Determination of tilmicosin residues in chicken, cattle, swine, and sheep tissues by liquid chromatography.

A method was developed and validated for determination and quantitation of tilmicosin residues in swine, cattle, and sheep edible tissues, as well as chicken fat, skin, and muscle over a concentration range of 0.025 microg/g-20 microg/g. For chicken kidney and liver, the method was validated over a range of 0.060 microg/g-20 microg/g. The tissue sample was extracted with methanol and a C18 cartridge was used for solid-phase extraction cleanup. A reversed-phase gradient liquid chromatographic method with detection at 280 nm was used to separate the tilmicosin from matrix components in 30 min run time. The limit of quantitation (LOQ) of the method was 0.025 microg/g for all tested tissues except chicken kidney and liver, for which the LOQ was 0.06 microg/g. Average recoveries for tissue samples ranged from 73 to 98%. Relative standard deviation values ranged from 0.6 to 14.7%.

CPR: a systematic design for change.

Major changes such as prospective payment and managed care are rocking the boat for today's home care providers. To stay afloat a number of providers are redesigning their organizations. Using Core Process Redesign, a team-based methodology for improving what, where, and how work is done in an organization, one agency saved nearly a half million dollars in one year.

Liquid chromatographic determination of monensin in premix and animal feeds: collaborative study.

An interlaboratory study of a liquid chromatographic (LC) method for determining monensin in premix (60-80 g/lb or 132-176 mg/g) and animal feeds (5-200 g/ton or 0.0055-0.22 mg/g) was conducted in laboratoriesin the United States, Canada, France, and Germany. The LC system used a reversed-phase column, postcolumn derivatization with vanillin, and UV detection. The method separates monensin from other ionophores such as narasin and salinomycin. Each laboratory analyzed a total of 20 samples of premix, liquid feed supplements, poultry, and cattle feeds. Concentrations of monensin in all samples ranged from 0 to 176 mg/g (80 g/lb). Reproducibility relative standard deviation (RSDR) for premix ranged from 2.8 to 3.4%. For feed samples containing monensin, repeatability standard deviation (sr) ranged from 0.9 to 7.0. Reproducibility standard deviation (sR) ranged from 1.2 to 11. Repeatability relative standard deviation (RSDr) ranged from 6.1 to 21% and RSDR values-ranged from 8.6 to 25%. Sample preparation for the LC method is less labor intensive than that for the microbiological assays. The LC assay is more efficient than the microbiological assays. This LC method for determination of monensin in premix and animal feeds has been adopted first action by AOAC INTERNATIONAL.

Determination of tilmicosin in bovine and porcine sera by liquid chromatography.

A liquid chromatographic (LC) assay is described for determining tilmicosin in bovine and porcine blood sera. Tilmicosin is isolated from the serum matrix and purified by solid-phase extraction with C18 sorbent. Sample is analyzed by LC using a gradient system with a phenyl reversed-phase column that separates tilmicosin from the matrix in 30 min. Tilmicosin is measured by UV absorbance at 280 nm. Validation of assay included evaluation of accuracy, precision, linearity, specificity, sensitivity, range, and sample stability. The method has a limit of quantitation of 0.1 ppm and a validated range of 0.1 to 10.0 ppm. Recoveries were 91-95% for bovine serum and 85-93% porcine serum. The limit of detection was 0.05 microgram/mL. Limits of detection and quantitation were based on 3 and 6 times the baseline noise of control serum samples, respectively. Relative standard deviations of precision samples (n = 6) were 2% or less for both sera. The method has better specificity and analysis time than previous microbiological methods for tilmicosin in sera.

Determination of monensin in high-moisture cattle rations by liquid chromatography with postcolumn derivatization.

An existing liquid chromatographic method using postcolumn derivatization has been used extensively to quantitate monensin in animal feeds. Because of the relatively high moisture content of many cattle feed rations, some modifications were made to this method. Several sample-processing steps were evaluated to determine optimum sample-processing procedure. The sample weight/sample diluent ratio was modified, and method linearity was validated for the lower monensin concentrations anticipated in high-moisture cattle rations. The accuracy and precision of data generated at these lower concentrations were also determined. Because of the high moisture content of these rations, data analysis for this method required correction of feed potency for loss on drying. With these modifications, monensin can be accurately determined in high-moisture cattle rations.

Microbiological plate assay for determination of tilmicosin in bovine serum.

A microbiological agar plate assay is described for determination of tilmicosin in bovine blood serum. The serum or serum dilution is added directly to wells cut in the agar plates. Tilmicosin activity is determined by measuring the zone of bacterial growth inhibition in agar medium inoculated with Micrococcus luteus, ATCC 9341. The assay was validated by evaluating the following parameters: accuracy, precision, linearity, parallelism, ruggedness, storage stability, and relative activity of isomers. Accuracy was evaluated with freshly collected bovine serum and with commercially available sera. Recoveries ranged from 93.4 to 97.5% across a fortification range of 0.08 to 1.28 micrograms/mL. Precision was estimated over a 6-day period with serum obtained from a tilmicosin-treated animal. Relative standard deviations were 0.63 to 3.13% within day and 5.23% across 6 days. Standard curves were linear with little variation in slope. No parallelism was observed between tilmicosin in serum and tilmicosin in buffered saline. The limit of detection was estimated to be 0.05 micrograms/mL, and the validated limit of quantitation was 0.08 micrograms/mL. Ruggedness was evaluated with different lots of antibiotic medium, different lots of sera, and different analysts. These variables did not affect method performance. Analyses of tilmicosin in frozen sera demonstrated that tilmicosin is stable for up to 16 days when stored at -20 degrees C. A comparison of the relative microbiological activities of the purified cis and trans isomers of tilmicosin to that of the reference standard indicated no differences in microbiological activities, and showed a parallel response among the 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of monensin in edible bovine tissues and milk by liquid chromatography.

A method is described for detection and quantitation of monensin in bovine tissues by liquid chromatography (LC) with postcolumn derivatization (PCD) with vanillin. Monensin is extracted from the tissues by homogenization with methanol-water and is isolated and concentrated by liquid-liquid partition and sorbent extraction with silica gel. Monensin is mixed postcolumn with vanillin under acidic conditions and heated, and the resulting products are measured by a variable-wavelength detector at 520 nm. The method has a limit of quantitation of 5 ppb monensin in milk and 25 ppb monensin in bovine muscle, liver, kidney, and fat. Standard recovery over the levels and matrixes tested ranged from 80 to 88%. The method is an improvement in specificity, accuracy, and analysis time over existing monensin residue methods for bovine tissues.

Determination of monensin in chicken tissues by liquid chromatography with postcolumn derivatization.

A method is described for the detection and quantitation of monensin in chicken tissues by liquid chromatography with postcolumn derivatization with vanillin. Monensin is extracted from the tissues by homogenization with methanol-water and is isolated and concentrated by liquid-liquid partition and sorbent extraction with silica gel. Monensin is mixed postcolumn with vanillin under acidic conditions and heated, and the resulting products are measured by a variable-wavelength detector operating at 520 nm. The method has a limit of quantitation of 0.025 microgram/g and is validated for use in the analyses of chicken muscle, liver, and skin (with adhering fat tissues) for monensin. Standard recoveries from the 3 tissue types tested at 3 levels ranged from 82 to 96%. The method represents an improvement in specificity, accuracy, and analysis time over existing methods, which use microbiological techniques.

The need for larger analytical samples with granulated feed additives.

Sampling an animal feed containing a granulated analyte is extremely difficult. A granulated feed additive, Tylan (Elanco, tylosin), was used to demonstrate how a granulated product can increase assay variability if steps are not taken to reduce and control this variability. The number of tylosin granules was determined for Granulated Tylosin Concentrate, the active ingredient of Tylan Premix. Multiple weighings of 3 lots of the concentrate were prepared, and the average number of granules per gram of concentrate was determined with the aid of image analyses. The total weight of Tylan Premix in varying analytical samples was calculated. Finally, the average number of tylosin granules per analytical sample was calculated based on the average number of granules per gram. A 10 g analytical sample obtained from an animal ration containing 8 ppm tylosin activity would contain an average of 1.16 granules of tylosin, and a 100 g analytical sample would contain 11.6 granules. These calculations demonstrate the need to increase the analytical sample size in analyzing an animal feed containing a granulated feed additive.

Branched-chain amino acid transport in Streptococcus agalactiae.

The transport of the branched-chain amino acids in Streptococcus agalactiae was characterized. Glucose-grown cells were able to utilize only glucose as an energy source for transport of L-leucine, whereas lactose-grown cells could utilize both glucose and lactose. It was determined from metabolic inhibitor studies that energy from glycolysis and substrate level phosphorylation was required for active transport. Energy was found to be coupled to transport by the action of adenosine triphosphatase and the generation of a proton motive force. The branched-chain amino acids were found to share a common transport system that may consist of multiple components.

Stability of the Plasma Membrane in Saccharomyces rouxii and Its Relationship to Glucose Tolerance.

The stability of spheroplasts from the osmotrophic yeast Saccharomyces rouxii was studied in buffered solutions of mannitol and glucose. The plasma membranes from cells grown in high glucose concentrations were more stable to osmotic lysis than were membranes from cells grown in lower glucose concentrations. Mannitol was a better osmotic stabilizer than glucose, except when the cells were grown in a high glucose concentration. Spheroplasts from a glucose tolerant-deficient mutant were much less stable than the corresponding spheroplasts from the parent strain, especially when suspended in glucose solutions. These results suggest an involvement of the plasma membrane in the glucose-tolerant mechanism of S. rouxii.

Effect of sugars on D-arabitol production and glucose metabolism in Saccharomyces rouxii.

The effect of sugars on the production of d-arabitol and on the glucose catabolic pathways was investigated in the osmotrophic yeast Saccharomyces rouxii. The activity of d-arabitol dehydrogenase, which served as a measure of total d-arabitol production, increased when cells were grown in the presence of increasing glucose concentrations. Growth in sucrose had no effect on the enzyme activity. A high intracellular concentration of d-arabitol could be demonstrated when the cells were grown in a 60% glucose medium and could be eliminated by anaerobic growth or growth in the presence of 4 mg of chloramphenicol per ml. A mutant was isolated that would not grow in 60% glucose; although the regulation of d-arabitol dehydrogenase was altered in this strain, the production of d-arabitol was not eliminated. The activity of d-arabitol dehydrogenase followed the growth phases of the parent strain when the cells were preadapted to 30% glucose. If the cells were adapting from 1 to 30% glucose, a large increase in enzyme activity was detected before growth occurred. Protein synthesis was found to be involved in this increase in activity. There was an increased participation of the pentose phosphate pathway when the cells were grown in the presence of increasing glucose concentrations. The mutant strain had only an 11% pentose phosphate pathway participation compared with 20% for the parent strain in glucose. The results suggest that the active pentose phosphate pathway is involved in glucose tolerance by providing a plentiful supply of reduced nicotinamide adenine dinucleotide phosphate which is necessary for cell survival.

Effect of potential water pollutants and enzyme inhibitors on an automated rapid test for Escherichia coli.

The effect of selected potential inhibitors that may be found in water or wastewater on the activity of glutamate decarboxylase (EC 4.1.1.5) from Escherichia coli was determined. Several classes of compounds inhibited the enzyme, but those expected to be most frequently encountered were the heavy metal ions, the phosphates, and possibly the sulfates. From the results, it was judged that these compounds should not adversely affect the routine usage of this enzyme in an automated rapid test for E. coli.