Ion-exchange separation of amino acids with postcolumn orthophthalaldehyde detection

Moore's and Stein's classical ion-exchange separation of amino acids remains the standard by which all methods are judged. The adaptation of liquid chromatography (LC) equipment to amino acid analysis was inevitable because microprocessor control of gradients allowed almost infinite variation in gradient shape, producing superior resolution with only 2 buffers. The versatility of LC equipment allowed the instruments to be used for other assays. Adaptation of orthophthalaldehyde (OPA) to amino acid analysis increased detection sensitivity to the picomole range. A method for essential amino acids analysis in mechanically separated red meat and poultry products has been adapted to liquid chromatography using postcolumn hypochlorite oxidation, OPA derivatization, and fluorescence detection. Separation is achieved with 2 sequential concave exponential gradients combining ionic strength and pH increases with halide-containing buffers. Hydroxyproline and proline are detected with increasing sensitivity through the use of 3-mercaptopropionic acid in a stabilized OPA reagent. Sample preparation is a critical part of the method. A defatting procedure removes fat and other nonprotein nitrogenous substances. The hydrolysis procedure is designed to protect tryptophan which can be routinely assayed in hydrolysates with a modified flow program. Corrosion damage to the equipment by halide buffers has brought about a search for alternative methodology.     

Quantitative analysis of cystine, methionine, lysine, and nine other amino acids by a single oxidation--4 hour hydrolysis method

The sulfur-containing amino acids cystine and methionine play important roles in animal, especially avian, nutrition. Because these sulfur-containing amino acids are destroyed to varying extents by 6N HCl hydrolysis, oxidation and hydrolysis of cystine to cysteic acid and methionine to methionine sulfone have been widely used for determination of cystine and methionine. Lysine is considered the next limiting amino acid after the sulfur amino acids in poultry nutrition; therefore, determination of the amino acid content of rations focuses first on these 3 amino acids. The objective of this investigation was to establish whether lysine and other amino acids could be accurately determined in proteinaceous materials which had undergone performic acid oxidation. To perform this evaluation, lysine was determined in a variety of protein-containing materials both with and without performic acid oxidation. Performic acid oxidation followed by 6N HCl hydrolysis at 145 degrees C for 4 h allows accurate measurement of 3 amino acids especially important to poultry nutrition, cystine, methionine, and lysine, in a single preoxidized hydrolysate; this method can be extended to another 9 protein amino acids.     

Rapid determination of free fatty acids in poultry feed lipid extracts by SB-ATR FTIR spectroscopy

A simple, rapid, and reproducible method has been developed for the quantitative determination of free fatty acid (FFA) content in lipids extracted from poultry feeds by Fourier transform infrared (FTIR) spectroscopy with the use of a single-bounce attenuated total reflectance (SB-ATR) accessory. An FTIR calibration curve was prepared by gravimetrically adding oleic acid (15-37%) to pure refined, bleached, and deodorized (RBD) canola oil and measuring the area of the COOH absorption band at 1710 cm-1. The oil from each of 12 poultry feed formulations was extracted using conventional Soxhlet extraction, and after evaporation of the solvent, the FFA content was determined by the conventional AOCS titrimetric procedure and by the SB-ATR/FTIR method. The SB-ATR/FTIR FFA predictions were related to those determined by the AOCS titrimetric method by linear regression, producing an R value of 0.999 and a SD of +/-0.28% FFA. Time-course spectra collected as lipids extracted into hexane indicated that a 15 min extraction was adequate to obtain a representative sample for FFA determination, with further extraction resulting in little, if any, change in the proportion of FFA in the lipid extract. Only a small volume of the hexane extract ( approximately 20 mL) yielded sufficient material for the SB-ATR/FTIR analysis. Thus, by shortening the extraction time and taking a small sample so as to reduce solvent removal time, the SB-ATR/FTIR procedure provides a very simple and rapid means of determining the FFA content of poultry feed lipids.     

Preparation and analysis of cyclotri- and cyclotetraphosphate and their hydrolysis products in soil

Cyclotriphosphate (Na(3)P(3)O(9)) and cyclotetraphosphate (Na(4)P(4)O(12)) are not strongly sorbed by soil constituents. Potential movement and efficient plant utilization of P from these compounds are dependent on the hydrolysis of the cyclophosphate ring structure and their hydrolysis products. The objectives of this study were to prepare pure useable quantities of these cyclophosphates and their hydrolysis products and to extract, separate, and analyze these compounds after application to diverse soils. Cyclophosphates of high purity (>99.0%) were prepared, and improved methods of extraction and analysis by ion chromatography were developed. Cyclophosphates and their hydrolysis products were extracted from soil using a sequential water/0.5 M H(2)SO(4)/1.0 M NaOH extraction that maximized P recovery and minimized hydrolysis of cyclic and linear phosphates during the extraction procedure. Gradient elution chromatography separated cyclic phosphates and their hydrolysis products. Separation and direct quantitative analysis of the applied cyclophosphates and their hydrolysis products were accomplished in <15 min.     

A screening method for determining nitrofuran drug residues in animal tissues.

A method was developed for measuring low levels of total nitrofurans in animal tissues and milk. The antimicrobial nitrofurans (5 or more products) used in agriculture are extracted from tissue with aqueous acid in the presence of ethyl acetate. After centrifugation and evaporation, the organic residue is washed with hexane and the nitrofurans are hydrolyzed to 5-nitrofuraldehyde in aqueous acid at 70 degrees C. The hydrolysis product is extracted with benzene and measured by gas-liquid chromatography with electron capture detection. Recoveries of nitrofurazone and furazolidone from fortified poultry and swine tissues at the levels of 0.5 and 0.1 ppm are 75 and 65%, respectively. This procedure can be used to detect the total nitrofuran content of as little as 10 ppb muscle tissues and milk, 100 ppb liver, and 50 ppb fat with no interference from related veterinary nitrodrugs.     

Determination of sulfur amino acids and tryptophan in foods and food and feed ingredients: collaborative study

Samples of 4 foods, 1 animal feed, isolated soy protein, and beta-lactoglobulin were analyzed by 9 laboratories to determine concentrations of cysteine as cysteic acid, methionine as methionine sulfone, and tryptophan. Sulfur amino acids were determined by AOAC method 43.A08-43.A13 for food and feed ingredients, in which samples are oxidized with performic acid before protein hydrolysis with 6N HCl. Tryptophan was determined after protein hydrolysis with 4.2N NaOH. In both methods, free amino acids were separated by ion-exchange or reverse-phase chromatography. Each laboratory was provided with detailed methods and with sealed vials containing solutions of standards. Samples were analyzed in duplicate, and variation between laboratories was determined. Coefficients of variation between laboratories for the 6 samples ranged from 5.50 to 11.8% for methionine as methionine sulfoxide, 8.59 to 17.3% for cysteine as cysteic acid, and 3.87 to 16.1% for tryptophan. Amino acid recoveries were determined by analysis of beta-lactoglobulin and were based on expected levels of each amino acid obtained from amino acid sequence data. The mean recovery of cysteine was 97% with a range of 88-119%. For methionine, mean recovery was 98% (range 89-115%) and for tryptophan, 85% (range 59-102%). Method 43.A08-43.A13 for food and feed ingredients has been adopted official first action for determination of cysteine and methionine in processed foods. The alkaline hydrolysis method has been adopted official first action for determination of tryptophan in foods and food and feed ingredients.     

Simultaneous determination of oxytetracycline, tetracycline, and chlortetracycline in milk by liquid chromatography

A simple, rapid procedure is described for the simultaneous quantification of 3 tetracycline drugs in bovine milk. Samples are prepared by dilution with an EDTA/phosphate buffer solution and filtration through a molecular weight cutoff filter. Analytes are concentrated on-column using a reverse-phase gradient elution system of oxalic acid, acetonitrile, and methanol. The limits of quantitation are approximately 15-50 ng/mL and the limits of detection are 10-20 ng/mL, depending on the compound. For oxytetracycline, over the range 50-1200 ng/mL, the average recovery and intralaboratory coefficient of variation were 97% and 4.1%, respectively. Over the same range, these parameters were, respectively, 97% and 5.0% for tetracycline, and 89% and 6.4% for chlortetracycline. The applicability of this procedure is demonstrated by separation and detection of incurred tetracycline residues in milk from treated animals.     

脱酰胺与双酶协同作用提高小麦面筋蛋白酶解效率

为了探讨了不同脱酰胺处理和双酶协同作用方式对小麦面筋蛋白酶解效率及其产物抗氧化活性的影响,该文研究了小麦面筋蛋白在各种预处理方式和酶解条件下的蛋白回收率、水解度、抗氧化性能及肽分子量分布情况。结果显示,单独热处理(90℃,30 min)小麦面筋蛋白对其酶解效率无显著影响,而采用添加0.5 mol/L柠檬酸溶液进行热处理(质量分数为5%,90℃,30 min)可显著(P0.05)提高其蛋白回收率。此外,酶制剂添加顺序及双酶共同水解作用时间对酶解效率均具有较大影响:加入谷氨酰胺酶预先水解对小麦面筋蛋白的深度水解有促进作用;一定时间内的双酶协同作用有利于酶解的进行,但较长时间的双酶作用反而会抑制酶解效率。采用谷氨酰胺酶(质量分数为0.2%)对经柠檬酸加热处理的小麦面筋蛋白作用12 h后再加入胰酶(质量分数为0.6%)共同作用7 h可使蛋白回收率达70.74%,水解度达到9.88%;另外,酶解产物的自由基清除能力ABTS+(2,2’-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)+)值与氧化自由基吸收能力(ORAC,oxygen radical absorbance capacity)值分别达到478.95 mmol/g和213.85μmol/g,提示该酶解产物是一种潜在优秀食品抗氧化剂。研究结果可为拓宽小麦面筋蛋白的应用领域,以及高效制备抗氧化活性肽提供方法和理论指导。     

Determination of ergosterol in ganoderma spore lipid from the germinating spores of Ganoderma lucidum by high-performance liquid chromatography

A gradient reversed-phase high-performance liquid chromatography (HPLC) method was developed for the separation and determination of free ergosterol in ganoderma spore lipid (GSL) extracted from the sporoderm-broken germinating spores of Ganoderma lucidum. Sodium hydroxide in methanol was added for the hydrolysis of ergosteryl esters to determine the total content of ergosterol in GSL by HPLC. A 0.04 M concentration of sodium hydroxide in reaction mixtures was appropriate for the complete hydrolysis of ergosteryl esters without a significant loss of ergosterol during saponification. In addition, the ergosterol content in four commercial GSL softgel supplements from four different firms was determined. The results showed that the ergosterol content in these samples had significant differences. Ergosterol content may be a suitable marker for evaluating the quality of GSL products.     

Liquid chromatographic determination of amprolium in poultry feed and premixes using postcolumn chemistry with fluorometric detection

Two extraction and liquid chromatographic procedures are presented which separate amprolium from compounds in poultry feed or premixes that could interfere with its fluorometric determination. The procedures are based on earlier work on the determination of thiamine in food samples. Amprolium is extracted from feed with a hexane-aqueous sulfosalicylic acid mix, separated on a C18 column, and detected fluorometrically after postcolumn derivatization. For premixes, water extraction is used. Values for the amprolium content of poultry feed obtained with these procedures are in good agreement with those obtained with AOAC official methods. It is suggested that these methods with suitable modifications may be of use for routine analysis of amprolium in feeds. The overall methods are rapid and appear to give reasonable results.     

An automated anion-exchange chromatographic procedure for the estimation of saccharides in acid hydrolysates of soil

It is well known that the major neutral monosaccharide components released from soil by acid hydrolysis are glucose, galactose, mannose, arabinose, xylose, ribose, rhamnose and fucose. A colorimetric determination of the saccharide mixture released from soil is unsatisfactory for determining an accurate figure for soil saccharides. More precise information can be obtained by the determination of monosaccharides after separation by chromatography. Paper and thin-layer chromatography for quantitative analysis are rather time consuming and laborious. The gas chromatographic procedure was applied successfully for the analysis of sugars in soil hydrolysates by OADES et at. (7). Preparation of the various derivatives for gas chromatography still requires many steps, much handling of the sample, and considerable time, although final analysis of the product derivative is accomplished in an hour or two.     

Simultaneous detection of residues of 25 β₂-agonists and 23 β-blockers in animal foods by high-performance liquid chromatography coupled with linear ion trap mass spectrometry

A sensitive method has been developed for the simultaneous determination of residues of 25 β?-agonists and 23 β-blockers in animal foods by high-performance liquid chromatography coupled with linear ion trap mass spectrometry (HPLC-LIT-MS). This method is based on a new procedure of hydrolysis and extraction by 5% trichloracetic acid, and then cleaned up by mixed strong cation exchange (MCX) cartridges coupled with a novelty cleanup step by methanol. Methanol and 0.1% formic acid were used as mobile phases for gradient elution, while a Supelco Ascentis Express Rp-Amide column was used for LC separation. ESI positive ion scan mode was used with consecutive reaction monitoring (CRM, MS3). Nine β?-agonists labeled by the deuterium isotope were used as internal standards for quantification. The linear ranges of 48 analytes were from 5 to 200 μg/L; the coefficient of correlation was not less than 0.995. Blank pork muscle, blank liver, and blank kidney were selected as representative matrix for spiked standard recovery test. The recoveries of each compound were in the range of 46.6-118.9%, and the relative standard deviations were in the range of 1.9-28.2%. Decision limits (CCα, α = 0.01) of 48 analytes in muscles, liver, and kidney samples ranged from 0.05 to 0.49 μg/kg, and the detection capability (CCβ, β = 0.05) ranged from 0.13 to 1.64 μg/kg. This method was successfully applied to 110 real animal origin food samples including meat, liver, and kidney of pig and chicken samples.     

Rapid method for determination of 2-hydroxy-4-(methylthio)butanoic acid in poultry feeds by capillary isotachophoresis

Capillary isotachophoresis, which involves the separation of charged species under an electric field, has been applied to the rapid determination of 2-hydroxy-4-(methylthio)butanoic acid at 0.04-0.50% concentration levels in corn/soy-based poultry feeds, using conductivity detection. The procedure merely involves a 15 min cold water extraction of the sample and a 15 min analysis after injection of the filtrate into the instrument. Since only charged species migrate and non-ionic species stay virtually at the front of the column, extraordinary selectivity can be achieved. The isotachophoresis method is an order of magnitude faster than the gas chromatographic method reported recently and also provides information on HMB-monomer/dimer ratio in the same run. The sample recoveries exceeded 90% in all concentration ranges studied with coefficients of variation less than +/- 10%.     

Amino acid analysis of feeds in The Netherlands: four-year proficiency study

To improve accuracy and precision of amino acid analysis in 12 Dutch feed laboratories, a proficiency study was organized twice annually over a 4-year period. The method used included reflux acid hydrolysis for 22 h followed by evaporation, separation on a cation-exchange resin in an amino acid analyzer, and photometric detection after post-column derivatization with ninhydrin. For the determination of sulfur-containing amino acids, samples were oxidized prior to hydrolysis. For the determination of tryptophan, samples underwent an alkaline hydrolysis excluding oxygen, were separated by liquid chromatography on a Hypersil ODS analytical column, and were assayed by UV or fluorescence detection. The average relative standard deviations within (CVr) and between (CVR) laboratories were 3 and 7%, respectively. For some mixed feed samples, the results from the proficiency study were compared with those obtained by the International Analytical Group. For those samples, the relative standard deviation of the difference between IAG and Dutch groups was only 1.9%. For samples that were analyzed twice during this 4-year period, relative standard deviation of between-series differences was only 2.1%.     

Investigation of enzymatic hydrolysis conditions on the properties of protein hydrolysate from fish muscle (Collichthys niveatus) and evaluation of its functional properties

This study was carried out to investigate the enzymatic hydrolysis conditions on the properties of protein hydrolysate from fish muscle of the marine fish species Collichthys niveatus. About 160 fish samples were tested, and the analyzed fish species was found to be a lean fish with low fat (1.77 ± 0.01%) and high protein (16.76 ± 1.21%). Fish muscle of C. niveatus was carefully collected and hydrolyzed with four commercial enzymes: Alcalase, Neutrase, Protamex, and Flavourzyme under the conditions recommended by the manufacturers. Among the tested proteases, Neutrase catalyzed the hydrolysis process most effectively since the hydrolysate generated by Neutrase has the highest content of sweet and umami taste amino acids (SUA). The effect of hydrolysis conditions was further optimized using response surface methodology (RSM), and the optimum values for temperature, pH, and enzyme/substrate ratio (E/S ratio) were found to be 40.7 °C, 7.68, and 0.84%, respectively. Finally, the amino acid composition of the hydrolysate was analyzed by AccQ·Tag derivatization and HPLC-PDA determination. Major amino acids of the muscle of C. niveatus were threonine, glutamic acid, phenyalanine, tryptophan, and lysine, accounting for respectively 10.92%, 10.85%, 10.79%, 9.86%, and 9.76% of total amino acid content. The total content of essential amino acids was 970.7 ng·mL(-1), while that of nonessential amino acids was 709.1 ng·mL(-1). The results suggest that the fish muscle and its protein hydrolysate from C. niveatus provide a versatile supply of the benefits and can be incorporated as supplements in health-care foods.     

Determination of ammonium,amides, and soluble carboxylates in plant tissues

Methods for extraction and determination of ammonium, amides and soluble carboxylates in plant tissues are compared and discussed. The procedure recommended involves extraction of finely ground plant tissues with 0.2 M formic acid, determination of ammonium and amides in the extract by overnight alkaline hydrolysis and distillation in Conway microdiffusion dishes, measurement of extracted ammonium using Carlson's rapid diffusion‐conductivity instrument, and estimation of soluble organic acids by titration after separation and purification with ion exchange resins. The method described is reasonably rapid, and gives complete recovery and highly reproducible results. The final solution of organic acids is suitable for direct chromatographic analysis.     

Effect of hydrolysis time on the determination of amino acids in samples of soybean products with ion-exchange chromatography or precolumn derivatization with phenyl isothiocyanate

Accurate determination of amino acid levels in soy products facilitates optimum diet formulation and amino acid supplementation. A study was carried out to investigate the effect of hydrolysis time and method of amino acid measurement on amino acid levels. Correction factors to standardize amino acid levels to 24 h of hydrolysis were also determined. Six different soybean products were evaluated. Hydrolysis was carried out for 10 different periods of time. Amino acids were analyzed by both ion-exchange chromatography and precolumn derivatization with phenyl isothiocyanate. Both hydrolysis time and measurement method affected (P < 0.05) amino acid levels. Standard hydrolysis conditions (hydrolysis in 6 M HCl at 110 degrees C for 24 h) rarely provide the maximal amino acid values. Therefore, sequential hydrolyses curves were very useful and should be used.     

Determination of water-insoluble cell walls in feeds: interlaboratory study

A collaborative study was conducted to test a new rapid procedure for determination of water-insoluble cell wall (WICW) content in feeds. In the method, starch is solubilized near boiling temperature with Termamyl, a heat-stable alpha-amylase, and proteins are solubilized at 40 degrees C with sodium dodecylsulfate and Pronase. Then, the organic matter of the residue is determined by incineration. Three hours were required to treat 12 different samples, including solubilization treatments, filtrations, and rinses. Eleven unknown products including 9 common feedstuffs of various origin and 2 mixed diets for poultry were analyzed by 7 analysts in France. Coefficients of variation ranged from 2.3 to 6.1%. The results were compared to those for water-insoluble dietary fiber (WIDF), total dietary fiber, and neutral detergent fiber. Agreement was best with the water-insoluble dietary fiber procedure. For most samples, the ratios of WIDF/WICW ranged from 0.981 to 0.842. The differences between WICW and WIDF values correspond to cell wall protein which is accounted for in WICW, but not in WIDF.     

Determination of polybrominated biphenyl residues in dry animal feeds.

A new procedure is described for the determination of polybrominated biphenyls (PBBs) in dry animal feeds and developmental results are discussed. Finely ground feed is packed into a chromatographic column containing Celite and then eluted with methylene chloride. The concentrated extract is cleaned up by elution with petroleum ether through Florisil before gas-liquid chromatographic quantitation. Chromatograms thus obtained were essentially free of the interfering peaks encountered when using AOAC methods for pesticide residues in dry products. Results of feed analyses by the proposed procedure averaged 30% higher than those obtained by AOAC procedures. Recoveries of PBBs from samples fortified at levels of 0.04 to 0.4 ppm ranged from 90 to 104%, with an average of 98%.     

NOTE: Hydrolysis Temperature Affects the Phloroglucinol Assay for Arabinoxylan Quantification in Wheat Flour

Phloroglucinol has a long history of use for the determination of pentose monomer content. Its application to cereal chemistry has been developed over several decades; however, no studies on the potential influence of hydrolysis temperature have yet been reported. We demonstrate the effect of hydrolysis temperature on the phloroglucinol assay for the measurement of pentosans in both refined and wholemeal wheat flours. In refined flour, monosaccharide degradation and interfering reactions from starch appear to effectively reduce the absorbance difference used to determine pentosan content. The presence of glucose in the reaction is also shown to provide stability to the reaction products. The potential impact of these factors on the determination of pentosan content needs to be considered when interpreting the results obtained using phloroglucinol‐based methods.