromatography-positive chemical ionization mass spectrometry.     

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.     

Rapid screening method for aflatoxins and zearalenone in corn.

The methanol-water extraction system used in AOAC Method II for aflatoxins extracts both the aflatoxins and zearalenone from corn. Using this methanol-water extraction system as a base, a rapid screening procedure has been developed for these mycotoxins. The methanol-water extract is defatted with hexane and the pigments are precipitated with copper carbonate. The aflatoxins and zearalenone are subsequently extracted into chloroform and are then detected by half-plate TLC. An elapsed time of about 1 hr is required to analyze 1 sample. The sensitivity of the method is about 2 mu-g/kg for aflatoxin B-1 and 100 mu-g/kg for zearalenone.