Determination of sulpiride by capillary electrophoresis with end-column electrogenerated chemiluminescence detection

Background: Capillary electrophoresis (CE) with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)(3)(2+)]-electro-generated chemiluminescence (ECL) detection is a promising method for clinical analysis. In this study, a method combining CE with Ru(bpy)(3)(2+) ECL (CE-ECL) detection that can be applied to amine-containing clinical species was developed, and the performance of CE-ECL as a quantitative method for determination of sulpiride in human plasma or urine was evaluated. Methods: Sulpiride was separated by capillary zone electrophoresis in uncoated fused-silica capillaries [510 cm x 25 mum (i.d.)] filled with phosphate buffer (pH 8.0 and a driving voltage of +15 kV, with end-column Ru(bpy)(3)(2+) ECL detection. A platinum disc electrode was used as working electrode. Sulpiride in human plasma or urine samples (100 muL) was extracted by a double-step liquid-liquid extraction procedure, dried under nitrogen at 35 degreesC in a water bath, and reconstituted with 100 muL of filtered water. The extraction solvent was ethyl acetate-dichloromethane (5:1 by volume). Results: Under optimum conditions (pH 8.0 phosphate buffer, injection for 6 s at 10 kV, and +1.2 V as detection potential), separation of sulpiride was accomplished within 4 min. The calibration curve was linear over a concentration range of 0.05-25.0 mumol/L, and the limit of detection was 2.9 x 10(-8) mol/L for sulpiride. Intra- and interday CVs for ECL intensities were <6%. Extraction recoveries of sulpiride were 95.6-101% with CVs of 2.9-6.0%. The method was,clinically validated for patient plasma and urine samples. Conclusions: CE combined with Ru(bpy)(3)(2+) ECL is reproducible, precise, selective, and enables the analysis of sulpiride in human plasma and urine. It thus is of value for rapid and efficient analysis of amine-containing analytes of clinical interest.

Application of inductively coupled plasma mass spectrometry in biological samples analysis

Recent important applications of inductively coupled plasma mass spectrometry in biological samples analysis are reviewed. The sample preparation, sample introduction techniques, interference correction and typical applications are introduced in detail with 154 references.

Chemiluminescent determination of luminol and hydrogen peroxide using hematin immobilized in the bulk of a carbon paste electrode

A new method for immobilization of a chemiluminescent reagent is presented. It is based on immobilizing hematin, a catalyst for luminol reaction, in the bulk of a carbon paste electrode. Bulk-immobilization allows renewal of the surface by simple polishing or cutting to expose anew and fully active surface in the case of fouling or deactivation by other means. By using a hematin-modified carbon paste electrode, the applied potential shifted negatively compared with that of unmodified carbon paste electrode or a glassy carbon electrode. The shift in potential changed the reaction processes and effectively stabilized the chemiluminescent signal during successive measurements. Under this condition, the signal was stable during 3 hours of continuous operation. The log-log plots of the emitted light intensity vs. luminol concentration and hydrogen peroxide concentration were linear over the region 10(-8)-10(-3) mol L-1 with a correlation coefficient of 0.999 and 3.9 x 10(-6)-10(-3) mol L-1 with a correlation coefficient of 0.994, respectively. Application of this method for other chemiluminescent and bioluminescent systems is suggested.

End-column amperometric detection of 5-fluorouracil by capillary zone electrophoresis with a carbon fiber microelectrode

A rapid and sensitive detection method for the determination of 5-fluorouracil(5-FU) in real samples such as human urine and bovine serum albumin (BSA) was described. A carbon fiber microdisk electrode was used to perform end-column amperometric detection in capillary zone electrophoresis. The detection limit was as low as 2.5x10(-7) M and the wider linear range for the concentration was between 5x10(-6) and 1x10(-4) M with a correlation coefficient of 0.995.

Voltammetric study of vitamin K-3 at interdigitated array microelectrodes

Cyclic voltammetry of Vitamin K-3 (V-K3) was measured with Pt disk electrode, platinum interdigitated array (Pt-IDA) and Au-IDA microelectrodes in single and dual modes. The effects of pH, scan rate and collector potential on the current of generator and collector were studied. The collection efficiency of V-K3 at IDA electrodes was measured. The linear ranges for current response as a function of V-K3 concentration were found to he 10 mu M-1 mM (i(g)) and 1 mu M - 1 mM (i(c)) for the generator and collector of the Pt-IDA electrode, respectively. The effects of waiting time, potential difference and pulse electrolysis time in differential pulse voltammetry (DPV) on the peak current of V-K3 were studied to get the optimal condition at 0.1 M Na2HPO4, pH 11.50 and 11.0 for Au-IDA and Pt-IDA, respectively.

Permselectivity of 4-aminophenol, paracetanol and phenacetin on the self-assembled n-alkanethiol monolayer modified gold electrode

The gold electrodes coated by n-alkanethiol with various chain lengths were used to study the permeability of uric acid, ascorbic acid, 4-aminophenol, paracetanol and phenacetin by means of linear sweep voltammetry. The results show that the optimum chain length is n=10. The improvements in the selectivity and the stability of the amperometric detection of these compounds in a flow stream were obtained by n-alkanethiol self assembled monolayers modified electrodes based on their differences in the hydrophobicity and the permeability.

Chromatographic separation and C-13 NMR characterization of aromatic diester-diacids isomers

Reverse phase high performance liquid chromatography (HPLC) was used to separate and quantify aromatic diester-diacids isomers which arise from the opening selectivity of anhydride rings towards methanol. C-13 NMR spectroscopy was a supplementary tool to characterize the isomer structure. It was found that a meta-position attack is slightly preferred in pyromellitic dianhydride (PMDA), while the preferred position of an attack in bridged dianhydrides is determined by the chemical nature (donors or accepters) of the bridged group. The stronger its electron-withdrawing abilities, the lower the probability of a meta-position attack.

ELECTROCATALYTIC OXIDATION AND FLOW DETECTION OF CYSTEINE AT AN AQUOCOBALAMIN ADSORBED GLASSY-CARBON ELECTRODE

A chemically modified electrode (CME) constructed by adsorption of aquocobalamin (VB12a) onto a glassy carbon electrode surface was demonstrated to catalyze the electro-oxidation of cysteine, a sulfhydryl-containing compound. The sulfhydryl oxidation occured at 0.54-0.88 V vs. Ag/AgCl depending on pH value (3.0-10.0). The electrocatalytic behavior of cysteine is elucidated with respect to solution pH, operating potential and other variables as well as the CME preparation conditions. When used as the sensing electrode in flow injection amperometric detection, the CME permitted detection of the compound at 0.8 V. The detection limit was 1.7 pmol. The linear response range went up to 1.16 nmol. The stability of the CME was shown by RSD (4.2%) over 10 repeated injections.

CHEMICAL-IONIZATION MASS-SPECTROMETRY OF SOME NITRO-CONTAINING BIFUNCTIONAL AROMATIC-COMPOUNDS - LOCATION OF PROTONATION SITE

It is found that the nitro substituent of some aromatic bifunctional compounds shows unusual reactivity towards protonation. In the chemical ionization mass spectra of nitrobenzoic acids and their esters and amides, and of nitrophenols and their ethers, protonations on the carboxyl, ester, amide, hydroxyl or alkoxyl groups are highly suppressed by that on the nitro group. As a result, fragmentations based on protonation on these groups unexpectedly become negligible. Ortho effects were observed for all the ortho isomers where the initial protonation on the nitro group is followed by an intramolecular proton transfer reaction, which leads to the expected 'normal' fragmentations. Protonation on the nitro substituent is much more favourable in energy than on any of the other substituents. The interaction of the two substituents through the conjugating benzene ring is found to be responsible for this 'unfair' competitive protonation. The electron-attracting nitro group strongly destabilizes the MH+ ions formed through protonation on the other substituent; although the COR (R = OH, OMe, OEt, NH2) groups are also electron-withdrawing, their effects are weaker than that of NO2; thus protonation on the latter group produces more-stable MH+ ions. On the other hand, an electron-releasing group OR (R = H, Me, Et) stabilizes the nitro-protonated species; the stronger the electron-donating effect of this group the more stable the nitro-protonated ions.

CATALYTIC-OXIDATION AND FLOW DETECTION OF ACETAMINOPHEN AT A DICYANOBIS(1,10-PHENANTHROLINE)IRON(II)-MODIFIED ELECTRODE

Dicyanobis(1,10-phenanthroline)iron(II)-modified glassy carbon electrodes were shown to exhibit an electrocatalytic response for the oxidation of acetaminophen with a decrease of 100 mV in the potential required. It can also inhibit the oxidation of ascor

AMPEROMETRIC DETECTION OF AMINO-ACIDS IN A FLOW-INJECTION SYSTEM WITH A NICKEL(II)-MODIFIED ELECTRODE WITH AN EASTMAN-AQ POLYMER FILM

Amperometic flow measurements were made at +0.55 V (vs. Ag/AgCl) in 0.1 mol l-1 KOH electrolyte with an Ni(II) chemically modified electrode (CME) with an Eastman-AQ polymer film. The use and characteristics of a Ni(II)-containing crystalline and polymer-modified electrode obtained by a double coating step as a detector for amino acids in a flow-injection system using reversed-phase liquid chromatography are described. The detection of these analytes is based on the higher oxidation state of nickel (NiOOH) controlled by the applied potential. The electroanalytical parameters and the detection current for a series of amines and amino acids were investigated. The use of such a CME in the flow-injection technique was found to be suitable in a solution at low pH. The linear range for glycine is 5 X 10(-6)-0.1 mol 1-1 with a detection limit of 1.0 X 10(-6) mol l-1. A 1 X 10(-4) mol 1-1 mixture of serine and tyrosine was also detected after separation on an Nucleosil C18 column.

AMPEROMETRIC DETECTION AT A CARBON-FIBER ARRAY RING GLASSY-CARBON DISK ELECTRODE IN A WALL-JET CELL

A wall-jet cell incorporating a carbon fibre array ring/glassy-carbon disk electrode has been constructed, and characterized by the cyclic voltammetry and flow-injection techniques. The ring (composed of several microdisks) and glassy-carbon disk electrode, can be used separately for different purposes, e.g., detection in solution without a supporting electrolyte, collection/shielding detection with dual-electrode and voltammetric/amperometric detection with series dual-electrode. The electrode shows better collection and shielding effects than usual ring-disk electrode in quiescent solution and the series dual-electrode in a thin-layer flow-through cell. The detection limit at the ring electrode is comparable with that at a conventional-size electrode, and has been used in the mobile phase without a supporting electrolyte, proving to be a promising detector for normal-phase liquid chromatography.

Characterization of the main light-harvesting chlorophyll a/b-protein complex of green alga, Bryopsis corticulans

The main light-harvesting chlorophyll a/b -protein complex (LHC II) has been isolated directly from thylakoid membranes of shiphonous green alga, Bryopsis corticulans Setch. by using two consecutive runs of anion exchange and gel-filtration chromatography. Monomeric and trimeric subcomplexes of LHC 11 were obtained by using sucrose gradient ultracentrifugation. Pigment analysis by reversed-phase high performance liquid chromatography showed that chlorophyll a (Chl a), chlorophyll b (Chl b), neoxanthin, violaxanthin and siphonaxanthin were involved in LHC 11 from B. corticulans. The properties of electronic transition of monomeric LHC II showed similarities to those of trimeric LHC II. Circular dichroism spectroscopy showed that strong intramolecular interaction of excitonic dipoles between Chl a and between Chl b exist in one LHC II apoprotein, while the intermolecular interaction of these dipoles can be intensified in the trimeric structure. The monomer has high efficient energy transfer from Chl b and siphonaxanthin to Chl a similarly to that of the trimer. Our results suggest that in B. corticulans, LHC II monomer has high ordered pigment organization that play effective physiological function as the trimer, and thus it might be also a functional organization existing in thylakoid membrane of B. corticulans.

The complete amino acid sequence of growth hormone and partial amino acid sequence of prolactin and somatolactin from sea perch (Lateolabrax japonicus)

Growth hormone (GH), prolactin (PRL) and somatolactin (SL) were purified simultaneously under alkaline condition (pH 9.0) from pituitary glands of sea perch (Lateolabrax japonicas) by a two-step procedure involving gel filtration on Sephadex G-100 and reverse-phase high-performance liquid chromatography (rpHPLC). At each step of purification, fractions were monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting with chum salmon GH. PRL and SL antisera. The yields of sea perch GH, PRL and SL were 4.2, 1.0 and 0.28 mg/g wet tissue, respectively. The molecular weights of 19,200 and 20,370 Da were estimated by SDS-PAGE for sea perch GH and PRL, respectively. Two forms of sea perch SL were found: one (28,400 Da) is probably glycosylated, while the other one (23,200 Da) is believed to be deglycosylated. GH bioactivity was examined by an in vivo assay. Intraperitoneal injection of sea perch GH at a dose of 0.01 and 0.1 mug/g body weight at 7-day intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. The complete sea-perch GH amino acid sequence of 187 residues was determined by sequencing fragments cleaved by chemicals and enzymes. Alignment of sea-perch GH with those of other fish GHs revealed that sea-perch GH is most similar to advanced marine fish, such as tuna, gilthead sea bream, yellowfin porgy, red sea bream, bonito and yellow tail with 98.4, 96.2%, 95.7%, 95.2%, 94.1% and 91% sequence identity, respectively. Sea-perch GH has low identity to Atlantic cod (76.5%), hardtail (73.3%), flounder (68.4%), chum salmon (66.3%), carp (54%) and blue shark (38%). Partial amino-acid sequences of 127 of sea-perch PRL and the N-terminal of 16 amino-acid sequence of sea-perch SL have been determined. The data show that sea-perch PRL has a slightly higher sequence identity with tilapia PRL( 73.2%) than with chum salmon PRL(70%) in this 127 amino-acid sequence. (C) 2001 Elsevier Science B.V. All rights reserved.

Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 column was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus pluvialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The results showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (> 16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingiensis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a remarkably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%).