Collaborative study of an automated method for the determination of crude protein in animal feeds.

An automated macro Kjeldahl instrument determines per cent protein at the rate of 20 samples/hr. The methodology involved is similar to the present official final action Kjeldahl method, sec. 7.016. The 2 methods were compared in a collaborative study. Sixteen animal feeds, 4 meats, tryptophan, ammonium dihydrogen phosphate NBS standard, and ammonium sulfate primary standard were analyzed by the participating laboratories. The data were agreement between the 2 methods. The automated method has been adopted as official first action for the determination of crude protein in feeds, plants, and cereal foods.     

Collaborative study of a semiautomated method for the determination of crude protein in animal feeds.

A semiautomated method consisting of digestion of animal feeds in a block digestor and determination of ammonia by ammonia-salicylate reaction has been studied collaboratively, along with the official final action Kjeldahl method, sec. 7.016. Each collaborator analyzed 16 feed samples, tryptophan, ammonium dihydrogen phosphate NBS standard, and ammonium sulfate primary standard. Statistical analysis showed that the 2 methods agreed. The semiautomated method has been adopted as official first action.     

Modified method for carbadox in feeds: collaborative study

The official first action method for carbadox in swine feed, 42.C01-42.C04, was modified in 2 respects. First, the samples were leached overnight at room temperature instead of boiled for 1 hr. This change avoided problems with overheating and excessive evaporation. Second, the dilution scheme for samples spiked with carbadox standard solution was changed to give absorbance values that were within the optimum working range of all types of spectrophotometers. The modified procedure was collaboratively studied by 21 laboratories. The repeatability standard deviation (sigma0) and reproducibility standard deviation (sigmax) were sigma0 = 0.00029% and sigmax = 0.00056% (8.9% of grand mean) for feeds containing 0.00617% carbadox; and sigma = 0.0012% and sigmax = 0.0019% (9.3% of grand mean) for feeds containing 0.0198% carbadox. The between-laboratory variance ratio was significant for feeds containing 0.0198% carbadox. The mean per cent of intent values for feeds containing 0.00617% carbadox and 0.0198% carbadox were 102% and 104%, respectively. In general, the statistical results were comparable to those previously obtained for the official first action method. Consequently, the modified procedure is not recommended as a replacement for the official first action method.     

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.     

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.     

Comparison of HgO and CuSO4 as digestion catalysts in manual Kjeldahl determination of crude protein in animal feeds: collaborative study

The official AOAC manual Kjeldahl method for determining crude protein in animal feeds, 7.015, uses HgO as a catalyst in the digestion step. Because of environmental considerations, there is considerable interest in alternative catalysts. A collaborative study compares the official HgO-catalyzed method and an alternative using CuSO4. Fifty-four samples consisting of blind duplicates of closely matched pairs, representing a range of animal feed materials and 2 standard materials, were analyzed once by each method. Results were returned by 22 laboratories. Means and standard deviations between methods were comparable. The CuSO4-catalyzed method has been adopted official first action.     

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.     

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.     

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.     

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.     

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.     

Optimized Monier-Williams method for determination of sulfites in foods: collaborative study

A collaborative study was conducted of the Food and Drug Administration (FDA)-optimized Monier-Williams method for determining sulfites in foods. Twenty-one industry and government laboratories participated in the study, which was jointly sponsored by the National Food Processors Association and FDA. Familiarization samples were shipped to each collaborator. Collaborators were permitted to proceed to the main study only after they demonstrated ability to perform the method to ensure that the study tested the performance of the method itself and not that of the individual laboratories. The study design involved 3 food matrixes (hominy, fruit juice, and protein [seafood]). Each matrix was prepared at 3 sulfite levels--the regulatory level, half the regulatory level, twice the regulatory level--and as a blank. All test samples were analyzed as blind duplicates, which gave each collaborator a total of 24 test portions. Collaborative recoveries gave a reproducibility (among-laboratories) coefficient of variation that ranged from 15.5 to 26.6% for sulfite determined as SO2 by weight in the 3 foods at the 10 ppm level. The optimized Monier-Williams method has been approved interim official first action to replace the AOAC modified Monier-Williams method, 20.123-20.125.     

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.     

Enzymatic-spectrophotometric method for determination of cholinesterase activity in whole blood: collaborative study

Ten collaborating laboratories assayed 4 blind duplicate pairs of whole bovine blood for cholinesterase activity. The 4 sample pairs ranged from normal (100%) to severely organo-phosphorus-inhibited (less than 10%) activity. Collaborators also received commercially available human lyophillized serum as an external control and a chromate solution to evaluate spectrophotometer performance. The Ellman kinetic assay was performed on a 1:1000 dilution of the whole blood in pH 8.0 phosphate buffer. The method monitors the increase in absorbance at 412 nm caused by formation of 5-thio-2-nitrobenzoic acid (yellow reaction product). Repeatability standard deviations (RSDr) ranged from 4.30 to 14.2%; reproducibility standard deviations (RSDR) ranged from 6.99 to 19.3%. The lower limit of detection was estimated to be 0.10 mumole/mL/min. The method has been approved interim official first action by AOAC.     

Thin layer chromatographic method for determination of deoxynivalenol in wheat: collaborative study

A collaborative study of a rapid method for the determination of deoxynivalenol (DON) in winter wheat was successfully completed. The method involves sample extraction with acetonitrile-water (84 + 16), cleanup using a disposable column of charcoal, Celite, and alumina, and detection by thin layer chromatography after spraying with an aluminum chloride solution. Each of the 15 collaborators analyzed 12 samples, 2 of which were naturally contaminated, and 10 to which DON was added, in duplicate, at levels of 0, 50, 100, 300, and 1000 ng/g. Average recoveries of DON ranged from 78 to 96% with repeatabilities of 30-64% and reproducibilities of 33-87%. The results of the study show that false positives were not a problem and that all of the analysts could detect DON at the 300 ng/g level or higher. The method has been adopted official first action.     

Liquid chromatographic method for determination of azinphos-methyl in formulated products: collaborative study

A liquid chromatographic method for determination of azinphosmethyl (Guthion) in formulated products has been developed. Samples are dissolved in acetonitrile and analyzed by reverse-phase chromatography using n-butyrophenone as an internal standard. The method was subjected to a collaborative study involving 15 participating laboratories. Each collaborator was furnished with reference standard, internal standard, and blind duplicate samples of Guthion 50% wettable powder (50WP), 3 flowable (3F), and emulsifiable concentrate (2L and 2S) formulations. Collaborators were instructed to evaluate the method by peak height measurements only. Relative standard deviations for reproducibility (RSDR) were 1.11, 6.28, 2.47, and 1.17% for the 50WP, 3F, 2L, and 2S formulations, respectively. The method has been approved interim official first action for determination of azinphos-methyl in the 50WP, 2L, and 2S formulations.     

Temperature-independent pectin gel method for coliform determination in dairy products: collaborative study

Ten laboratories participated in a collaborative study to compare a pectin-based violet red bile (VRB) method with the VRB agar-based standard method for estimation of coliform bacterial counts in 7 different dairy food groups: cream, cheddar cheese, cottage cheese, homogenized milk, raw milk, sour cream, and yogurt. Each laboratory analyzed 8 samples of each food group as sample pairs prepared at high, medium, and low inoculum levels, and an uninoculated control pair. Overall mean log counts were higher for the pectin gel method in 18 of 21 cases (7 samples at 3 inoculum levels); 12 results were significantly higher (P less than 0.01) than those for the VRB agar method. Of the 3 higher VRB agar method means, 2 were not significant at P less than 0.10, and one was of borderline significance (0.05 less than P less than 0.06). Repeatability variation (sr) favored the pectin gel method in 14 of 21 cases; 7 were highly significant differences (P less than 0.01). None of the 7 results favoring the VRB agar method was statistically significant. Reproducibility variation (sR) favored the pectin gel method in 14 of 21 results. These data indicate that the pectin gel method gives higher recovery of coliforms with better precision than does the VRB agar method, and strongly support the suitability of the pectin gel method as an alternative to the agar-based VRB method for coliform counts in dairy products. The pectin gel method has been adopted official first action.     

CuSO4-TiO2 as Kjeldahl digestion catalyst in manual determination of crude protein in animal feeds

The official AOAC manual Kjeldahl methods for determining crude protein in animal feeds have several disadvantages. For the HgO catalyst method, there are environmental concerns and a lengthy digestion. For the CuSO4 catalyst method, the digestion period is shorter, but still 90 min. A different catalyst combination, CuSO4-TiO2, makes 40 min digestion feasible. Comparison of these catalysts on a group of representative feeds resulted in a mean difference, Cu-Ti minus HgO, of 0.034% protein. Standard deviation of the differences was 0.36. A Student's t-test showed no significant difference. The method will be collaboratively studied.