Simultaneous determination of aminothiols, ascorbic acid and uric acid in biological samples by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection has been employed for the separation and determination of homocysteine, cysteine, reduced glutathione, ascorbic acid and uric acid. Effects of several important factors such as the acidity and concentration of the running buffer, separation voltage, injection time and detection potential were investigated to acquire the optimum conditions. The detection electrode was a 500 microm diameter platinum disk electrode at a working potential of +1.05 V (vs saturated calomel electrode). The five analytes were well separated within 10 min in a 50 cm long fused silica capillary at a separation voltage of 18 kV in a 100 mm phosphate buffer (pH 7.8). The relation between peak current and analyte concentration was linear over about 3 orders of magnitude with the detection limits (S/N = 3) ranging from 0.83 to 2.58 microm. The proposed method was successfully applied to determine cysteine, reduced glutathione, ascorbic acid and uric acid in human whole blood and rat brain tissues with satisfactory assay results and should find a wide range of bioanalytical applications.     

A validated HPLC method with electrochemical detection for simultaneous assay of 5-aminosalicylic acid and its metabolite in human plasma

A high-performance liquid chromatographic method was developed, validated and applied to the simultaneous determination of 5-aminosalicylic acid (5-ASA) and its acetylated metabolite (acetyl-5-ASA) in human plasma. The method involves liquid-liquid extraction with methanol followed by isocratic reversed-phase chromatography on a Kromasil KR100 C(18) column with electrochemical detection. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma samples. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of 5-ASA, acetyl 5-ASA and internal standard were investigated. Limits' of detection were 5 ng/mL for 5-ASA and 10 ng/mL for acetyl-5-ASA, respectively. The method can be used for supporting therapeutical drug monitoring and pharmacokinetic studies.     

A specific sensitive HPLC method for determination of plasma dopamine

Summary We describe here a sensitive, selective and rapid method to quantitate plasma catecholamines, especially dopamine, using high-performance liquid chromatography with electrochemical detection. This method requires a 10-minute run time and has a threshold for detection of 2 picograms, (10pg/ml).A number of commonly employed mobile phases for catecholamine analysis have been tested and have failed to detect dopamine in biological samples. Neither acetonitrile (3–7%) or methanol, (5–8%) in the mobile phase has produced consistently interpretable data either due to inability to detect or interference from co-eluting substances. Optimal detection was achieved with a mobile phase containing sodium acetate (6.8g), citric acid (5.9g), EDTA (48mg), di-n-butylamine (270l), Na-1-octane sulfate (850mg), methanol (100 ml) (amounts refer to 1 liter aqueous solution) (pH 4.3). The mobile phase was passed through a Waters 5 resolve C₁₈ column using a Waters 590 pump and m460 electrochemical detector and 740 data module, Flow rate was 0.9ml/min. Using this method, normal values in human and swine left ventricular myocardium and human and swine plasma have been established for norepinephrine, epinephrine, and dopamine.     

Exploiting micellar environment for simultaneous electrochemical determination of ascorbic acid and dopamine

A simple and reliable method for simultaneous electrochemical determination of ascorbic acid (AA) and dopamine (DA) is presented in this work. It was based on the use of the cationic surfactant cetylpyridinium chloride (CPC) that enables the separation of the oxidation peaks potential of AA and DA. Cyclic voltammetry (CV) as well as pulse differential voltammetry (PDV) were used in order to verify the voltammetric behaviour in micellar media. In the cationic surfactant CPC, a remarkable electrostatic interaction is established with negatively charged AA, as a consequence, the oxidation peak potential shifted toward less positive potential and the peak current increased. On the other hand, the positively charged DA is repelled from the electrode surface and the oxidation peak potential shifts toward more positive potential in comparison to the bare electrode. Therefore, the common overlapped oxidation peaks of AA and DA can be circumventing by using CPC. Parameter that affects the E_pa and I_pa such as CPC concentration and pH were studied. Under optimised conditions, the method presented a linear response to AA and DA in the concentration range from 5 to 75 μmol L⁻¹ and 10 to 100 μmol L⁻¹, respectively. The proposed method was successfully applied to the simultaneous determination of AA and DA in dopamine hydrochloride injection (DHI) samples spiked with AA.     

A quick method for the simultaneous determination of ascorbic acid and sorbic acid in fruit juices by capillary zone electrophoresis

A method of quickly determining ascorbic acid and sorbic acid by capillary zone electrophoresis with ultraviolet detection was developed. The choice of background electrolyte, wavelength, injection time and applied voltage were discussed. Ascorbic acid and sorbic acid were well separated in 80 mmol L⁻¹ boric acid-5 mmol L⁻¹borax (pH = 8.0) in 5 min at the detecting wavelength of 270 nm. Under the optimum condition, the method has linear ranges of 2.54-352.00 mg L⁻¹ for ascorbic acid and 1.08-336.39 mg L⁻¹ for sorbic acid with the detection limit of 1.70 mg L⁻¹ for ascorbic acid and 0.54 mg L⁻¹ for sorbic acid, respectively. Other organic acids in fruit juices have no effect on the detection. This method is very feasible and simple and can be used to detect ascorbic acid and sorbic acid in fruit juices.     

Determination of ascorbic acid in human plasma with a view to stability using HPLC with UV detection

A method for the measurement of ascorbic acid using HPLC with UV detection and investigation into the protein precipitation techniques with regard to stability and recovery are described. The effectiveness of various protein precipitants was tested. Stability of ascorbic acid samples for analysis was investigated over 10 h. Ascorbic acid samples extracted with metaphosphoric acid were stable on a cooled autosampler (4 degrees C) for at least 10 h (with a decline of 1.8% for ascorbic acid solution and 2.8% for plasma). Perchloric acid as protein precipitant for ascorbic acid was unsuitable (with a decline of 36.0% for ascorbic acid solution and 7.3% for plasma). Analytical performance of this method is satisfactory. The intra- and interassay coefficients of variation were 2.1% (n = 10) and 5.8% (n = 12), respectively. The calibration curve was linear with the tested range of 2.0-250.0 micromol/L. The recovery was 96.1% with CV = 4.8% (n = 6) and the LOD was 3 micromol/L. The preliminary reference ranges of ascorbic acid in a group of blood donors are 50.8 +/- 22.4 micromol/L. This assay is a highly sensitive and reproducible HPLC method for the determination of ascorbic acid in human plasma.     

动力学荧光法测定抗坏血酸

基于在硫酸介质中 ,抗坏血酸能活化钒 ( )催化溴酸钾氧化藏红 T的反应 ,使其荧光猝灭 ,建立了动力学荧光法测定抗坏血酸的新方法。方法的检出限为 5 .8× 1 0 -3 μg/m L,线性范围为 0～ 0 .5 6μg/m L。可用于药品、蔬菜、尿液中抗坏血酸含量的测定     

Printed electrochemical sensor for ascorbic acid determination

Simple, strip-type sensors based on 7,7,8,8-tetracyanoquinodimethane-modified graphite were prepared using screen printing techniques. The electrochemical strips operated at low potentials [50 mV at pH 7.0 or 100 mV at pH 4.8 vs. Ag/AgCl (printed)] and had a sensitivity of 3.5–7.1 μA 1 mmol^?1L-ascorbic acid. Determination of ascorbic acid concentration was achieved in 30 s and required samples of ca. 30 μl. The current output of the electrodes was found to be relatively insensitive to variations in pH over the range 5.0–8.5. Between 15 and 35 °C, the temperature coefficient was 2.7% °C^?1. The printed electrodes were suitable for single determinations but demonstrated adequate stability for periodic re-use. The ascorbic acid concentration in the juice of fresh fruit was determined using the electrochemical printed electrodes and a commercially available enzymatic test kit. Close agreement was observed between the two methods [r=0.9997 (n=12),slope=0.9798]. The limit of detection using the printed sensor for real samples was calculated as 4mg l^?1(22 μM).     

动力学荧光法测定抗坏血酸

基于在pH 10.7的NH3-NH4Cl缓冲溶液中,抗坏血酸能活化氯化血红素酶催化H2O2氧化L-酪氨酸的反应,使其反应速率增大,将时间驱动技术和动力学中斜率法相结合,建立了一种新的测定抗坏血酸的动力学荧光分析方法。在最佳实验条件下,方法的线性范围为0.1~4.8μg/mL,相对标准偏差3.8%,检出限为1.48μg/L。并考察了环境介质和常见物质的干扰情况。方法可用于实际样品的测定。     

A simple capillary electrophoresis with electrochemical detection method for determination of the hydrolysis rate constant of chlorogenic acid

A method based on the kinetics stability study on hydrolysis of chlorogenic acid by capillary zone electrophoresis with electrochemical detection (CE-ED) has been developed in this paper. Both cyclic and hydrodynamic voltammograms of chlorogenic acid and its hydrolysis product caffeic acid have been investigated. The conditions for separation of chlorogenic acid and caffeic acid, such as the buffer pH and concentration, the separation voltage, and the injection time have been optimized. Under the optimum CE running conditions, the effects of reaction temperature and pH values of the hydrolysis solutions on the hydrolysis rate constants were further studied. The hydrolysis rate constants of chlorogenic acid were obtained from the concentration change of hydrolysis during the process of hydrolysis. Based on the fact, a simple and economical method for the determination of the hydrolysis rate constant and activation energy of hydrolysis reaction has been developed.     

Zeolite A functionalized with copper nanoparticles and graphene oxide for simultaneous electrochemical determination of dopamine and ascorbic acid

A novel Cu-zeolite A/graphene modified glassy carbon electrode for the simultaneous electrochemical determination of dopamine (DA) and ascorbic acid (AA) has been described. The Cu-zeolite A/graphene composites were prepared using Cu²⁺ functionalized zeolite A and graphene oxide as the precursor, and subsequently reduced by chemical agents. The composites were characterized by X-ray diffraction, Fourier transform infrared spectra and scanning electron microscopy. Based on the Cu-zeolite A/graphene-modified electrode, the potential difference between the oxidation peaks of DA and AA was over 200 mV, which was adequate for the simultaneous electrochemical determination of DA and AA. Also the proposed Cu-zeolite/graphene-modified electrode showed higher electrocatalytic performance than zeolite/graphene electrode or graphene-modified electrode. The electrocatalytic oxidation currents of DA and AA were linearly related to the corresponding concentration in the range of 1.0 × 10⁻⁷–1.9 × 10⁻⁵ M for DA and 2.0 × 10⁻⁵–2.0 × 10⁻⁴ M for AA. Detection limits (<!-- no-mfc -->S/N<!-- /no-mfc --> = 3) were estimated to be 4.1 × 10⁻⁸ M for DA and 1.1 × 10⁻⁵ M for AA, respectively.     

A highly sensitive HPLC method with automated on-line sample pre-treatment and fluorescence detection for determination of reboxetine in human plasma

A fully automated, rapid and highly sensitive HPLC method with automated sample pre-treatment by column-switching system and fluorescence detection has been developed for the trace quantitative determination of the new antidepressant reboxetine (RBX) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. Paroxetine (PXT) was used as an internal standard. Plasma samples containing both RBX and PXT, after filtration, were derivatized by heating with NBD-F in borate buffer of pH 8 at 70 °C for 30 min. The derivatized plasma samples were injected into the HPLC system where an on-line sample clean up was achieved on the pre-treatment column (Co-sense Shim-pack MAYI-ODS) with a washing mobile phase (acetonitrile:2% acetic acid; 40:60, v/v) at a flow rate of 5 mL min⁻¹ for 1 min. After an automated on-line column switching to the analytical Hypersil phenyl 120A column (250 mm × 4.6 mm, 5 μm), the separation of the derivatized RBX and PXT was performed using a mobile phase consisting of sodium acetate buffer (pH 3.5):tetrahydrofuran:acetonitrile (55:35:10, v/v/v) at a flow rate of 2.0 mL min⁻¹. The eluted derivatives were monitored by a fluorescence detector set at an excitation wavelength of 470 nm and an emission wavelength of 530 nm. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r = 0.9995, n = 5) was found between the peak area ratio of RBX to PXT and RBX concentration in the range of 2-500 ng mL⁻¹, with limits of detection and quantification of 0.5 and 1.7 ng mL⁻¹, respectively. The intra- and inter-day precisions were satisfactory; the relative standard deviations were 2.25 and 3.01% for the intra- and inter-day precisions, respectively. The accuracy of the method proved as the mean recovery values were 100.11 ± 2.24% and 100.99 ± 2.98% for the intra- and inter-day assay runs, respectively. The proposed method involved simple and minimum sample preparation procedure and short run-time (<12 min) and therefore it can be applied to the routine therapeutic monitoring and pharmacokinetic studies of RBX.     

Separation and determination of homovanillic acid and vanillylmandelic acid by capillary electrophoresis with electrochemical detection

A method of separation and determination of homovanillic acid (HVA) and vanillylmandelic acid (VMA) was developed based on capillary zone electrophoresis/amperometric detection with high sensitivity, good resolution and selectivity. In order to achieve complete separation and good response, several factors including pH, buffer concentration, separation voltage, detection potential and the length of separation capillary, were studied in detail. The method has been used to determine both HVA and VMA in human urine. Uric acid (UA) in human urine did not interference with their determination. The limit of detection of the method was 1.3×10⁻⁶ mol/l (1.4 fmol) for HVA and 7.9×10⁻⁷ mol/l (0.87 fmol) for VMA at a signal-to-noise ratio of 3.     

Quantitative determination of terbutaline in human plasma after administration of bambuterol using coupled columns and electrochemical detection

Summary A method is presented for quantitative determination of terbutaline after administration of its prodrug Bambuterol. Terbutaline is extracted from plasma by liquid-solid extraction on small C₁₈-cartridges. The extract is then analysed by coupled column liquid chromatography with amperometric detection. To inhibit the esterase catalyzed hydrolysis of bambuterol to terbutaline an analogue of bambuterol is added to the plasma sampling tubes. The within-day variation on spiked samples were 2.1% and 2.4% at 8.0 and 40.0 nmol/L respectively. The between-day variation on spiked samples (8.0 nmol/L) was 3.6% and on authentic samples 5.9% at the 11 nmol/L level. The absolute recovery was in the range 83–94% for terbutaline and the internal standard. The limit of quantitation was set at 4.0 nmol/L (C_v=3.4%, n=46). Subsidiary of AB Astra.     

New microdialysis-electrochemical device for simultaneous determination of ascorbic acid and 5-hydroxyindole-3-acetic acid in rat striatum

A new device combining microdialysis with electrochemical microsensor was developed. It can be applied to monitor the biomolecules in the brain for biological and pharmaceutical research. In this paper, the device was applied to simultaneously determine ascorbic acid (AA) and 5-hydroxyindole-3-acetic acid (5-HIAA) in rat striatum. The microsensor used for the device was poly (sulphosalicylic acid) microsensor, which exhibited a good electrocatalytic effect on oxidization of AA and 5-HIAA. The oxidation currents measured by differential pulse voltammetry (DPV) were linear for AA in the range of 0.02-1.0 mmol l⁻¹, and for 5-HIAA from 0.5 to 10.0 μmol l⁻¹ (r=0.9998 and 0.9991, respectively). The detection limits were calculated to be 0.01 mmol l⁻¹ for AA and 0.25 μmol l⁻¹for 5-HIAA (S/N=3). Studies also showed that co-existing substances in biological fluids did not interfere with AA and 5-HIAA determination when using this microsensor. Since, the substances in the microdialysate are easily oxidized by air, the microdialysate in this device was under the protection of N₂. It was found that the concentrations of AA and 5-HIAA in rat striatum were 215±5 and 6.21±0.61 μmol l⁻¹ (mean±S.E.M., n=7), respectively with this device under the protection of N₂. In addition, the effect of the nitric oxide donor, sodium nitroprusside (SNP), on 5-HIAA in the rat striatum was investigated. It was found that a high concentration of SNP (1.0 mmol l⁻¹) resulted in a 34% increase in 5-HIAA level.     

A simple electroanalytical methodology for the simultaneous determination of dopamine,serotonin and ascorbic acid using an unmodified edge plane pyrolytic graphite electrode

A simple method using an unmodified edge plane pyrolytic graphite electrode (EPPGE) is reported for the simultaneous determination of dopamine (DA), serotonin (ST) and ascorbic acid (AA). The performance of this electrode is superior to other unmodified carbon-based electrodes and also to many modified electrodes in terms of detection limit, sensitivity and peak separation for determination of DA, ST and AA. Using this method, detection limits of 90 nM, 60 nM and 200 nM were obtained for DA, ST and AA respectively. No electrode fouling is observed during a set of experiments and good sensitivity is obtained for the simultaneous determination of DA, ST and AA. The peaks for the three species are well resolved from each other and the electrode is successfully utilised for their determination in standard and real samples.      

Automated analysis of terbutaline (BricanylR) in human plasma with liquid chromatography and electrochemical detection using column-switching (multidimensional chromatography)

Summary An automated method based on liquid chromatography has been developed for the determination of terbutaline in human plasma in the range of 5–50 pmole·ml^–1. The necessary sensitivity and selectivity was obtained by using electrochemical detection and a microprocessor-controlled column switching system. A combination of three columns was used: a C₈ type for pre-separation, a C₁₈ type for trapping and, for final separation, a strongly acidic ion exchanger. The accuracy of the method was examined by comparison with a method based on gas chromatography — mass spectrometry. The overall precision was ±3.5% and ±2.2% respectively at 5 and 50 pmole·ml^–1. The total absolute recovery for terbutaline and internal standard at the above concentration levels were in the range 85–106%.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Selective determination of catechin, epicatechin and ascorbic acid in human urine using chiral capillary electrophoresis

Chiral CE was successfully applied to the separation and quantification of catechin, epicatechin and ascorbic acid in some commercial drinks and human urine. Analysis involved the separation of analytes in less than 5.0 min at 240 nm with an untreated fused-silica capillary under hydrodynamic injection mode. The running buffer consisted of 50 mM borate buffer with 3 mM beta-CD at pH 8.35. Detection limits for catechin, epicatechin and ascorbic acid were 0.028, 0.011 and 0.004 microg/mL, respectively. Linearity was investigated by selecting the ranges of calibration according to the amount of analytes in urine giving correlation coefficient percent (% r(2)) ranging between 99.4 and 99.6 at 99% confidence level. The maximum urinary excretion of catechin and epicatechin were noted at 2.0 and 4.0 h of the administrated dose. Unchanged catechin, epicatechin and ascorbic acid amounted to about 1.500, 8.696 and 0.003% of the administered dose in the 48.0 h urine collection. The proposed method achieved 99.2% completeness (n = 20) in urine media.     

Solvent dependent dimercaptothiadiazole monolayers on gold electrode for the simultaneous determination of uric acid and ascorbic acid

Dimercaptothiadiazole compound, 2,5-dimercapto-1,3,4-thiadiazole (DMcT) forms ‘thin’ monolayers on Au electrode when it was adsorbed from methanol, ethanol or DMSO solutions while it forms ‘thick’ layers on Au electrode from an aqueous solution under identical experimental conditions. Thick DMcT layers formed from aqueous solution effectively blocks the redox reaction of couple in contrast to thin DMcT monolayers. The monolayer thickness did not vary when structurally related DMcT compounds, 5-methyl-1,3,4-thiadiazole-2-thiol or 5-amino-1,3,4-thiadiazole-2-thiol was adsorbed from aqueous and non-aqueous solutions. This indicates that the presence of two thiol groups in DMcT plays a crucial role in the formation of thick and thin DMcT layers on Au electrode when it was adsorbed from aqueous and non-aqueous solutions. Methanol, ethanol, or DMSO solution of DMcT is considered as strong acid because these solvents are able to deprotonate DMcT into DMcT⁻ and thus thin monolayers formed on Au electrode. The deprotonating ability of these solvents was further verified from the observed absorption spectrum characteristic of DMcT⁻ species. On the other hand, an aqueous solution of DMcT is less acidic due to weak deprotonation of DMcT by water and thus DMcT forms thick layer on Au electrode. Interestingly, thin DMcT monolayers formed from non-aqueous solvents separates the voltammetric signals of uric acid and ascorbic acid while thick DMcT layers formed from aqueous solution fails to separate them.     

Determination of asymmetric and symmetric dimethylarginines in human plasma by HPLC with electrochemical detection

A new HPLC method was developed and validated for the determination of asymmetric and symmetric dimethylarginines and l ‐arginine in human plasma. After SPE and evaporation of the eluate, the samples were derivatised with an o‐phthaldialdehyde reagent containing 3‐mercaptopropionic acid. The derivatives formed were analysed by isocratic RP‐HPLC with electrochemical detection at +320 mV. The mobile phase consisted of 50 mM phosphate buffer (pH 6.1) containing 10% v/v acetonitrile, the flow rate was 1 mL/min. The retention times of all compounds including monomethylarginine (internal standard) were <24 min. The LODs (S/N 3:1) were 0.012 μM for both dimethylarginines and 0.013 μM for l ‐arginine; the linearity of the method was from 0.1 to 20 μM for both dimethylarginines and from 1 to 200 μM for l ‐arginine. Absolute extraction recoveries measured for all analytes ranged from 85 to 88%.