共查询到20条相似文献,搜索用时 625 毫秒
1.
A simple, rapid microwave digestion procedure for protein hydrolysis preceding the determination of amino acids in yeast using gas chromatography–mass spectrometry (GC–MS) is described. Protein hydrolysis was performed in a focused microwave using 4 M methanesulfonic acid (MAS). Amino acids were derivatized with methyl chlorofomate (MCF) and extracted into chloroform prior to GC–MS analysis. The microwave parameters, including power, temperature and heating time, were optimized. It was found that temperature and heating time were the most influential factors. A total of 17 amino acids were determined in selenium-enriched yeast with use of standard addition calibration. Limits of detection and quantitation (LODs/LOQs) of the amino acids measured were in the sub-nmol range, suitable for monitoring of amino acids in yeast and other food products. 相似文献
2.
Damm M Holzer M Radspieler G Marsche G Kappe CO 《Journal of chromatography. A》2010,1217(50):7826-7832
An efficient microwave-assisted high-throughput protein hydrolysis protocol was developed utilizing strongly microwave absorbing silicon carbide-based microtiter platforms. The plates are equipped with 20 bore holes having the proper dimensions for holding standard screw-capped HPLC/GC vials. Due to the possibility of heating up to four heating platforms simultaneously (80 vials), parallel microwave-assisted acid hydrolyses can be performed under carefully controlled conditions significantly reducing the overall time required for protein hydrolysis and the subsequent evaporation step required for larger volumes of acid. An extensive optimization of the hydrolysis conditions has demonstrated that 5min irradiation at 160°C with 6N HCl leads to comparable results in terms of total and individual amino acid recovery as the traditional method requiring 24h heating at 110°C. Complete hydrolysis of several proteins and synthetic peptides was performed using 25μg of sample material and 100μL of 6N HCl in a dedicated low-volume HPLC/GC vial. Since the hydrolysis and subsequent analysis can be performed from the same vial, errors caused by sample transfer can be minimized. Control experiments have demonstrated that the observed rate enhancements are the result of a purely thermal/kinetic effect as a consequence of the considerable higher reaction temperatures. 相似文献
3.
4.
Microwave-oven-assisted acid hydrolysis of albumin in 7.5 M HCl was carried out in only 12 min in a pressurized Teflon reactor using a power of 390 W. The effect of experimental conditions on the hydrolysis of albumin were evaluated and the results obtained were compared with those found by conventional hydrolysis, using in both cases the ninhydrin reaction for the spectrophotometric determination of free amino acids. A chromatographic study showed that microwave oven heating did not change the nature of the amino acids obtained from the hydrolysis of the protein. 相似文献
5.
微波水解衍生高效液相色谱法测定饲料中的氨基酸 总被引:1,自引:0,他引:1
建立了一种微波水解衍生高效液相色谱同时测定饲料中17种氨基酸含量的方法。采用2,4-二硝基氯苯作为柱前衍生试剂,利用C_(18)色谱柱分离。二极管阵列检测器进行检测,检测波长为360 nm,并对微波水解时间及温度进行优化。17种氨基酸在2.5~50 mg/L范围内呈良好线性关系,相关系数为0.990 7~0.999 9;相对标准偏差为0.88%~4.2%;加标回收率为90.6%~107.2%;检出限为0.15~2.37 mg/L。研究结果表明,150℃下微波水解16 min的结果与传统加热水解(110℃,24 h)的效果基本相同。该方法分析时间较短,灵敏度较高,可用于饲料中氨基酸含量的检测。 相似文献
6.
Optimization and application of microwave-assisted acid hydrolysis for rapid quantification of protein oxidation markers using LC-MS 总被引:1,自引:0,他引:1
Simple and efficient microwave-assisted acid hydrolysis (MAAH) of proteins was used for rapid quantification of α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) as major protein oxidation markers. The precursor amino acid residues corresponding to AAS and GGS in oxidized proteins were derivatized by reductive amination with sodium cyanoborohydride (NaCNBH3) and p-aminobenzoic acid (ABA) followed by MAAH to generate the marker derivatives AAS-ABA and GGS-ABA. The quantification was performed using electrospray ionization liquid chromatography-mass spectrometry (ESI LC-MS). The important parameters for hydrolysis were optimized, which include the temperature, the reaction time, the acid concentration and volume as well as the microwave power. Compared to the conventional acid hydrolysis of 18-24 h using 6-12 M HCl at 110 °C applied commonly in the literature and also in this work, MAAH of proteins can be completed as fast as in only 2-10 min and, additionally, with a 3-5 times higher yield of the final derivatization products. Furthermore, a better agreement between the ratio of the detected derivatization products and the theoretical yields from the studied protein has also been achieved, which indicates that MAAH may serve as a more reliable method of acid hydrolysis for this purpose than that with conventional thermal heating. The MAAH method is demonstrated to be a time-saving, reproducible and efficient technique for studying AAS and GGS as protein oxidation markers using LC-MS. 相似文献
7.
Analysis of amino acids in blood samples is an important tool for the diagnosis of neonatal amino acid metabolism disorders. In the work, a novel, rapid and sensitive method was developed for the determination of amino acids in neonatal blood samples, which was based on microwave-assisted silylation followed by gas chromatography/mass spectrometry (GC/MS). The amino acids were derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) under microwave irradiation. The controlled reaction was carried out employing BSTFA under conventional heating at 120 degrees C for 30 min. Experimental results show that microwave irradiation can accelerate the derivatization reaction of amino acids with BSFTA, and much shorten analysis time. The method validations (linear range, detection limit, precision and recovery) were studied. Finally, the method was tested by determination of amino acids in neonatal blood by the measurement of their trimethylsilyl derivatives by GC/MS in electron impact (EI) mode. Two biomarkers of L-phenylalanine and L-tyrosine in phenylketonuria (PKU)-positive blood and control blood were quantitatively analyzed by the proposed method. The results demonstrated that microwave-assisted silylation followed by GC/MS is a rapid, simple and sensitive method for amino acid analysis and is also a potential tool for fast screening of neonatal aminoacidurias. 相似文献
8.
The rapid determination of protein in plant material based on spectrophotometric determination of amino acids in protein hydrolysates with trinitrobenzenesulfonic acid has been adapted to flow injection analysis. With the manifold described, a routine sampling rate of 120 samples/h is possible, though this, as well as sensitivity, can easily be varied. The method was calibrated against the Kjeldahl method and a good correlation was obtained between the two methods over a wide range of protein values for beans. 相似文献
9.
A rapid method for the derivatization of phenolic antioxidants using microwave irradiation has been developed. Six antioxidatively active phenolic components of wines and fruits, namely gallic acid, gentisic acid, vanillic acid, caffeic acid, ferulic acid and p-coumaric acid were used in the model study. The solution of phenolic acids was evaporated to dryness on a rotary evaporator followed by further drying under microwave irradiation (600 W, 30 s). The resultant residue was dissolved in pyridene and treated with bis(trimethylsilyl)acetamide while irradiated by microwave using high power for 30 s. Controlled reaction was carried out employing bis(trimethylsilyl)trifluoroacetamide under conventional heating for 30 min. The trimethylsilyl derivatives were identified and quantified on a gas chromatography/mass selective detector. The mass spectral fragmentation patterns of the derivatives obtained by microwave irradiation were identical to those prepared by heating. The yields of microwave-assisted silylation were comparable to those from conventional heating. The rsd were less than 8% for six replicates. The linearity in wine matrix was nearly perfect. This method is a useful protocol to examine the phenolic constituents in wines and agricultural products. 相似文献
10.
Beneito-Cambra M Herrero-Martínez JM Simó-Alfonso EF Ramis-Ramos G 《Rapid communications in mass spectrometry : RCM》2008,22(22):3667-3672
A method for the rapid classification of proteases, lipases, amylases and cellulases used as enhancers in cleaning products, based on precipitation with acetone, hydrolysis with HCl, dilution of the hydrolysates with ethanol, and direct infusion into the electrospray ion source of an ion-trap mass spectrometer, has been developed. The abundances of the ([M+H]+ ions of the amino acids, from the hydrolysates of both the enzyme industrial concentrates and the detergent bases spiked with them, were used to construct linear discriminant analysis models, capable of distinguishing between the enzyme classes. For this purpose, the variables were normalized as follows: (A) the ion abundance of each amino acid was divided by the sum of the ion abundances of all the amino acids in the corresponding mass spectrum; (B) the ratios of pairs of ion abundances were obtained by dividing the ion abundance of each amino acid by each one of the ion abundances of the other 17 amino acids in the corresponding mass spectrum. Using normalization procedure B, excellent class-resolution between proteases, lipases, amylases and cellulases was achieved. In all cases, enzymes in industrial concentrates and manufactured cleaning products were correctly classified with >98% assignment probability. 相似文献
11.
Summary A reversed phase high performance liquid chromatographic (RP-HPLC) method for the analysis of amino acids in kelp, using precolumn derivatization, is described. Phenylisothiocyanate (PITC) was used as the reagent for derivatization. The kelp samples were prepared by microwave hydrolysis in only 5 min; seventeen PTC amino acids were separated after hydrolysis and derivatization within 12 min. The coefficients of variation were >1.94 and the correlation coefficients for concentration versus response were >0.999 for all derivatives. The ratio of branched amino acid (BAA) to aromatic amino acid (AAA) was also studied. The method has the advantage of shorter hydrolysis and analysis times with optimum separation. In addition, it gives high repeatability of retention times and peak areas for all the amino acids present. 相似文献
12.
Hassan M. Al-Hazimi Ayman El-Faham Mohamed Ghazzali Khalid Al-Farhan 《Arabian Journal of Chemistry》2012,5(3):285-289
We describe herein a fast and rapid technique for preparation of N-phthaloyl amino acids under microwave irradiation. The microwave methodology is rapid, convenient, proceeds under mild conditions, and gives a better yield (81–98%) and high purity of the title compounds. The spectral data as well as the X-ray analysis established the structure of the prepared N-protected amino acids. 相似文献
13.
Determining the amino acid content of a protein involves the hydrolysis of that protein, usually in acid, until the protein-bound amino acids are released and made available for detection. Both the variability in the ease of peptide bond cleavage and differences in the acid stability of certain amino acids can significantly affect determination of a protein's amino acid content. By using multiple hydrolysis intervals, a greater degree of accuracy can be obtained in amino acid analysis. Correction factors derived by linear extrapolation of serial hydrolysis data are currently used. Compartmental modeling of the simultaneous hydrolysis (yield) and degradation (decay) of amino acids by nonlinear multiple regression of serial hydrolysis data has also been validated and applied to determine the amino acid composition of various biological samples, including egg-white lysozyme, human milk protein, and hair. Implicit in the routine application of serial hydrolysis in amino acid analysis, however, is an understanding that correction factors, derived either linearly or through the more accurate nonlinear multiple regression approach, need to be determined for individual proteins rather than be applied uniformly across all protein types. 相似文献
14.
Experiments were undertaken to verify a method for complete amino acid analysis of plant and animal tissues and waste products from a single hydrolysis and high-performance liquid chromatographic run. Using methanesulfonic acid, hydrolysis of cytochrome c at 115 degrees C for 22 h yielded recoveries equal to or higher than hydrolysis at 115 degrees C for 70 h or at 150 degrees C for 22 h. Triple evacuation of the hydrolysis tube alternated with nitrogen flush gave recovery improvements over single evacuation. Refrigerated storage of samples under vacuum for up to 4 days between hydrolysis and further analysis was not different from immediate analysis. However, recoveries of several amino acids were reduced by refrigerated storage in air. Recoveries of individual amino acids were determined by hydrolysis of biological samples with and without added cytochrome c. Although recoveries from biological samples were lower for several amino acids, precision was sufficient to allow quantitation after correction for incomplete recoveries. Derivatization with 9-fluorenylmethylchloroformate (FMOC) was chosen because derivatives are formed with both primary and secondary amino acids, derivatives are quite stable, and detection may be either UV absorbance or fluorescence. Derivative yield is sensitive to the pH of the reaction mixture. A pH of 8.0 gave reproducible derivative yield for all physiological amino acids. Solvent extraction of excess FMOC, when compared to addition of amantadine to react with excess FMOC, gave both higher recoveries and greater precision. Following derivatization, samples could be kept at 4 degrees C for at least 24 h before high-performance liquid chromatographic analysis without loss of response. Derivative yield and detector response were constant across a wide range of molar ratio of FMOC to total amino acids. Gradient elution was required to separate FMOC derivatives on a reversed-phase column. The capability of the pumping system to produce exponential gradients permitted rapid and easy fine-tuning of the gradient. 相似文献
15.
16.
A method for the determination of 3-methylhistidine using an automatic amino acid analyser has been developed. A single column system with lithium buffer (pH 3.950, 0.500 mol/l lithium and 0.067 mol/l citrate) was used for elution. The standard amino acid mixture of basic amino acids and some dipeptides usually present in physiological fluids was analysed for the development of the method. 3-Methylhistidine eluted in 46.7 +/- 0.049 min and the peak area coefficient of variation for the same sample was 1.07%. As 3-methylhistidine is completely resolved from the other basic amino acids and some dipeptides (anserine and carnosine), this method is suitable for the analysis of urine and muscle extracts as well as skeletal muscle protein hydrolysates where this amino acid is present in much lower concentrations than other amino acids. 相似文献
17.
Amino acids are the building blocks of proteins and the analysis of their 13C abundances is greatly simplified by the use of liquid chromatography (LC) systems coupled with isotope ratio mass spectrometry (IRMS) compared with gas chromatography (GC)‐based methods. To date, various cation exchange chromatography columns have been employed for amino acid separation. Here, we report strong anion exchange chromatography (SAX) coupled to IRMS with a Liquiface interface for amino acid δ13C determination. Mixtures of underivatised amino acids (0.1–0.5 mM) and hydrolysates of representative proteins (prawns and bovine serum albumin) were resolved by LC/IRMS using a SAX column and inorganic eluents. Background inorganic carbon content was minimised through careful preparation of alkaline reagents and use of a pre‐injector on‐line carbonate removal device. SAX chromatography completely resolved 11 of the 16 expected protein amino acids following acid hydrolysis in underivatised form. Basic and neutral amino acids were resolved with 35 mM NaOH in isocratic mode. Elution of the aromatic and acidic amino acids required a higher hydroxide concentration (180 mM) and a counterion (NO, 5–25 mM). The total run time was 70 min. The average δ13C precision of baseline‐resolved peaks was 0.75‰ (range 0.04 to 1.06‰). SAX is a viable alternative to cation chromatography, especially where analysis of basic amino acids is important. The technology shows promise for 13C amino acid analysis in ecology, archaeology, forensic science, nutrition and protein metabolism. Copyright © 2011 John Wiley & Sons, Ltd. 相似文献
18.
Yao Ren Hui Wu Xiaofeng Li Furao Lai Guanglei Zhao Xinglong Xiao 《Applied biochemistry and biotechnology》2014,172(3):1227-1240
Biocatalytic hydrolysis reactions were designed for preparation of bioactive hydrolysate of duck egg white protein (DEWP) employing two enzymes in one pot. Firstly, the fresh DEWP was thermal treated at 95 °C, for 40 min at pH 10, to effectively deactivate enzyme inhibitors thus facilitating the following two-step enzymatic hydrolysis. Compared with single-enzyme processes, the two-step enzymatic procedures showed much higher reaction efficiency. The first enzymatic step (in the presence of Alcalase or hydrolase SEEP) allowed to hydrolyze DEWP with degree of hydrolysis (DH) of 8.8–10 % and soluble peptide yield (SEP) of 60.5–70.2 % in a short period (4 h). The second enzymatic step (in the presence of Trypsin or Alcalase) gave a further degradation of DEWP with DH and SEP being more than 26.2 % and 90.4 %, respectively. The final hydrolysates exhibited high antioxidant activity in an evident DH dependent manner. The hydrolysates achieved by sequential addition of the proteinase SEEP and Alcalase at DH value 21 % gave the highest antioxidant activity, which was mainly ascribed to the changes in the amino acid compositions that the contents of some key amino acids and total hydrophobic amino acids were significantly improved by the enzymatic hydrolysis. 相似文献
19.
A novel beaker shaped electrodeless ultraviolet lamp, excited by the radiation of a conventional microwave oven, has been employed in the breakdown of organophosphate compounds in preparation for colorimetric phosphate determination. This new approach offers a highly rapid method of organophosphate decomposition prior to their analysis, with complete breakdown being achieved within 3 min. When evaluated using a number of inorganic and organophosphate compounds, quantitative release of phosphate from a carbon oxygen bond was achieved. Photo-oxidation of the organic triphosphate adenosine 5'-triphosphate results in the release of triphosphate that can then be broken down to three orthophosphate units by acid hydrolysis. 相似文献
20.
The content of protein, moisture content and essential amino acids in conventional and genetically modified soybean grain and selected soybean products (soybean pâté, soybean drink, soybean dessert, tofu) was analyzed in this paper. The following comparative analysis of these products has not yet been carried out. No differences were observed in the amino acid profiles of soybeans and soybean products. The presence of essential amino acids was confirmed except for tryptophan. Its absence, however, may be due not to its absence in the raw material, but to its decomposition as a result of the acid hydrolysis of the sample occurring during its preparation for amino acid determination. Regardless of the type of soybean grain, the content of protein, moisture content and essential amino acids was similar (statistically insignificant difference). Thus, the type of raw material did not determine these parameters. There was a significant imbalance in the quantitative composition of essential amino acids in individual soybean products. Only statistically significant variation was found in genetically modified and conventional soybean pâté. Moreover, in each soy product their amount was lower irrespective of the raw material from which they were manufactured. Therefore, the authors indicate the necessity of enriching soybean products with complete protein to increase their nutritional value. 相似文献