Fluorimetric determination of amino acids and photochemical stability of their reaction products with ortho-phthalic aldehyde under irradiation by high-power pulsed laser

The opportunity of the highly sensitive determination of amino acids using the fluorescence of their derivatives that form in the reaction with aqueous solutions of ortho-phthalic aldehyde and nucleophilic agents, potassium cyanide, sodium sulfite, and N-acetyl-L-cysteine was studied as an example. The chemical stability of forming fluorescent compounds (substituted isoindoles) and their photochemical stability under the action of power pulsed laser radiation is higher, and the analytic characteristics of reagents are highly competive with a standard applied reagent for determining amino acids and a mixture ortho-phthalic aldehyde with 2-mercaptoethanol.     

Analysis of amino acids in human serum by isocratic reversed-phase high-performance liquid chromatography with electrochemical detection

A simple, sensitive and reproducible isocratic high-performance liquid chromatography (HPLC) method has been developed for the determination of amino acids in human serum. The method involves precipitation of the serum proteins with methanol followed by pre-column derivatization of amino acids with o-phthalaldehyde-2-mercaptoethanol or o-phthalaldehyde-sodium sulfite. HPLC separation of the derivatives was performed using an ODS column with an isocratic mobile phase system and electrochemical detection (+0.75 V). The response was linear over the range 5-300 microM for all amino acids. The method allows quantitative determination of glutamic acid, asparagine, serine, glutamine, histidine, taurine, alanine, arginine, methionine, isoleucine, ornithine, leucine, phenylalanine, lysine and tryptophan at concentrations as low as 0.5-5.0 pmol (signal-to-noise ratio=2). Using this method, the levels of amino acids in serum from healthy donors and patients with ischemic stroke were determined.     

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.     

Determination of Amino Acid Enantiomers in Pharmaceuticals by Reversed-Phase High-Performance Liquid Chromatography

Precolumn derivatization with the reagent o-phthalic aldehyde/N-acetyl-L-cysteine (OPA/NAC) was used for the determination of amino acid enantiomers by reversed-phase high-performance liquid chromatography. The influence of the composition and pH of the eluent on the separation of the resulting derivatives was studied with the example of four amino acids. It was found that the highest selectivity and efficiency of the separation of OPA/NAC derivatives of amino acids is attained with the use of the eluent methanol–0.01 M Na₂HPO₄ (pH 6.0). The optimum composition of the mobile phase and conditions of the gradient elution were selected for the separation of a mixture of 20 amino acid derivatives. A procedure was developed for the determination of amino acid enantiomers in parenteral nutrition preparations. The procedure was used for the determination of D-isomers of arginine, alanine, methionine, phenylalanine, and leucine in the preparation Polyamine.     

Capillary gas chromatography of amino acids as their tert-butyldimethylsilyl derivatives in the analysis of serum amino acids in metabolic diseases

Reaction of amino acids with N-methyl-N-(tert-butyldimethylsilyl)trifluoroaceamide (MTbSTFA) in acetonitrile affords good yields of amino acid derivatives with excellent gas chromatographic and mass spectrometric properties. The single-step derivatization procedure is highly reproducible. The TBDMS amino acids are stable at room temperature for at least three days. Only a single peak is observed for each amino acid. The procedure allows simultaneous analysis of asparagine and glutamine together with other serum amino acids. Separation is achieved on a borosilicate glass capillary coated with OV-1. The mass spectra of the TBDMS amino acids possess characteristic diagnostic ions. These properties were used in the sensitive detection by GC-MS and SIM-GC-MS of GABA and pipecolic acid in the serum of a newborn suspected of a Zellweger-type syndrome, which could not be detected by other methods.     

Spectrophotometric and Fluorimetric Determination of Amino Acids by Their Reaction with o-Phthalic Aldehyde in the Presence of Sulfite and Cyanide Ions

Optimum conditions were selected for the determination of amino acids by their reaction with o-phthalic aldehyde in the presence of sodium sulfite and potassium cyanide using glycine as an example. It was shown that the analytical characteristics of the proposed procedure are highly competitive with those of a widely employed procedure using 2-mercaptoethanol and that the stability of the analytical form (substituted isoindols) was much higher.     

Homocyclic o-dicarboxaldehydes: Derivatization reagents for sensitive analysis of amino acids and related compounds by capillary and microchip electrophoresis

Amino acids are essential compounds for living organisms, and their determination in biological fluids is crucial for the clinical analysis and diagnosis of many diseases. However, the detection of most amino acids is hindered by the lack of a strong chromophore/fluorophore or electrochemically active group in their chemical structures. The highly sensitive determination of amino acids often requires derivatization. Capillary electrophoresis is a separation technique with excellent characteristics for the analysis of amino acids in biological fluids. Moreover, it offers the possibility of precapillary, on-capillary, or postcapillary derivatization. Each derivatization approach has specific demands in terms of the chemistry involved in the derivatization, which is discussed in this review. The family of homocyclic o-dicarboxaldehyde compounds, namely o-phthalaldehyde, naphthalene-2,3-dicarboxaldehyde, and anthracene-2,3-dicarboxaldehyde, are powerful derivatization reagents for the determination of amino acids and related compounds. In the presence of suitable nucleophiles they react with the primary amino group to form both fluorescent and electroactive derivatives. Moreover, the reaction rate enables all of the derivatization approaches mentioned above. This review focuses on articles that deal with using these reagents for the derivatization of amino acids and related compounds for ultraviolet-visible spectrometry, fluorescence, or electrochemical detection. Applications in capillary and microchip electrophoresis are summarized and discussed.     

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.     

Potencies of ligand-exchange capillary electrophoresis in the determination of biologically active compounds

The analytical potencies of ligand-exchange capillary electrophoresis (LECE) in regard to the determination of bioactive substances are discussed. As it is shown, complexation with copper(II) in the electrophoretic determination with UV detection makes possible the determination of amino acids, amines, and sugars, which do not absorb in the UV region, with the limits of detection 5–10 mg/L, and reduce the detection limits of the absorbing analytes, such as tryptophane, tyrosine, and histamine by 2–3 times. Prospects for using online preconcentration for the reduction of c _min are shown on an example of aliphatic amino acids, i.e., thirtyfold preconcentration is attained. LECE is shown to be applicable to the simultaneous determination of glucose, Na(⁺), and K(⁺) in human blood serum. Also versions of ligand-exchange capillary electrophoresis with direct UV detection and capillary zone electrophoresis with indirect one are compared in terms of efficiency, time of analysis, and limit of detection. The potencies of the LECE method are compared with those of ligand-exchange chromatography.     

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.     

Some observations on the automation by flow injection analysis of the spectrophotometric determination of amino acids and proteins witho-phthalaldehyde

Automation by flow injection analysis with Spectrophotometric detection of the determination of total amino acids and proteins witho-phthalaldehyde is not straightforward. The use of spectrophotometry, instead of spectrofluorimetry, and of N-acetyl-L-cysteine, instead of the conventional mercaptoethanol is advantageous because of the lower variability of absorptivities with respect to fluorescence yields, and the larger stability of the derivatives. Under adequate working conditions and with leucine as reference, the procedure can be used for the evaluation of total amino acids. A similar procedure is proposed for the analysis of proteins in a sample. Limits of detection are 1 × 10^–5 M for amino acids, and 1 × 10^–6 M for proteins, respectively.     

Derivatization of chiral carboxylic acids with (S)‐anabasine for increasing detectability and enantiomeric separation in LC/ESI‐MS/MS

A simple and practical derivatization procedure for increasing the detectability and enantiomeric separation of chiral carboxylic acids in LC/ESI‐MS/MS has been developed. (S)‐Anabasine (ANA) was used as the derivatization reagent and rapidly reacted with carboxylic acids [3‐hydroxypalmitic acid (3‐OH‐PA), 2‐(β‐carboxyethyl)‐6‐hydroxy‐2,7,8‐trimethylchroman (γ‐CEHC), and etodolac] in the presence of 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholium chloride. The resulting ANA‐derivatives were highly responsive in ESI‐MS operating in the positive‐ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using selected reaction monitoring; the detection responses of the ANA‐derivatives were increased by 20–160‐fold over those of the intact carboxylic acids and the limits of detection were in the low femtomole range (1.8–11 fmol on the column). The ANA‐derivatization was also effective for the enatiomeric separation of the chiral carboxylic acids; the resolution was 1.92, 1.75, and 2.03 for 3‐OH‐PA, γ‐CHEC, and etodolac, respectively. The derivatization procedure was successfully applied to a biological sample analysis; the derivatization followed by LC/ESI‐MS/MS enabled the separation and detection of trace amounts of 3‐OH‐PA in neonatal dried blood spot and γ‐CEHC in human saliva with a simple pretreatment and small sample volume.     

Determination of amino acids in the hepatic and brain tissue of the rat by gas chromatography

A procedure is described for the quantitative determination of amino acids in hepatic and brain tissue samples from the rat. Because the presence of certain matrix components in the tissue material led to interference with chromatographic analysis they were removed by a prechromatographic “clean-up” step. Quantitative analysis of amino acids, as their N-heptafluorobutyryl iso-butyl ester derivatives, was achieved by high resolution gas chromatography on an apolar fused silica open tubular column. Reproducibility data from the complete procedure are presented; coefficients of variation for arginine and histidine in hepatic tissue varied between 7.1 and 10.1% whereas those for most other amino acids were better than 5%, with a mean recovery of 90%.     

Gas chromatography-mass spectrometry analysis of amino acid enantiomers as methyl chloroformate derivatives: application to space analysis

This work describes a GC-MS method for enantioselective separation of amino acids. The method is based on a derivatization reaction which employs a mixture of alkyl chloroformate-alcohol-pyridine, as reagents to obtain the N(O,S)-alkyl alkoxy carbonyl esters of amino acids. Various reaction parameters are investigated and optimized to achieve a reproducible derivatization procedure suitable for separation of amino acid enantiomers on Chirasil-L-Val chiral stationary phase. In particular, the following topics are investigated for 20 proteinogenic amino acids: (i) the proper reagent and reaction conditions to obtain the highest derivative yield; (ii) the amino acid reactivity and the MS properties of the obtained derivatives; (iii) the linearity and sensitivity of the analytical method; (iv) the retention behavior of the derivatives and their enantiomeric separation on the Chirasil-L-Val chiral stationary phase. By combining the resolution power of the Chirasil-L-Val column and the high selectivity of the SIM MS detection mode, the described procedure enables the enantiomeric separation and quantification of 16 enantiomeric pairs of amino acids. The procedure is simple and fast and reproducible. It displays a wide linearity range at ppb detection limits for quantitative determinations: these properties make this derivatization method a suitable candidate for amino acid GC-MS analysis on board of the spacecrafts in space exploration missions of solar system body environments.     

Study of a new derivatizing reagent that improves the analysis of amino acids by HPLC with fluorescence detection: application to hydrolyzed rape bee pollen

A simple and sensitive method for evaluating the chemical compositions of protein amino acids, including cystine (Cys)₂ and tryptophane (Try) has been developed, based on the use of a sensitive labeling reagent 2-(11H-benzo[α]-carbazol-11-yl) ethyl chloroformate (BCEC–Cl) along with fluorescence detection. The chromophore of the 1,2-benzo-3,4-dihydrocarbazole-ethyl chloroformate (BCEOC-Cl) molecule was replaced with the 2-(11H-benzo[α]-carbazol-11-yl) ethyl functional group, yielding the sensitive fluorescence molecule BCEC–Cl. The new reagent BCEC–Cl could then be substituted for labeling reagents commonly used in amino acid derivatization. The BCEC–amino acid derivatives exhibited very high detection sensitivities, particularly in the cases of (Cys)₂ and Try, which cannot be determined using traditional labeling reagents such as 9-fluorenyl methylchloroformate (FMOC-Cl) and ortho-phthaldialdehyde (OPA). The fluorescence detection intensities for the BCEC derivatives were compared to those obtained when using FMOC-Cl and BCEOC-Cl as labeling reagents. The ratios I _BCEC/I _BCEOC = 1.17–3.57, I _BCEC/I _FMOC = 1.13–8.21, and UV_BCEC/UV_BCEOC = 1.67–4.90 (where I is the fluorescence intensity and UV is the ultraviolet absorbance). Derivative separation was optimized on a Hypersil BDS C₁₈ column. The detection limits calculated from 1.0 pmol injections, at a signal-to-noise ratio of 3, ranged from 7.2 fmol for Try to 8.4 fmol for (Cys)₂. Excellent linear responses were observed, with coefficients of >0.9994. When coupled with high-performance liquid chromatography, the method established here allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids including (Cys)₂ and Try from bee-collected pollen (bee pollen) samples.     

Über Farbreaktionen mehrfach acylierter Aromaten mit Aminosäuren

The colour reactions of 10ortho-acylated benzene derivatives with amino acids are described and compared with ninhydrin. The preparation of the new compounds: 1-ethyl-2,3,5-triacetyl- resp. 1,3-diethyl-2,4,5-triacetyl-benzene and 1-acetyl-2-propionylbenzene is given, including the NMR and MS data.

28. Mitt.: Acylverbindungen isocyclischer Verbindungen.     

Laser-induced fluorescence detection in liquid chromatography after preliminary derivatization of carboxylic acid and primary amino groups

The development of selective derivatization for the determination of carboxylic acids, amino acids and peptides in aqueous solutions is described as a preliminary study for the determination of these compounds in biological materials. The derivatization reactions are completed before the liquid chromatographic separation and laser-induced fluorescence detection for which a continuous-wave argon-ion gas laser is used in the ultraviolet or visble mode. Carboxylic acid groups arre derivatized with 9-hydroxymethylathracene and primary amino groups are derivatized with fluorescein isothiocyanate. Detection limits, in aqueous solutions, for the carboxylic acid derivatives are ca. 190 fg (ultraviolet mode). In the visible mode, the detection limits are ca. 1 fg for the primary amino derivatives of amino acids and peptides. In al the chromatographic analyses, the derivatization mixtures are injected onto a standard reversed-phase or reversed- phase ion-pair system and conventional flow cells are used without expensive photon counting or optical systems.     

High sensitivity laser induced fluorescence detection in capillary zone electrophoresis

Laser induced fluorescence is used for the detection of labeled amino acids. A preliminary comparison is made of three fluorescence pre-column labeling reagents, ortho-phthaldialdehyde, naphthalene dicarboxaldehyde, and fluorescein isothiocyanate, and data on phenylalanine detection limits are given.     

Quantitative analysis of 17 amino acids in tobacco leaves using an amino acid analyzer and chemometric resolution

A method was developed for quantifying 17 amino acids in tobacco leaves by using an A300 amino acid analyzer and chemometric resolution. In the method, amino acids were eluted by the buffer solution on an ion‐exchange column. After reacting with ninhydrin, the derivatives of amino acids were detected by ultraviolet detection. Most amino acids are separated by the elution program. However, five peaks of the derivatives are still overlapping. A non‐negative immune algorithm was employed to extract the profiles of the derivatives from the overlapping signals, and then peak areas were adopted for quantitative analysis of the amino acids. The method was validated by the determination of amino acids in tobacco leaves. The relative standard deviations (n = 5) are all less than 2.54% and the recoveries of the spiked samples are in a range of 94.62–108.21%. The feasibility of the method was proved by analyzing the 17 amino acids in 30 tobacco leaf 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.