Generic combustion method for determination of crude protein in feeds: collaborative study

Nine laboratories participated in a collaborative study on determination of crude protein in animal feeds to compare a generically described combustion method with the AOAC mercury catalyst Kjeldahl method (7.015). The combustion method was written in general terms of method principle, apparatus specifications, and performance requirements. The sample set comprised closely matched pairs of feed ingredients and mixed products ranging from 10 to 90% protein. Ten pairs ground to 0.5 mm were the focus of the study; 4 pairs were ground to 1.0 mm for comparison. Nicotinic acid and lysine monohydrochloride were included as standards. Collaborators were instructed to report their results for performance checks using materials supplied. Only one laboratory failed to meet the proposed limits. Seven laboratories used the LECO Model FP-228 analyzer and 2 used the LECO CHN 600 analyzer. For the 0.5 mm pairs, repeatability standard deviations (Sr) ranged from 0.09 to 0.58 for the Kjeldahl method and from 0.14 to 0.33 for the combustion method, with a pooled Sr value of 0.28 and relative standard deviation (RSDr) of 0.59%. Reproducibility standard deviations (Sg) ranged from 0.23 to 0.86 (Kjeldahl) and from 0.30 to 0.61 (combustion), with a pooled Sg value of 0.52 and RSDg of 1.10%. Grand means for the samples ground to 0.5 mm were 47.65% protein by the combustion method and 47.41% protein by the Kjeldahl method. For samples ground to 1.0 mm, corresponding values were 31.82 and 31.50% protein. The generic combustion method has been approved interim official first action.     

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.     

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.     

Microbiological determination of neomycin in feeds: collaborative study

A modification of the AOAC microbiological determination of neomycin in feeds was collaboratively studied by 12 laboratories. The official method was modified by substituting a constant salt concentration diluent for the feed extract diluent, preparing the agar medium in tris buffer, and performing the test with a monolayer plating system. Each laboratory performed single assays on 8 samples in a randomized sequence. The samples included duplicates of a cattle and swine feed at 2 different marketed concentrations. The mean recovery across all laboratories was 110.7% of theory with a range of means of 69.4-128.6 across the 12 laboratories. The results of one laboratory and 2 additional values from different laboratories were deemed outliers and excluded from statistical analysis. The statistical analysis gave a confidence interval of +/- 26% for individual assays.     

Pyridine extraction method for determination of bacitracin in feeds: collaborative evaluation.

Seventeen laboratories evaluated the pyridine extraction method and neomycin-sensitized agar for the determination of zinc and MD bacitracin in swine and broiler rations at 10 and 100 g/ton. The method was also applied to the analysis of 2 premixes labeled 50 g/lb (MD bacitracin) and 40 g/lb (zinc bacitracin). Bacitracin activity was determined on each of 2 days with 2 dilutions on each day. No significant difference was found between dilutions within a day or between days for each sample. The type of bacitracin or type of feed did not significantly affect results. The difference in results between MD and zinc bacitracin in premixes approached significance. The large coefficients of variation for premixes (ca 13%) and complete feeds (ca 15--30%) indicate operational problems. The main difficulty was evaporation of pyridine. Some laboratories were not able to evaporate it completely, whereas others lost bacitracin activity, probably due to high temperature of drying. The pyridine extraction method as in 42.200 and 42.204 should be discontinued.     

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%.     

Spectrophotometric determination of pyrantel tartrate in swine feeds by method of standard additions: collaborative study

The spectrophotometric method for pyrantel tartrate in swine feeds was collaboratively studied. Twenty-seven laboratories assayed feeds containing 0.0103, 0.0965, and 0.7902% pyrantel tartrate. Repeatability (sigmao) and reproducibility (sigmax) standard deviations were: sigmao = 0.00068%, sigmax = 0.00105% (10% of grand mean) for 0.0103% pyrantel tartrate level; sigmao - 0.0065%, sigmax = 0.0090% (10% of grand mean) for 0.0965% pyrantel tartrate level; and sigmao = 0.0415%, sigmax = 0.0743% (10% of grand mean) for 0.7902% pyrantel tartrate level. The mean theoretical recovery values for feeds containing 0.0103, 0.0965, and 0.7902% were 100, 97, and 96%, respectively. The method was adopted as official first action for feeds or concentrates containing 0.0106-0.8811% pyrantel tartrate.     

Microbiological plate and turbidimetric assays of chlortetracycline in feeds: collaborative study.

The manual and automated turbidimetric assays and a modified official plate assay for chlortetracycline (CTC-HCl) in feed were collaboratively studied. Three feed samples (swine feed, 100 g CTC-HCl/ton; premix I, 20 g each of CTC-HCl and sulfamethazine/lb, and 10 g penicillin/lb; and premix II, 50 g CTC-HCl/lb) were analyzed at 2 dilutions. Twelve laboratories conducted the plate assay; 8 laboratories the manual turbidimetric method; and 7 laboratories, the Autoturb analysis. Within a method, there was no significant difference between dilutions. Between methods, there was a significant difference between the manual turbidimetric plate assays only for swine feed. However, the same sample dilutions or the average values of the 2 dilutions for both methods showed no statistical difference. Among the collaborators, the slope of CTC-HCl standard curve varied between about 2.0 and 3.0 for the plate method. The turbidimetric assay has been adopted as official first action for feeds containing larger than or equal to 20 g CTC-HCl/lb.     

Semiautomated method for riboflavin in food products: collaborative study

A collaborative study was conducted comparing a semiautomated riboflavin method with a manual riboflavin method for 10 food products. Six laboratories provided results from the semiautomated method and 16 laboratories used the manual technique. The semiautomated method was more repeatable within a laboratory than was the manual method. The semiautomated method results compared favorably with the manual method for all 10 products.     

Enzymatic-ultraviolet method for measuring lactose in milk: collaborative study

Collaborators in 8 dairy and food industry laboratories performed one lactose determination on each of 8 unknown samples of milk, lowfat milk, or skim milk, as 3 pairs of blind duplicates. Two known samples were provided to gain experience prior to analysis of the unknown samples. All of the above samples were also analyzed for lactose content by the official AOAC gravimetric method (16.507) by a commercial laboratory. From the overall mean of results on all samples, determinations by the enzymatic method averaged 0.49% lower than by the AOAC method. This difference was significant by the t-test (P = 0.05), which indicated a lack of agreement between the compared methods in determining lactose content. Standard deviations were similar for the 3 sets of blind duplicates which ranged between 3.67 and 4.55% lactose content. F-values revealed that variations between means obtained by laboratories differed significantly as compared with variations within laboratory means. The method has been adopted official first action.     

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.     

Semiautomated method for determining ferrous sulfate in pharmaceuticals: collaborative study

A semiautomated colorimetric method is described for determining ferrous sulfate in tablets, capsules, and elixirs. The method involves the formation of an orange-red complex between 1,10-phenanthroline and ferrous ion. Collaborators were supplied with 3 solid composites and one liquid sample. Two of the solid composites were prepared from commerical tablets of different dosage and one from commercial timed-release capsules; the fourth sample was an elixir. The results of the automated analyses for ferrous sulfate agreed well with those of the applicable USP method. No interference was found from ferric iron or from the red and green coloring used on coated tablets. The method has been adopted as official first action.     

Anion-exchange method for determination of phytate in foods: collaborative study

Phytate, a naturally occurring organic compound found in plant seeds, roots, and tubers, was determined in a collaborative study using a modified anion-exchange method. Seven samples (peanut flour, oats, rice, isolated soybean protein, a vegetarian diet composite, wheat bran, and whole wheat bread), supplied as blind duplicate samples, were analyzed in triplicate by 7 collaborators. Phytate concentrations in the samples ranged from 2.38 to 46.70 mg/g. Relative standard deviations (RSD = CV) for repeatability ranged from 2.5 to 10.1%, and for reproducibility, from 4.5 to 11.0%. The method has been adopted official first action.     

Preliminary collaborative study of modified spectrophotometric determination of pyrantel tartrate in swine feeds by the method of standard additions

A modified spectrophotometric method for determining pyrantel tartrate in swine feeds was subjected to a preliminary collaborative study. Two small-scale commercial pyrantel-medicated feed samples (0.0881 and 0.0106%) were assayed in replicate by 4 collaborators. The mean results of all laboratories were 0.0862 and 0.0112%. The mean coefficients of variation were 10.57 and 6.48%, repectively. Suggestions for improving recovery include the following: complete dissolution of standard, use of analytical grade KI, careful phase separation, thorough mixing, and minimum exposure of compound to light.