The Separation of Pre-Column O-Phthalaldehyde Derivatized Amino Acids by High Performance Liquid Chromatography

Abstract

A method is presented in which a pre-column fluorescent derivative of primary amino acids is formed using o-phthalaldehyde (OPA). After a one minute derivatization period, the OPA amino acids are separated by high performance liquid chromatography using a ternary gradient in the reversed phase mode and fluorometric detection. The complete analysis time, including the derivatization step is less than 40 minutes with an average minimum detectable quantity of 40 picograms.     

LC-Fluorescence Detection Analysis of Amino Acids from Stellera chamaejasme L. Using 2-[2-(Dibenzocarbazole)-ethoxy] Ethyl Chloroformate as Labeling Reagent

A pre-column derivatization method for the sensitive determination of amino acids using the tagging reagent 2-[2-(dibenzocarbazole)-ethoxy] ethyl chloroformate (DBCEC) followed by liquid chromatography with fluorescence detection has been developed. Identification of DBCEC-amino acids derivatives was by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS–MS). DBCEC can easily and quickly label amino acids, and derivatives are stable enough to be efficiently analyzed by LC. Separation of the derivatized amino acids had been optimized on Hypersil BDS C₁₈ column. A perfect baseline separation for 20 amino acid derivatives was achieved with a ternary gradient elution program. The chromophore of dibenzocarbazole group, which comprise a large rigid planar structure with p–π conjugation system, resulted in a sensitive fluorescence detection for amino acid derivatives. The derivatized amino acids were detected with fluorescence detector with excitation maximum and emission maximum at 300 and 390 nm, respectively. Excellent linear responses were observed with coefficients of >0.9993, and detection limits were in the range of 0.78–5.13 fmol (signal-to-noise ratio of 3). The mean accuracy ranged from 83.4 to 98.7% for fluorescence detection. The mean inter-day precision for all standards was <4.2% of the expected concentration. Therefore, the proposed method was a highly sensitive and specific method for the quantitative analysis of amino acids from biological and natural environmental samples.     

HPLC Determination of α-Amino Acids in Phytoplanktonic Marine Cells

Abstract

A procedure for the quantitative determination of 17 amino acids in a marine matrix using HPLC is reported. Pre-column derivatization with o-phthalaldehyde, separation on C₁₈-bonded silica with phosphate buffer (pH 7.2)-acetonitrile as eluent and fluorescence detection have been used. The good variation coefficient (average 2% with working curves in real matrix) and the low detection limit (1-5 fmoles) make the procedure suitable for the determination of total or free amino acids in matrix cultures.     

Reversed Phase Chromatographic Analysis of 13 Amino Acids in Honey Samples

A simple method using reversed phase high-performance liquid chromatography (RP-HPLC) was developed for the simultaneous analysis of 13 amino acids. Amino acids were pre-column derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl) before analysis by RP-HPLC. Experimental parameters affecting the derivatization and chromatographic separation were investigated. Amino acids were derivatized with FMOC-Cl under alkaline condition in 0.1 mol/L borate buffer pH 10.0 at room temperature. The FMOC-amino acid derivatives were separated on an Atlantis C18 column under the gradient elution of 0.05 % trifluoroacetic acid and acetonitrile and UV detection at 265 nm. Linear ranges were 0.2–100.0 μg/mL with the correlation coefficients greater than 0.992. Limits of detection and limits of quantitation were in the range of 0.05–2.0 and 0.2–5.0 µg/L, respectively. The intra-day precision (n = 3) of retention time was less than 1 %, while for the peak area was less than 4 %. The inter-day precision (n = 3 × 3) of retention time was less than 2 % and the peak area was less than 8 %. This method was applied in honey samples and the results showed that proline is the major amino acids in honey samples.

     

Reversed-Phase Ion-Pairing Liquid Chromatographic Separation and Fluorimetric Detection of Polyamines

Abstract

A rapid and specific reversed-phase ion-pairing high performance liquid chromatographic procedure for putrescine, spermidine and spermine is reported. The ion-pairing reagent, heptanesulfonate, was employed and o-phthalaldehyde and 2-mercaptoethanol were used for on-line post-column derivatization and subsequent fluorescence detection. Experiments were carried out to determine the effects of several variables such as pH, concentration of the aqueous buffer, counter-ion concentration, and the percentage of organic modifier in the moving phase. The minimum detection limits for the polyamines ranged from 120 pmoles for spermine to 12 pmoles for putrescine. The method includes a gradient program which provides complete separation from amino acids and specificity for the three polyamines. The procedure was applied successfully to urine and serum samples.     

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 citratesodium chloride buffer. Enantioseparation is by subsequent injection of 3 l 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.     

A Chemiluminiscence Detector for Trace Determination of Fluorescent Compounds

Abstract

A detector making full use of the advantages offered by chemiluminiscence has been constructed. Light measurement was performed with modern electronic equipment using photon counting and the system was tested using dansyl derivatives of adrenaline, noradrenaline and of some amino acids. The detection limits for the two catecholamines were 6 and 16 fmol, respectively, and less than 0.5 fmol for the amino acids. To reach these low levels all reagents were purified or selected for ultra-high purity. The effects of reagent contaminants and of various optical filters have been studied using adrenaline as the test substance.     

Determination of trans-fatty acids in food samples based on the precolumn fluorescence derivatization by high performance liquid chromatography

Trans-fatty acids are unsaturated fatty acids that are considered to have health risks. 1,3,5,7-Tetramethyl-8-butyrethylenediamine-difluoroboradiaza-s-indacene is a highly sensitive fluorescent labeling reagent for carboxylic acids developed by our lab. In this study, using this precolumn fluorescent derivatization reagent, a rapid and accurate high-performance liquid chromatography with fluorescence detection method was developed for the determination of two trans-fatty acids in food samples. Under the optimized derivative conditions, two trans-fatty acids were tagged with the fluorescent labeling reagent in the presence of 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide at 25°C for 30 min. Then, the baseline separation of trans- and cis-fatty acids and their saturated fatty acid with similar structures was achieved with less interference using a reversed-phased C₁₈ column with isocratic elution in 14 min. With fluorescence detection at λ_ex/λ_em = 490 /510 nm, the linear range of the TFAs was 1.0-200 nM with low detection limits in the range of 0.1–0.2 nM (signal-to-noise ratio = 3). In addition, the proposed approach was successfully applied for the detection of trans-fatty acids in food samples, and the recoveries using this method ranged from 96.02 to 109.22% with low relative standard deviations of 1.2–4.3% (n = 6).     

Rapid and Efficient Microwave‐Assisted Synthesis of N‐Carbamoyl‐L‐amino Acids

A rapid and efficient method for the synthesis of N‐carbamoyl‐L‐amino acids is reported. The procedure, involving the reaction between urea and α‐amino acids sodium salts, was performed under microwave conditions using an unmodified domestic microwave oven. A careful study of the operative conditions indicated proline (1d) as the less reactive substrate and phenylglycine (1e) as the more reactive one among all the α‐amino acids tested. Substitution of urea with potassium cyanate produced a low conversion into the corresponding N‐carbamoyl derivative, and a possible explanation of this result is reported.     

A ChemiluminesClent Precolumn Labelling Reagent for High-Performance Liquid Chromatography of Amino Acids

Abstract

A chemiluminescent tag for precolumn derivatization of amino acids has been developed. The tag, 4-isocyanatophthalhydrazide, couples with 17 amino acids tested, including proline and hydroxyproline, in less than ten minutes. Twelve derivatized amino acids have been separated and detected with an average detection limit of 10 femptomoles per 20 μL injection.     

Rapid determination of p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript> values of 20 amino acids by CZE with UV and capacitively coupled contactless conductivity detections

A rapid and universal capillary zone electrophoresis (CZE) method was developed to determine the dissociation constants (pK _a) of the 20 standard proteogenic amino acids. Since some amino acids are poorly detected by UV, capacitively coupled contactless conductivity detection (C⁴D) was used as an additional detection mode. The C⁴D coupling proved to be very successful on a conventional CE-UV instrument, neither inducing supplementary analyses nor instrument modification. In order to reduce the analysis time for pK _a determination, two strategies were applied: (i) a short-end injection to reduce the effective length, and (ii) a dynamic coating procedure to generate a large electroosmotic flow (EOF), even at pH values as low as 1.5. As a result, the analysis time per amino acid was less than 2 h, using 22 optimized buffers covering a pH range from 1.5 to 12.0 at a constant ionic strength of 50 mM. pK _a values were calculated using an appropriate mathematical model describing the relationship between effective mobility and pH. The obtained pK _a values were in accordance with the literature. Figure a UV (1) and C⁴D (2) detectors placed on-line on the CE capillary. b Curve of effective mobility as a function of pH for histidine     

High Performance Liquid Chromatographic Analysis for Free Valine in Plasma

Abstract

A precolumn derivatization method is presented with the use of a fluorescent derivative, 1-dimethylaminonaphhalene-5-sulfonyl-chloride, dansyl chloride, for the detection of free valine in plasma. Dansylated amino acids were determined in deproteinized samples by reverse-phase liquid chromatography. The level of detection is 100 femtoraoles (10^?15). Sample preparation required precipitation of proteins with trichoroacetic acid and removing the excess acid with water saturated ether. The deproteinized sample was adjusted to pH 9.0 and reacted with dansyl chloride. The dansylated products were detected by ultraviolet and fluorescence spectrometry. Elution time for valine subsequent to injection is 25 minutes, while the total assay requires less than 50 minutes.     

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.     

Direct UV detection of underivatized amino acids using capillary electrophoresis with online sweeping enrichment

This is an original report proposed a CE method for direct analysis of the underivatized amino acids using UV detection with relatively higher sensitivity, which was based on coordination interactions between amino acids and Cu (II) ions. In addition, an online sweeping preconcentration technique was easily combined to improve the detection sensitivity. Satisfying separations of the amino acids were obtained under optimized conditions: 50 mmol/L CuSO₄–0.05% HAc–H₂O (pH 4.5), and the separation voltage of 15 kV. The LODs for the analytes ranged from 0.1 to 0.5 μmol/L. The linearity of detection for all analytes was two orders of magnitude with the correlation coefficients greater than 0.99. The repeatability was displayed with an RSD less than 3% for migration time and peak height (n = 5). Moreover, some amino acids in real samples of human saliva and green tea were analyzed by this direct UV detection CE method with acceptable sensitivity.     

Fast Amperometric Determination of Enzymatic Activity of Glutaminase

ABSTRACT

The activity of the enzyme glutaminase has been measured using a glutamate electrochemical biosensor based on H₂O₂ detection. Calibration curves for glutamate detection and for glutaminase activity using standard glutaminase from Escherichia coli demonstrated the high sensitivity and the rapid analysis time of this novel amperometric procedure, which was 100 times more sensitive than that reported in liternature.

Porcine liver and kidney tissue and human kidney tissue samples have been tested for glutaminase activity, demonstrating the possibility to perform measurement directly on whole tissues, with no need of sample extraction and purification.     

Capillary gas chromatographic analysis of amino acids in geological environments with electron capture detection

Studies have been performed on the analysis of 21 amino acids using a fused-silica open tubular (FSOT) capillary column, and electron-capture detection (ECD) or flame-ionization detection (FID). It was shown with the N(O)-heptafluorobutyryl (HFB) amino acid isobutyl esters that the ECD response was several hundred times more sensitive than the FID response. The relative standard deviation (RSD) of retention relative to norleucine is determined with the ECD. RSD values for all N(O)-HFB amino acid isobutyl esters are relatively small (≦ 0.5%). This method has been successfully applied to trace analysis of most of the amino acids from fossil brachiopods and black shales.     

An HPLC method for the determination of selected amino acids in human embryo culture medium

A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra‐cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at −80°C. Filtered medium samples were derivatized with ortho ‐phthalaldehyde (naphthalene‐2,3‐dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse‐phase columns (LichroCART, Purospher STAR RP_18e or Ascentis Express C₁₈) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra‐assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos.     

High-Performance Liquid Chromatographic Analysis of Amino Acids in Edible Seaweeds after Derivatization with Phenyl Isothiocyanate

Summary A reversed-phase high-performance liquid-chromatographic method has been used for analysis of the amino acids in edible seaweed. Sample proteins were hydrolysed with hydrochloric acid and the amino acids produced were derivatized with phenyl isothiocyanate. The resulting phenylthiocarbamyl amino acids were chromatographed on an ODS2 column with UV detection at 254 nm. The mobile phase was a mixture of 0.14 M ammonium acetate buffer, pH 6.4, containing 0.05% triethylamine (A) and 60:40 (v/v) acetonitrile–water (B), at a flow rate of 1.1 mL min^–1; the elution gradient (min:A%) was: 0:90, 8:90, 10:70, 12:70, 18:52, 20:0, 25:0, 28:90, 35:90. Method precision for the different amino acids was between 1.33 and 3.88% (relative standard deviation); detection limits were between 6.9 and 14.3 ng mL^–1. The amino acid content of the algae analysed ranged from 22.4 ± 1.9 to 138.0 ± 5.6 mg g^–1 d.w. The amino acids present at highest concentrations were glutamic acid, alanine, and phenylalanine.     

Liquid‐Chromatography Quantitative Analysis of 20 Amino Acids after Derivatization with FMOC‐Cl and Its Application to Different Origin Radix isatidis

We developed a simple, rapid and reliable method for determination of 20 common amino acids based on derivatization with 9‐fluorenylmethyl chloroformate (FMOC‐Cl) and RP‐LC/UV, this method was first introduced into quantitative analysis of amino acids. The amino groups of amino acids were trapped with FMOC‐Cl to form amino acid‐FMOC‐Cl adducts which can be suitable for LC‐UV. Chromatographic separation was performed on a C₁₈ column with a mobile phase gradient consisting of acetonitrile and sodium acetate solution. This method was shown to be sensitive for 20 common amino acids. In the intra‐day precisions assay, the range of RSDs was 3.21‐7.67% with accuracies of 92.34‐102.51%; for the inter‐day precisions assay, the range of RSDs was 5.82‐9.19% with accuracies of 90.25‐100.63%. The results also indicated that solutions of amino acids‐FMOC‐Cl can be kept at room temperature for at least 24 h without showing significant losses in the quantified values. The validated method was successfully applied to the determination of major four kinds of amino acids in R. isatidis samples (Arg, Pro, Met and Val). The total content of amino acids in different origin R. isatidis was 13.32‐19.16 mg/g. The differences between R. isatidis samples were large using HCA.     

Analytical Separation of Reduced and Oxidized Forms of Glutathione from Amino Acid Mixtures by Overpressured Thin-Layer Chromatography

Abstract

A new method has been developed for the separation of reduced and oxidized forms of glutathione from amino acid mixtures. The samples were spotted on Kieselgel plates and developed in phenol-water = 7:3 containing SDS. The separation was performed in pressurized ultramicro chamber. The running time was shorter than that in a normal chamber, decreasing the possibility of diffusion. In addition to the two forms of glutathione, nine different kinds of amino acids could be separated. The method affords a possibility for rapid analysis of two forms of glutathione both in the clinical and industrial practice.