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.     

Turbidimetric assay for tetracyclines in feeds using a microtiter plate system

The microtiter plate system for turbidimetric assay of chlortetracycline (CTC) and oxytetracycline (OTC) levels in feeds uses a 96 well microtiter plate, a multichannel pipette, and an ELISA reader to measure turbidity. Feeds are extracted for both tetracyclines using AOAC extraction systems. For CTC, the range of the standard curve is 0.001-0.005 microgram CTC/mL; for OTC, the range is 0.004-0.016 microgram OTC/mL. Repeatability of CTC assays, as shown by the coefficient of variation (CV), ranged from 0.54 to 5.65% for same-day assays and from 2.01 to 9.39% for assays on different days. For OTC, CVs ranged from 2.69 to 10.01% for same-day assays and 3.24 to 9.08% for different-day assays. Average recoveries for CTC were 108.7% for same-day assays and 106.8% for different-day assays; for OTC, average recoveries were 112.4% and 106.5% for same-day and different-day assays, respectively.     

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%).     

Liquid chromatography and fluorescence detection of vitamin A in animal feeds, finished feeds, and premixes

A simple liquid chromatographic method for vitamin A (retinol) in animal feeds is described. The feed is saponified, diluted to minimize interferences, and extracted into petroleum ether with a single step. The analysis is sensitive and specific with liquid chromatography and a fluorescence detector. The minimum level of detection is 15 ng/mL, which is equivalent to 10,000 units/lb vitamin A. The method includes a stable and reproducible standardization of vitamin A that is used to calibrate standard peak heights in terms of units retinol/mL. Guarantees of 10,000 units/lb up to premix levels can be analyzed with good recoveries and precision.     

Ion-pair reverse-phase liquid chromatographic determination of amprolium in complete feeds and premixes

Amprolium is extracted with methanol-water (2 + 1) containing 5mM dioctylsulfosuccinate (DOSS) and 10mM CaCl2. The analyte is separated from coextracted materials by isocratic ion-pair reverse-phase liquid chromatography, following removal of late-eluting materials on an acid alumina cleanup column, and is detected at 270 nm. The mobile phase contains 4mM DOSS with 0.3% diethylamine and 1% acetic acid in 40% acetonitrile. Linearity is satisfactory over the range of 2.5-50 micrograms/mL. Mean recovery, as determined by standard addition to commercial samples, is 100.1%. Accuracy was further tested in studies comparing the LC method to the official AOAC colorimetric method, using commercial samples, and was found to be satisfactory. Studies show that common poultry feed additives, grass meals, and some pelletization aids do not interfere with the analysis; however, when bentonite is present, recovery is decreased. The precision of the method, measured over several experiments on commercial samples, is satisfactory as indicated by coefficients of variation ranging from less than 1 to 4.5. A ruggedness test resulted in an overall CV of 3.2%, indicating the probable success of the method in a collaborative study.     

Liquid chromatographic method for determination of furazolidone in premixes and complete feeds: collaborative study

A liquid chromatographic (LC) method for determining furazolidone in finished feeds and premixes was collaboratively studied. Finished feed sample is extracted with acetone-water (93 + 7) on a Goldfisch apparatus, extracting solvent is removed, and the residual material is dissolved in warm DMF. A solution of tetraethylammonium bromide is added, the fat layer is removed, and the sample is clarified by filtration and injected onto a reverse phase LC system with detection at 365 nm. Premixes, extracted by shaking with DMF and diluted so that the final furazolidone concentration is about 55 micrograms/mL, are chromatographed and detected the same as finished feed samples, using a mobile phase of acetonitrile-2% acetic acid (20 + 80). Ten commercial feed samples were preweighed and supplied to 14 collaborators. The 5 matched pairs were chosen to represent the following allowed levels: 0.0055, 0.022, 0.033, 2.2, and 22%. Two familiarization samples at the 0.0055 and 11% levels were also supplied. Instructions called for a single analysis of each sample. Two results were eliminated by the Dixon test. The coefficients of variation, following treatment by the ranking test, ranged from 2.0 at the 22% level to 6.5 at the 0.0055% level. Calculated F-values are not significant (P greater than 0.01) except for the 0.0055% level samples extracted overnight. This method has been adopted official first action.     

Liquid chromatographic determination of carbadox in complete feeds, premixes, and concentrates: interlaboratory study

A liquid chromatographic (LC) method previously published for the determination of carbadox in finished feeds and premixes was slightly modified and tested in an interlaboratory study. The feed samples are extracted with methanol-acetonitrile (50 + 50) after wetting with water. The extracts are purified over a short alumina column. An aliquot of the eluate is analyzed with reverse phase liquid chromatography with ultraviolet detection. Before the actual interlaboratory study, a prestudy with 2 familiarization feed samples was performed. For the interlaboratory study, 2 series of meal and pelleted samples were prepared with carbadox from different suppliers. Eight collaborating laboratories received 6 feed samples previously milled and ground and 4 pelleted samples which had to be ground by the collaborator's in-house method. Collaborators also received 3 carbadox concentrates (about 10% w/w) and 4 premix samples derived from the concentrates (about 1% w/w). Coefficients of variation under reproducibility conditions were 8.3% for meal samples and 4.9% for pellets. A minor but significant effect was noted for the influence of pelleting temperature on the carbadox content. A minor and insignificant effect was observed for the influence of the milling and grinding procedure on the carbadox content. Alumina cleanup of 1% premixes was not essential, although the resulting chromatograms were cleaner. A slight difference in reproducibility was observed with concentrates (10%) when 0.2 or 0.5 g sample size was used, although the average carbadox concentration found was the same. For premixes and concentrates, coefficients of variation under reproducibility conditions were low, ranging from 2.9 to 7.5%.     

Reverse-phase liquid chromatographic determination of lysine in complete feeds and premixes, using manual precolumn derivatization

A sample portion is hydrolyzed with 6N HCl for 23 h and cooled, the pH is adjusted to 7.7 with NaOH, and the solution is diluted with pH 7.7 borate buffer. An aliquot of the sample extract is derivatized with 9-fluorenylmethyl chloroformate (9-FMC). Lysine is separated from other amino acids by isocratic reverse-phase liquid chromatography (LC) using fluorescence detection: 260 nm excitation and 313 nm emission. The mobile phase is acetonitrile-0.1M acetic acid (pH 4.2) buffer (53 + 47). Linearity is satisfactory over a range of 0.4-24 micrograms/mL. Results from 9 feed samples (1.1-2.7% lysine) analyzed by both the LC method and an amino acid analyzer were not significantly different statistically. Recovery of standard lysine, spiked just before derivatization on these same 9 samples (in duplicate), was 100.9% with a coefficient of variation (CV) of 2.4%. A study of within-day and between-day method precision resulted in CVs of 1.1 and 1.8%, respectively. The variation of results was negligible when the method was tested for ruggedness on 7 factors.     

Microbial diffusion assay for antibiotics in feeds using a simplified design

Results are compared for the microbiological analysis of antibiotics in feeds using the AOAC plate diffusion assay and the simplified 2-plate assay. Five antibiotics, bacitracin, chlortetracycline, oxytetracycline, penicillin, and streptomycin, were used to supplement feed extracts at levels of 100 and 25 micrograms antibiotic/g feed (bacitracin at 100 micrograms/g only). For bacitracin at the one level and for penicillin at both levels, the 2-plate design yielded significantly more accurate results than those of the AOAC assay. The same was true for the 25 micrograms/g level of oxytetracycline and the 100 micrograms/g level of streptomycin. For streptomycin at the 25 micrograms/g supplementation, the AOAC assay results showed better accuracy. There was no significant difference in results between the 2 designs for oxytetracycline at 100 micrograms/g and chlortetracycline at either level. The accuracy and precision of the results for the 2-plate design are equivalent to or better than those obtained using the AOAC design; in addition, the 2-plate assay is less labor-intensive, is more cost-effective, and is able to determine reasonable conditions of similarity.     

Turbidimetric microbiological assay results calculated by a BASIC computer program

A BASIC computer program was used to calculate the results of microbiological vitamin assays. The program, which incorporates the official AOAC guidelines for microbiological methods, reduces the uncertainty inherent in manual curve plotting and interpolation and minimizes the human error of repetitive calculations. Because the BASIC programming language is well suited for use on self-contained personal computers, it can be easily adapted by small laboratories.     

Improved turbidimetric assay of tylosin in premix and animal feeds not containing urea

A turbidimetric method is described for the determination of tylosin in premix and animal feeds not containing urea. This method includes several modifications of existing tylosin turbidimetric and AOAC plate assays to remove interferences from the feed, to concentrate low levels of tylosin, and to reduce the variability of the assay results. An acidic alumina column cleanup step has been incorporated into the method to remove interferences from feed ingredients. A disposable C18 column was used to concentrate the tylosin from low-level feeds, and the use of a larger analytical sample size has decreased the variability of the assay results. Average recoveries of tylosin added to chicken and swine rations were 98 and 101%, respectively.