Methods of sample preparation for detecting alkaline phosphatase in casein: collaborative study

A collaborative study was conducted in which 2 different sample preparation techniques were used to determine alkaline phosphatase in casein by the rapid colorimetric test. Seven collaborators tested 10 unknown casein products containing different amounts of residual phosphatase. Results indicated that the phosphatase contents of casein prepared by the 2 methods were not significantly different. The collaborators correctly analyzed 100% of the test samples that were ground and 98% of the test samples that were unground. The alternative rapid sample preparation method has been adopted official first action.     

Rapid screening method for alkaline phosphatase activity in cheese: collaborative study

The method developed for developed for determining alkaline phosphatase activity in cheese, in which phenolphthalein monophosphate is used as the substrate, was collaboratively studied. A 7.5% butanol extract of cheese is reacted with phenolphthalein monophosphate; phenolphthalein is released and yields a red solution that is compared visually with a standard (s) prepared from the same extract. Seven collaborators analyzed 8 samples of cheese, in duplicate, by the screening method and Scharer I method. Of the 208 observations returned, only 4 were incorrect. The alkaline phosphatase method has been adopted as official first action.     

Colorimetric determination of alkaline phosphatase as indicator of mammalian feces in corn meal: collaborative study

In the official method for rodent filth in corn meal, filth and corn meal are separated in organic solvents, and particles are identified by the presence of hair and a mucous coating. The solvents are toxic, poor separation yields low recoveries, and fecal characteristics are rarely present on all fragments, especially on small particles. The official AOAC alkaline phosphatase test for mammalian feces, 44.181-44.184, has therefore been adapted to determine the presence of mammalian feces in corn meal. The enzyme cleaves phosphate radicals from a test indicator/substrate, phenolphthalein diphosphate. As free phenolphthalein accumulates, a pink-to-red color develops in the gelled test agar medium. In a collaborative study conducted to compare the proposed method with the official method for corn meal, 44.049, the proposed method yielded 45.5% higher recoveries than the official method. Repeatability and reproducibility for the official method were roughly 1.8 times more variable than for the proposed method. 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.     

Determination of phosphorus in processed cheese: collaborative study

A second interlaboratory collaborative study of the determination of phosphorus in processed cheese products by the molybdenum blue method verifies that this method is prone to producing a laboratory-induced systematic error. It would be useless to continue to make minor modifications in the details of the method, which will improve only the within-laboratory precision, until an accuracy control of the final measurement step is incorporated into the method.     

Determination of zearalenone in corn: collaborative study.

Corn samples spiked at levels of 100, 300, 1000, and 2000 mug zearalenone/kg were sent to 22 collaborators for analysis by the Eppley method. All samples were yellow corn except one white corn sample spiked at 2000 mug/kg. Results from 16 collaborators were statistically analyzed. Only 4 of 16 collaborators detected zearalenone in the sample containing 100 mu/kg, but 11 detected the toxin in the sample containing 300 mug/kg. Average recoveries from all samples were 129% at 300 mug/kg, 101% at 1000 mug/kg, and 88% at 2000 mug/kg. The between-laboratory coefficients of variation were 53.0% at 300 mug/kg, 38.2% at 1000 mug/kg, and 27.0% at 2000 mug/kg. Five naturally contaminated corn samples, one in triplicate, were also provided. The mean level of zearalenone in the naturally contaminated samples ranged from 431 to 7622 mug/kg. The mean coefficient of variation for all samples was 40.5%. Two collaborators measured quantities of zearalenone on thin layer chromatographic plates densitometrically. Their results were not included in the statistical analysis, but the results indicated that densitometric measurement, given proper dilutions of solutions, could be used. The method has been adopted as official first action.     

Determination of N-nitrosodimethylamine in nonfat dry milk: collaborative study

Ten laboratories participated in a collaborative study of a method for the determination of N-nitrosodimethylamine (NDMA) in nonfat dry milk. NDMA is eluted with dichloromethane from a mixture of Celite, acidic sulfamic acid, and nonfat dry milk (all packed in a chromatography column), concentrated in a Kuderna -Danish concentrator, and finally analyzed by a GC-thermal energy analyzer technique. Ten samples were studied: 6 were naturally contaminated (NDMA levels 0.38-3.56 ppb) and 4 were spiked with known levels (0.96 and 3.2 ppb) of NDMA. The coefficients of variation (CV) of the complete data for the naturally contaminated samples (excluding the 2 samples containing the lowest levels) were 8.5% and 22.5% for repeatability and reproducibility, respectively. The corresponding CVs for the spiked samples were 14.4% and 20.4%, respectively. The percent recoveries of the added NDMA in the spiked samples (at the 2 levels indicated above) were 101.6 +/- 3.2 (omitting 1 outlier) and 95 +/- 2.1, respectively. The method has been adopted official first action.     

Determination of melengestrol acetate in bovine tissue: collaborative study.

Seven laboratories collaboratively studied a method for the assay of melengestrol acetate at the 0, 10, and 20 ppb levels in bovine fat, liver, muscle, and kidney. The study included fortification of tissue by each laboratory and analysis of fat samples taken from treated heifers which had endogenous levels of 0, 10, and 20 ppb melengestrol acetate. The multistep cleanup procedure used included extraction, solvent partition, column chromatography, and electron capture gas-liquid chromatographic, determination. Results of the study for muscle, liver, kidney, and fat showed that the method gave satisfactory recoveries and accuracy. In fat, the most critical tissue, recovery was greater than 93%. A statistical comparison of the results reported for fat tissue from treated heifers demonstrated that 5 of the 7 laboratories obtained similar results. The results produced by the method can be expected to be repeatable within and among laboratories. On the basis of the collaborative results the method has been adopted as official first action.     

Determination of internal insect infestation of oats: collaborative study

An improved method has been developed for determining internal insect infestation of oat kennels. The method involves alcohol defatting and acid hydrolysis of the cracked oats, wet sieving to remove the acid, transfer to a 2 L Wildman trap flask, deaeration by boiling, and treatment with Tween 80-Na4EDTA. Insects are extracted with light mineral oil. Reports from 6 collaborators showed that recoveries averaged 88.98% for adult insect heads and 97.22% for larvae. The method has been adopted official first action.     

Determination of diagnostic levels of arsenic in animal tissue: collaborative study

The method chosen for this collaborative study is a modification of the AOAC method for As residues, 41.009-41.012. The tissue is dry-ashed overnight at 500 degrees C, and then dissolved in dilute HCl. The solution is diluted and an aliquot is reacted with zinc metal to evolve arsine gas. The gas is trapped in AgDDC solution and As is quantitated at 540 nm. Nine collaborating laboratories performed single analyses on 4 blind duplicate pairs of bovine liver samples which were spiked at 0, 4.3, 10.8, or 21.6 mg As/kg liver. A National Bureau of Standards control (SRM 1566 Oyster Tissue, 13.4 +/- 1.9 mg As/kg) and a 1000 mg As/L standard were also submitted to the collaborators. Intralaboratory coefficients of variation ranged from 7.7 to 17.8%; interlaboratory coefficients of variation ranged from 10.9 to 19.0%. The method has been adopted official first action.     

Determination of natamycin in cheese and cheese rind: interlaboratory collaborative study

A collaborative test on the determination of natamycin in cheese and cheese rind was conducted. Participants were from 37 laboratories in 13 countries. Eight samples, consisting of 4 duplicates, were investigated by a spectrometric method and a liquid chromatographic (LC) method. The spectrometric method gave good results (coefficient of variation [CV] = 12%) and the LC method with ultraviolet detection gave reasonable results (CV = 25%) for levels down to 15 mg/kg (0.9 mg/dm2). For very low levels, a preconcentration step is necessary, but even then quantitation is poor (CV = 35-37%) for both methods at 1.7 mg/kg, although the presence of natamycin can be detected qualitatively. For a level of 0.3 mg/kg, quantitation is poor (CV = 39%) for the LC method and impossible (CV = 60%) for the spectrometric method.     

Rapid methods for differentiating reactivated from residual phosphatase in milk and cream: collaborative study.

Three methods for differentiating reactivated from residual phosphatase in milk and cream were collaboratively tested using both magnesium acetate and magnesium chloride for reactivating phosphatase. The methods evaluated were the modified Scharer rapid test, the rapid colorimetric test, and the Rutgers method. Nine collaborators tested 6 unknown milk samples containing reactivated and/or residual phosphatase, and 16 collaborators tested 6 unknown cream samples containing reactivated and/or residual phosphatase. Results indicated that use of magnesium acetate in place of magnesium chloride for reactivating phosphatase improved test results. Visual tests (modified Scharer rapid and Rutgers) predicted correct results when the samples contained high levels of reactivated or residual phosphatase. In borderline cases where the reactivated phosphatase contents of the undiluted control sample and the diluted sample containing Mg were very close, the test results of the visual methods were significantly different from 100% correct results at the alpha=0.05 level. Use of a photoelectric colorimeter or its equivalent for measuring the absorbance in conjunction with the modified Scharer rapid test improved results considerably. The modified Scharer rapid test was adopted official first action.     

Determination of total dietary fiber in foods and food products: collaborative study

A collaborative study was conducted to determine the total dietary fiber (TDF) content of food and food products, using a combination of enzymatic and gravimetric procedures. The method was basically the same as published earlier (J. Assoc. Off. Anal. Chem. (1984) 67, 1044-1052), with changes in the concentration of alcohol and buffers, time of incubation, sample preparation, and some explanatory notes, all with the intent of decreasing the coefficient of variation (CV) of the method. Duplicate blind samples of soy isolate, white wheat flour, rye bread, potatoes, rice, wheat bran, oats, corn bran, and whole wheat flour were analyzed by 9 collaborators. TDF was calculated as the weight of the residue minus the weight of protein and ash. CV values of the data from all laboratories for 7 of the samples ranged from 1.56 to 9.80%. The rice and soy isolate samples had CV values of 53.71% and 66.25%, respectively; however, each sample contained only about 1% TDF. The enzymatic-gravimetric method for determining TDF has been adopted official first action.     

Determination of nitrate in forages by using selective ion electrode: collaborative study

Each of 10 collaborating laboratories analyzed 4 blind duplicate pairs of forage samples for nitrate, by using a potentiometric method. Two forage controls and a 100 000 mg KNO3/L standard were also provided. Nitrate was extracted into an aqueous Al2(SO4)3 solution containing 70 mg KNO3/L and quantitated with a nitrate-selective electrode. Standards were prepared using extracting solution as diluent. Nitrate concentrations in forage samples ranged from less than 0.50 to 4.35% KNO3. Repeatability coefficients of variation (CVo) ranged from 1.74 to 3.61%, and reproducibility coefficients of variation (CVx) ranged from 6.92 to 7.66%. Mean recovery of a 0.55% KNO3 spike was 94.5%. The method has been adopted official first action.     

Solvent-mediated disruption of bovine casein micelles at alkaline pH

The disruption of casein micelles at alkaline pH was investigated using turbidity measurements. The rate and extent of disruption of casein micelles at alkaline pH (8.0-11.0) increased with pH. Furthermore, the extent of alkaline disruption increased with increasing temperature (5-40 degrees C). Preheating milk for 10 min at 90 degrees C did not influence the extent of alkaline disruption of casein micelles, suggesting that whey proteins do not influence the alkaline disruption process. Levels of ionic calcium and serum calcium and phosphate decreased in a logarithmic fashion with increasing pH, indicating precipitation of calcium phosphate onto the casein micelles. A mechanism for alkaline disruption of casein micelles is proposed, in which increasing the milk pH improves the solvent quality for the caseins, thereby leading to the disruption of casein micelles into their constituent nanoclusters; increases in the net-negative charge on the caseins on increasing pH may contribute to micellar dissociation.     

Determination of fluoride in fluoride tablets and solutions by ion-selective electrode: collaborative study

A proposed method using the fluoride (F) ion-selective electrode has been developed for determining the fluoride ion concentration in tablets and solutions containing sodium fluoride. The method has been subjected to collaborative study. Eight samples consisting of 2 authentic fluoride solutions, 2 commercial fluoride solutions, and 4 commercial fluoride tablets were sent to each of 11 collaborators together with a copy of the method. Single assays on the authentic fluoride solutions known to contain 1 mg F/5 mL were performed with average recoveries of 99.5 and 99.6% and relative coefficients of variation (CV) of 2.11 and 1.91%, respectively. Single assays of 2 commercial fluoride solutions declared at 1 mg F/5 mL gave mean values of 0.994 and 0.990 mg and relative CV values of 1.88 and 2.36%, respectively. Single assays of 2 commercial fluoride tablet preparations declared at 0.5 mg F gave mean values of 0.485 and 0.478 mg and relative CV values of 3.12 and 3.71%, respectively. Single assays of 2 commercial fluoride tablet preparations declared at 1 mg F gave mean values of 0.991 and 0.981 mg and relative CV values of 2.99 and 2.85%, respectively. The method has been adopted official first action.     

Determination of ethanol in wine by titrimetric and spectrophotometric dichromate methods: collaborative study

A dichromate-spectrophotometric method for the determination of ethanol in wine was compared in a collaborative, matched pair study with the AOAC dichromate-titrimetric method, 11.008-11.011. Both methods require distillation of the sample into dichromate. The titrimetric method measures ethanol by titrating the excess dichromate with ferrous ammonium sulfate after conversion of ethanol to acetic acid; the spectrophotometric method directly measures the reduced dichromate formed after oxidation. In addition to comparing the 2 methods, the collaborative study also compared the use of 2 types of assemblies for obtaining the ethanol distillate: the Scott-type, which is used in 11.008-11.011, and the electric Kirk-type. Results of the collaborative study indicated that the repeatability and reproducibility of the official titrimetric method were generally far superior to those of the spectrophotometric method; therefore, adoption of the spectrophotometric method is not recommended. Comparison of titrimetric method results obtained using the 2 types of stills indicated that repeatability and reproducibility were somewhat better when Scott apparatus was used, but measurements using Kirk-type compared well in the range of ethanol concentrations found in table and fortified wines. The Kirk-type distillation apparatus has been adopted official first action as an alternative to Scott apparatus in the dichromate oxidation method for ethanol in wine, 11.008-11.011.