Determination of Amino Acids in an Individual Erythrocyteby Capillary Electrophoresis with Intracellular FITC-derivatization and Laser-induced Fluorescence Detection

A novel approach for analysis of amino acids in individual erythrocytes was established.In this method, the derivatization reagent was in~oduced into the living cells by electroporation.After derivatization, the amino acids in a single cell were determined by capillary electrophoresis with laser-induced fluorescence detection.     

猪毛水解母液中氨基酸的反相高效液相色谱分析

A new method for separation and quantification of amino acids by pre-column derivatization withphenylisothiocyanate(PITC ) and reversed-phase HPLC is described. Seventeen amino acids were separatedin 30 min at room temperature.The method has been successfully applied to the determination of amino acidcomposition in the solution which was prepared by hydrolyzing pig hair and extration of cystine.     

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.     

Determination of Amino Acids in Single Human Lymphocytes after On-capillary Derivatization by Capillary Zone Electrophoresis with Electrochemical Detection

Sensitive and selective methods for the detection of amino acids (AAs) in single cells are of increasing importance. Capillary zone electrophoresis (CZE) with electrochemical detection (ED) is suitable for analysis of electroactive contents in single cells. However, most native AAs have no electroactivity. Derivatization with an electroactive tag is an attractive method. An on-capillary derivatization scheme was reported1. Naphtha- lene-2, 3-dicarboxaldehyde (NDA) can react with pri…     

HPLC Determination of the Major Non-protein Amino Acids and Common Biogenic Amines in Lathyrus sativus Using a Novel Extraction Method

An assay is presented for simultaneously determining 5 biogenic amines and the major non-protein amino acids: the toxin β-N-oxalyl-L-α，β-diaminopropanoic acid (β-ODAP), its isomer α-ODAP and homoarginine in Lathyrus sativus extracts using the HPLC system after derivatization with para-nitrobenzyloxycarbonyl chloride (PNZ-Cl). However, it is more worthy of noting that this paper also describes a new extraction method using 0.2 mol/L HClO4. The new method has some advantages: shorter extraction-time, simultaneous extraction of free amino acids and polyamines, better inhibiting the isomerization of β-ODAP to α-ODAR and so on.     

Determination of metoprolol in rabbit blood using capillary electrophoresis with laser-induced fluorescence detection

This work described a sensitive method for determination of metoprolol in rabbit plasma.The method involved purification by ultrafiltration,derivatization with fluorescein isothiocyanate,determination by capillary electrophoresis(CE) coupled with laser-induced fluorescence(LIF) detector.Other components in plasma including a variety of amino acids and proteins did not interfere with the determination of metoprolol in experimental condition.The assay had a wide range(2.0-500 ng/mL) of linearity and a detection limit of 0.8 ng/mL.The intra- and inter-day precisions were satisfactory with relative standard deviation(RSD) less than 10.0%and accuracy within 10.0%.This method was successfully applied to pharmacokinetic study of metoprolol in rabbit blood.     

Rapid Determination of Amino Acids in Ginseng by High Performance Liquid Chromatography and Chemometric Resolution

Ginseng is one of the most important traditional Chinese medicines and functional foods.A method for the fast determination of amino acids in ginseng samples using high performance liquid chromatography（HPLC） was developed,in which strong isocratic elution was employed for simplifying the separation and speeding up the analysis.All amino acids were eluted within 3 min with the chromatogram composed of overlapped peaks from the interferences.Then,non-negative immune algorithm（NNIA） was adopted to resolve the chromatographic signals of the components from the chromatogram measured.The results show that the signals of the amino acids can be correctly extracted by NNIA and the signal extracted can be used for the quantitative analysis.The method was validated via determining six amino acids of four different samples of ginseng.The recoveries of the spiked samples are in a range of 96.6％-106.3％.     

A STUDY ON SERUM CANCER-SUPPRESSIVE PEPTIDE

A peptide which was isolated from serum of cancer-bearing mice has an activity to killcancer cells in vitro, but little effect on bone marrow cells. This peptide was named SerumCancer-Suppressive Peptide (SCSP). SCSP can be crystallized and still has the activity tokill the cancer cells. It consists of more than ten kinds of amino acids with molecularweight about 2000-3000 daltons estimated by SDS-polyacrylamide gel electrophoresis. Anantiserum was prepared from rabbits immunized with SCSP and the corresponding IgG couldbe obtained from the antiserum. The titers of SCSP in some cells of the normal andcancer-bearing mice were estimated by passive hemagglutination method. The results showedthat the SCSP titer in spleen is much higher than those in muscle, liver and ascites tumorcells. The results suggest that SCSP is probably produced by spleen cells.     

Determination of Amino Acids by Ru(bpy)_3~(2+) Cathodic Electrochemiluminescence with High Sensitivity

The unique cathodic electrochemiluminescence(ECL) emission of Ru(bpy)32+(bpy=2,2′-bipyridine) was observed via Nafion film at Au electrode[Au/Nafion/Ru(bpy)32+] at about 0.20 V(vs. Ag/AgCl) and applied to the determination of several amino acids without prior derivatization with high sensitivity. The cathodic electrochemilumi-nescence(ECL) exhibits the detection limits and linear ranges of several amino acids comparable to or better than those of capillary electrophoresis with conventional ECL detection method(at 1.10—1.20 V vs. Ag/AgCl) based on precolumn derivatization. The results suggest that the cathodic ECL is promising for the detection of amino acids in bioanalysis.     

Determination of D- and L-amino acids in biological samples by two-dimensional column liquid chromatography

Summary A two-dimensional, column liquid chromatographic system is used for the determination of the D- and L-enantiomers of amino acids in biological samples. Separation of the amino acids is first on ion-exchange column by gradient elution with a sodium citrate-sodium chloride buffer. Enantioseparation is by subsequent injection of 3 μl heart-cuts of the individual amino acids onto a second column with a chiral crown ether stationary phase. Finally, fluorescence detection is after post-column labelling of the amino acids using ano-phthalaldehyde-2-mercaptoethanol reagent solution. The high separation power and selectivity of the system allow processing of complex samples with hardly any additional treatment and the determination of small quantities of D-amino acids in the presence of excess L-form. Applicability of the system is illustrated by the determination of D- and L-aspartate, serine, glutamate and alanine in various complex biological samples, such as protein hydrolysates, urine and biotechnological and food samples. Data are given on detectability, repeatability and linearity.     

Method of intracellular naphthalene-2,3-dicarboxaldehyde derivatization for analysis of amino acids in a single erythrocyte by capillary zone electrophoresis with electrochemical detection

A novel method of intracellular derivatization was developed. In this method, the derivatization reagents [naphthalene-2,3-dicarboxaldehyde (NDA) and CN-] were introduced into living cells by electroporation for the derivatization reaction. After completion of derivatization reaction in cells, a single cell was drawn into the capillary tip by electroosmotic flow. Then the lysing solution was introduced into the capillary by diffusion. Once the individual cell was lysed, the derivatized amino acids in the individual cell were separated by capillary zone electrophoresis and detected by end-column amperometric detection at the outlet of the capillary. This method of intracellular NDA derivatization confined the analytes and the derivatization reagents to the volume of a single cell expanded. For an 8-microm erythrocyte, the contents were diluted by a factor of only ca. 1.6. The method was used to determination of amino acids in single erythrocytes. Six amino acids were identified and quantified.     

Analysis of amino acids in individual human erythrocytes by capillary electrophoresis with electroporation for intracellular derivatization and laser-induced fluorescence detection

A method for monitoring amino acids in single erythrocytes is described. For intracellular derivatization, reagent fluorescein isothiocyanate (FITC) was introduced into living cells by electroporation. For an 8 microm erythrocyte, the analytes were diluted by a factor of only 1.6. After completion of the derivatization reaction, a single cell was injected into the separation capillary tip and lysed there. The derivatized amino acids were separated by capillary electrophoresis, followed by laser-induced fluorescence detection. Nine amino acids were quantitatively determined, with amounts of amino acids ranging from 3.8-32 amol/single cell.     

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.     

Rapid analysis of amino acids using pre-column derivatization

A new approach to the pre-column derivatization and analysis of amino acids is described. The method is based upon formation of a phenylthiocarbamyl derivative of the amino acids. The derivatization method is rapid, efficient, sensitive, and specific for the analysis of primary and secondary amino acids in protein hydrolyzates. The liquid chromatographic system allows for the rapid, bonded-phase separation with ultraviolet detection of the common amino acids with 12-min analysis time and a 1-pmol sensitivity.     

Complete separation of ephedrines by graphite-layer open-tubular (GLOT) column

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.     

氨基酸和多肽在碱性硅胶载体上的固相衍生研究

为进行复杂体系中痕量生理活性物质 (如氨基酸和多肽等 )的高灵敏度分析 ,往往需要对其进行柱前或柱后的荧光衍生 -高效液相色谱或毛细管电泳分析 [1] .在一个存在着竞争反应的体系中 ,为保证样品有足够的反应产率 ,往往需使衍生试剂过量很多 ,这就使得衍生后的样品中必然含有高浓度的衍生试剂及其水解后形成的副产物 ,从而大大地干扰了分离与分析 .为解决这一问题 ,通常可采取溶剂萃取[2～ 5] 或加入 1 -金刚烷胺 [6 ,7] 或羟胺 [8,9] 等方法除去过量试剂 .但这些额外的处理使衍生方法更加烦琐 ,有时还导致收率的降低 .也有使用固相化的衍…     

Determination of free amino acids by precolumn derivatization with phenylisothiocyanate. Application to wine samples

Summary A method to determine free amino acids by pre-column derivatization with phenylisothiocyanate is discussed. The method has been applied to determine free amino acids in wine samples, and the results have been compared with those obtained by means of an automatic orthophthal-aldehyde-9-fluorenylmethyloroformate (OPA-FMOC) method.     

Amino acid analysis using DABS-CL

Summary Pre-column derivatization followed by reversed-phase HPLC has become widely used as a sensitive and speedy method of amino acid analysis.The technique has been improved by the development of simple and reproducible strategies for derivatization of the amino acids with DABS and for the HPLC analysis. Results obtained indicate that the method has a high sensitivity is well and suited to generate precise amino acid composition information from peptides and proteins.