Analysis of amino acids in complex samples by using voltammetry and multivariate calibration methods

A voltammetric method is proposed for the simultaneous determination of tryptophan, cysteine, and tyrosine using multivariate calibration techniques. Various electrodes and voltammetric techniques were explored to ascertain the optimum measurement strategy. Among them, differential pulse voltammetry (DPV) with a Pt electrode was selected as analytical technique since it provided a suitable compromise between sensitivity and reproducibility while allowing the oxidation peaks of the three compounds to be reasonably discriminated. The sensitivity of DPV with Pt electrode for Trp standards was 8.4×10⁻² A l mol⁻¹, the repeatability 3.7% and the detection limit below 10⁻⁷ M. The lack of full selectivity of the voltammetric data was overcome using multivariate calibration methods on the basis of the differences in the voltammetric waves of each compound. The accuracy of predictions was evaluated preliminarily from the analysis of three-component synthetic mixtures. Subsequently, this method was applied to the analysis of oxidizable amino acids in feed samples. Results obtained were in good concordance with those given by the standard method using an amino acid analyzer.     

Electrochemical investigations of amino acids at solid electrodes: Part II. Amino acids containing no sulfur atoms: Tryptophan,tyrosine, histidine and derivatives

The electrochemical properties of amino acids containing no sulfur atoms have been investigated using stationary or rotating solid electrodes such as Au and vitreous carbon. Among the 20 amino acids present in the proteins only tryptophan and tyrosine are specifically oxidizable at a gold, platinum or carbon electrode. Histidine is oxidizable only at a carbon electrode. In the case of tryptophan and tyrosine the oxidation phenomena are very similar whatever the nature of the electrode. Oxidation can be performed on a smooth surface electrode, and at the level of metal oxide formation it leads to a mixture of monomeric species (oxindolalanyl with tryptophan and hexacyclodienone alanyl with tyrosine) and polymeric species (filming effect: polytryptophan with tryptophan, polyphenylene oxide alanyl with tyrosine). According to experimental features (pH, concentrations, electrode angular velocity, etc.) either the first of the second electrochemical oxidation pathway can be favored.     

Voltammetric determination of tyrosine based on an L-serine polymer film electrode

A novel film electrode for the voltammetric determination of tyrosine has been constructed based on electropolymerization of L-serine on a glassy carbon electrode. Voltammetric behaviour of tyrosine on the poly-L-serine film electrode was investigated with cyclic voltammetry and linear sweep voltammetry, and electrochemical parameters were calculated from chronocoulometry. In optimal conditions, the oxidation peak current of tyrosine on the poly-L-serine film electrode was enhanced greatly. A sensitive oxidation peak at 0.90 V was employed to determine tyrosine. A linearity between the oxidation peak current and the tyrosine concentration was obtained in the range of 3.0 x 10(-7) to 1.0 x 10(-4) mol L(-1) with a detection limit of 1.0 x 10(-7)mol L(-1). The practical application of the film electrode in the determination of tyrosine in a commercial amino acid oral solution demonstrated that it has good selectivity and high sensitivity.     

Chemical analysis of single neurons by open tubular liquid chromatography

A method, based on open tubular liquid chromatography with voltammetric detection, has been developed for the determination of trace levels of organic compounds in single cells. Individual neurons from the subesophageal ganglia ofHelix aspersa were analyzed using this method. The putative neurotransmitters, dopamine and serotonin, and their precursor amino acids, tyrosine and tryptophan, were identified and quantified in the cells.     

Zeolite-Modified Carbon-Paste Electrode as a Selective Voltammetric Sensor for Detection of Tryptophan in Pharmaceutical Preparations

Abstract

Several synthetic zeolites such as mazzite, mordenite, zeolite L, zeolite beta, and MCM-41 were tested as electrode modifiers in voltammetric determination of tryptophan. It was found that addition of zeolite beta to the carbon paste would generate the peak current of Trp because of its catalytic effect. The anodic peak currents were proportional to Trp concentrations in the range of 5.0 × 10^?7 to 5.0 × 10^?3 M. The detection limit was 1.0 × 10^?7 M. The influence of several species, especially other amino acids, were tested. The proposed method was applied successfully to the determination of tryptophan in pharmaceutical formulations.     

Electrochemical sensor for amino acids and albumin based on composites containing carbon nanotubes and copper microparticles

This work reports on the analytical performance of composites obtained by dispersing copper microparticles and multi-wall carbon nanotubes within a mineral oil binder (CNTPE-Cu) for the determination of amino acids and albumin. The strong complexing activity of amino acids towards copper makes possible an important improvement in the sensitivity for the determination of amino acids and albumin. This new electrode permits the highly sensitive amperometric detection of amino acids, even the non-electroactive ones, at very low potentials (0.000 V) and physiological pH (phosphate buffer solution pH 7.40). The response of the electrode is highly dependent on the amount of copper, demonstrating the crucial role of the metal in the analytical performance of the sensor. The best analytical performance is obtained for the electrode containing 6.0% (w/w) copper. The resulting sensor shows a fast response (7 s) and a sensitivity that depends on the nature of the amino acid. The electrode surface demonstrates an excellent resistance to surface fouling, with R.S.D. of 4% for the sensitivities of 10 successive calibration plots. Albumin is determined with CNTPE-Cu using a protocol based on the accumulation of the protein for 10 min at −0.100 V, followed by the square-wave voltammetric analysis. The quantification of albumin concentration in lyophilized control serum gives excellent agreement with the classical spectrophotometric methodology and with the value informed for the supplier.     

Use of 4-chloro-7-nitrobenzofurazane as a derivative reagent for the determination of amino acids by cathodic stripping square wave voltammetry in biological fluids

The rapid and simple reaction of 4-chloro-7-nitrobenzofurazane with amino acids allows the determination of amino acids in urine using cathodic stripping square wave voltammetry. The obtained compounds are adsorbed on a hanging mercury drop electrode for determination by voltammetric methods using phosphate buffer at pH 2.0 as supporting electrolyte. The proposed method allows determination with an error of 5.68%. A limit of detection (3sigma) of 3.64 nM (0.766 ng ml(-1)) and a limit of determination (10sigma) of 12.12 nM (2.55 ng ml(-1)) are obtained for arginine determination. The proposed method has been applied to the determination of amino acids in urine.     

Selective determination of amino acids using flow injection analysis coupled with chemiluminescence detection

The determination of the amino acids proline, histidine, tyrosine, arginine, phenylalanine and tryptophan using flow injection analysis (FIA) with chemiluminescence detection is described. Proline was the only amino acid to exhibit chemiluminescence with the tris(2,2-bipyridyl)ruthenium(III) reaction at pH 10. While, histidine was found to selectively enhance the reaction of luminol with Mn(II) salts in a basic medium. Acidic potassium permanganate chemiluminescence was able to selectively determine tyrosine at pH 6.75. Low pressure separations using a C18 guard column allowed the simultaneous determination of tyrosine and tryptophan or phenylalanine and tryptophan with acidic potassium permanganate and copper(II)-amino acid-hydrogen peroxide chemiluminescence, respectively. Precision for each method was less than 3.9% (R.S.D.) for five replicates of a standard (1×10⁻⁵ M) and the detection limits ranged between 4×10⁻⁹ and 7×10⁻⁶ M. Preliminary investigations revealed that the methodology developed was able to selectively determine the individual amino acids in an equimolar mixture of the 20 naturally occurring amino acids.     

The electrochemical behavior and direct determination of tyrosine at a glassy carbon electrode modified with poly (9-aminoacridine)

9-Aminoacridine was firstly immobilized on the surface of a glassy carbon electrode to form a poly (9-aminoacridine) film modified electrode. The results demonstrated that the modified electrode exhibited a high degree of catalytic activity towards the oxidation of tyrosine and can resolve the interference of tryptophan in the determination of tyrosine. Compared with the bare electrode, the peak current had obviously increased, and the peak potential had shifted in a negative direction. Under the optimum conditions, a linear response to tyrosine was observed in the concentration of 1.0 x 10(-6) -2.8 x 10(-3) M, with a correlation coefficient of 0.9987, and a detection limit (S/N = 3) of 1.0 x 10(-7) M. The modified electrode has been successfully applied to determine the concentration of tyrosine in composite amino acid injections; and it displays excellent repeatability and high sensitivity. The proposed sensor has promising features such as ease of fabrication, good reproducibility, high stability and low cost. And most of all, it has good selectivity.     

Simultaneous and Direct Determination of Tryptophan and Tyrosine at Boron‐Doped Diamond Electrode

A facile method for the simultaneous measurement of tryptophan (Trp) and tyrosine (Tyr) was firstly exploited at unmodified boron‐doped diamond (BDD) electrode. The experimental results indicated that by using differential pulse voltammetry, the oxidative peaks of these two kinds of amino acids could be completely separated at BDD electrode. The peak separation of Trp and Tyr was developed to be 0.64 V when Na₂PO₄/NaOH buffer solution with the optimized pH 11.2 was employed. The detection limit of Trp was obtained to be 1×10^?5 M, while that of Tyr was achieved to be 1×10^?6 M. The present method was also evidenced to be available to the determination of real samples of amino acids.     

A Simple Method for Melatonin Determination in the Presence of High Levels of Tryptophan using an Unmodified Carbon Paste Electrode and Square Wave Anodic Stripping Voltammetry

Electrochemical determination of melatonin in the presence of tryptophan is a challenge because of the coincidence of voltammetric signals of these compounds when executing a voltammetric technique. The new method for selective determination of melatonin based on the square wave anodic stripping voltammetry determination of an electroactive product of melatonin was suggested here. This product is produced by previously applied positive pre-potential to a carbon paste electrode, immersed in the test solution. By this means, the electrochemical signal of melatonin was separated effectively from that of tryptophan, making it possible to determine melatonin in the presence of a high concentration of tryptophan. The effect of important parameters on electrode performance was studied and optimized. The optimum response was obtained at pH=2 and utilizing the pre-potential magnitude of +0.8 V, applied for 10 s. A linear relationship was found between peak current intensity and melatonin concentrations over the range of 5.00×10⁻⁷ to 8.00×10⁻⁵ mol L⁻¹. A detection limit of 8.30×10⁻⁸ mol L⁻¹ was calculated for the method (S/N=3). The selectivity of the method was considerably high, because of the independence of melatonin signal to the presence of higher amounts of some potentially interfering agents such as ascorbic acid, tryptophane glucose, etc. As an analytical application, the proposed sensor was used for the determination of melatonin in pharmaceutical and food samples.     

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.     

Simultaneous determination of tyrosine and tryptophan by mesoporous silica nanoparticles modified carbon paste electrode using H-point standard addition method

A simple, selective and sensitive sensor based on mesoporous silica nanoparticles modified carbon paste electrode (MSNs/CPE) is introduced for electrochemical determination of tyrosine (Tyr) and tryptophan (Trp). Compared with the unmodified electrode and commercial SiO₂ modified electrode (SiO₂/CPE), the oxidation peak current significantly improved for both amino acids. Under optimized experimental conditions, the oxidation peak current of Trp was linear over a concentration range of 5.0 × 10⁻⁸ to 4.0 × 10⁻⁴ M with a detection limit of 3.4 × 10⁻⁸ M. The oxidation peak current of Tyr was linear over a concentration range from 5.0 × 10⁻⁷ to 6.0 × 10⁻⁴ M with a detection limit of 1.5 × 10⁻⁷ M. For simultaneous determination Trp and Tyr, H-point standard addition method was applied to resolve the overlapping of differential pulse voltammetric peaks of Trp and Tyr. The results showed that the method was successfully applied to the simultaneous determination of Trp and Tyr in some synthetic samples. Moreover, the applicability of the method was demonstrated by the recovery tests of Trp and Tyr in artificial urine.     

Preparation of multiwall carbon nanotubes film modified electrode and its application to simultaneous determination of oxidizable amino acids in ion chromatography

A multiwall carbon nanotubes (MWNTs) film modified electrode was prepared and used as an amperometric sensor for the simultaneous determination of oxidizable amino acids including cysteine, tryptophane and tyrosine. The electrochemical behaviors of these amino acids at this modified electrode were studied by cyclic voltammetry (CV). The results indicated that the MWNTs chemically modified electrode (CME) exhibited efficient electrocatalytic activity towards the oxidation of these amino acids with relatively high sensitivity, stability and long-life. Following separation by ion chromatography (IC) with 2.0×10⁻³ mol l⁻¹ citric acid buffer solution (pH 6.5) as eluent, cysteine, tryptophane and tyrosine could be determined by the MWNTs CME successfully. Under the optimal conditions, the detection limits were 7.0×10⁻⁷ mol l⁻¹ for cysteine, 2.0×10⁻⁷ mol l⁻¹ for tryptophane and 3.5×10⁻⁷ mol l⁻¹ for tyrosine at the signal-to-noise of 3, respectively. The method was applied successfully to the determination of these substances in plasma.     

Ultraviolet thermal lensing detection of amino acids

Thermal lensing (TL) permits ultra-sensitive measurements of optical absorption of analytes in very small liquid volumes. We report the construction and use of a TL detector based on pulsed ultraviolet (UV) laser excitation (266 nm). We applied this detector to quantitate amino acids using capillary electrophoresis (CE) as a means of separation. Sixteen individual amino acids are readily detected, but the signal has a complex dependence on intensity caused by the combination of (1) one-photon absorption; (2) two-photon absorption (TPA); and (3) photodestruction of amino acid molecules in the focus of the laser beam. An aqueous solution containing tyrosine, tryptophan, and cysteine is electrophoretically separated and the individual amino acids are detected by UV TL. The estimated limit of detection is 7 microM for tyrosine, 2.5 microM for tryptophan and 33 microM for cysterine, which translates into 0.35 fmol for tyrosine, 0.125 fmol for tryptophan, and 1.65 fmol for cysteine in the 140pL detection volume. It is found that two-photon absorption of water and the formation of color centers in the fused silica walls of the flowcell can contribute a significant, drifting background signal, but this interference can be minimized by selecting an appropriate focus condition and excitation-detection geometry. We suggest that as UV laser sources become available, UV TL may become a method of choice for measuring the concentrations of many analytes in different separation formats in which the volume is highly limited.     

Electrocatalytic oxidation and flow-injection determination of sulfur amino acids on a glassy carbon electrode modified by a nickel(II) polytetrasulfophthalocyanine film

The electrochemical oxidation of sulfur amino acids, i.e., cysteine, cystine, and methionine, is studied on a glassy carbon electrode modified by a film of nickel(II) polytetrasulfophthalocyanine (poly-NiTsPc). Poly-NiTsPc demonstrates a selective mediator activity in the oxidation of sulfur amino acids, depending on the pH of solution. The proper conditions for fabricating a polymer film on the surface of glassy carbon are found and the conditions of registering the maximal electrocatalytic effect on the modified electrode are determined. A procedure is proposed for the voltammetric determination and amperometric detection of cysteine, cystine, and methionine on an electrode coated by a poly-NiTsPc film under the conditions of flow-injection analysis (FIA). The linear relation of the electrocatalytic response of a composite electrode to amino acid concentration is observed to the level n × 10^?6 M in the static mode and n × 10^?9 M under FIA conditions.     

Turbulent hydrodynamic voltammetry: Part III. Analytical investigations with a turbulent voltammetric cell and applications to amino acid analysis

The analytical applicability of a turbulent voltammetric cell in a flowing stream has been studied. A silicone rubber-based graphite electrode is used. Under optimal conditions, the distortion of the signal in the cell as well as the fluctuations of the signal cause a relative standard deviation of 1% in the results. An examination of the amino acids which occur in protein hydrolyzates is reported. Proline, histidine and arginine can be determined with standard deviations of about 4%; the standard deviations range from 10% to 16% for the other amino acids, except threonine, serine and tyrosine which cannot be determined.     

Amperometric determination of underivatized amino acids at a nickel-modified gold electrode by anion-exchange chromatography

A nickel-based composite electrode obtained by anodic electrodeposition of nickel (III) oxyhydroxide film on the gold electrode substrate was characterized as an amperometric sensor and successfully applied to the determination of underivatised amino acids in flow-through systems. The electrodeposition of nickel oxyhydroxide films was obtained by cycling a gold electrode between 0.0 V and +1.0 V vs. a saturated calomel electrode in a 80 microM Ni2+ solution buffered at pH 10 with NaHCO3/Na2CO3. The resulting Au-Ni composite electrode exhibits good stability in alkaline medium and can be used as an amperometric sensor of underivatised amino acids at a fixed applied potential (+0.55 V vs. Ag/AgCl). The detection limits (S/N=3) for all investigated compounds ranged between 5 and 30 pmol injected, while the linear ranges spanned over two or three orders of magnitude. The contents of several free amino acids in two sample cheeses from different brands were evaluated by calibration graphs.     

Electrocatalytic Oxidation of Cysteine and Cystine at a Carbon-Paste Electrode Modified with Ruthenium(IV) Oxide

The electrocatalytic activity of ruthenium(IV) oxide incorporated into a carbon-paste electrode was studied in the oxidation of cysteine and cystine. The oxidation potentials of the amino acids decreased and the current peaks of their oxidation increased at a modified electrode as compared to an unmodified one. Procedures for the voltammetric determination of cysteine and cystine with the use of electrodes chemically modified with ruthenium(IV) oxide were proposed.     

Effect of surface modification of indium tin oxide by nanoparticles on the electrochemical determination of tryptophan

The effect of surface modification of indium tin oxide (ITO) by multi wall carbon nanotube (MWNT) and gold nanoparticles attached multi wall carbon nanotube (AuNP-MWNT) has been studied to determine tryptophan, an important and essential amino acid for humans and herbivores. A detailed comparison has been made among the voltammetric response of bare ITO, MWNT/ITO and AuNP-MWNT/ITO in respects of several essential analytical parameters viz. sensitivity, detection limit, peak current and peak potential of tryptophan. The AuNP-MWNT/ITO exhibited a well defined anodic peak at pH 7.2 at a potential of ∼669 mV for the oxidation of tryptophan as compared to 760 mV at MWNT/ITO electrode. Under optimum conditions linear calibration curve was obtained over tryptophan concentration range 0.5-90.0 μM in phosphate buffer solution of pH 7.2 with detection limit and sensitivity of 0.025 μM and 0.12 μA μM⁻¹, respectively. The oxidation of tryptophan occurred in a pH dependent, 2e⁻ and 2H⁺ process and the electrode reaction followed adsorption controlled pathway. The method has been found selective and successfully implemented for the determination of tryptophan in human urine and plasma samples using standard addition method. The electrode exhibited an efficient catalytic response with good reproducibility and stability.