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.     

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.     

Comparative study on the use of ortho-phthalaldehyde, naphthalene-2,3-dicarboxaldehyde and anthracene-2,3-dicarboxaldehyde reagents for alpha-amino acids followed by the enantiomer separation of the formed isoindolin-1-one derivatives using quinine-type chiral stationary phases

Cinchona alkaloid based chiral stationary phases (CSPs) were evaluated and compared for the enantiomer separation of a set of alpha-amino acid derivatives as selectands (SA), using ortho-phthalaldehyde (OPA), naphthalene-2,3-dicarboxaldehyde (NDA) and anthracene-2,3-dicarboxaldehyde (ADA) as reagents in the presence of acetonitrile. Protocols have been developed for the derivatization of most common amino acids in the absence of the usual thiol components (2-mercaptoethanol, mercaptosulphonic acid, sodium sulfite) under acidic and neutral conditions providing the corresponding isoindolin-1-one (phthalimidine) derivatives. They are stable for hours at various reaction conditions compared to thiol or sulfide modified isoindoles resulted by the OPA-thiol reaction type. Among the derivatizing agents, ADA afforded the highest retention factors (k) and for the majority of the analytes also resolution (Rs) and enantioselectivity (alpha) values (i.e. for tryptophan k1 = 23, Rs = 4.93 and alpha = 1.43). Structure variation of the CSPs and selector (SO), respectively indicates that steric arrangement around the binding cleft plays a major role in the enantiodiscriminating events. To provide more detailed information about the derivatization reaction itself, the proposed mechanism for the formation of the OPA derivative (isoindolin-l-one) was further evaluated by deuterium labeling and LC-MS analysis.     

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.     

Assay of amino acids in individual human lymphocytes by capillary zone electrophoresis with electrochemical detection

Amino acids in individual human lymphocytes were determined by capillary zone electrophoresis with electrochemical detection (ED) at a carbon fiber bundle electrode after on-column derivatization with naphthalene-2,3-dicarboxaldehyde (NDA) and CN⁻. In order to inject cells easily, a cell injector was designed. In this method, a single human lymphocyte and then the lysing/derivatizing buffer were electrokinetically injected into the front end of the separation capillary as a chamber to lyse the lymphocyte and derivatize amino acids in the cell. Four amino acids (serine (Ser), alanine (Ala), taurine (Tau), and glycine (Gly)) in single human lymphocytes have been identified. Quantitation has been accomplished through the use of calibration curves.     

Stacking, derivatization, and separation by capillary electrophoresis of amino acids from cerebrospinal fluids

This paper describes the in-column derivatization, stacking, and separation of amino acids by CE in conjunction with light-emitting diode-induced fluorescence using naphthalene-2,3-dicarboxaldehyde (NDA). According to the relative electrophoretic mobilities and the migration direction in tetraborate solution (pH 9.3), the injection order is cyanide, then amino acids, then NDA. Once poly(ethylene oxide) (PEO) migrates through the capillary under EOF, the amino acid.NDA derivatives, amino acids, and CN- ions migrating against the EOF enter the PEO zone. As a result of increases in viscosity and possible interactions with PEO molecules, the reagents/analytes slow down such that they become stacked at the boundary. In comparison with the off-column approach to the analysis of amino acids, our proposed method provides a lower degree of interference from polymeric NDA compounds and other side products. As a result, the plot of the peak height as a function of gamma-aminobutyric acid (GABA) concentration is linear over the range from 10(-5) to 10(-8) M, with the LOD being 4 nM. We demonstrate the diagnostic potential of this approach for the determination of amino acids, including GABA and glutamine, in biological samples through the analysis of large volumes of cerebral spinal fluids without the need for sample pretreatment.     

Performance and selectivity of polymeric pseudostationary phases for the electrokinetic separation of amino acid derivatives and peptides

Two polymeric pseudostationary phases, one an acrylamide polymer and the second a siloxane polymer, have been investigated for the separation of naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized amino acids and small peptides. The dervatized amino acids were detected by UV absorbance and laser-induced fluorescence (LIF) detection. The polymers provided very high efficiency and good selectivity for the separation of the amino acids. The separation selectivity using the polymers was significantly different from that of SDS micelles, and there were subtle differences in selectivities between the polymers. Although very good detection limits were obtained with LIF detection, a significant background signal was observed when the polymers were not washed to remove fluorescent impurities. The polymers did not separate the peptides very well. It is postulated that the fixed covalent structure of the polymers prevents them from interacting strongly or efficiently with the peptides, which are large in relation to the analytes typically separated by electrokinetic chromatography using polymers.     

Electrophoresis PDMS/glass chips with continuous on-chip derivatization and analysis of amino acids using naphthalene-2,3-dicarboxaldehyde as fluorogenic agent

In this work, we developed a PDMS electrophoresis device able to carry out on-chip derivatization and quantification of amino acids (AAs) using naphthalene-2,3-dicarboxaldehyde (NDA) as a fluorogenic agent. A chemical modification of the PDMS surface was found compulsory to achieve the derivatization of AAs with NDA and a limit of detection (LOD) of 40 nM was reached for glycine. Finally, we suggested the applicability of this microdevice for the analysis of real biological samples such as a rat hippocampus microdialysate.     

Quantitation of branched-chain amino acids in ascites by capillary electrophoresis with light-emitting diode-induced fluorescence detection

Branched-chain amino acids (BCAAs) are one of the important biomarkers for monitoring liver disease such as hepatitis or hepatoma. In this communication, we present the determination of the concentrations of BCAA in ascites by CE light-emitted diode-induced fluorescence (LEDIF) using 1.5% m/v poly(ethylene oxide) (average M(v) : ~8?000?000?g/mol) that was prepared in 10?mM sodium tetraborate solution (pH 9.3). Naphthalene-2,3-dicarboxaldehyde was used to derivatize 15 amino acids (AAs) to form naphthalene-2,3-dicarboxaldehyde (NDA)-AA derivatives prior to CE analysis. The separation of 15 NDA-AA derivatives was accomplished within 15?min, with RSD values of <5.8% (within-day) and 7.4% (between-days) with respect to their migration times. The limits of detection for the tested BCAAs ranged from 10.6 to 10.9?nM. We determined the concentrations of three BCAAs--leucine, isoleucine and valine--in ascites by applying a standard addition method, with recovery percentages ranging from 93.9 to 111%. The results obtained from this CE-LEDIF method is in good agreement with those by a gold standard method using an AA analyzer. We have found that the concentrations of the three BCAAs in ascites obtained from patients suffering from liver diseases were lower than those from healthy individuals. Our approach is highly efficient, sensitive, and cost-effective, which holds great potential for the diagnosis of liver diseases.     

Determination of amino acids in tea leaves and beverages using capillary electrophoresis with light-emitting diode-induced fluorescence detection

The combination of capillary electrophoresis (CE) and light-emitting diode-induced fluorescence (LED-IF) detection has been demonstrated in the analysis of major amino acids in tea leaves and beverages. The separation efficiency of amino acids, which were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA), depended on the capillary length and PEO concentration. We suggested that the interactions between the NDA derivatives and poly(ethylene oxide) (PEO) molecules are based on hydrogen bonding, hydrophobic patches, and Van der Waals forces. The magnitude of EOF and the interactions between them can be further controlled by the capillary length. The separation of 17 NDA-amino acids derivatives was completed within 16 min using 0.5% PEO and 60 cm capillary length. The relative standard deviations (R.S.D.) of their migration times (n = 5) were less than 2.7%. Additionally, the limits of detection at signal-to-noise ratio 3 for the tested amino acids ranged from 3.6 to 28.3 nM. Quantitative determination of amino acids in tea leaves and beverages was accomplished by our proposed method. This study showed that amino acid present in highest concentration in tea leaves and beverages is γ-aminobutyric acid and theanine, respectively. The experimental results suggest that our proposed methods have great potential in the investigation of the biofunction of different tea samples.     

氨基酸的2,3-二甲醛基喹喔啉荧光衍生及高效液相色谱分离分析

将荧光试剂2,3-二甲醛基喹喔啉首次应用到氨基酸的高效液相色谱分离分析中。考察了该荧光试剂与伯胺氨基酸衍生反应的pH值、比例、反应时间等影响因素,确定衍生反应的最佳条件为:100 mmol/L硼砂缓冲液(pH 9.5),二巯基乙醇、衍生试剂与氨基酸的比例为9∶3∶1,室温下反应5 min。采用C18色谱柱,经二元梯度洗脱,荧光检测,17种伯胺蛋白氨基酸得到较好分离。氨基酸的浓度在0.01～1.00 mmol/L范围内与其衍生物峰面积呈较好的线性关系,氨基酸衍生物的检出限为0.02～0.07μg/L。应用该方法对麦冬中游离氨基酸的含量进行测定,结果满意。此方法快速,高效,灵敏度高,精密度好。     

Post-column reactor of coaxial-gap mode for laser-induced fluorescence detection in capillary electrophoresis

A post-column reactor with coaxial-gap mode is developed for laser-induced fluorescence detection (LIF) in capillary electrophoresis (CE). The reactor can be assembled simply and conveniently, in which a thin polyimide sleeve of 10-mm length obtained from the capillary coating is used to align separation and reaction capillary with a 20 microm gap. Naphthalene-2,3-dicarboxaldehyde and 2-mercaptoethanol are used as derivatization reagents and delivered into the reaction capillary through the annulus between the separation capillary and polyimide sleeve and the gap of two capillaries by gravity. A reaction distance from the gap to detection point is 5mm. For the post-column reactor of CE-LIF, several configuration parameters are optimized, including liquid level difference between the derivatization solution and outlet buffer, annular dimension between the outer diameter of etched separation capillary and the inner diameter of polyimide sleeve, and reaction distance, etc. The detection limits in the range from 8.0x10(-8) to 1.0x10(-6) mol/L and linear calibration range more than two orders of magnitude are obtained for amino acids. The separation efficiency ranges from 1.35x10(5) to 1.67x10(5) theoretical plates.     

Analysis of amino acids by capillary zone electrophoresis with electrochemical detection

The precapillary derivatization of 20 amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) and CN(-) was investigated. All these derivatized amino acids could be oxidized on the carbon fiber microdisk bundle electrode except proline. Capillary zone electrophoresis with electrochemical detection was employed for the analysis of 19 amino acids. The optimum conditions of separation and detection were borate, pH 9.48, for the electrolyte, 18 kV for the separation voltage and 1.15 V versus a saturated calomel electrode for the detection potential. Limits of detection of concentration or mass for individual amino acids were between 1.7 x 10(-7) and 1.8 x 10(-6) mol/L or 84 and 893 amol (according to the signal-to-noise ratio of 3) for the injection voltage of 6 kV and injection time of 10 s. The relative standard deviations were between 0.80 and 2.3% for the migration times and 1.4 and 6.4% for the electrophoretic peak currents. From a mixture of 19 amino acids, 10 amino acids (Arg, Lys, Orn, Try, Ser, Ala, Gly, Cys, Glu, Asp) could be well separated. The other 9 amino acids appeared on three electrophoretic peaks. From the samples, in which the nine amino acids do not exist simultaneously, some of them could also be detected. The method was applied to the determination of amino acids in beer by the standard addition method. The recovery for the amino acids in beer was 91-109%.     

C-acylations of polymeric phosphoranylidene acetates for C-terminal variation of peptide carboxylic acids

C-Acylations of polymer-supported 2-phosphoranylidene acetates ("linker reagents") with protected amino acids yielded 2-acyl-2-phosphoranylidene acetates as flexible intermediates for the C-terminal variation of carboxylic acids: peptidyl-2,3-diketoesters, peptidyl vinyl ketones, peptidyl-2-ketoaldehydes, and 1,3-diamino-2-hydroxy-propanes were obtained as products. [reaction: see text]     

Selective determination of gamma-aminobutyric acid, glutamate and alanine by mixed micellar electrokinetic chromatography and fluorescence detection

A mixed micellar electrokinetic chromatography method with fluorescence detection was developed to simultaneously monitor gamma-aminobutyric acid (GABA), glutamate (Glu) and alanine (Ala) in biological samples. Amino acids were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA). The separation of three NDA-labeled isomers (GABA, alpha-ABA, beta-ABA) was studied in detail with different micelles solutions such as sodium dodecyl sulfate (SDS), beta-cyclodextrin (beta-CD) and sodium cholate (SC). Simultaneous resolution of GABA, Glu and Ala from 21 amino acids was achieved within 5 min using 20 mM phosphate buffer at pH 8.7 containing 24 mM SC and 26 mM SDS. The detection limits were 4.0 x 10(-8), 1.1 x 10(-8) and 1.3 x 10(-8) M, for GABA, Glu and Ala, respectively, with S/N = 2. The method was applied to monitor the changes of amount of GABA, Glu and Ala in tobacco leaf in response to cold and dark stress.     

The use of naphthalene-2,3-dicarboxaldehyde for the analysis of primary amines using high-performance liquid chromatography and capillary electrophoresis

This paper reviews analytical methods, instrumental developments and applications for derivatization of primary amines with naphthalene-2,3-dicarboxaldehyde using fluorescence and chemiluminescence detection with capillary electrophoresis (CE) and high performance liquid chromatography (HPLC). The use of lasers as well as lamps as the excitation source for fluorescence detection is discussed. The detection limit observed with naphthalene-2,3-dicarboxaldehyde derivatization is often lower and better than those obtained with other analytical separations and other fluorescent dyes. In addition, this paper describes the crucial points that influence the stability of NDA primary amine derivatives, and summarize the separation, derivatization and migration conditions of the different techniques, with their advantages and drawbacks.     

Optical fiber light-emitting diode-induced fluorescence detection for capillary electrophoresis

A highly sensitive optical fiber light-emitting diode (LED)-induced fluorescence detector for CE has been constructed and evaluated. In this detector, a violet or blue LED was used as the excitation source and an optical fiber with 40 microm OD was used to transmit the excitation light. The upper end of the fiber was inserted into the separation capillary and was situated right at the detection window. Fluorescence emission was collected by a 40 x microscope objective, focused on a spatial filter, and passed through a cutoff filter before reaching the photomultiplier tube. Output signals were recorded and processed with a computer using in-house written software. The present CE/fluorescence detector deploys a simple and inexpensive optical system that requires only an LED as the light source. Its utility was successfully demonstrated by the separation and determination of amino acids (AAs) labeled with naphthalene-2,3-dicarboxaldehyde (NDA) and FITC. Low detection limits were obtained ranging from 17 to 23 nM for NDA-tagged AAs and 8 to 12 nM for FITC-labeled AAs (S/N=3). By virtue of such valuable features as low cost, convenience, and miniaturization, the presented detection scheme was proven to be attractive for sensitive fluorescence detection in CE.     

Manganese(II) and palladium(II) complexes containing a new macrocyclic Schiff base ligand: antibacterial properties

Complexes of tetradentate macrocyclic Schiff base ligand, L, with Mn^II and Pd^II ions have been synthesized by the template condensation of 1,10-phenanthroline-2, 9-dicarboxaldehyde, 2,3-diamino-1,4-naphthoquinone and 1,2-dibromoethane in EtOH. The complexes were characterized by physicochemical and spectroscopic methods and an octahedral geometry is suggested for their structure. They have been screened for antibacterial activity against several bacteria, and the results are compared with the activity of penicillin.     

临床化学上氨基酸、有机酸及核苷类的二维薄层色谱多相分离

在同一块薄层板上多相二维分离３０余种氨基酸、４０余种非挥发性有机酸和２０余种碱基。核苷类化合物。应用一对新的展开剂,用紫外灯－吖啶试剂－镉茚三酮试剂组合显色定位分析,改良了尿液非挥发性有机酸的溶剂萃取。尿液中此三类化合物提取物的多相二维色谱分离效果良好。     

Reaction of Amino Acid Esters and Amides with o-Phthalaldehyde. Limitations in Fluorescent Yield

Abstract

Amino acid esters and amides fail to produce strong fluorescence following their reaction with o-phthalaldehyde (OPA) in the presence of β-mercaptoethanol. The lack of luminescence appears to arise from a quenching phenomenon rather than from reduced reactivity of these substrates toward OPA. The amido hydrogen in the peptide linkage has been implicated in the loss in fluorescence yield.

o-Phthalaldehyde (OPA) has proven to be a very useful reagent for converting amino acids to fluorescent isoindole derivatives¹. This derivatization process permits the visualization of femtomolar quantities of amino acids. Unfortunately, this technique has not been applied successfully to the derivatization of peptides^2,3, except for those containing lysine residues, in which the terminal amino group is attacked by the reagent⁴. It is the purpose of this note to more clearly define the limitations of OPA as a fluorogenic reagents for peptides and amino acid esters.