Rapid method for determination of protein content in cereals and oilseeds: validation, measurement uncertainty and comparison with the Kjeldahl method

The objective of this research was to test suitability of the Dumas combustion method to completely substitute the Kjeldahl method in routine laboratory determination of crude protein content in cereals and oilseeds. The validation of the method demonstrated that it is able to determine crude protein content in cereals and oilseeds in an efficient and accurate manner, with a detection limit w(N) = 0.006%, quantification limit w(N) = 0.019%, repeatability precision RSD _r = 0.41%, intra-laboratory reproducibility precision RSD _R = 0.74%, trueness, expressed in terms of bias b = 0.43%, and linear response between (2.36–19.2) mg N. Measurement uncertainty, expressed as relative expanded uncertainty (coverage factor k = 2, confidence level 95%), was calculated from validation data (U _rel = 2.24%). In order to examine the relationship between two methods, 15 cereal grain and oilseed samples were analyzed using Dumas and Kjeldahl procedure. The Kjeldahl procedure gave slightly lower w(N) values than the Dumas procedure: w _K(N) = 0.9905 w _D(N) = 0.0376 (R ²  = 0.9996). Relative standard deviations and results of homogeneity test obtained during analysis of complex cereal products (cereal breakfast and muesli bars) show that the Dumas combustion method may be less suitable for analysis of such samples compared to Kjeldahl method.     

A visual detection of protein content based on titration of moving reaction boundary electrophoresis

A visual electrophoretic titration method was firstly developed from the concept of moving reaction boundary (MRB) for protein content analysis. In the developed method, when the voltage was applied, the hydroxide ions in the cathodic vessel moved towards the anode, and neutralized the carboxyl groups of protein immobilized via highly cross-linked polyacrylamide gel (PAG), generating a MRB between the alkali and the immobilized protein. The boundary moving velocity (V_MRB) was as a function of protein content, and an acid–base indicator was used to denote the boundary displacement. As a proof of concept, standard model proteins and biological samples were chosen for the experiments to study the feasibility of the developed method. The experiments revealed that good linear calibration functions between V_MRB and protein content (correlation coefficients R > 0.98). The experiments further demonstrated the following merits of developed method: (1) weak influence of non-protein nitrogen additives (e.g., melamine) adulterated in protein samples, (2) good agreement with the classic Kjeldahl method (R = 0.9945), (3) fast measuring speed in total protein analysis of large samples from the same source, and (4) low limit of detection (0.02–0.15 mg mL⁻¹ for protein content), good precision (R.S.D. of intra-day less than 1.7% and inter-day less than 2.7%), and high recoveries (105–107%).     

High-performance liquid chromatographic separation of noreximide and norendimide in bulk material, tablets and animal feed

Noreximide, a sedative, is generally contaminated to some extent with its endo-isomer, norendimide, which produces excitation. A high-performance liquid chromatographic assay was developed to separate and quantitate these compounds on a 5-microns Ultrasphere ODS column with methanol-water (20:30) as mobile phase and detection at 254 nm. Assay of mixtures of these compounds in bulk material and tablets utilized isoniazide as internal standard. Peak area ratios were linear (r = 0.9999) over 1.4-66.2 micrograms of injected noreximide and 0.2-8.4 micrograms of injected norendimide. Overall percent recovery from simulated tablets containing noreximide alone was 99.6 +/- 0.8% (S.D., n = 3). Overall percent recoveries (+/- S.D.) from tablets containing a mixture of these compounds were 98.9 +/- 0.5% and 102.3 +/- 1.1% for noreximide and norendimide, respectively (n = 3). Noreximide in animal feed for long-term pharmacological studies was isolated by ether extraction and after work up, subjected to the same procedure, except that theophylline was the internal standard. Peak area ratios were linear over 0.2-19.3 micrograms of injected noreximide (r = 0.9999). Overall percent recoveries (+/- S.D., n = 3) of noreximide from spiked animal feed were 97.4 +/- 1.4% and 99.0 +/- 0.5% at the 500- and 5000-ppm levels, respectively. Limits of detection at the 95% confidence level (0.01 a.u.f.s., 20-microliters sample volume injected) were 1.67 microgram/ml and 2.56 micrograms/ml of noreximide and norendimide, respectively, in the final test solution.     

Repeatability and reproducibility of determination of the nitrogen content of fishmeal by the combustion (Dumas) method and comparison with the Kjeldahl method: interlaboratory study

Ten fishmeal samples (hidden duplicates of 4 meals plus 2 high-protein meals as a Youden pair), tryptophan, and nicotinic acid were analyzed by 18 laboratories using the Dumas method. Thirteen of the laboratories also analyzed the same 12 samples using their current Kjeldahl method. Recoveries (+/-SR) of tryptophan and nicotinic acid were 99.3+/-1.04 and 98.8+/-2.11% by Dumas and 97.1+/-3.03 and 74.6+/-26.76% by Kjeldahl. The Dumas method gave significantly greater values (P < 0.001) than the Kjeldahl method. For fishmeals, Kjeldahl N = 0.989 of Dumas N (P < 0.001). A similar proportionate difference (0.984 of Dumas N) was observed with tryptophan. Most laboratories failed to determine nicotinic acid correctly by Kjeldahl. For fishmeals, the relative standard deviations for repeatability and reproducibility were for Dumas 1.48 and 2.01% and Kjeldahl 1.62 and 2.37%, respectively. A single analysis conducted in 2 laboratories should not differ by more than 5.63% of the mean value when measured by Dumas or by more than 6.64% by Kjeldahl. It is concluded that with fishmeal, Dumas gives a more reliable measure of organic nitrogen than Kjeldahl, and, therefore, Dumas should be the method of choice.     

Determination of the total nitrogen content of hard, semihard, and processed cheese by the Kjeldahl method: collaborative study

The objective of this collaborative study was to determine interlaboratory performance statistics for a modified and optimized version of AOAC Method 920.123 for the determination of the total nitrogen content of hard, semihard, and processed cheese by Kjeldahl analysis. Details included addressing the issues of material homogeneity, test portion size (1 g), quantitative transfer (weighing on to filter paper), ensuring system suitability (nitrogen recoveries), and using AOAC Method 991.20 as the basis for nitrogen analysis. Fifteen laboratories tested 18 pairs of blind duplicate cheese materials with a crude protein content between 18 and 36%. Materials represented hard, semihard, and processed commercial cheeses with a wide range of composition. Statistical performance parameters expressed as crude protein (nitrogen x 6.38), g/100 g, with invalid and outlier data removed were mean = 26.461, repeatability standard deviation (Sr) 0.111, reproducibility standard deviation (S(R)) = 0.153, repeatability relative standard deviation (RSDr) = 0.42%, reproducibility relative standard deviation (RSDR) = 0.58%, repeatability (r) = 0.312, and reproducibility (R) = 0.428. The interlaboratory study results were acceptable and comparable to those for the milk Kjeldahl nitrogen method on a relative nitrogen basis. The Study Directors recommend that this modified method for the determination of total nitrogen in hard, semihard, and processed cheese by Kjeldahl analysis be adopted First Action as an improved method to replace Method 920.123.     

Kjeldahl nitrogen analysis as a reference method for protein determination in dairy products

Measurement of total nitrogen by Kjeldahl analysis is the historical reference method for determination of the protein content of dairy products and is used for both calibration and validation of alternative methods for protein determination. Accurate evaluation of alternative methods is not possible if there is large uncertainty regarding the reference values. When Kjeldahl analysis is used to establish reference values, the performance of the Kjeldahl testing must be verified and within established expectations. Advice is given for Kjeldahl system optimization, evaluation of test results, and trouble-shooting. Techniques for successful Kjeldahl nitrogen analysis of dairy products other than milk are discussed.     

全自动凯氏定氮仪测定化肥中的含氮量

建立全自动凯氏定氮仪检测化肥中氮含量的分析方法。称取总氮量在0.03~0.3 g范围的样品,经消解仪消解及全自动凯氏定氮仪碱化蒸馏,以硼酸为接收液,用0.15~0.20 mol/L的盐酸标准溶液进行自动滴定。该方法加标回收率为99.0%~101.5%,测定结果的相对标准偏差小于1.0%(n=6)。与国标GB/T 8527–2010法测定结果无显著性差异,全自动凯式定氮仪法的准确度、精密度均优于国标方法,而且所用试剂少、分析时间短,满足实验室快速检测大批量化肥中含氮量的需要。     

Gravimetric determination of amylase-treated neutral detergent fiber in feeds with refluxing in beakers or crucibles: collaborative study

As an important constituent of animal feeds, fiber represents the portion of feeds that is bulky and difficult to digest. The neutral detergent fiber (NDF) method, developed over 30 years ago, is the method of choice for measuring total fiber in forages and other feeds. Several modifications that were made to improve its general applicability to all feeds and others developed in individual laboratories often resulted in variability among laboratories in measuring NDF. The amylase-treated NDF (aNDF) method, therefore, was developed as an accurate and precise method of measuring total insoluble fiber in feeds. A collaborative study was conducted to evaluate the repeatability and reproducibility of the aNDF method over the full range of animal feed materials. Twelve laboratories representing research, feed company, regulatory, and commercial feed testing laboratories analyzed 11 materials as blind duplicates. The materials represented feed matrixes, including animal products; high-protein, high-fat, and high-pectin feeds; oil seeds; grains; heated by-product feeds; and legume and grass hays and silages. Materials selected varied in chemical composition and contained 0-90% aNDF, 1-16% ash, 1-20% crude fat, 1-40% crude protein, and 0-50% starch. Correcting results for changes in blanks and reporting results as ash-free aNDF organic matter (aNDFom) improved the repeatability and reproducibility of results when aNDF was <25%. The within-laboratory repeatability standard deviation (Sr) for percentage aNDFom in feeds varied from 0.21 to 1.82 and among-laboratory reproducibility standard deviation (S(R)) varied from 0.37 to 2.24. The HORRAT was <2 for all materials except feed materials containing >10% fat. However, standard deviations of repeatability and reproducibility for feeds with >10% fat were similar to those of other materials. It is recommended that the aNDF method be accepted for Official First Action status.     

DNA sequencing and multiplex STR analysis on plastic microfluidic devices

The ability of plastic microfluidic devices with separation channel lengths of 6, 10 or 18 cm to perform high-quality and high-performance ssDNA analysis was evaluated. Specifically, four-color DNA sequencing separation of a terminator sequencing standard using replaceable, urea-denaturing linear polyacrylamide (LPA) solution as a sieving matrix, yielded read lengths of 410 bases in 15 min with base calling accuracy of 99.2% on a 6-cm device, and 640 bases in 35 min with accuracy of 98.0% on a 18-cm device. A two-color sizing analysis of four-locus (CSF1PO, TPOX, TH01, vWA) short tandem repeats (STRs) allelic ladder on a 10-cm device indicated a mean SD of +/- 0.08 base pairs (bp) between runs, and single bp resolution of spiked TH01 allele 9.3 (198 bp) from TH01 allele 10 (199 bp) of the CTTv ladder with R = 0.81. A four-color multiplex sizing analysis of three different AmpFlSTR allelic ladders consisting of nine loci (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) and gender alleles (Amelogenin) on a 10-cm device had a mean SD of +/- 0.15 bp between runs for sizing three loci, i.e., FGA, D18S51 and D3S818; alleles differing by 2 bp in size were resolved with resolutions close to baseline. This work demonstrates that plastic microfluidic devices are capable of quality sequencing and STR sizing comparable to that of glass devices of similar separation lengths.     

Spectrophotometric determination of organic nitrogen by a modified Lassaigne method and its application to meat products and baby food

A modified Lassaigne method was developed for N determination based on fusion of the organic substance with metallic Na, conversion of the cyanide in the aqueous leachate to thiocyanate by ammonium polysulfide treatment, and colorimetric measurement of the thiocyanate formed by the addition of excessive ferric ions in acidic medium. The mean molar absorptivity of the Fe(NCS)2+ complex at 480 nm is 2.96 x 10(3) L/mol x cm, enabling quantitation of 0.25-7.72 ppm N (linear range) in the final solution. The relative amounts of Na, (NH4)2S2, and Fe(III) with respect to nitrogen in the analyte were optimized. The developed method was successfully applied to the determination of N in various brands of baby food, and it was compared statistically with the conventional Kjeldahl and elemental analysis methods. Protein nitrogen in a number of meat products was also precisely determined by the developed method. Thus, the total digestion time of the conventional Kjeldahl method was reduced considerably (e.g., to approximately 15 min for a dried sample) with a relatively simple spectrophotometric method requiring no sophisticated instrumentation.     

Validation study of a lateral-flow immunoassay for detection of ruminant by-product material in animal feeds and feed ingredients

An immunoassay with a lateral flow format has been developed for the detection of ruminant by-product material in animal feeds and feed ingredients. The test is designed for the analysis of animal feeds destined for feeding to ruminants to ensure that they do not contain ruminant by-products in violation of the ruminant-to-ruminant feed ban established by the U.S. Food and Drug Administration in 1997. This feed ban was established as a firewall against exposure of ruminant livestock animals to the prion agents responsible for neurological diseases such as bovine spongiform encephalopathy and scrapie. The test is designed for field use, e.g., at a feed mill, and yields a qualitative (presence/absence) result in 15-20 min. The objective of the study was to validate the lateral-flow test for detection of ruminant by-product material in a variety of finished animal feeds and feed ingredients. Results indicate that the test is specific for ruminant material and can detect as little as 1% ruminant material in these commodities.     

A comparison of the Kjeldahl and Dumas methods for the determination of protein in foods,using data from a proficiency testing scheme

Both the Kjeldahl and the Dumas methods for the determination of protein in foodstuffs are currently in use, but the empirical nitrogen factors used to convert the determined nitrogen content to protein content are based on the Kjeldahl method alone. Non-equivalence between the two methods could therefore result in some laboratories reporting an incorrect protein content. We report here a study using data accumulated over several years in the results of a proficiency testing scheme. On average the Dumas method provided results that were relatively higher by about 1.4% than the Kjeldahl method, but the difference between the methods depended on the type of foodstuff. The methodology of looking for bias between analytical methods is critically discussed.     

Increasing the Value of Hominy Feed as a Coproduct by Fermentation

Hominy feed is a low value ($83.7/metric ton) coproduct of the corn dry milling process that accounts for nearly 35% of the starting corn quantity. The average composition of hominy feed on a dry basis is 56.9% starch, 25.2% neutral detergent fiber, 11.1% protein, and 5.3% fat. Starch in hominy feed can be fermented to ethanol thus increasing its levels of protein and fat. The increase in protein and fat percentages may increase the market competitiveness and price of hominy feed. Hydrolysis and fermentation were performed on nine hominy feed samples collected from three corn dry milling plants in the USA. The original hominy feed samples and postfermentation solids were analyzed for starch, protein, fat, and fiber content. Compared to the original hominy feed, the percentage increase in protein, fat and fiber in postfermentation solids of nine samples ranged from 10.4 to 21.3, 6.78 to 10.6, and 12.6 to 28.7% (dry basis), respectively. Ethanol yields varied from 271.7 to 380.2 l/metric ton for the nine hominy feed samples. These results indicate that the value of hominy feed as an animal feedstock can potentially be increased with fermentation and can produce more profit per metric ton than currently being derived by its sale as a low protein feed ingredient.     

磷酸铵镁沉淀法测定聚马来酸酐氨化体系中的铵离子

通过甲醛法、四苯硼钠沉淀法以及磷酸铵镁沉淀法(MAP)测定模拟体系中铵离子含量,分析比较确定了聚马来酸酐(PMA)与氨气反应产物中铵离子的测定方法,即利用磷酸氢二钠和氯化镁结合铵离子形成磷酸铵镁沉淀,并通过凯式定氮法确定体系中铵离子含量。结果表明,pH值为9.0时,铵离子测定得到了较好的结果,测定平均值为5.50mmol/g,测定平均误差为2.1%,方法有很好的精密度和准确度,能够满足实际测试要求。     

Development of a matrix solid-phase dispersion method for the simultaneous determination of pyrethroid and organochlorinated pesticides in cattle feed

A matrix solid-phase dispersion (MSPD) method was developed for the simultaneous extraction of 36 common pesticides and breakdown products (mostly pyrethroids and organochlorines) in cattle feed. Different parameters affecting the extraction efficiency (such as dispersing phase, clean-up adsorbent and elution volume) were investigated. The experimental procedure was optimized using a multivariate statistical approach and the final analyses were carried out by GC-muECD. Several protocols for extract purification were also studied. As far as we know, this is the first application of MSPD for the extraction of most of the target pesticides from animal feed. Using the optimized extraction conditions, the method was validated in terms of accuracy, and precision (within-a-day and among-days), using a certified reference material (CRM 115) as well as spiked cattle feedingstuffs at different concentration levels. A matrix effect study was also carried out using various real samples. The recoveries were satisfactory (>75% in most cases) and the quantification limits, at the sub-ngg(-1) or low-ngg(-1) level, complied with the regulated maximum residue levels (MRLs) in animal feed and in main crops used in the preparation of cattle feeding materials. Finally, the MSPD-GC-muECD methodology was applied to the analysis of real cattle feed samples collected in farms of dairy cattle from NW Spain.     

Classification and determination of total protein in milk powder using near infrared reflectance spectrometry and the successive projections algorithm for variable selection

This paper proposes a methodology for the classification and determination of total protein in milk powder using near infrared reflectance spectrometry (NIRS) and variable selection. Two brands of milk powder were acquired from three Brazilian cities (Natal-RN, Salvador-BA and Rio de Janeiro-RJ). The protein content of 38 samples was determined by the Kjeldahl method and NIRS analysis. Principal component regression (PCR) and partial least squares (PLS) multivariate calibrations were used to predict the total protein. Soft independent modeling of class analogy (SIMCA) was also used for full-spectrum classification, resulting in almost 100% classification accuracy, regardless of the significance level adopted for the F-test. Using this strategy, it was feasible to classify powder milk rapidly and nondestructively without the need for various analytical determinations. Concerning the multivariate calibration models, the results show that PCR, PLS and MLR-SPA models are good for predicting total protein in powder milk; the respective root mean square errors of prediction (RMSEP) were 0.28 (PCR), 0.25 (PLS), 0.11 wt% (MLR-SPA) with an average sample protein content of 8.1 wt%. The results obtained in this investigation suggest that the proposed methodology is a promising alternative for the determination of total protein in milk powder.     

Development of an analytical method based on microwave-assisted extraction and solid phase extraction cleanup for the determination of organochlorine pesticides in animal feed

A method to determine 21 organochlorine pesticides in animal feed samples using microwave assisted extraction and solid phase extraction cleanup was optimised regarding its main parameters. After extraction with hexane-acetone (50:50), three different sorbents (alumina/ENVI-Florisil, ENVI-Carb and ENVI-Carb II/PSA) were assayed for the cleanup step. Analytes were eluted with hexane-ethyl acetate (80:20) and determined by gas chromatography and electron capture detection followed by gas chromatography-mass spectrometry. ENVI-Carb and ENVI-Carb II/PSA provided colourless eluates but fewer interferent compounds were found in ENVI-Carb II/PSA chromatograms, so this system was selected to carry out the purification of the extracts. The analytical recoveries obtained with this method were close to 100% in most cases with relative standard deviations lower than 10%. These percentages were similar to those obtained with the Soxhlet extraction procedure, which shows the method suitable for the determination of organochlorine pesticides in animal feed material. The method was also validated with the analysis of a certified reference material (CRM-115 BCR), and the results obtained were in good accordance with the certified values.     

基于整体材料搅拌棒固相萃取高效液相色谱联用测定饲料和水样中硝基呋喃类药物残留

利用自制的以聚(乙烯基咪唑-二乙烯基苯)(VIDB)整体材料为涂层的固相萃取搅拌棒(VIDB-SBSE)萃取3种硝基呋喃类药物,然后与高效液相色谱-二极管阵列检测器联用建立了测定饲料和水样品中硝基呋喃类药物残留的方法。详细考察了萃取过程中萃取和解吸时间、样品基质的pH值以及离子强度等实验条件对萃取效率的影响。在最佳条件下,呋喃唑酮的线性范围为0.5~200μg/L,呋喃妥因和呋喃西林的线性范围为0.25~200μg/L,3种目标物的检出限(LO D)(S/N=3)在0.068~0.11μg/L之间,所建方法具有理想的日内和日间重现性(R SD值均小于6%)。在对饲料和实际水样的测定中,不同加标浓度呋喃唑酮、呋喃妥因和呋喃西林的回收率在80.6%~108%之间。研究表明,所建立的方法具有简便、灵敏、环境友好等特点。     

Fast and selective determination of total protein in milk powder via titration of moving reaction boundary electrophoresis

In this paper, moving reaction boundary titration (MRBT) was developed for rapid and accurate quantification of total protein in infant milk powder, from the concept of moving reaction boundary (MRB) electrophoresis. In the method, the MRB was formed by the hydroxide ions and the acidic residues of milk proteins immobilized via cross‐linked polyacrylamide gel (PAG), an acid‐base indicator was used to denote the boundary motion. As a proof of concept, we chose five brands of infant milk powders to study the feasibility of MRBT method. The calibration curve of MRB velocity versus logarithmic total protein content of infant milk powder sample was established based on the visual signal of MRB motion as a function of logarithmic milk protein content. Weak influence of nonprotein nitrogen (NPN) reagents (e.g., melamine and urea) on MRBT method was observed, due to the fact that MRB was formed with hydroxide ions and the acidic residues of captured milk proteins, rather than the alkaline residues or the NPN reagents added. The total protein contents in infant milk powder samples detected via the MRBT method were in good agreement with those achieved by the classic Kjeldahl method. In addition, the developed method had much faster measuring speed compared with the Kjeldahl method.     

Liquid chromatographic method for the quantification of zearalenone in baby food and animal feed: interlaboratory study

An interlaboratory trial for determination of zearalenone (ZON) in baby food and animal feed was conducted. The study involved 39 participants in 16 European Union member states, as well as Turkey, Uruguay, and China, representing a cross-section of industry, and official food control and research institutes. The method is based on immunoaffinity column cleanup followed by high-performance liquid chromatography using fluorimetry (HPLC-FI). The test portion of the sample is extracted with methanol-water (75 + 25, v/v). The sample extract is filtered, diluted, and passed over an immunoaffinity column. ZON is eluted with methanol. The separation and determination of ZON is performed by reversed-phase HPLC-FI with an excitation wavelength of 274 nm and an emission wavelength of 446 nm. Test portions of the samples were spiked at levels of 20 and 30 microg/kg ZON in baby food and at levels of 100 and 150 microg/kg ZON in animal feed. Mean recoveries from each participant ranged from 78 to 119% with an average value of 92% for baby food and from 51 to 122% with an average value of 74% for animal feed. Based on results for spiked samples (blind duplicates at 2 levels), as well as naturally contaminated samples (blind duplicates at 3 levels), the relative standard deviation for repeatability (RSDr) in baby food ranged from 2.8 to 9.0%. For animal feed, this value ranged from 5.7 to 9.5%. The relative standard deviation for reproducibility (RSDR) in baby food ranged from 8.2 to 13.3%, and for animal feed this value ranged from 15.5 to 21.4%. The Horwitz ratio (HorRat) in baby food ranged from 0.3 to 0.4, and for animal feed this value ranged from 0.6 to 0.9. The method showed acceptable within- and between-laboratory precision for each matrix, as required by European legislation.