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.     

Towards the chiral metabolomics: Liquid chromatography–mass spectrometry based dl-amino acid analysis after labeling with a new chiral reagent, (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate,and the application to saliva of healthy volunteers

A novel triazine-type chiral derivatization reagent, i.e., (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl) pyrrolidine-2-carboxylate (DMT-(S)-Pro-OSu), was developed for the highly sensitive and selective detection of chiral amines and amino acids by UPLC–MS/MS analysis. The enantiomers of amino acids were easily labeled with the reagents at room temperature within 40 min in an alkaline medium containing triethylamine. The diastereomers derived from proteolytic amino acids, except serine, were well separated under isocratic elution conditions by reversed-phase chromatography using an ODS column (R_s = 1.2–9.0). dl-Serine was separated by use of an ADME column which has relatively higher polar surface than the conventional ODS column. The characteristic product ions, i.e., m/z 195.3 and m/z 209.3, were detected from all the diastereomers by the collision-induced dissociation of the protonated molecule. A highly sensitive detection on the amol–fmol level was obtained from the selected reaction monitoring (SRM) chromatogram. The chiral amines (e.g., adrenaline and noradrenaline) labeled with DMT-(S)-Pro-OSu were also well separated and sensitively detected by the present procedure. The method using DMT-(S)-Pro-OSu was used for the determination of dl-amino acids in the human saliva from healthy volunteers. Various l-amino acids were identified in the saliva. Furthermore, d-alanine (d-Ala) and d-proline (d-Pro) were also detected in relatively high concentrations (>5%). The ratio was higher in male saliva than in female saliva. However, the difference in the ratio of d-Ala for one day was not very high and the effect of foods and beverage seemed to be negligible. Based on the results using l-Ala-d₃, the d-Ala in saliva seemed to be produced due to the racemization with some enzymes such as racemase. The racemization reaction was reversible, i.e., d-Ala-d₃ was also racemized to l-Ala-d₃ in saliva. Thus, care should be taken during the analysis of dl-amino acids in saliva. The present method using DMT-(S)-Pro-OSu may be applicable for the determination of chiral amine metabolomics, because the resulting derivatives produce the same product ions without relation to the compounds and show highly sensitive detection in the SRM mode of MS/MS. Consequently, DMT-(S)-Pro-OSu seems to be a useful chiral derivatization reagent for the determination of amines and amino acids in biological samples.     

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.     

Pyrolysis of Amino Acid Derived Stabilised Ylides as a Route to Chiral Acetylenic Amines and Amino Acids and Gaba Analogues

Abstract

We recently reported the pyrolysis of stabilised ylides as a method for overall conversion of carboxylic acids to homologous acetylenic esters and terminal alkynes.^1,2 This has now been applied successfully to amino acids. A wide range of alkoxycarbonyl protected amino acids have been converted to the stable crystalline ylides 1. These have been fully characterised, and upon FVP, eliminate Ph₃PO to afford the protected acetylenic amino acids 2 in good yield and without significant racemisation. Subsequent reactions of these extremely versatile intermediates have been used to gain access to a wide variety of chiral amine and amino acids of great interest as potential selective enzyme inhibitors and components for modified peptide structures.     

Characterization and application of a new ultraviolet derivatization reagent for amino acids analysis in capillary electrophoresis

A new ultraviolet (UV) labeling reagent, p-acetamidobenzenesulfonyl fluoride (PAABS-F), was designed and synthesized to label and determine the amino acids by capillary electrophoresis (CE) with diode-array detector (DAD). PAABS-F is very stable and easy to synthesize. It reacted with primary or secondary amino acids very quickly under facile conditions to give corresponding derivatives in high yield with excellent sensitivity and stability. No by-products were observed in amino acid derivatives when stored at room temperature under natural daylight for at least 7 days. Both amino acids standard solution and real samples reacted with this new UV labeling reagent smoothly to form high UV-absorption derivatives. The labeled 20 standard amino acids were efficiently separated by CE and the mass detection limits (S/N = 3) were ranged from 59.3 fmol for l-tryptophan to 1.70 pmol for l-histidine.     

Ion-Exchange Chkomatographic Determination of Aming Acids On Impresgnated Papers

Abstract

The modified optical density method of Moore and Stein has been used for the estimation of amino acids present in biological samples. the mixture of amino acids were chromatographad on bismuth oxide impregnated papers, the identified spots were cut out and eluted, the optical densities were measured at 570 nm. With the help of a calibration curve, the amino acids present in the samples were estimated.     

Analysis of amino acids in human vascular endothelial (ECV-304) cells by microchip electrophoresis with fluorescence detection

A rapid and sensitive method was developed for the analysis of amino acids by microchip electrophoresis with Hg-lamp excitation fluorescence detection. Fluorescein-isothiocyanate (FITC) was chosen to estimate the sensitivity of this system, and the detection limit (S/N = 3) with FITC was 1.7 nM, which showed that the system was sensitive as well as simple. Two derivatizing agents, FITC and ortho-phthalaldehyde (OPA) were employed to label amino acids and were compared in the same fluorescence detection system with an Hg lamp as the excitation source. The separation parameters were optimized in detail. Optimum separation of OPA-labeled amino acids was obtained in less than 200 s with 20 mM borate buffer (pH 9.0) containing 20% acetonitrile and 10 mM beta-cyclodextrin. Detection limits for amino acids (alanine (Ala), taurine (Tau), glycine (Gly), glutamic acid (Glu), and aspartic acid (Asp)) of 0.38-1.0 muM were achieved. The method was successfully applied to analysis of amino acids in human vascular endothelial cells (ECV-304). The average amount of amino acids in single ECV-304 cells is estimated to be 5.84 fmol for Ala, 2.78 fmol for Tau, 1.15 fmol for Gly, 3.10 fmol for Glu, and 1.30 fmol for Asp.     

4:3-β-naphthapyrone-4-acetic acidN-hydroxysuccinimidyl ester as a fluorescent labeling reagent for amino acids and oligopeptides in high-performance liquid chromatography

Summary 4:3-β-Naphthapyrone-4-acetic acidN-hydroxysuccinimidyl ester (NPA-OSu) is a highly sensitive and moderately reactive derivatizing reagent with a naphthapyrone moiety as fluorophore and anN-hydroxysuccinimidyl active ester as reactive group toward amino compounds. It is readily prepared in two steps. The fluorescence properties of NPA-OSu and its hydrolysis product have been studied in detail, and the conditions for derivatization and separation of the NPA-OSu derivatives of some amino acids and oligopeptides have been investigated. Atλ _ex = 352 nm andλ _em = 422 nm the detection limits (signal-to-noise ratio=3) for amino acids and oligopeptides reached fmol levels, for injection of 20 μL; this sensitivity was comparable with that obtained by use of 7-(diethylamino)coumarin-3-carboxylic acid succinimidyl ester as derivatizing reagent in the analysis of amino acids by capillary electrophoresis with laser-induced-fluorescence detection.     

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.     

Comparison of different amino acid derivatives and analysis of rat brain microdialysates by liquid chromatography tandem mass spectrometry

The efficiencies of three derivatisation reagents that react with either the amine (9-fluorenylmethyl chloroformate (FMOC)) or the carboxylic acid group (butanol) of amino acid or with both types of functional groups (propyl chloroformate) were compared in the analysis of amino acids by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS). Separation of 20 amino acids derivatised with these three reagents was studied on reversed-phase chromatography. Linearity, repeatability and limits of detection of the LC-ESI-MS/MS method were determined by analysing FMOC-, butanol- and propyl chloroformate-derivatised lysine, β-aminobutyric acid, threonine and glutamic acid. The limits of detection for the derivatised amino acids (7.5-75 fmol) were as much as 2-60 times lower than those of the corresponding underivatised molecules. The best linearity was observed for amino acids derivatised with propyl chloroformate or butanol (r² = 0.996-0.999, range = 100-8500 nmol L⁻¹). Propyl chloroformate was the best suited of the reagents tested for the analysis of amino acids with LC-MS/MS and was used for the analysis of amino acids in rat brain microdialysis samples.     

Evaluation of Fluorescamine as a Phosphogenic Derivatizing Reagent of Primary Amines

Abstract

Fluorescamine has been shown to be a promising derivatizing reagent for the room temperature phosphorescence analysis of primary amino acids. The fluorescamine concentration used for derivatization in solution was 600 μg/ml and for direct derivatization, 1160 μg/ml. For derivatized primary amino acids the limits of detection were found to be between 0.8 ng and 2.9 ng with linear dynamic ranges between 2 and 3 orders of magnitude.     

Chemiluminescence Detection of Amino Acids,Peptides, and Proteins Using Tris-2,2′-Bipyridine Ruthenium (III)

Abstract

The feasibility of using the tris-2-2′-bipyridine ruthenium (III) (Ru(bpy)₃ ^{3 +}) chemiluminescent (CL) reaction for the detection of amino acids, peptides, and proteins has been studied.

Detection limits of the amino acids as determined by flow injection analysis (FIA) ranged from 20 pmol of proline to 50 nmol of asparagine. In general, amino acids containing secondary amine groups yielded the strongest responses. A reaction mechanism for Ru (bpy)₃ ^{3 +} chemiluminescence of aliphatic amines has been proposed. Studies of peptide molecules and poly-prolines showed that the peptide bond barely contributes to the detection signals. The separation of hydroxyproline and proline in synthetic collagen by HPLC with Ru (bpy)₃ ^{3 +} chemiluminescence detection has been shown to be possible.     

HPLC Analysis of Amino Acids with Ion Exchange Chromatography and O-Phthalaldehyde Post-Column Derivatization: Applications to the Assay of Endogenous Free Amino Acids and Their Transport in Human Placental Villus

Abstract

A method using high performance liquid chromatography (HPLC) for the analysis of primary amino acids in human placenta is described. This method involves separation of primary amino acids by high performance ion-exchange chromatography followed by post column derivatization using O-phlthalaldehyde (OPA) and 2-mercaptoethanol and fluorescence (excitation 340 nm and emission 410 nm) detection of derivatives. Waters 840 HPLC Amino Acid System was used for this purpose.

For analysis, villus tissue was extracted with acetonitrile, and the recovered amino acids were reconstituted in a sodium diluent (pH 2.2). The complete profile of the primary amino acids in the sample could be constructed in about 90 minutes. Up to 44 samples can be analyzed without special attention. Using this method, essential amino acids (threonine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine) and nonessential amino acids (aspartic acid, serine, glutamic acid, glycine, alanine, arginine) were detected and quantified in human placental villus in pmol quantities. Plots of peak heights (or areas) were linear for several amino acids. The same method was also used for (a) the assay of free primary amino acids in umbilical bloods, (b) the efflux of amino acids from isolated human placental villus, and (c) to study the uptake of α-aminoisobutyric acid (AIB), a non-metabolizable amino acid, by the isolated placental villus.     

TLC Resolution of Amino Acids in a New Solvent and Effect of Alkaline Earth Metals

Abstract

A new solvent system for the resolution of 15 component mixture of amino acids and the effect of alkaline earth metals on the resolution is reported, the TLC was carried out using plain and impregnated silicagel plates and, solutions of amino acids in tris buffer and pretreated with metal hydroxides.     

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 Thin Layer Chromatography of Amino Acids

Abstract

The use of reversed phase layers for the thin layer chromatography of amino acids is described. Only when a modifier was added to the mobile phase was clear separation of the amino acids achieved. Ion paring with trifluoroacetic acid overcame problems with streaking and poor separation on C₂ or C₁₈ reversed phase layers. All amino acids could not be separated with a single mobile phase. Thus, three different combination of acetonitrile-0.4% trifluoroacetic acid were used to separate eighteen amino acids with derivatization. No derivative was required.     

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.     

TLC Separations of Amino Acids on Silica Gel Impregnated Layers

Abstract

Copper sulphate and polyamide were tried as impregnants for improving the separation of twenty amino acids on silica gel ‘G’ layers using a new solvent system MeOH-BuOAc-AcOH-Pyridine(20:20:10:5). Tables are presented to illustrate the improvement in resolution of amino acids on silica gel plates.     

HPLC-Analysis of PTH-Amino Acids

Abstract

Some of the problems that affect the usefulness of HPLC-analysis of PTH-amino acids have been examined. Four different C₁₈-reversed phase packings have been tested, together with different mobile phases. The separation problems of the Met/Val-group, the Phe/Leu/Ile/Lys-group and of the basic amino acids His and Arg on the reversed phase packings have been examined and solutions to some of the problems have been demonstrated. The sensitivity of the quantitative analysis of amino acids from a hydrolysate was determined.     

Isotopic variants of light and heavy l-pyroglutamic acid succinimidyl esters as the derivatization reagents for dl-amino acid chiral metabolomics identification by liquid chromatography and electrospray ionization mass spectrometry

l-Pyroglutamic acid succinimidyl ester (l-PGA-OSu) and its isotopic variant (l-PGA[d₅]-OSu) were newly synthesized and evaluated as the chiral labeling reagents for the enantioseparation of amino acids, in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The enantiomers of amino acids were easily labeled with the reagents at 60 °C within 10 min in an alkaline medium containing triethylamine. Although all the diastereomers derived from 18 proteolytic amino acids could not be satisfactorily separated, the pairs of 9 amino acids were completely separated by reversed-phase chromatography using the small particle (1.7 μm) ODS column (Rs = 1.95–8.05). The characteristic daughter ions, i.e., m/z 84.04 and m/z 89.04, were detected from all the derivatives by the collision induced dissociation of the protonated molecular ions. A highly sensitive detection at a low-fmol level (0.5–3.2 fmol) was also obtained from the selected reaction monitoring (SRM) chromatograms. An isotope labeling strategy using light and heavy l-PGA-OSu for the differential analysis of the dl-amino acids in different sample groups is also presented in this paper. The differential analysis of biological sample (i.e., human serum) and food product (i.e., yogurt) were tried to demonstrate the efficiency of the proposed method. The ratios of the dl-amino acids in human serum samples, spiked with the different concentrations of d-amino acids, were determined by the procedures using l-PGA-OSu and l-PGA[d₅]-OSu. The d/l ratios in the two sample groups at different concentrations of amino acids were similar to the theoretical values. Furthermore, the ratios of d/l-alanine values in different yogurt products were comparable to the ratios obtained from the d/l values using only light reagent (i.e., l-PGA-OSu). Consequently, the proposed strategy is useful for the differential analysis not only in biological samples but also in food products.