阴离子交换-积分脉冲安培检测法分离测定氨基酸注射液中的氨基酸和葡萄糖

 用阴离子交换 积分脉冲安培检测法测定了氨基酸注射液中 1 7种氨基酸和葡萄糖。研究了氨基酸和葡萄糖在阴离子交换中的保留行为。采用了优化的水、NaOH和NaAc三元梯度淋洗条件。在优化的梯度淋洗条件和积分脉冲安培检测条件下 ,氨基酸和葡萄糖的检出限为 0 3pmol～ 1 0 3pmol,线性范围约为 2个数量级。样品加标回收率为 88 3 %～ 1 0 4 6 %。方法简单、灵敏、准确。     

Ligand-exchange chromatography of amino acids on copper-, cobalt- and zinc-chelex 100.

Procedures for the ligand-exchange chromatography of amino acids on copper-, cobalt-and zinc-Chelex 100 have been examined. Ligand exchange on the copper complex affords a simple and rapid method for the removal of amino acids (except for aspartic and glutamic acids) from dilute solutions. The influence of the pH on the binding of amino acids to the metal complex was also studied. The bound amino acids could be eluted with ammonium hydroxide which also causes a slight metal leakage. Chromatography on cobalt- and zinc-Chelex 100 showed that only the basic amino acids were quantitatively attached to these complexes at pH 8.3-9.5, whereas the others were predominantly EXCLUDED. This procedure can be used for the selective concentration and removal of basic amino acids in the presence of other amino acids.     

Determination of free intracellular amino acids in single mouse peritoneal macrophages after naphthalene-2,3-dicarboxaldehyde derivatization by capillary zone electrophoresis with electrochemical detection.

A method is described for the direct identification and quantification of amino acids in individual mouse peritoneal macrophages by capillary zone electrophoresis with electrochemical detection after on-column derivatization with naphthalene-2,3-dicarboxaldehyde (NDA) and CN-. In this method, individual macrophages and then the lysing/ derivatizing buffer are injected into the front end of the separation capillary by electromigration with the aid of an inverted microscope. The front end of the separation capillary is used as a chamber to lyse the macrophage and derivatize its contents, which minimizes dilution of amino acids of a single macrophage during derivatization. Six amino acids (serine, alanine, taurine, glycine, glutamic acid, and aspartic acid) in single mouse peritoneal macrophages have been identified. Quantitation has been accomplished through the use of calibration curves, where the concentration ratios of these standard amino acids are similar to the concentration ratios of amino acids in macrophages. Cellular levels of the amino acids in these cells range from 0.27 +/- 0.20 fmol/ cell for alanine to 6.4 +/- 4.6 fmol/cell for taurine.     

Use of capillary electrophoresis and laser-induced fluorescence for attomole detection of amino acids

A capillary electrophoresis and laser-induced fluorescence (CE-LIF) method was developed to identify and quantitate at amol (10(-18)) concentration. Amino acids were derivatized with 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde prior to CE-LIF analysis. The assay was developed by varying the sodium borate concentration, buffer pH, operating voltage, and operating temperature. A run buffer system containing 6.25 mM borate, 150 mM sodium dodecyl sulfate, and 10 mM tetrahydrofuran (pH 9.66) at 25 degrees C, and 24 kV provided analysis conditions for a high-resolution, sensitive, and repeatable assay of amino acids. The rate of derivatization, stability of the labeled amino acids, and amino acid quantitation varied for each amino acid. Amino acids were detected with greater efficiency by this method than automated HPLC amino acid analysis. The repeatability of the assay ranged from 0.3 to 0.9% within a day and 0.7 to 1.5% between analysis days. Bacterial amino acid utilization in a chemically defined medium was successfully monitored using this method. This work defines a sensitive and repeatable method for the detection of amino acids during bacterial metabolism.     

Rapid determination of amino acids in foods by hydrophilic interaction liquid chromatography coupled to high-resolution mass spectrometry

This study describes a rapid and sensitive analytical method for the determination of amino acids in foods and drinks. The method entailed dilution or extraction of amino acids from foods using the mixture of acetonitrile and 0.1% aqueous formic acid (50:50, v/v). Chromatographic separation of underivatized amino acids was performed using a hydrophilic interaction liquid chromatography within a runtime of 6 min. Both hydrophobicity and charge of the side chain played important roles on the elution order of amino acids under the chromatographic conditions. High-resolution mass spectrometry allowed qualitative and quantitative detection of amino acids in complex food matrices. Its response was found linear over a concentration range of 0.25-10 μg/ml. The method could be successfully applied to various foods and drinks to profile individual amino acids. Mean percentage recoveries of amino acids from different matrices were 88.5% or higher with residual standard deviation of less than 5.0%.     

Quantitation of blood and plasma amino acids using isotope dilution electron impact gas chromatography/mass spectrometry with U-(13)C amino acids as internal standards.

A method to quantitate blood and plasma amino acids by isotope dilution gas chromatography/mass spectrometry (GC/mS) is described. Samples were spiked with U-(13)C amino acids as internal standards and the tert-butyldimethylsilyl derivatives (tBDMS) separated by capillary column gas chromatography. Linear regression curves, generated for individual amino acids, gave correlation coefficients of 0.9999. The reproducibility of the method was assessed from the analysis of 10 replicate blood and plasma samples. For most amino acids a coefficient of variance (CV) of 相似文献   

反相高效液相色谱法测定深层培养含硒构菌菌丝体中的硒蛋氨酸

本文用苯异硫氰酸酯作柱前衍生化试剂，衍生的硒蛋氨酸及其它非硒代氨基酸用反相高交液相色谱进行分离分析，分析表明，加无机硒培养的构菌菌丝体中含一定量的硒蛋氨酸，且这一方法广泛适于生物物质中其它硒代氨基酸的分析。     

Simultaneous determination of amino acids and carbohydrates in culture media of Clostridium thermocellum by valve-switching ion chromatography

An improved method for the simultaneous determination of 20 amino acids and 7 carbohydrates using one-valve switching after injection, ion chromatography, and integrated pulsed amperometric detection is proposed. The resolution of the amino acids and carbohydrates in the cation trap column was investigated. In addition, parameters including flow liquid type, flow rate, concentration, and valve-switch timing were optimized. The method is time-saving, effective, and accurate for the simultaneous separation of amino acids and carbohydrates, with a mean correlation coefficient of >0.99 and repeatability of 0.5–4.6% for eight replicates. The method was successfully applied in the analysis of amino acids and carbohydrates in aseptic media and in extracellular culture media of three phenotypes of Clostridium thermocellum.     

二维阀切换离子色谱法测定海带中游离氨基酸

建立一种测定海带中游离氨基酸的阀切换高效阴离子交换色谱耦合脉冲安培检测器法。采用一根阳离子交换柱对氨基酸进行富集,而后经阀切换至氨基酸分析柱Amino Pac~PA-10(250 mm×2 mm)上分离并进入安培检测器检测。在最佳分离条件下,20种氨基酸的质量浓度在0.1~20.0 mg/L范围内与其色谱峰面积线性关系良好,线性相关系数r~20.99,20种氨基酸的检出限为0.01 mg/L,加标回收率为83.12%~117.34%,测定结果的相对标准偏为1.02%~13.05%(n=8)。该方法样品前处理简单,无基底杂质干扰,适用于海带样品中游离氨基酸的测定。     

Study on Production of Amino Acids from Bean Dregs by Hydrolysis in Sub-critical Water

This study investigated the production of value‐added amino acids from bean dregs by hydrolysis in subcritical water. It was investigated that the effect of reaction temperature, reaction time and CO₂ on amino acid concentration in the hydrolysates of bean dregs. The product of amino acid was determined by Amino Acid Analyzer. The results show that a variety of amino acids are produced. The concentrations of arginine, lysine and alanine were relatively high in the hydrolysate. Temperature and time have a great influence on the hydrolysis reaction. The effects of reaction temperature and time on concentration of different amino acids vary. The addition of carbon dioxide led to an increase in amino acid yield due to the acceleration of acid hydrolyzed catalysis steps. The highest yield of total amino acids is 22% (0.22 g/g of dry bean dregs) at 330°C and 30 min. This method may provide a practical and economical solution for the disposal of bean dregs wastes.     

Analysis of amino acids by capillary zone electrophoresis with electrochemical detection

The precapillary derivatization of 20 amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) and CN(-) was investigated. All these derivatized amino acids could be oxidized on the carbon fiber microdisk bundle electrode except proline. Capillary zone electrophoresis with electrochemical detection was employed for the analysis of 19 amino acids. The optimum conditions of separation and detection were borate, pH 9.48, for the electrolyte, 18 kV for the separation voltage and 1.15 V versus a saturated calomel electrode for the detection potential. Limits of detection of concentration or mass for individual amino acids were between 1.7 x 10(-7) and 1.8 x 10(-6) mol/L or 84 and 893 amol (according to the signal-to-noise ratio of 3) for the injection voltage of 6 kV and injection time of 10 s. The relative standard deviations were between 0.80 and 2.3% for the migration times and 1.4 and 6.4% for the electrophoretic peak currents. From a mixture of 19 amino acids, 10 amino acids (Arg, Lys, Orn, Try, Ser, Ala, Gly, Cys, Glu, Asp) could be well separated. The other 9 amino acids appeared on three electrophoretic peaks. From the samples, in which the nine amino acids do not exist simultaneously, some of them could also be detected. The method was applied to the determination of amino acids in beer by the standard addition method. The recovery for the amino acids in beer was 91-109%.     

Determination of amino acids in tobacco samples by capillary electrophoresis/indirect absorbance detection with isolation of the electrolysis compartment and p-Aminobenzoic acid as a background electrolyte.

A fast, convenient and sensitive method of capillary zone electrophoresis (CZE) and indirect UV detection was proposed for the determination of 16 amino acids. p-Aminobenzoic acid (PAB) was selected as a background electrolyte (BGE). An isolated cell included a BGE buffer part and an electrode buffer one, which were jointed with a glass frit. The isolated cell can prevent PAB from the electrode reaction and improve the stability of the detection baseline. The separation conditions of amino acids were investigated, such as different BGEs, BGE concentration, buffer pH and electroosmotic flow (EOF) modifiers. Under the selected separation conditions, 14 amino acid peaks could be separated in 12 min. The detection limits of the amino acids were in the range of 1.7 - 4.5 micromol/L. The isolated cell is suitable for reagents reacting on the electrodes in capillary electrophoresis. The proposed method has been successfully applied to the determination of the amino acids in tobacco samples.     

HPLC with In-Capillary Optical Fiber Laser-Induced Fluorescence Detection of Picomolar Amounts of Amino Acids by Precolumn Fluorescence Derivatization with Fluorescein Isothiocyanate

In this paper, a fluorescein isothiocyanate (FITC) precolumn derivatization technique in conjunction with an HPLC-in-capillary optical fiber laser-induced fluorescence (HPLC-ICOF-LIF) detection method has been developed for determination of amino acids. The HPLC separation of FITC-labeled amino acids and the ICOF-LIF detection system are studied and optimized. Optimum separation conditions were obtained with a gradient elution program of acetonitrile and phosphate buffer (10 mM, pH 6.8). The ICOF-LIF detection system comprises a 530-??m capillary and a 380-??m optical fiber. The analyses of amino acids display excellent linear relationship between peak area and concentration with correlation coefficients greater than 0.999 and the method also provides good repeatability with RSD < 3%. The detection limits for FITC-tagged amino acids are very low and the lowest LOD for tyrosine is 51 pM. The proposed method has been successfully applied to determination of amino acids in human serum. Our developed HPLC-ICOF-LIF system is cheap, simple, stable, and sensitive which is potentially useful for the formulation analysis and bioanalysis.     

High-performance liquid chromatographic determination of selected amino acids in rat brain by precolumn derivatization with phenylisothiocyanate

We describe here a simple, sensitive, selective and reproducible assay method for quantitative determination of aspartate, glutamate, serine, glutamine, glycine and gamma-aminobutyric acid in rat brain using reversed-phase high-performance liquid chromatography. The method is based upon formation of phenylthiocarbamyl derivatives of the amino acids. Good resolution of the six amino acids and the internal standard norvaline is achieved within 40 min. Other amino acids which have been reported to be present in rat brain do not interfere with the analysis. Standard curves for each of the amino acids exhibited good linearity (r greater than 0.9993) over the range 0.5-20 nmol. The coefficient of variation for the intra-day and inter-day determinations ranged from 0.4% at the highest to 11% at the lowest concentration limit. Storage of whole brains at -0 degrees C for up to 8 weeks did not affect mean concentrations of the six amino acids.     

Some factors affecting separation and detection of amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Some factors influencing the separation and detection of amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection were investigated. These factors include eluent concentration, column temperature, and detection waveform. The selectivity changes in weakly retained amino acids are slight with changing sodium hydroxide eluent concentration. When sodium acetate eluent concentration is changed, the selectivity variations between strongly retained amino acids containing two carboxyl groups and containing only one carboxyl group are obviously different. Significant but slight selectivity changes in weakly retained amino acids can be achieved through changing the column temperature. Sodium hydroxide and sodium acetate eluent concentration affect the detection of amino acids. Detection sensitivity of amino acids can be improved by increasing the concentration of sodium hydroxide and sodium acetate in a certain concentration range. The detections of amino acids at two different detection waveforms were compared. The hydroxyl amino acids can be selectively detected by choosing a modified detection waveform. The optimized gradient elution condition and column temperature for analyzing 19 amino acids were obtained. The time for the gradient elution program was 60 min. The column temperature was 35 degrees C. Under the optimized conditions, detection limits for 19 amino acids were 0.15-4.52 pmol. The calibration graphs of peak area for all the analytes were linear for about three orders of magnitude. The RSDs (n=5) of peak area were 0.6-5.6%. The determination of trace amino acid impurities in valine product is shown as an application example.     

Amino acid analysis by ion-exchange chromatography using a lithium elution gradient. Influence of methanol concentration and sample pH.

The separation of amino acids has been achieved on a short column of Chromo-Beads C2 resin, with a lithium gradient-elution system. The analysis took 8 h. The separation of asparagine and glutamine from glutamic acid was highly dependent on the sample pH and on the methanol concentration in the first buffer of the gradient. The method has been applied to analysis of human plasma and granulocytes for amino acids.     

烟叶和烟草料液中氨基酸的直接检测及碳水化合物的去除

建立了高效阴离子交换色谱-积分脉冲安培法(HPAEC-IPAD)直接检测烟叶和烟草料液中氨基酸的方法。利用离线除糖的方法,去除烟叶和烟草料液中大量干扰糖类,包括葡萄糖、果糖和蔗糖及麦芽低聚糖等。采用AminoPac PA10阴离子交换柱,以NaOH和NaAc的强碱性溶液为淋洗液,采用梯度洗脱,流速为0.25 mL/min;积分脉冲安培法对氨基酸进行检测,回收率可达76%~105%。此方法可以有效的解决烟叶、烟草料液等含糖量高的样品中糖类化合物的干扰,对氨基酸实现灵敏、准确的定量分析。     

氨基酸衍生物在毛细管区带电泳下的分离研究

采用新合成的荧光试剂咔唑-9-乙基氯甲酸酯作为柱前衍生试剂,利用毛细管区带电泳法对衍生氨基酸进行分离,考察了该试剂用于毛细管区带电泳法进行氨基酸分离的关键条件,实现了12种氨基酸的快速基线分离.     

A capillary electrophoresis detection scheme for underivatized amino acids based on luminol-BrO- chemiluminescence system

A novel chemiluminescence (CL) detection scheme has been developed for detecting underivatized amino acids following capillary electrophoresis (CE) separation. This detection was based on the inhibitory effect of amino acids on the CL reaction between luminol and BrO⁻ in alkaline aqueous solution. Detection of amino acids was accomplished with a borate-based background electrolyte at pH 9.2. The luminol was used as a component of the separation carrier electrolyte. Parameters affecting CE-CL separation and detection, such as the pH value, the concentration of electrolyte and CL reagent on the resolution were optimized. The relative standard deviation for the analysis of amino acids was less than 1.5% for the migration time and 4% for the peak height. The mass limits of detection were from 7 to 144 fmol for the 7 amino acids. This method has been applied of 7 amino acids in amino acid injection.     

Effect of amino acids on aggregation behaviors of sodium deoxycholate at air/water surface: surface tension and oscillating bubble studies

The aggregation behaviors of sodium deoxycholate (NaDC) at the air/water surface were investigated via surface tension and oscillating bubble measurements in the absence and presence of three alkaline amino acids, namely, L-Lysine (L-Lys), L-Arginine (L-Arg), and L-Histidine (L-His). The results of surface tension measurements show that NaDC has a lower ability to reduce the surface tension of water, because NaDC molecules orient at the surface in an oblique direction and tend to aggregate together, which is approved by molecular dynamics (MD) simulation. L-Lys is the most efficient of the three amino acids in reducing the critical aggregation concentration (cac) of NaDC in aqueous solution. The influence of amino acids on the dilational rheological properties of NaDC was studied using the drop shape analysis method in the frequency range from 0.02 to 0.5 Hz. The results reveal that the absolute modulus passes through a maximum value with increasing NaDC concentration. The addition of amino acids increases the absolute modulus of NaDC, and the maximum value is observed at much lower concentration. From the perspective of structures of amino acids, the performance of L-Arg is similar to that of L-His, and both of them bring out a smaller effect on the absolute modulus than that of L-Lys. From the above results, it may be presumed that electrostatic and hydrophobic effects are important impetus during the interaction between amino acids and NaDC at the air/water surface. Hydrogen bonding is so ubiquitous in the system that the difference of hydrogen bonding between NaDC and amino acid is ignored.