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.     

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.     

Automated instrumental analysis of carbon and nitrogen in plant and soil samples

Abstract

An automated CHN Analyzer was compared with the Walkley‐Black and Kjeldahl methods for organic carbon (C) and nitrogen (N). Four organic compounds, twenty nine plant materials and five soils were tested. The CHN Analyzer gave C and N values that were not significantly different (P<0.05) to the theoretical weight percents of the organic compounds. The Walkley Black method gave soil C values significantly lower (P<0.05) than those obtained with the CHN Analyzer. The Kjeldahl method gave soil N values significantly lower (P<0.05) than the CHN Analyzer on three of five soils tested. The discrepancies observed between methods appear to be due to different oxidation efficiencies. CHN Analyzer and Kjeldahl N analyses were not significantly different (P<0.05) for the plant materials except where samples contained greater than 0.7% NO3‐N. Potassium nitrate was also added as a spike to a tall fescue sample. Based on recovery of the spiked NO3‐N, the Kjeldahl method was a poor measure of total N for plant materials containing greater than 0.7% NO₃‐N. The findings suggest the CHN Analyzer can be used for the rapid, accurate and simultaneous determination of C and N in plant and soil samples.     

Comparison of HgO and CuSO4/TiO2 as catalysts in manual Kjeldahl digestion for determination of crude protein in animal feed: collaborative study

Because of environmental concerns about HgO, and because of lengthy digestion requirements for HgO and CuSO4, interest in alternative catalysts for the Kjeldahl determination of animal feeds remains high. A digestion system using a mixed CuSO4/TiO2 catalyst has been found to reduce digestion times to 40 min. A collaborative study was carried out to compare this system to the official AOAC HgO method, 7.015. Thirty-eight samples, consisting of blind duplicates of closely matched pairs and 2 standard materials, were analyzed once by each method. Results were received from 13 laboratories. Means and standard deviations of individual samples were comparable, with an overall difference of grand means of 0.005% protein. With only one exception, analyses of variance showed no significant method difference at the 95% confidence level. The CuSO4/TiO2 method has been approved interim official first action as an alternative method for determination of crude protein in animal feed.     

Analysis of fat-soluble vitamins. XIX. Collaborative studies on the chemical assay for vitamin D concentrates

In 1971, a chemical method for the assay of vitamin D in concentrates containing only vitamin D was collaboratively studied by 14 laboratories, using 6 different samples from 2 European manufacturers. On the basis of these results, the laboratories were divided into 2 groups: 5 with significant laboratory biases of greater than or equal to 2%, and 9 laboratories with nonsignificant bias. The 9 laboratories were subdivided into 2 groups which differed significantly as to reproducibility within laboratories. The reproducibility between laboratories, expressed as a standard deviation in per cent with 95% confidence limits, was 1.2% (confidence range 0.6-7.3) and 4.7% (confidence range 2.4-29.3) for 3 and 6 laboratories, respectively. A second collaborative test was performed in 1974, using 12 vitamin D resin samples in oil from 3 United States manufacturers, to compare 2 chemical vitamin D assay methods (with and without maleic anhydride) and to compare results from the chemical and biological methods; 9 laboratories participated in the chemical method study and 3 in the rat bioassay study. The correlation of results of the chemical method including maleic anhydride treatment and the rat bioassays was satisfactory. The reproducibility of the chemical method was about the same as that in the first collaborative test.     

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.     

Pollution-reduced Kjeldahl method for crude protein.

Pollution from the Kjeldahl method for crude protein has been reduced by substituting a low level of copper (0.04 g CuSO4) for the mercury (0.7 g HgO) specified in the AOAC official method, 2.049. Adjustments were made in the salt-acid ratio so the new system could handle hard-to-digest samples in a reasonable time. The new method was rugged for lysine. HCl. It is designed to be used for crude protein in feeds or similar Kjeldahl work. Precision and accuracy were equal to or better than that for the official method in a study of 17 samples analyzed in duplicate on 3 different days. The following samples were used in the study: lysine. HCl, tryptophan, NBS standards, urea, meals, mixed feeds, grains, and forage. The average per cent nitrogen found was 9.52 by the official method and 9.53 by the copper method. The average standard deviation was 0.038 by the official method and 0.033 by the copper method, giving the corresponding relative standard deviations of 0.40 and 0.35%.     

Interlaboratory study on determination of 2,3,7,8-tetrachlorodibenzo-p-dioxin in fish

An interlaboratory round robin study was carried out to estimate the reliability of data on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in fish. Using different methods, 13 laboratories (4 Canadian, 9 American) agreed to analyze 4 fish samples; 3 were Great Lakes salmonids containing bio-incurred levels of TCDD below 100 ppt and the fourth was an ocean fish fillet containing no measurable TCDD. Samples were sent as freeze-dried portions as it was shown that no change of TCDD occurred by this sample preparation. Results were normalized between laboratories by supplying each with an aliquot of the same 2,3,7,8-TCDD standard. Eight laboratories reported a set of results of which one set was rejected. Values from the 7 remaining laboratories for the 3 positive fish showed mean concentrations in pg/g (ppt) and (CV, %) of 61.2 (13.9), 30.4 (18.4), and 32.3 (25.4). Detection limits averaged 3.6 ppt and ranged between 1 and 10 ppt. No significant differences appeared in the concentration of 2,3,7,8-TCDD in fish samples from methods differing in the use of: (i) digestion or extraction techniques, (ii) high or low resolution mass spectrometry, and (iii) isomer specific or nonspecific separations. Overall recovery values using internal standards varied greatly (29-109%) even within the same laboratory and pointed to the need to use an internal standard to obtain precise results. Agreement among laboratories was good considering the level quantitated (ppt) and the diverse methodology.     

Does the nitrate fraction account for differences between dumas‐N and Kjeldahl‐N values in vegetable leaves?

With recent advances in nitrogen (N) analyzers, the Dumas method may replace the traditional Kjeldahl method for the routine diagnosis of N in plants. Because of its nature, the Dumas method truly determines total N. The Kjeldahl method only converts protein N and some nitrate (NO₃‐N) into ammonium. Therefore, the N‐NO₃ ^‐ fraction may explain the difference observed between Kjeldahl‐N (Kn) and Dumas‐N (Dn) values. This study was conducted to (1) determine the Kn:Dn ratio for vegetable crops and (2) evaluate the effect of the size of the nitrate fraction on the Kn:Dn ratio. Over the 0.9–7.0% N range, Dn was a good predictor of Kn in vegetable samples. The Kn may be estimated from Dn as Kn=0.68 Dn (n=134 obs., R²=0.71, p<0.01). For all vegetable crops combined, the mean Kn:Dn ratio was 0.75. This ratio suggests that approximately 25% of N in the samples was recovered by the Dumas method but not by Kjeldahl digestion. This percentage is much higher than the actual N‐NO₃ foliar content. These results suggest that when N‐NO₃ is not known (as in most routine samples), Kn may be estimated from Dn as Kn=0.75 Dn. These results also suggest that under a wide range of NO₃‐N, NO₃‐N alone does not account for the difference between Kjeldahl‐N and Dumas‐N.     

Precision parameters of methods of analysis required for nutrition labeling. Part I. Major nutrients

Major components of foods and feeds are fat, protein, and carbohydrates. Fat and protein are determined by direct measurements that are interpreted as the quantity of the constituent. Carbohydrates are usually calculated by difference. For this calculation, values for moisture/solids, ash, and "fiber" are also needed. The readily available collaborative studies for the determination of these major components are reviewed in an attempt to assign precision parameters to validated methods of analysis. When a number of studies for the same analyte, in the same food, by the same method are available, it is seen that the precision parameters among laboratories (standard deviations, SR; relative standard deviations, RSDR) and the ISO maximum tolerable difference functions (repeatability value, r; reproducibility value, R) are not characterized by any conventional distribution. The precision data are best summarized as a median or average parameter and the interval containing the centermost 90% of reported values. Typically, the precision of methods of analysis can be expressed as a function of concentration only, independent of analyte, matrix, and method. The average RSDR value from each collaborative data set can then be used as the numerator in a ratio containing, as the denominator, the value calculated from the Horwitz equation: RSDR = 2 exp (1 - 0.5 log C) where C is the concentration as a decimal fraction. A series of ratios consistently above 1, and especially above 2, probably indicates that a method is unacceptable with respect to precision. By this criterion, only the protein (Kjeldahl) determination is unqualifiedly acceptable with a 90% interval for RSDR of 1 to 3% at C values above about 0.01 (1 g/100 g). Fat, moisture/solids, and ash are acceptable down to limiting concentrations in the region of 1 to 5 g/100 g, if a test portion large enough to provide at least 50 mg of weighable residue or volatiles is specified. Measurements of individual carbohydrates and fiber-related analytes have unexpectedly poor precisions among laboratories. The variability, although high, may still be suitable for nutrition labeling. Reliability of analyses for the control of labeling of the primary nutrients must be achieved through quality assurance programs that require strict adherence to the directions of empirical methods and the use of suitable reference materials for absolute methods.     

Using Interlaboratory Proficiency Data to Guide NIR/MIR Calibrations

There is growing interest in the use of near-range and/or midrange infrared (IR) diffuse reflectance spectroscopy (NIR and MIR) as nondestructive alternatives to chemical testing of soils. This trend is supported by research on how best to correlate IR spectral data with results obtained by conventional laboratory measurements. While for soils there is growing interest in developing local and national calibrations using “legacy” data, the proven analytical performance of provider laboratories now and earlier, the moisture status of reported results, and the method of soil preparation warrant greater attention. Examples for soil carbon (C) and total soil nitrogen (N) from Australasian interlaboratory proficiency testing across multiple years from 1993 are provided to demonstrate the magnitude of past and present measurement uncertainties, including the effects of method and different concentrations. The evidence is sufficient to require those commissioned to develop NIR and MIR calibrations to subject their prototype calibrations to external peer review by participating in credible, independent interlaboratory proficiency testing programs for ≥12 months, including checks on soil moisture status and possible effects of sample preparation. To rate as credible for most uses, the prototype results should be within the interquartile range for each sample and ideally there should be no outliers and few stragglers. Across the period of assessment (1993–2008), users of Walkley and Black organic C and Kjeldahl digestion for total soil N (Kjeldahl method does not measure total N, but most of the organic N plus an undetermined proportion of nitrate and nitrate present in the sample; quantitative inclusion of both requires a modification of the Kjeldahl procedure) declined as use of furnace technologies for soil C and N increased linearly. There is a strong case to commission two or three well-performing and experienced laboratories to reanalyze samples in “legacy” soil collections prior to finalizing predictive relationships with NIR/MIR spectra for the same samples.     

杜马斯燃烧法与凯氏法测定畜禽粪便中氮含量的比较

为了比较杜马斯燃烧法与凯氏法测定畜禽粪便氮含量结果的异同,以取自中国不同地区的5种畜禽粪便和磺胺嘧啶标准物为试验材料,分别采用凯氏法和杜马斯燃烧法测定各种畜禽粪便的氮含量,并对上述2种氮含量测定结果进行比较分析。结果表明:5类畜禽粪便凯氏氮的质量分数范围为0.51%～3.19%,杜马斯燃烧氮的质量分数范围为0.51%～3.35%,2种方法测定值之间不存在显著性差异,变异系数CV均小于5%.畜禽粪便样品凯氏法与杜马斯燃烧法的测定结果呈显著性相关(R2=0.987,p<0.05),拟合直线的斜率与1以及截距与0之间均不存在显著性差异。因此,杜马斯燃烧法可以代替凯氏法测定蛋鸡、肉鸡、猪、奶牛和肉牛粪便中的氮含量。     

Comparison of Two Digestion Methods for Determining Total Phosphorus in Farm Canal Water

Abstract

Rapid and accurate determination of low‐level (0.01 to 1.0 mg L^?1) phosphorus (P) concentrations in farm canal water is important in evaluating water quality in the Everglades Agricultural Area (EAA) canals in south Florida. Two U.S. Environmental Protection Agency methods, persulfate digestion (365.1) and Kjeldahl digestion with mercury oxide (365.4), were used to analyze total P (TP) and total dissolved P (TDP) in two sets of representative canal water samples collected at low‐flow conditions in 2003 and high‐flow conditions in 2004. Quality assurance samples (blanks, duplicates, and spikes) were included to evaluate differences between the two digestion methods. Precision analysis had a mean of less than 5% for both TP and TDP using both methods. The high coefficient of correlations (r>0.98) indicated that the two methods were significantly correlated in determining TP and TDP of the samples. Low detection limits (0.004 mg L^?1) were achieved by the persulfate method. This method offers many other advantages over the mercury digestion: it produces no toxic mercury waste, uses less time, and uses a lower temperature. High suspended solids in canal water samples were not proven to be a problem when using the persulfate digestion, though lower spike recoveries were observed than those when using the mercury digestion. We conclude that persulfate digestion is a more sensitive and environmentally responsible alternative to and is, as precise as, the mercury method for routine determination of TP and TDP in water samples. This information is useful to environmental laboratories in monitoring P concentrations in surface and groundwater.     

Addressing the Dilemmas of Measuring Amylose in Rice

Amylose content is a parameter that correlates with the cooking behavior of rice. It is measured at the earliest possible stages of rice improvement programs to enable breeders to build the foundations of appropriate grain quality during cultivar development. Amylose is usually quantified by absorbance of the amylose‐iodine complex. The International Network for Quality Rice (INQR) conducted a survey to determine ways that amylose is measured, reproducibility between laboratories, and sources of variation. Each laboratory measured the amylose content of a set of 17 cultivars of rice. The study shows that five different versions of the iodine binding method are in use. The data show that repeatability was high within laboratories but reproducibility between laboratories was low. The major sources of variability were the way the standard curve was constructed and the iodine binding capacity of the potato amylose used to produce the standard. Reproducibility is much lower between laboratories using a standard curve of potato amylose alone compared with those using calibrated rice cultivars. This study highlights the need to standardize the way amylose is measured, and presents research avenues for doing so.     

Interlaboratory and Intralaboratory Testing of Soil Sulfate Analysis in Mollisols of the Pampas

Sulfur (S) deficiencies in grain and forage crops have been detected in many agricultural regions of the world, but soil tests are not commonly used as the basis for S fertilizer recommendation programs. Errors of measurements of soil sulfate were determined to assess whether the variation among and within soil-testing laboratories could be a factor that prevent the adoption of soil testing to assess soil sulfate availability. Subsamples of 10 selected soils (Mollisols) from the Pampas (Argentina) were sent in two batches to five soil-testing laboratories. Laboratories were unaware of the existence of subsamples and performed routine sulfate analysis as if these soils came from 60 different fields. Soil sulfate ranged from 3.3 to 20.6 mg kg^?1. One laboratory reported sulfate values greater than the other ones, having a mean bias of 4.1 mg kg^?1 S sulfate (SO₄). The other four laboratories reported similar sulfate values when soils had low sulfate availability (less than 10 mg S kg^?1), even when they used different extractants. Considering only these four laboratories, average interlaboratory coefficients of variations ranged from 6 to 24% for the 10 soils. Within-laboratory mean coefficients of variation (CVs) ranged from 12 to 22%. However, mean absolute errors of all laboratories were less than 1.2 mg kg^?1 S-SO₄. Two laboratories reported different sulfate values for the two batches of shipment (an average difference of 4.7 and 3.8 mg kg^?1 of S-SO₄). Laboratories using different extractants obtained similar results, suggesting that using the same extractant is not a prerequisite to standardize laboratory results in these soils. Differences between laboratories in our study were smaller than in other interlaboratory comparisons for soil sulfate. These differences could be easily detected and corrected if laboratories participate in an interlaboratory control system. The observed low mean absolute errors suggested that, in general, all laboratories achieve acceptable precision when evaluating within the same batch of determinations. Differences between batches of shipment (within laboratory error) stressed the importance of using reference material for internal quality control.     

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.     

Analysis of foods for iodine: interlaboratory study

An accurate, reproducible method for less than or equal to 1 ppm iodine in foods is required for nutritional labeling. In order to ascertain the current status of iodine analysis in foods, 7 samples, representing different food classes, were analyzed by 8 laboratories. Six laboratories used their modifications of the Ce-As-I catalytic method preceded by alkaline dry ashing. Two laboratories used neutron activation analysis (NAA), with differing radiochemical separations. The study showed wide discrepancy in analytical results. Mean relative standard deviation for all laboratories was 77.9% between laboratories; 19.1% within-laboratories. Laboratories using NAA had only slightly better precision than did laboratories using the chemical method. The lowest level reported on the entire group of samples ranged among laboratories from 0.0089 to 0.65 ppm. Figures reported by a laboratory are, in general, consistently high or consistently low. The only differences in methodology which may possibly correlate with level of iodine obtained are the use of NAA technique and use of manual, rather than automated, colorimetry.     

Preparation of whey protein hydrolysates using a single- and two-stage enzymatic membrane reactor and their immunological and antioxidant properties: characterization by multivariate data analysis

An initial 5% (w/v), followed thereafter with replacement aliquots of 3% (w/v), whey protein isolate (WPI) (ca. 86.98% Kjeldahl N x 6.38), was hydrolyzed using Protease N Amano G (IUB 3.4.24.28, Bacillus subtilis) in an enzymatic membrane reactor (EMR) fitted with either a 10 or 3 kDa nominal molecular weight cutoff (NMWCO) tangential flow filter (TFF) membrane. The hydrolysates were desalted by adsorption onto a styrene-based macroporous adsorption resin (MAR) and washed with deionized water to remove the alkali, and the peptides were desorbed with 25, 50, and 95% (v/v) ethyl alcohol. The desalted hydrolysates were analyzed for antibody binding, free radical scavenging, and molecular mass analysis as well as total and free amino acids (FAA). For the first time a quantity called IC50, the concentration of peptides causing 50% inhibition of the available antibody, is introduced to quantify inhibition enzyme-linked immunosorbent assay (ELISA) properties. Principal component analysis (PCA) was used for data reduction. The hydrolysate molecular mass provided the most prominent influence (PC1 = 57.35%), followed by inhibition ELISA (PC2 = 18.90%) and the antioxidant properties (PC3 = 10.43%). Ash was significantly reduced in the desalted fractions; the protein adsorption recoveries were high, whereas desorption with alcohol was prominently influenced by the hydrophobic/ hydrophilic amino acid balance. After hydrolysis, some hydrolysates showed increased ELISA reactivity compared with the native WPI.     

Analyses of the seed protein contents on the cultivated and wild buckwheat <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> resources

The seed protein contents of 179 accessions belonging to ten species of genus Fagopyrum were determined by means of the Kjeldahl method. The results indicated that there are significant differences of seed protein content among different accessions within the same species. The average protein content of F. esculentum is 12.94%, ranging from 8.81–18.71%, and the protein content of Sibano is the highest, up to 18.71%, Gantian 1 the lowest with 8.81%. The average protein content of tartary buckwheat is 12.17%, with the range from 7.82–18.94%, and the protein content of cultivated tartary buckwheat Qianku 2 is the highest (18.59%), and that of Ganku 1 (7.82%) the lowest. Moreover, there are significant differences of seed protein content among different buckwheat species and between the different buckwheat types (between common buckwheat and tartary buckwheat, between the diploid and the tetraploid, between the cultivated and the wild, and between the big-achene group and the small-achene group). The average seed protein content of F. giganteum is the highest in all tested species, up to 17.81% with the range of 13.91–21.27%, and that of F. callianthum the lowest, only 8.31%. This study was financed by the Natural Science Foundation of China (30270852, 30471116), Program for New Century Excellent Talents in University (NCET-2004-0913), Guizhou Key International Cooperation Project (Qianke Hewai G Zi 2005#400108), Guizhou Oversea Talent Project (Qianren Xiangmu Zizhu Hetong 2004#02), and Key Project of Students in Guizhou Normal University (GNU-SP-2005-SJXY-A01)     

Microbiological method for assaying lincomycin in animal feed: collaborative study.

A microbiological assay for determining lincomycin in swine feed, supplement, and a vitamin-mineral premix was studied collaboratively in 16 laboratories. The design of the study involved a complete feed, feed supplement, and a vitamin-mineral premix covering a range of fortification from 20 to 80 g/ton and 80 to 2600 g/ton. Two methods of sample preparation were used depending on the concentration of lincomycin in the sample. Statistical evaluation of the results from the 2 methods indicated that 10 and 11 collaborators, respectively, had mean recoveries which were not significantly different from one another. Ten laboratories obtained a mean recovery of 112.2% (range 102.3--123.5%) for the lower level, and 11 laboratories obtained a mean recovery of 104.4% (range 100.0--107.7%) for the higher level. The method has been adopted as official first action.