Determination of polybrominated biphenyl residues in dairy products.

A method for the determination of polybrominated biphenyls (PBBs) in dairy products is described. Fat is extracted from the products by the official AOAC method. The PBB residues are separated from the fatty material by gel permeation chromatography prior to gas-liquid chromatographic (GLC) quantitation. An additional cleanup using petroleum ether elution through a miniature Florisil column is necessary for thin layer chromatographic (TLC) confirmation. Recoveries of PBBs from samples fortified at levels from 0.1 to 0.5 ppm ranged from 94 to 104% with an average of 99%. GLC sensitivity permits the estimation of PBB residue levels as low as 0.007 ppm. Routine TLC confirmation is limited by sensitivity to greater than or equal to 0.2 ppm.     

Confirmation of polybrominated biphenyl residues in feeds and dairy products, using an ultraviolet irradiation-gas-liquid chromatographic technique.

An ultraviolet (UV) irradiation-gas-liquid chromatographic (GLC) detection procedure was used to confirm the presence of polybrominated biphenyl (PBB) residues in sample extracts after GLC quantitation. Chromatograms of PBB standard and sample extract solutions showed similar photodecomposition peak patterns dependent on time and intensity of UV irradiation. Confirmation of PBBs in sample extracts by this procedure was possible for any amount detectable by the GLC system employed. Prolonged UV irradiation resulted in complete disappearance of all PBB peaks from the chromatogram, permitting their distinction from background peaks due to extraneous sample material unaffected by UV irradiation.     

Liquid chromatographic determination of tetracycline residues in animal feeds

A liquid chromatographic method for the multiresidue determination of tetracyclines (TCs) in feeds is described. The levels of quantitation were 10 ppm each for tetracycline-HCl (TC), oxytetracycline (OTC), and chlortetracycline-HCl (CTC); the detection limit was 40 ppb for each. The calibration curves were linear between 2.5 and 100 ppm. The procedure involved double extraction with pH 2.0 and pH 4.5 McIlvain buffers, cleanup on a Sephadex LH-20 column, separation on a Nova-Pak C18 column, and detection at 370 nm. Recoveries of 10 micrograms/g of each TC in multiresidue feed samples ranged from 55.8 to 75.5% for OTC, 71.6 to 100% for TC, and 22.4 to 60.6% for CTC. The identities of the TCs were confirmed by thin layer chromatography.     

Identification of nosiheptide in feeds and detection of residues in animal tissues

Nosiheptic is determined in fermentation broths of Streptomyces actuosus either by a microbiological method using Staphylococcus aureus or, more easily, by an automated colorimetric method. The results obtained with both methods correspond well for concentrations greater than 100 microgram/mL with a standard deviation of 1-3%. For determination of nosiheptide as a feed additive, the microbiological assay is made more specific by pretreatment with petroleum ether and 1N HCl. Standard deviation is less than 4%, and the assay is sensitive to 1 ppm. Nosiheptide is identified in feed containing other frequently used antibiotics by thin layer chromatography with bioautography; sensitivity is 1 ppm. The absence of traces of nosiheptide in tissues of treated swine and broiler is confirmed by microbiological diffusion, sensitive to 0.025 ppm.     

Determination of vitamin B12 in dry feeds by atomic absorption spectrophotometry.

Vitamin B12 was determined in dry feeds by atomic absorption spectrophotometry (AAS). Samples containing B12 were extracted with an assay solution, 5 g EDTA was added to the filtrate, the pH was adjusted to 7 with NH4OH, and 5 g charcoal was added. The charcoal was removed by filtering through ashless paper which was then placed in a beaker and ashed at 600 degrees C. After dissolving the cobalt oxide from the ash in 5N HNO3, cobalt content was determined by using AAS. To determine mg B12/lb feed, ppm cobalt in the feed is multiplied by 10.43. The sensitivity of the proposed procedure is 1 mg vitamin B12/lb. The procedure is rapid and precise, and results compare favorably with AOAC method 43.109.     

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.     

Detection of low-level carbadox residues in animal feeds by high pressure liquid chromatography

The high pressure liquid chromatographic (HPLC) method is capable of detecting from 1 to 0.024 ppm methyl 3-(2-quinoxalinyl-methylene)carbazate-N1,N4-dioxide (carbadox). Carbadox is extracted from the feed with 2% NH4OH in acetone, passed through a liquid-liquid partition, subjected to HPLC, and detected by using a 365 nm detector. No feed materials or other active drug ingredients produced false positive results.     

Determination of biphenyl residues in citrus fruit by derivative infrared spectrophotometry

A method for determination of biphenyl residues in whole citrus fruit is described. The mascerated fruit was distilled in an acid medium, the distillate was extracted with cyclohexane, and biphenyl was determined in the extract using various measures obtained by first and second derivative infrared (IR) spectrophotometry. Calculations were performed by PE680 and SNGLE programs on data obtained using a Perkin-Elmer Model 3600 data station. The relative precision of the determinations at 70 ppm was 1.8 to 2.0%; the detection limit was 5 ppm in all measurements, and recovery of spiking concentrations of 20 to 80 ppm ranged from 90.2 +/- 5.5% (for the amplitude of the 739 cm-1 peak of the first derivative) to 97.5 +/- 2.0% (for the trough-to-peak difference from the 737 cm-1 minimum to the 743 cm-1 maximum of the second derivative.     

Detection of arsanilic acid in animal feeds.

The animal feed sample is extracted with alcohol, the extract is evaporated to a small volume, and an aliquot is spotted on a silica gel H plate. After development of the plate in chloroform-methanol (80+20), the spotting area is scraped and extracted with ethanol, which is evaporated to dryness. The residue is dissolved in 3 ml alcohol, and an aliquot is reacted with p-dimethylaminobenzaldehyde solution; another aliquot of the extract is used as a sample blank. A control is prepared by spiking another portion of the same feed with about 5 ppm arsanilic acid. The method is applicable in the presence of procaine and sulfonamides.     

Determination of virginiamycin in feeds.

Virginiamycin was extracted from the feed by ethanol-pH 2.5 phosphate buffer (1 + 1). The pH during extraction was adjusted (when necessary) to between 4 and 5. Sample dilutions and the standard dose response line were prepared to contain ethanol pH 6 phosphate buffer (2 + 8), and the test organism was Sarcina lutea. Three feeds (a poultry ration, a swine finishing ration, and a swine starter ration) showed virginiamycin recovery of 88.8--108.9% when standard solutions were added at concentrations of 4.54--90.8 g/ton. The coefficient of variation (4--20%) was larger for low potency feeds (10 g/ton) compared to the higher feeds (100 g/ton). Similarly, excellent recovery was obtained when the swine starter feed was fortified by a commercial premix. Amprolium, roxarsone, and monensin can be present at 20 times the concentration of virginiamycin with little or no interference in the antibiotic determination. Lasalocid at 10 times the concentration of virginiamycin caused a slightly positive bias (recovery, 107.4%).     

Agar well technique for antibiotics in animal feeds.

The agar well technique compares favorably with the cylinder plate assay in accuracy, sensitivity, and precision. It is more flexible and more rugged, and growth of seed organism is not inhibited. The wells are precision cut with Grafar gel punch assembly with sets (6) of 10, 7, 5, 4, and 3 mm cutters. The wells are easily and rapidly filled with short, disposable Pasteur pipets fitted with rubber bulbs. The smaller wells are filled with capillary pipets. The diameter of the well (independent of volume) appears to be a function of concentration. For every decrease in the diameter size of the well, concentration can be increased, at least to the next higher level of the standard response line. Extracts of chlortetracycline containing as much as 3.2 mu-g/ml can be analyzed if the 3 mm wells are used and with no sacrifice in accuracy or precision. This works especially well for antibiotic premixes. The technique has been used successfully for penicillin, streptomycin, chlortetracycline, oxytetracycline, oleandomycin, and tylosin.     

High-speed liquid chromatographic determination of biphenyl residues in citrus products.

A simple, direct, and rapid method is given for the analysis of citrus oils for the fungistat biphenyl by high-speed liquid chromatography. The method is extended to orange juice and various "dry flavors" by an extraction procedure. Analytical limits are less than 1 ppm without need for any cleanup or concentration steps.     

Determination of xanthomegnin in grains and animal feeds by liquid chromatography with electrochemical detection

A sensitive, highly selective liquid chromatographic (LC) method is described which uses electrochemical (EC) reduction of the analyte in the determinative step. The method is capable of determining xanthomegnin in mixed animal feeds and grains at levels ranging from 15 to 1200 ng/g. The method can detect as little as 0.5 ng xanthomegnin injected on the LC column. Xanthomegnin is extracted with chloroform and 0.1M phosphoric acid. An aliquot of the crude extract is purified by silica gel column chromatography using a Sep-Pak silica gel cartridge. A novel feature of the method is that xanthomegnin is "backed off" the column by reversing the flow of the eluant through the column. LC is then used to separate xanthomegnin from other interfering substances. Xanthomegnin is detected by EC reduction at -0.16 V. Recoveries of xanthomegnin added to samples at levels ranging from 15 to 1200 ng/g averaged 79% with a coefficient of variation of 7.9%. Results also demonstrate that this LC system can separate the related metabolites viomellein and rubrosulphin from each other and from xanthomegnin and that the same EC detection system can be used to detect these metabolites.     

Determination of amprolium in feeds: collaborative study.

The official final action method, 42.028--42.032, for determining amprolium in feeds was modified by a change in the preparation of aluminum oxide for chromatography. A premix containing 0.5% amprolium was collaboratively studied by the modified and the official methods. Compared with the modified method, 87.7% of the drug was recovered from the premix by using the official method. The modification makes possible the assay of premixes as well as finished feeds. The official final action method has been modified to incorporate this change.     

Determination of crude protein in animal feeds, using block digestion followed by steam distillation: collaborative study.

A method consisting of digesting animal feeds in a block digestor and determining ammonia by steam distillation followed by titration has been evaluated and compared with the official final action Kjeldahl method, 7.016. Fifteen laboratories analyzed 5 feed samples and lysine monohydrochloride. Statistical analysis showed that results from the 2 methods were comparable. The distillation technique has been adopted as official first action as an alternative technique for ammonia determination from the digest of the official final action block digestor method, 7.B11.     

Determination of penicillin G residues in edible animal tissues by liquid chromatography

An improved method has been developed for the determination of benzyl penicillin in animal tissues. Tissues are fortified with a known amount of penicillin V (internal standard) and extracted with water. The extract is deproteinized with sulfuric acid and sodium tungstate, filtered, and concentrated on a conditioned C18 solid phase extraction column. Penicillin V and benzyl penicillin are then eluted from the column with 1 mL 60% acetonitrile-35% water-5% 0.2M phosphate buffer solution and derivatized with 1 mL 1,2,4-triazole-mercuric chloride solution at 65 degrees C for 30 min. An aliquot of this sample is analyzed by reverse phase liquid chromatography with UV detection at 325 nm. The limit of detection is 5 micrograms/kg (ppb) penicillin G (8.4 IU/kg) in liver, kidney, and muscle tissues).     

Determination of polybrominated diphenyl ethers and polybrominated dibenzo-p-dioxins/dibenzofurans in marine products

Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants in plastics and textile coatings, and these compounds have been recognized as ubiquitous environmental contaminants. Furthermore, it is considered a serious problem that polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/DFs), having toxicities similar to those of chlorinated dioxins, are generated by the manufacture of brominated flame retardants (BFRs) such as PBDEs, and formed by the combustion of substances containing BFRs. Several congeners of PBDD/DFs and PBDEs have been detected in the adipose tissue of the Japanese. Although food is suspected as an exposure source, little information is available regarding the levels of these brominated compounds in food, as compared with information regarding dioxin or polychlorinated biphenyls. It is necessary to investigate the levels of these brominated organic compounds in various foods and to estimate their influence in the case of human exposure. We developed an efficient method of analyzing PBDEs and PBDD/DFs contents in food samples using accelerated solvent extraction and determined the concentrations in several marine products such as raw fish, processed foods, and seaweed purchased in Japan. A recovery test (n = 5) using the method and involving dried fish showed acceptable recoveries of 57.7-78.5% (RSD 5.4-15.9%) for PBDEs and 50.0-56.4% (RSD 1.5-7.9%) for PBDD/DFs. In the analysis of marine product samples, several congeners of PBDEs were detected in raw fish, processed fish, and seaweed; the highest concentration of sigmaPBDEs was detected in yellowtail (1161 pg/g whole basis), followed by mackerel (553.5 pg/g whole basis). The most dominant congener present in these marine samples was 2,2',4,4'-tetraBDE (#47).     

Turbidimetric assay of tylosin in animal feeds containing urea

A turbidimetric method is described for determination of tylosin in animal feeds containing urea. This method includes several modified or new steps to existing turbidimetric and AOAC plate assays that improve the extraction of tylosin, remove interferences from feeds, free tylosin activity, concentrate tylosin from low-level feeds, and reduce variability of assay results. A larger analytical sample size has been incorporated into the assay to decrease variability of assay results. A methanol-phosphate buffer extraction solution has replaced the hot buffer and methanol extraction solution. A hydrolysis step, which is not contained in the AOAC plate assay, was developed to free tylosin from the tylosin urea adduct that forms over time in feeds containing urea. A disposable C18 column was used to concentrate tylosin from feeds at levels less than 15 ppm. By increasing the analytical sample size from 25 to 100 g, the coefficient of variation for 12 weighings of cattle feed was reduced from 28.4 to 9.3%. Average recoveries from cattle rations containing tylosin at levels of 8, 10, and 100 ppm were 94, 94, and 91%, respectively.