Profiling and screening analysis of 27 aromatic amino acids by capillary electrophoresis in dual modes

An efficient capillary electrophoretic (CE) profiling and screening system based on dual modes of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) was developed for the simultaneous determination of 23 nonprotein amino acids (NPAAs) and 4 protein amino acids with aromatic moiety. It involves separation by an uncoated fused-silica capillary under phosphoric acid buffer in CZE mode and by another uncoated fused-silica capillary under neutral sodium dihydrogen phosphate buffer containing sodium dodecyl sulfate in MEKC mode. Migration orders of the amino acids studied on the two separation modes under each optimum condition were very different. The repeatability of migration times measured by the CZE and MEKC was found to be better than 4.8 and 3.4%, respectively, thereby enabling to cross-check the identification of each amino acid. The method linearity and limit of detection of the CZE for each amino acid were found to be adequate for the assay of aromatic amino acids. When the present CE profiling and screening analysis in dual modes was applied to plant seeds, NPAAs such as mimosine from Mimosa pudica Linné, and 2-phenylglycine from Lindera erythrocarpa Makino were positively detected along with tryptophan, phenylalanine and tyrosine.     

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.     

Pre-capillary derivatisation and capillary zone electrophoresis for amino acids analysis in beverages

A simple and rapid method for the simultaneous analysis of amino acids has been developed. Amino acids were derivatised based on pre-capillary derivatisation with 1,2-naphthoquinone-4-sulfonate (NQS) in basic medium (pH 10.0) and developed reaction at 70 degrees C. Their derivatives were analysed by capillary zone electrophoresis (CZE). The parameters affecting CZE separation were investigated including buffer (pH, type and concentration), organic modifier and separation voltage. The optimum condition was 70 mmol L(-1) borate (pH 10.0) containing 10% acetonitrile, separation voltage of 12 kV, and sample injection (0.5 psi, 5s) and on-capillary detection at 240 nm. The separation of seven amino acids was achieved within 17 min. The detection limit was 1.0 mg L(-1) for all studied amino acids. The calibration curves were linear in the concentration range of 1.0-100.0 mg L(-1). The repeatability, intra-day and inter-day analysis were < or = 1.0% and < or = 2.0% for migration time and < or = 5.0% and 6.0% for peak area. The proposed method has been applied to several beverage samples with only a simple dilution and filtration treatment of sample before derivatisation and analysed by CZE.     

Microchip capillary electrophoresis with electrochemical detector for fast measurements of aromatic amino acids

A method based on microchip capillary electrophoresis with amperometric detection was developed for the rapid separation and direct detection of oxidizable aromatic amino acids (without prior derivatization). The working electrode was a thick-film carbon strip electrode positioned opposite the outlet of the separation channel. Factors influencing the separation and detection processes were examined and optimized. The five aromatic amino acids, tyrosine, 5-hydroxytryptophan, tryptophan, p-aminobenzoic acid, and m-aminobenzoic acid, can be well separated within 5 min using a separation voltage of 2000 V and a 25 mM phosphate buffer (pH 7.0) run buffer containing 50 mM sodium dodecylsulfate. Most favorable amperometric detection was obtained at +0.95 V. Linear calibration plots are observed for micromolar concentrations of the oxidizable amino acids. The new protocol offers good stability and for reproducibility, with relative S.D. of less than 5% for both migration times and peak currents (n=8). It should be useful for the analysis of aromatic amino acids, as desired for life sciences.     

One-pot labeling-based capillary zone electrophoresis for separation of amino acid mixture and assay of biofluids

A fast, simple and cost-effective one-pot labeling strategy coupled with capillary zone electrophoresis was developed for the complete separation of amino acid mixture. The strategy includes two steps of reactions: Cyanuric chloride was made to react first with 7-amino-1,3-naphthalenedisulfonic acid monopotassium salt at 0 °C for 10 min, and then with amino acids at 55 °C for 6 min. The resulted products, after diluted with water, were injected into capillary zone electrophoresis system for separation. Using a running buffer of 20 mM sodium tetraborate decahydrate at pH 10.1, nineteen amino acids were efficiently separated in 25 min, with relative standard deviation of 0.36–1.6% and 0.96–2.1% (within and between days, respectively) for migration time and 0.030–1.6% and 0.22–2.4% (within and between days, respectively) for peak area. The proposed method has been successfully applied to the determination of free amino acids in biofluids, including human serum, urine, and saliva. The linearity of quantification was over two orders of magnitude for most amino acids, with a correlation coefficient larger than 0.999. The average recovery, determined by spiking a known amount of amino acid standards into real samples, was in a range from 91.6% to 105.9%. This method can be a noninvasive means since it could directly assay the urine and saliva samples.     

Pressurized gradient capillary electrochromatographic separation of eighteen amino acid derivatives

A pressurized gradient capillary electrochromatography (pCEC) instrument was developed to separate 18 amino acid derivatives. A reversed-phase C18 column (3 microm, 130 mm x 75 microm I.D.) and an acetate buffer (50 mmol/l NaAc, pH 6.4) with an ion-pair reagent (1% N,N-dimethylformamide) were used to separate derivatized amino acids from a standard solution (2 microg/ml), and the wavelength of the UV-Vis detector was 360 nm. The pressure on the capillary column was kept at approx. 70 Pa and 3 kV positive voltage was added on the outlet end of column. The effect of voltage on the eluting order of amino acids and the resolution of separation were studied, and it was found that when the voltage was higher than 3 kV, the adsorption of amino acids in the porous C18 column occurred. The effect of salt concentration, injection volume, and column length on the separation of amino acids was determined. The amino acid sample was separated by pCEC, and RSDs of the migration times of each amino acid were all less than 2.5%.     

Trace analysis of oxidized, nitrated, and chlorinated aromatic amino acids by capillary electrophoresis with electroosmotic flow modification allowing large-volume sample stacking

A capillary electrophoresis method has been developed for the simultaneous analysis of the oxidized, nitrated, and chlorinated aromatic amino acids, as well as their parent compounds. These modifications of the aromatic amino acids in proteins or free form are induced by the attack of reactive, mainly free radical species generated during cell stress, and these stable products may serve as biomarkers of cell damage. The analytes tyrosine, phenylalanine, dihydroxyphenylalanine, tryptophan, 3-nitrotyrosine, 3-chlorotyrosine, ortho-tyrosine, meta-tyrosine, 3-hydroxyphenylacetic acid (internal standard 1), and alpha-methyltyrosine (internal standard 2) were separated in their anionic forms in alkaline borate buffer. The polyamine spermine was used as electroosmotic flow (EOF) modifier. Adsorbing to the capillary wall, spermine can either suppress or even reverse the EOF depending on its concentration and the pH. The effects of the pH of the separation buffer, the spermine concentration, the temperature, and the applied field strength on the separation were examined. The modified aromatic amino acids are present in biological fluids in a much lower concentration than their parent compounds, thus high detection sensitivity of the analytical method is required. To achieve good detection sensitivity, field-amplified sample stacking of large injection volumes was applied. Omitting polyamine from the sample buffer allowed local reversal of the EOF, thus removal of the low conductivity sample buffer at the capillary inlet. In this way, 100% of the capillary to the detection window could be filled with the sample, and the detection limits achieved for the modified aromatic amino acids were in the range of 2.5-10 nM.     

Proline-coated column for the capillary electrochromatographic separation of amino acids by in-column derivatization

With 3-trimethoxysilylpropyl chloride as the spacer, a proline-coated capillary column was prepared for the capillary electrochromatographic (CEC) separation of amino acids by in-column derivatization. Nine standard mixtures, including aspartic acid, glutamic acid, valine, phenylalanine, alanine, isoleucine, leucine, tyrosine, and tryptophan, were injected. o-Phthalaldehyde (OPA), OPA/2-mercaptoethanol (2-ME) and OPA/N-acetylcysteine (NAC) in borate buffer were tested as the derivatizing agent. Among them, OPA (50 mM) in borate buffer (pH 9.5, 50 mM) gave the best performance. The formation of isoindole could be detected by UV detection. The sandwich-type injection was carried out in hydrostatic mode (10 cm) with the program R(10 s)S(10 s) R(10 s)W(10 min) with R, S, and W being the reagent, sample, and waiting times. Mesityl oxide, benzyl alcohol, and acetone showed some interaction with the column. A current monitoring method was used instead of the determination of the electroosmotic flow (EOF). The direction of EOF was from anode to cathode even under acidic condition lower than the pI value (6.31) of the bonded group due to some unreacted silanol groups. Some parameters including pH, nature, and concentration of the mobile phase and the effect of organic modifier with regard to the CEC separation were investigated. With the proline-coated column (75 (50) cm x 75 microm ID) the best separation was performed in phosphate buffer (pH 4.00, 100 mM) with an applied voltage of -15 kV. The established method was also compared with those precolumn derivatized prior to the separation with proline-coated column as well as with in-capillary derivatization and separation with a bare fused-silica column.     

Potential of fermentation profiling via rapid measurement of amino acid metabolism by liquid chromatography-tandem mass spectrometry

Monitoring amino acid metabolism during fermentation has significant potential from the standpoint of strain selection, optimizing growth and production in host strains, and profiling microbial metabolism and growth state. A method has been developed based on rapid quantification of underivatized amino acids using liquid chromatography-electrospray tandem mass spectrometry (LC-MS-MS) to monitor the metabolism of 20 amino acids during microbial fermentation. The use of a teicoplanin-based chiral stationary phase coupled with electrospray tandem mass spectrometry allows complete amino acid analyses in less than 4 min. Quantification is accomplished using five isotopically labeled amino acids as internal standards. Because comprehensive chromatographic separation and derivatization are not required, analysis time is significantly less than traditional reversed- or normal-phase LC-based amino acid assays. Intra-sample precisions for amino acid measurements in fermentation supernatants using this method average 4.9% (R.S.D.). Inter-day (inter-fermentation) precisions for individual amino acid measurements range from 4.2 to 129% (R.S.D.). Calibration curves are linear over the range 0-300 microg/ml, and detection limits are estimated at 50-450 ng/ml. Data visualization techniques for constructing semi-quantitative fermentation profiles of nitrogen source utilization have also been developed and implemented, and demonstrate that amino acid profiles generally correlate with observed growth profiles. Further, cellular growth events, such as lag-time and cell lysis can be detected using this methodology. Correlation coefficients for the time profiles of each amino acid measured illustrate that while several amino acids are differentially metabolized in similar fermentations, a select group of amino acids display strong correlations in these samples, indicating a sub-population of analytes that may be most useful for fermentation profiling.     

Sensitive analysis of amino acids in injection liquor by reversed-phase HPLC

A convenient derivatization method of amino acids with l-fluoro-2,4-dimtrobenzene as reaction reagent and a separation system were described. The derivative amino acids were separated on a specific chemically bonded phase column with a simple linear gradient elution consisting of aqueous buffer and methanol. The eluate was detected by common ultraviolet absorption detector at 360 nm. The detection limits of amino acids were as low as 10 picomole. This method has been successfully applied to assay amino acid injection liquor used in hospital. It has good repro-ducibility and precision. The procedures avoid the requirements of particular derivative equipment and analyzer employed in conventional amino acid analysis.     

Sensitivity enhancement by on-line preconcentration and in-capillary derivatization for the electrophoretic determination of amino acids

This study describes an application of on-line preconcentration by large-volume stacking in combination with in-capillary derivatization for enhancing spectrophotometric detection sensitivity in capillary electrophoresis. The method is illustrated by an example dealing with the determination of amino acids with 1,2-naphthoquinone-4-sulfonate as a labelling agent. Samples are dissolved in water in order to create a stacking process based on differences in the conductivity between this medium and a concentrated running buffer. The in-capillary derivatization is accomplished following a sandwich procedure in which the sample is inserted between two segments of reagent. Amino acid derivatives are obtained and separated in a fused-silica capillary with a sodium borate electrolyte buffer using 2-propanol as an organic modifier. The method is applied to the analysis of amino acids in pharmaceutical and feed samples. A good concordance between the predicted values and those obtained with the standard method is observed, with overall quantification error below 5%. The proposed procedure allows the detection limits sensitivity to be enhanced in 1000-fold with respect to conventional precapillary derivatization.     

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.     

Determination of soil amino acids by high performance liquid chromatography-electro spray ionization-mass spectrometry derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate

Determination of amino acids by mass spectrometry (MS) is an important technique to investigate soil nitrogen transformation and cycling as amino acids being the major nitrogen-containing compounds in soil organic matter. However, researchers have long faced a critical problem in coupling an efficient separation technique to a sensitive MS detection system simultaneously. In this context, we established a new method of liquid chromatography coupled to mass spectrometry based on the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization method for convenient and accurate quantification of amino acids in soil samples. Baseline separation of 17 amino acid AQC-derivatives was achieved on an XTerra^R MS C₁₈ column using ammonium formate as a mobile phase modifier. The concentration of ammonium formate and the pH of the mobile phase were optimized in order to obtain sensitive MS signals. The response curves were linear over the range of 50-800 μmol L⁻¹ amino acids. The detection limits were 0.20-0.60 pmol μL⁻¹ on column and 0.07-0.24 μg g⁻¹ soil under the optimized conditions. The method has been applied successfully for the first time to determine amino acids in 4 types of soil samples, in which 15 amino acids were quantified by MS detector but methionine and cystine were below the detection limits. Both the recovery and the precision were satisfactory. Hence, this proposed technique shows a potential for the identification of amino acids in soil as well as tracing the transformation of soil amino acids with isotope dilution technique in nitrogen cycling investigation.     

Determination of amino acids in Sargassum fusiforme by high performance capillary electrophoresis

High performance capillary electrophoresis (HPCE) was developed for quantitative determination of 18 phenylthiohydantoin (PTH)-amino acids. The influence of electrolyte concentration, pH, organic modifier and applied voltage on HPCE performance was investigated. The HPCE separation of a PTH-amino acids mixture was much improved by adding organic modifier and Tris-boric acid buffer to the run buffer. After optimization of the method, 17 PTH-amino acids in a solution containing 18 PTH-amino acids could be separated using 400 mmol l⁻¹ Tris-boric acid, 1.0 mmol l⁻¹ diethylamine at pH 9.5 adjusted with 0.1 mol l⁻¹ NaOH as a run buffer, voltage of 25 kV was applied, temperature was maintained at 25 °C, detection wavelength was 254 nm. The precision (n = 7) of this method is less than 3.2% (peak area) and 1.1% (migration time) of relative standard deviation (R.S.D.). Linearity was established over the concentration range 50-1000 μM of each derivative, with correlation coefficients (r) ranging between 0.9904 and 0.9993. The detection limits (S/N = 3) range from 2 to 48 μmol l⁻¹. The method was applied to determine amino acids in Sargassum fusiforme, a marine algae collected from Tongtou County of Zhejiang Province in China with satisfactory results.     

High‐speed separation and detection of amino acids in laver using a short capillary electrophoresis system

A high‐speed separation method of capillary MEKC with LIF detection had been developed for separation and determination of amino acids in laver. The CE system comprised a manual slotted‐vial array (SVA) for sample introduction that could improve the separation efficiency by reducing injection volume. Using a capillary with 80 mm effective separation length, the separation conditions for amino acids were optimized. Applied with the separation electric field strength of 300 V/cm, the ten amino acids could be completely separated within 2.5 min with 10 mol/L Na₂HPO₄–NaOH buffer (pH = 11.5) including 30 mmol/L SDS. Theoretical plates for amino acids ranged from 72 000 to 40 000 (corresponding to 1.1–2.0 μm plate heights) and the detection limits were between 25 and 80 nmol/L. Finally, this method was applied to analyze the composition of amino acids in laver and eight known amino acids could be found in the sample. The contents of five amino acids, tyrosine, glutamic acid, glycine, lysine, and aspartic acid that could be completely separated in real sample were determined. The recoveries ranged from 82.3% to 123% that indicated the good reliability for this method in laver sample analysis.     

毛细管电泳-间接紫外检测法测定蜂蜜中的氨基酸

采用毛细管电泳-间接紫外检测法同时分离测定蜂蜜中的赖氨酸、色氨酸、谷氨酸等9种氨基酸。考察了磷酸浓度、进样方式和缓冲液pH对分离效率和重现性的影响。在分离电压为-15 kV、检测波长为220 nm条件下,以含有0.5 mmol/L十六烷基三甲基溴化铵、20 mmol/L烟酸、10%甲醇的10 mmol/L磷酸二氢钠缓冲溶液(pH 10.2)为运行缓冲液,9种组分在11 min内达到基线分离;检出限最低可达到0.3 mg/L;线性范围为1.0~1000 mg/L;日间及日内精密度为0.64%~5.83%。实际样品中除甲硫氨酸外的8种氨基酸的加标回收率为60.00%~118.37%。将该方法应用于不同蜜源植物和产地的蜂蜜样品的测定,在市售的5种蜂蜜中均检测到脯氨酸、丝氨酸和天冬氨酸,而只在荔枝蜜中检测到苏氨酸。该方法可以为蜂蜜的蜜源鉴别及质量评估提供借鉴方法。     

Analysis of free amino acids in Amur sturgeon by ultra‐performance liquid chromatography using pre‐column derivatization with 6‐aminoquinolyl‐carbamyl

A rapid, sensitive, and reliable ultra‐performance liquid chromatography (UPLC) coupled with photodiode array detection method was developed for the amino acid analysis of Amur sturgeon (Acipenser schrenckii Brandt). The method uses minimal sample volume and automated online precolumn derivitization of amino acids with fluorescent 6‐aminoquinolyl‐carbamyl reagent. The chromatographic separation was achieved by UPLC, which used a column with 1.7 μm particle packing that enabled higher speed of analysis, peak capacity, greater resolution, and increased sensitivity. Amino acid derivatives obtained under optimal conditions were separated on a Waters UPLC BEH C₁₈ column with Acetonitrile–acetate buffer as mobile phase. Matrix effects were investigated and good linearities with correlation coefficients better than 0.9949 were obtained over a wide range of 5–1000 μmol/L for all amino acids. The simple sample preparation and minimal sample volume make the method useful for the quantitation of 17 amino acids in Amur sturgeon samples. It is concluded that a rapid and robust platform based on UPLC was established, and a total of 17 amino acids of Amur sturgeon were tentatively detected. This method showed good accuracy and repeatability that can be used for the quantification of amino acids in real samples.     

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.     

Enantioseparation of amino acid derivatives by capillary zone electrophoresis using vancomycin as chiral selector

The separation of racemic derivatized amino acids (N-acetyl) into their enantiomers was achieved using capillary zone electrophoresis employing vancomycin as a chiral selector. Due to the strong absorption properties of the chiral selector at the low wavelengths used, the partial-filling countercurrent method was adopted in order to improve method sensitivity. In the separation system studied, the chiral selector filled only a part of the capillary and, due to the appropriate selection of the pH, was moving in the opposite direction of the analytes keeping the detector free from absorbing compounds. The effect of several experimental parameters on the enantioresolution of analytes was studied, e.g., vancomycin concentration (0-5 mM), pH of the background electrolyte (pH 4-7), capillary temperature (15-35 degrees C), and the presence of an organic modifier in the run buffer (methanol or ethanol or n-propanol). N-Acetyl glutamic acid, serine, cystine, tyrosine, and proline were all baseline-resolved into their enantiomers and the enantioresolution factor (R(s)) was increased by raising the vancomycin concentration. pH 4 allowed the baseline resolution of the five studied analytes in the presence of 2.5 mM of chiral selector and an increase in pH caused a decrease of R(s).     

A fully automated amino acid analyzer using NBD-F as a fluorescent derivatization reagent

A fully automated amino acid analyzer using NBD-F (4- fluoro-7-nitro-2,1,3-benzoxadiazole) as a fluorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re-evaluated and optimized. Amino acids were derivatized by NBD-F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and fluorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8-20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefficients of 0.999. The coefficients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method.