Determination of total homocysteine in human serum by capillary gas chromatography with sulfur-specific detection by double focusing ICP-MS

A selective and sensitive method for determination of total homocysteine (Hcy) in human serum, by gas chromatography coupled to ICP–MS(HR), has been developed. After reduction of the sample with sodium borohydride the liberated Hcy and other aminothiols, such as cysteine (Cys) and methionine (Met), were converted to their N-trifluoroacetyl (TFA)-O-isopropyl derivatives and these were injected into a gas chromatograph equipped with an HP-5 capillary column. Detection was carried out by means of a double-focusing inductively coupled plasma mass spectrometer (DF-ICP–MS) monitoring ³²S at m/m (resolving power)=3000. The transfer line used to transport the analytes from the GC column to the ICP–MS had previously been developed in our laboratory. The different parameters affecting the derivatisation process were optimised, as were the instrumental operating conditions. This optimised GC–ICP–MS(HR) method was successfully applied to the determination of total homocysteine in human serum—values obtained were in agreement with data reported in the literature. Quantitative recoveries and good precision were obtained for spiked human serum, demonstrating the suitability of the method for quantitative determination of total homocysteine in serum.     

Electrocatalytic detection of thiols using an edge plane pyrolytic graphite electrode

The first example of using an edge plane pyrolytic graphite electrode in electroanalysis is reported as the determination of homocysteine, N-acetylcysteine, cysteine and glutathione is studied. The response of the electrode in the direct oxidation of thiol moieties is explored and found to be electrocatalytic producing a reduction in the overpotential while having enhanced signal-to-noise characteristics compared to glassy carbon and basal plane pyrolytic graphite electrodes. The effectiveness of the methodology is examined in the determination of cysteine species in a growth tissue media that contains a high number of common biological interferences. The advantageous properties of this electrode for thiol determination lie in its excellent catalytic activity, sensitivity and simplicity.     

Indirect Simultaneous Kinetic Determination of L‐Cysteine and Homocysteine by ANNs

Abstract

Simultaneous determination of cysteine and homocysteine in binary mixtures was performed by application of neural networks on the spectral kinetic data. This method is based on the complexation of bivalent iron with 2,2′–bipyridin (bipy). Iron(III) is quantitatively reduced to iron(II) with cysteine and homocysteine in the presence of 2,2′–bipyridin producing iron(II)–bipy complex (λmax=522 nm), and it can be used as a visible spectrophotometric signal for indirect simultaneous determination of the cysteine and homocysteine concentrations. On the basis of the difference in the rate between the two reactions, these two amino acids can be determined simultaneously using principal component‐artificial neural networks (PC‐ANN). The parameters controlling behavior of the system were investigated and optimum conditions selected. Determinations were made over the concentration range 0.10–5.50 µg · mL^?1 of cysteine and 0.1–5.00 µg · mL^?1 of homocysteine. Applying this method satisfactorily to simultaneous determination of these amino acids with total relative standard error less than 5% validated the proposed method.     

Electrochemical studies of homocysteine and homocystine at mercury electrodes

The behaviour of homocysteine and cysteine at mercury electrodes is compared. The one-electron oxidation associated with thiols is shown to be the same for both compounds in acidic phosphate buffer, giving rise to an adsorbed thiol—mercury complex, (RS)₂Hg, at the electrode surface. Formation of this complex is utilized in the cathodic stripping voltammetric determination of homocysteine; the detection limit is 10^?9 M after a deposition time of 90 s at a hanging mercury drop electrode. The similar E_{$\text{1}\text{2}$} values for homocysteine and cysteine mean that prior separation is needed for their individual determination. Amperometric detection with a mercury-coated goal electrode after separation by cation-exchange liquid chromatography provides a method for the simultaneous determination of both compounds. Reduction of homocystine at the mercury electrode is also compared to that of cystine. The more negative reduction potential, and the maximum observed for homocystine on d.c. polarograms, which is not seen for cystine, is attributable to different reaction kinetics at the mercury electrode; the products of both the 2-electron reductions are the corresponding thiol-containing amino acids.     

Determination of different species of homocysteine in human plasma by high-performance liquid chromatography with ultraviolet detection

This assay measures reduced, free oxidized, protein-bound, and total homocysteine in human plasma. Oxidized species of homocysteine are converted to reduced form by sodium borohydride, and, after precolumn derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate, homocysteine 2-S-quinolinium derivative is separated from those of other plasma thiol derivatives, and quantitated by ion-paired reversed-phase high-performance liquid chromatography with ultraviolet detection. The reduced homocysteine sulfhydryl groups are trapped with minimal oxidation by derivatizing blood samples at the time of collection. With the use of this precise and sensitive HPLC method utilizing popular ultraviolet detection, homocysteine in plasma can be detected and quantitated at the level of 0.1 and 0.2 for reduced fraction, and 0.3 and 0.5 nmol/ml for total homocysteine, respectively. The method is applied for determination of different fractions of homocysteine in plasma of apparently healthy men and women.     

Detection of homocysteine by conventional and microchip capillary electrophoresis/electrochemistry

A method based on capillary electrophoresis (CE) with electrochemical (EC) detection for the determination of both total homocysteine (tHcy) and protein-bound homocysteine (pbHcy) in plasma is described. Both end-column and off-column amperometric detection were investigated. Off-column detection resulted in a more sensitive assay for the determination of homocysteine (Hcy). The detection limit for homocysteine was 500 nM using off-column EC detection and the response was linear over the range 1-100 microM. Therefore, this assay is appropriate for the quantification of Hcy over the physiological concentration ranges found in all disease states. Methodologies for the determination of tHcy and pbHcy in human plasma were investigated and optimized and the concentrations of both pbHcy and tHcy in plasma obtained from a healthy individual were determined to be 2.79+/-0.31 nuM (n = 4) and 3.37+/-0.15 microM (n = 3), respectively. The methodology was then transferred to a microchip CE-EC format and Hcy and reduced glutathione (GSH) were detected. Future work will focus on the development of ancillary methodologies to identify the other forms of Hcy in vivo.     

Les possibilités et limitations de l'atomisation électrothermique en spectrom/'etrie d'absorption atomique lors de l'analyse directe des metaux lourds dans l'eau de mer

This work shows the analytical possibilities of an electrothermal atomizer for the direct determination of trace metals in sea-water.The high background signals generated by the matrix perturb in particular volatile elements because of the low decomposition temperature allowed. In the case of cadmium, addition of ascorbic acid to the sample permits the modification of the atomization mechanism and the reduction of the optimum temperature. Under these conditions, absorption peak of the cadmium precedes the background absorption and consequently the analysis is no longer limited by the magnitude of the matrix signal: the determination of cadmium concentrations far below the μg^?1 level is easily possible. Although the direct determination of the other elements should be in principle less disturbed by the background, the analytical performance is poorer than for cadmium. Limits of determination of the order from 0.1 to 1 μg^?1 can be reached for chromium, copper and manganese. Lead and nickel appeared to be the most difficult elements; their direct determination is only possible in polluted coastal or estuarine waters.The injection of the sample as an aerosol into hot graphite tube showed to be well adapted to this kind of investigations. The simultaneous visualization of specific and background signals allows interpretations which until now were impossible with commercially available apparatus.     

Highly selective colorimetric sensor for cysteine and homocysteine based on azo derivatives

A simple colorimetric method for the determination of cysteine and homocysteine has been developed. The reaction of the azo dyes containing an aldehyde group with cysteine or homocysteine afforded very stable derivatives thiazolidines or thiazinanes under neutral pH conditions. The method is selective and sensitive for cysteine and homocysteine detection without the interference of other amino acids. Importantly, the recognition of Cys and Hcy could be observed by naked eyes.     

Direct (without degassing) determination of sulfur-containing compounds in unstable gas condensate by gas chromatography

A procedure has been developed for direct determination of sulfur-containing components in the concentration range 0.0002?C0.7 wt % in samples of unstable gas condensate under a pressure of 8 MPa using a gas chromatograph equipped with a flame photometric detector. The determination of the overall composition of samples (including hydrocarbons and inorganic gases) takes no more than 1 h, i.e., is 5 times more rapid than the standard procedure with preliminary sample degassing. In addition, direct (without degassing) determination results is lower errors (the repeatability for the main components is 0.01?C0.06; n = 10).     

Microdetermination of silicon in blood, serum, urine, and milk using furnace atomic absorption spectrometry

A simple, direct microanalytical method for quantitative determination of silicon in human whole blood, serum, urine, and milk by furnace atomic absorption technique has been developed. The method employs standard additions and combines the inherent specificity and simplicity of atomic absorption analysis with the greatly increased sensitivity possible with a heated graphite tube atomizer for the determination of silicon in microliter samples. The sensitivity of the method is 1.3 ng. The method is suitable for the direct analysis of silicon with no sample preparation other than dilution with deionized water, thereby minimizing contamination due to sample preparation. The relative standard deviation for 10 μl of blood (1:1), serum (1:1), urine (1:7), and milk (1:1) was 3.45% or less.     

Electrochemical determination of homocysteine at a gold nanoparticle-modified electrode

The construction of a colloidal gold-cysteamine-carbon paste electrode, Au_coll-Cyst-CPE, for the electrochemical determination of homocysteine is reported. The improved voltammetric behaviour of homocysteine at Au_coll-Cyst-CPE with respect to that observed at a gold disk electrode is attributed to an enhanced electron transfer kinetics as a consequence of the array distribution of gold nanoparticles immobilized onto the Cyst SAM. Cyclic voltammtery of homocysteine showed an adsorption-controlled current for scan rates between 500 and 5000 mV s⁻¹. The hydrodynamic voltammogram constructed for homocysteine allowed the selection of a potential value of +600 mV, where the background current is negligible, for the amperometric detection of the analyte at the Au_coll-Cyst-CPE. Using a flow rate of 0.8 ml min⁻¹, the R.S.D. value for i_p after 25 repetitive injections of homocysteine was of 4.3%, and one single electrode could be used for more than 15 days without any treatment or regeneration procedure of the modified electrode surface. An HPLC method for the separation and quantification of homocysteine and related thiols, using amperometric detection at the modified electrode has been developed. A mobile phase consisting of 2:98% (v/v) acetonitrile:0.05 mol l⁻¹ buffer solution of pH 2.0, and a detection potential of +0.80 V were selected. Separation with baseline resolution and retention times of 3.00, 3.60, 4.52, 5.71 and 7.79 min were obtained for cysteine, homocysteine, glutathion, penicillamine and N-acetyl-cysteine, respectively. Calibration graphs were constructed for all the separated compounds. Detection limits ranged between 20 nM for cysteine and 120 nM for penilcillamine, with a value for homocysteine of 30 nM. These values compare advantageously with those achieved with previously reported HPLC methods using electrochemical, UV, fluorescence and MS detection modes. The developed method was applied to the determination of cysteine and homocysteine serum samples with good results.     

On-column derivatization with o-phthaldialdehyde for fast determination of homocysteine in human urine

A new assay for urinary homocysteine is described. The assay relies on an on-column derivatization with o-phthaldialdehyde, using a reversed-phase HPLC column and detection/quantification by fluorescence. The analysis time for reduced and total homocysteine, including sample work-up, was 5 and 13 min, respectively. Quantification limit was 25 nM.     

Direct Determination of Aluminum in Archaeological Clays by Laser‐Induced Breakdown Spectroscopy

Abstract

Instrumental techniques that allow the direct analysis of solids with little or no sample preparation are particularly important for the evaluation of samples that are difficult to analyze such as refractory or geological materials. Laser‐induced breakdown spectroscopy (LIBS) is a promising technique for the direct, rapid analysis of elements in solid materials with minimal sample preparation. The main advantages over wet techniques are virtual nondestructiveness and analysis speed. The goal of this work is the direct determination of aluminum of archaeological pieces using laser‐induced breakdown spectroscopy. The corresponding signals of metals were interpolated from calibration graphs of different salts of the metals. The matrix effects from the direct determination of these elements were thoroughly investigated. The potential of this technique for direct quantitative analysis of real archaeological materials (from Department of Ancient Science, University of Zaragoza) was evaluated, and the reproducibility of LIBS spectra from different archaeological samples was measured as a function of the number of laser shots. Finally, the results from LIBS are compared with those obtained by laser ablation inductively coupled plasma mass spectrometry.     

临床样品微量元素分析过程中样品制备技术的进展

对临床样品微量元素分析过程中的样品制备技术的最近进展进行了评述,主要从直接稀释、湿法消解、干式灰化及微量元素形态分析的样品预处理技术等几方面进行了分别介绍。     

Direct determination of lead at sub-ppb levels in secondary coolants of pressurized water reactor by electrothermal atomic absorption spectrometry with multiple injections

The method of incorporating in-situ pre-concentration technique via multiple injections followed by electrothermal atomic absorption spectrometry (ETAAS) is described for the direct determination of lead at sub-ppb levels in secondary coolants of pressurized water reactor (PWR). Prior to ETAAS measurement, water samples were concentrated by means of multiple injections (9×90 ml) and evaporation in a pyrocoated graphite tube. Optimization of the in-situ pre-concentration via multiple injections with ETAAS analysis was investigated. The feasibility of the proposed method was tested for direct determination of lead in secondary coolants of pressurized water reactor. A SRM 1643c certified reference water sample has been used for quality control. The result are in good agreement with the certified value. By the use of 0.81 ml sample volume 0.012 ppb detection limit was obtained.     

Time-resolved laser-induced fluorimetry for screening polycylic aromatic hydrocarbons on solid-phase extraction membranes

The potential of solid-phase extraction (SPE) time-resolved laser-induced fluorimetry (TRLIF) is evaluated for screening polycyclic aromatic hydrocarbons (PAHs) in aqueous samples. Octadecyl membranes are used with the dual purpose of extracting the pollutants from the water sample and serving as the solid substrate for fluorescence detection. Excitation of fluorescence is performed with a Nd:YAG pumped tunable dye laser pumped with a pulsed source for time-resolving spectral interference. Wavelength time matrices (WTMs) and real time-resolved fluorescence spectra are recorded with a pulsed delay generator, a spectrograph and an intensified charge-coupled device (ICCD). In comparison to SPE solid-matrix luminescence (SML) with conventional instrumentation, this approach provides better limits of detection (LOD) and selectivity. The improvement in LOD is of one order of magnitude, reaching the parts-per-trillion level with 10 ml of water sample. The improvement in selectivity allows the direct determination of target compounds in complex samples. The direct determination of benzo[a]pyrene from a spiked river water sample of unknown composition is presented.     

Analytical determination of homocysteine: a review

The determination of homocysteine has gained high interest within the biomedical community over recent years as the molecule is an important biomarker for a wide range of diseases. The current report gives a brief overview into the biological significance of homocysteine and a rigorous account of the existing detection protocols for homocysteine in biological fluids, as well as trying to anticipate possible future trends in the development of rapid, low-cost and sensitive assays for its detection.     

A one-step selective fluorescence turn-on detection of cysteine and homocysteine based on a facile CdTe/CdS quantum dots–phenanthroline system

In this paper, we report a simple, selective, sensitive and low-cost turn-on photoluminescent sensor for cysteine and homocysteine based on the fluorescence recovery of the CdTe/CdS quantum dots (QDs)–phenanthroline (Phen) system. In the presence of Phen, the fluorescence of QDs could be quenched effectively due to the formation of the non-fluorescent complexes between water-soluble thioglycolic acid (TGA)-capped QDs and Phen. Subsequently, upon addition of cysteine and homocysteine, the strong affinity of cysteine and homocysteine to QDs enables Phen to be dissociated from the surface of QDs and to form stable and luminescent complexes with cysteine and homocysteine in solution. Thus, the fluorescence of CdTe/CdS QDs was recovered gradually. A good linear relationship was obtained from 1.0 to 70.0 μM for cysteine and from 1.0 to 90.0 μM for homocysteine, respectively. The detection limits of cysteine and homocysteine were 0.78 and 0.67 μM, respectively. In addition, the method exhibited a high selectivity for cysteine and homocysteine over the other substances, such as amino acids, thiols, proteins, carbohydrates, etc. More importantly, the sensing system can not only achieve quantitative detection of cysteine and homocysteine but also could be applied in semiquantitative cysteine and homocysteine determination by digital visualization. Therefore, as a proof-of-concept, the proposed method has potential application for the selective detection of cysteine and homocysteine in biological fluids.     

Applications of silver nanoparticles capped with different functional groups as the matrix and affinity probes in surface-assisted laser desorption/ionization time-of-flight and atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry for rapid analysis of sulfur drugs and biothiols in human urine

A strategy is presented for the analysis of sulfur drugs and biothiols using silver nanoparticles (AgNPs) capped with different functional groups as the matrix and affinity probes in surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS) and atmospheric pressure-matrix assisted laser desorption/ionization ion trap mass spectrometry (AP-MALDI-ITMS). Biothiols adsorbed on the surface of AgNPs through covalent bonding were subjected to ultraviolet (UV) radiation that enabled desorption and ionization due to the excellent photochemical property of NPs. The proposed method has been successfully applied for the determination of cysteine and homocysteine in human urine samples using an internal standard. The limit of detection (LOD) and limit of quantification (LOQ) for cysteine and homocysteine in urine sample are 7 and 22 nM, respectively, with a relative standard deviation (RSD) of <10%. The advantages of the present method compared with the methods reported in the literature for biothiol analysis are simplicity, rapidity and sensitivity without the need for time-consuming separation and tedious preconcentration processes. Additionally, we also found that the bare AgNPs can be directly used as the matrix in MALDI-TOF MS for the analysis of sulfur drugs without the addition of an extra proton source. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Simultaneous Detection of Homocysteine and Cysteine in the Presence of Ascorbic Acid and Glutathione Using a Nanocarbon Modified Electrode

The simultaneous electrochemical detection of homocysteine and cysteine using an absorbed ortho‐quinone species, catechol, at the nanocarbon modified glassy carbon electrode was achieved via 1,4‐Michael addition reaction. The detection was done in the presence and the absence of each other as well as with both glutathione and ascorbic acid present in order to investigate the selectivity of homocysteine and cysteine. A determination of homocysteine sensitivity is (0.882±0.296) nA nM^?1 with a LOD of ca. 11 nM and cysteine sensitivity is (7.501±0.202) mA µM^?1 with a LOD of ca. 5.0 µM within a range of 0–0.1 mM.