Comparative Study of Fluorometric and Electrochemical Detection of Catecholamine,Serotonin, and Metabolites in Rat Brain by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatograph connected to a newly designed fluorometer was used for the simultaneous determination of norepinephrine, dopamine, serotonin, 3-methoxy-4-hydroxyphenylglycol (MHPG), homovanillic acid, and 5-hydroxyindoleacetic acid by measuring native fluorescence. These amines and metabolites extracted from the frontal cortex of a rat were purified by adsorption on alumina and an extraction with ethyl acetate and separated in the chromatograph with an ODS column. The detection limits ranged from 0.5 to 3 pmoles. Compared to the electrochemical (EC) detection at an electrode potential of 0.8 V, the sensitivity was almost the same or somewhat lower, but MHPG was highly fluorescent. In the fluorometry, large front peaks observed in the EC detection did not appear, and interfering peaks were not observed.     

Mutual Binary Separations of Zinc,Cadmium and Mercury by Extraction with Tribenzylamine from Aqueous Bromide and Iodide Solutions

Abstract

The extraction of zinc, cadmium and mercury(II) with a 0. 1 M solution of tribenzylamine in chloroform was studied at varying concentrations of hydrobromic and hydroiodic acid solutions. The extractions of halo complexes of these elements are in accordance with the stability of their tetrahalo complexes in aqueous solution. Using the data mutual binary separations of zinc, cadmium and mercury with high separation coefficients are proposed.     

Retention Characteristics of Co+3, Fe+3, and Cu+2 4-(2-Pyridylazo)Resorcinol (PAR) Complexes on C-18 and Amino Silica Packings

Abstract

Chloroform extraction of Co-PAR, Fe-PAR, and Cu-PAR complexes in a pH 6.5 phosphate buffer implied the first two species were primarily monoanions, but the latter was a dianion. Examination of the literature and retention data of these complexes on C-18 and amino columns confirmed the more anionic nature of Cu-PAR. The Co⁺³ and Cu⁺² complexes were slightly resolved from each other, but the Fe⁺³ complex was retained longer on the C-18 silica. In contrast, the Co⁺³ and Fe⁺³ complexes were not resolved, but the Cu⁺² complex was well-retained on the weak anion exchange amino silica column. Use of short amino and C-18 columns in series provided a good separation of all three complexes. Detection of the metal complexes at 546 nm instead of 254 nm avoided interference by PAR and good detection limits were still maintained.     

Determination of Dirithromycin,LY281389 and Other Macrolide Antibiotics by HPLC with Electrochemical Detection

Abstract

Sensitive and rapid assays have been developed for the determination of the macrolide antibotics dirithromycin, erythromycylamine, and LY281389 in plasma. The methods utilize dichloromethane extraction of alkalinized plasma and isocratic reverse phase HPLC with electrochemical detection. The lower limit of detection is 10 ng/ml. Calibration curves are linear and highly reproducible over the range of 20–500 ng/ml. Precision of the calibration curves is very good having relative standard deviations of 5% or less over the dynamic range. These methods can be used for other macrolide antibiotics with minor modifications to the mobile phase. The electrochemical response of various macrolides was found to be dependent upon the functionality at C-9 of the macrolide ring.     

Direct Atomic Absorption Spectrophotometric Analysis of Anion Complexes of Platinum and Gold After their Concentration and Separation from Aqueous Solutions by Anion Exchange Chromatography

Abstract

A strongly basic anion exchange chromatography column (Dowex 2X-8) is utilized for the concentration and separation of platinum and gold anion complexes from aqueous solutions at pH 6. Atomic absorption spectrophotometry has shown to be a very sensitive and selective detection system for the determination of the metallic species. When the column effluent is fed directly to the nebulizer of the atomic absorption instrument detection limits, using a 10-ml sample, are 0.4 μg for Pt and 0.03 μg for Au. These detection limits can be proportionately improved by passing larger sample volumes through the column. A linear relationship between signal and concentration is readily achieved using the procedure.     

Microbore Liquid Chromatography For Analysis of Plasma Pindolol Concentrations

Abstract

Isocratic high-performance liquid chromatographic assay of pindolol with use of a microbore column was developed. the sample preparation involves extraction of alkalized plasma into ether and back extraction into 0.05 N H₂SO₄. Metoprolol was used as the internal standard. Chromatographic separation is performed on a microbore C18 (5 μm) column using acetonitrile-disodium hydrogenphosphate buffer (37:63) containing 20 mM sodium dodecyl sulfate as the mobile phase. the detection is achieved by using a fluorescence detector operated at the excitation and emission wavelengths of 260 and 310 nm, respectively. Acceptable reproducibility and accuracy data are presented over the concentration range normally encountered in human plasma samples. the lower detection limit is 2.5 ng/ml. This sensitivity has been found to be adequate for routine analysis of pindolol in human plasma samples, making the method applicable to pharmacokinetic studies and clinical trials.     

Routine Determination of a New 1–4 Dihydropyridine,Oxodipine, in Human Plasma by High Performance Liquid Chromatography with Electrochemical Detection

Abstract

A rapid, specific and reproducible high-performance liquid chromatographic routine assay with electrochemical detection was developed for the determination of Oxodipine in human plasma.

After extraction at alkaline pH by cyclohexane, Oxodipine and its internal standard were chromatographied on a reversed-phase column.

Calibration curves were linear over a concentration range of 1–50 ng/ml with relative errors within-day or between-day not exceeding 8% at any level.

The limit of detection was 30 pg injected based on a signal-to- noise ratio of 7. However, the reliable limit of quantification was 1 ng/ml using 1 ml of human plasma.

A dual-electrode coulometric detector was operated in a screening mode of oxidation, providing a greater specificity and reducing background noise.

This method allowed the complete follow-up of clinical pharmacokinetic studies and drug monitoring in patients.     

Determination of Doxorubicin,Daunorubicin and some of their Metabolites in Mouse Plasma by High-Performance Reversed-Phase Liquid Chromatography with Amperometric Detection

Abstract

A selective and sensitive reversed-phase liquid chromatographic method with electrochemical detection for the analysis of doxorubicin, daunorubicin and some of their metabolites in plasma is reported. A mobile phase consisting of acetonitrile-phosphate buffer solution-tetrahydrofuran (25–71,5–3,5) flowing at 1 ml/min through a Lichrocart RP 18 column was employed. The influence of various parameters on the separation (solvent composition, pH, tetrahydrofuran content) has been examined. An extraction of anthracyclines from plasma was performed using chloroform-ethanol mixture (4: 1) with high extraction efficiency; reproducible results were attained by working with a 1 M phosphate buffer which ensured a real buffering of the plasma samples. The sensitivity of amperometric detection makes this method suitable for analyzing small amounts of the parent drugs and their metabolites. The precision was better than 4% in the range 0.2 to 5 μg/ml plasma.     

Determination of Exogenously Administered Dopamine in Infant Plasma Using Liquid Chromatography with Electrochemical Detection

Abstract

A simple, rapid liquid chromatography method is described for the measurement of exogenously administered dopamine in infant plasma, using electrochemical detection and 3,4 dihydroxybenzylamine hydrobromide as the internal standard (IS).

The mobile phase consisted of a monochloroacetate buffer (pH=3.0) with sodium octyl sulfate as the ion-pairing agent, and 2.5% acetonitrile. A biophase II^R reverse phase column was used, and the electrochemical detector was set at +650 mV versus Ag/AgCl. The retention times of IS and dopamine were 3.3 and 6.5 minutes, respectively. The recovery of dopamine following an alumina extraction procedure ranged from 75 to 80%. The inter- and intra-day coefficients of variation using this method were less than 10% and less than 5%, respectively. The major advantages of this method include use of a very small volume of plasma sample (20 μl) and ability to determine very wide ranges of plasma concentrations of dopamine following its administration intravenously. The method was used to measure the plasma concentrations of dopamine in two newborn infants to demonstrate its clinical application.     

Chromatographic retention of molybdenum,titanium and uranium complexes for removal of some interference in inductively-coupled plasma mass spectrometry

Complexes of molybdenum(CI) or titanium(IV) with N-mehylfurohydroxamic acid (N-MFHA) are retained on a column packed with polystyrene/divinylbenzene. At the pH values chosen, copper , zinc and cadmium are washed rapidly through the column and are detected by inductively-coupled plasma mass spectrometry (i.c.p./m.s.) without interference from metal oxide ions of titanium or molybdenum. Detections limits are 1 to 2 μg 1^?1, and analyte recoveries are essentially 100%. the resin capacity for the titanium and molybdenum complexes is sufficient for several hundred injections and the complexes can be radily washed from the column. Uranium(IV) also forms a stable complex with N-MFHA, and ionizatin interference caused by excess of uranium can be avoided by chromatographic removal of the uranium complex. Various other potentially interfering elements with aqueous oxidations states of ⁺4 or higher (e.g., Sn, W, Hf or Zr) could also be separated by this technique.     

Quantitative Analysts of 5-Hydroxytryptamtne in Biological Material by High Perfornance Liquid Chromatography and Electrochmical Detection

Abstract

A quantitative method for the analysis of 5-hydroxytryptamine in biological material is described. The method is based on high performance liquid chromatography (HPLC) with electrochemical detection. A simple purification on a weakly acidic ion exchange resin prior to the analysis gives quite clean samples and permits concentration of diluted samples. The chromatographic separation is performed on a reverse phase column with organic modifier added to an aqueous eluent. With this analytical system 25 pg of 5-hydroxytryptamine can be detected.     

A Multicolumn Ion Chromatographic Determination of Nitrate and Sulfate in Waters Containing Humic Substances

Abstract

A three-column ion chromatographic system for the removal of humic substances from natural waters, and subsequent on-line concentration and determination of nitrate and sulfate using non-suppressed ion chromatography is presented. Humic substances are removed using disposable adsorption columns packed with chemically bonded amine silica material. The sample is directly transfered to an ion exchange column where the anions are concentrated ca 10 times. After reversing the flow, the ions are transferred to a third column where they are separated and quantified. The detection limit is less than 1 mg L^?1 of nitrate or sulfate in water containing 45mgL^?1 of humic acid.     

Ultrasound-assisted analyte extraction for the determination of sulfate and elemental sulfur in zinc sulfide by different liquid chromatography techniques

The speciation and determination of sulfate (SO4(2-)) and elemental sulfur (S degree) in zinc sulfide (ZnS) using ion-chromatography (IC) and reversed-phase liquid chromatography (RPLC) respectively is described. Three sample pretreatment approaches were employed with the aim of determining sulfate: (i) conventional water extraction of the analyte; (ii) solid-liquid aqueous extraction with an ultrasonic probe; and (iii) elimination of the zinc sulfide matrix via ion-exchange dissolution (IED). The separation of sulfate was carried out by an anion-exchange column (IonPac AS17), followed by suppressed conductivity detection. Elemental sulfur was extracted ultrasonically from the acid treated sample solution into chloroform and separated on a reversed phase HPLC column equipped with a diode array detector (DAD) at 264 nm. The achievable solid detection limits for sulfate and sulfur were 35 and 10 microg g(-1) respectively.     

Synergic extraction of zinc, cadmium and lead with hexafluoroacetylacetone and di-n-butylsulphoxide and tri-n-butyl phosphate, and the gas chromatography of the zinc adduct

The synergic solvent extraction of zinc(II), cadmium(II), and lead(II) with 1,1,1,5,5,5-hexafluoro-2,4-pentanedione, H(HFA), and tri-n-butyl phosphate (TBP) or di-n-butylsulphoxide (DBSO) as neutral donors, into cyclohexane has been investigated. Quantitative extraction occurs at pH 4.5-6.0 in extraction times of 10-30 min, depending on the metal species. The optimum pH, equilibration time, stoichiometry and stability of the extracted species, as well as the effect of fluorinated beta-diketone concentration, metal concentration and neutral donor concentration on the extraction are reported. The extracted species was found to be M(HFA)(2).2DBSO or M(HFA)(2).2TBP by mass-action studies. Thermogravimetric analysis of the complexes is reported. The gas chromatographic behaviour of the ternary complexes of the three metals has also been studied. A calibration plot of peak area vs. the amount of zinc injected was linear over the range 40-900 ng of zinc for the Zn(HFA)(2). 2DBSO species; the cadmium and lead species apparently decomposed on the column and useful chromatographic peaks were not observed. The calibration plot for zinc was determined on the basis of the averages of 3-5 replicate determinations for 14 different concentrations over the range stated. The average relative standard deviation was 2.9%.     

Off-Line and On-Line Preconcentration Techniques for the Determination of Phenylureas in Freshwaters

Abstract

Phenylurea herbicides are analysed by reversed-phase liquid chromatography using UV detection at 244 nm after a concentration step in order to determine ppb or sub-ppb levels in drinking and river waters. With an average UV detection limit of 5 ng, a 500 ml sample volume is necessary to reach the 10 ppt level for spiked LC grade water samples and enables easy determination of concentrations below the ppb level for river water samples. Off-line and on-line methods are compared for the concentration step. Off-line concentration consists in a liquid sorption on n-octadecyl silica (C18) and elution by a suitable organic solvent. Polar phenylureas have low retention volumes on C18 silica and consequently the length of the concentration column has to be 10 cm to concentrate them at the ppb level from 100 ml of water and longer for lower levels of detection. Nevertheless, we show that increasing the size of the concentration column does not improve the limits of detection because of the numerous interferences also concentrated when percolating high volumes of water. On-line technology can be used only with short precolumns and requires a sorbent with a great retention for phenylureas. The copolymer-based PRP-1 is found to be an excellent sorbent and it is then possible to apply on-line precolumn technology with preconcentration through two precolumns (10 × 21 mm ID) in series, the first one being packed with C18 silica and the other with the PRP-1 polymer. Interfering compounds are then trapped onto the first precolumn acting as a filter and common phenylurea-breakthrough volumes on the PRP-1 precolumn are higher than 500 ml. Knowing the amounts preconcentrated on both precolumns and using UV and electrochemical detection help the identification of phenylureas in river water.     

Assay of Yohimbine in Human Plasma Using High Performance Liquid Chromatography with Electrochemical Detection

Abstract

Yohimbine is a selective α₂ adrenoreceptor antagonist used in the study of α₂ adrenoreceptors in man. In order to better improve administration regimens for the study of yohimbine in man, we have developed an assay for the determination of yohimbine in plasma utilizing reverse phase high performance liquid chromatography with electrochemical detection. Using a C₁₈ column and a methanol:acetate (60:40) mobile phase, we detected yohimbine in plasma following a simple chloroform extraction. Reserpiline was used as an internal standard. The assay was linear over a concentration range of 50–250 ng/ml in spiked plasma and had a lower limit of sensitivity of 10 ng/ml. It was used to detect yohimbine in plasma sampled from 4 volunteers during an infusion of the alkaloid.     

Quantitative separation of zinc traces from cadmium matrices by solid-phase extraction with polyurethane foam

A system for separation of zinc traces from large amounts of cadmium is proposed in this paper. It is based on the solid-phase extraction of the zinc in the form of thiocyanate complexes by the polyurethane foam. The following parameters were studied: effect of pH and of the thiocyanate concentration on the zinc extraction, shaking time required for quantitative extraction, amount of PU foam necessary for complete extraction, conditions for the separation of zinc from cadmium, influence of other cations and anions on the zinc sorption by PU foam, and required conditions for back extraction of zinc from the PU foam. The results show that zinc traces can be separated from large amounts of cadmium at pH 3.0±0.50, with the range of thiocyanate concentration from 0.15 to 0.20 mol l⁻¹, and the shaking time of 5 min. The back extraction of zinc can be done by shaking it with water for 10 min. Calcium, barium, strontium, magnesium, aluminum, nickel and iron(II) are efficiently separated. Iron(III), copper(II) and cobalt(II) are extracted simultaneously with zinc, but the iron reduction with ascorbic acid and the use of citrate to mask copper(II) and cobalt(II) increase the selectivity of the zinc extraction. The anions nitrate, chloride, sulfate, acetate, thiosulphate, tartarate, oxalate, fluoride, citrate, and carbonate do not affect the zinc extraction. Phosphate and EDTA must be absent. The method proposed was applied to determine zinc in cadmium salts using 4-(2-pyridylazo)-resorcinol (PAR) as a spectrophotometric reagent. The result achieved did not show significant difference in the accuracy and precision (95% confidence level) with those obtained by ICP–AES analysis.     

Methoxy-isoporphyrins of water-soluble porphyrins: synthesis,characterization and electrochemical properties

Abstract

Methoxy-isoporphyrins of zinc [5,10,15,20-tetrakis(4-sulfonatophenyl)]porphyrin, ZnTSPP (1a) and zinc [5,10,15,20-tetrakis(4-carboxyphenyl)]porphyrin, ZnTCPP (1b) have been synthesized and characterized using standard spectroscopic techniques (Uv-visible, ¹H NMR) , ESI-mass spectrometry and powder X-ray diffraction studies. The isoporphyrins [5-(methoxy)-5,10,15,20-tetrakis(4-sulfonatophenyl)-5H,15H-porphinato]zinc(II) (2a) and [5-(methoxy)-5,10,15,20-tetrakis(4-carboxyphenyl)-5H,21H-porphinato]zinc(II) (2b) are formed due to nucleophilic attack of the methanol to the zinc porphyrin dication. Ceric ammonium nitrate (CAN) was used to oxidize zinc porphyrin and to form zinc porphyrin dication. The electronic spectra of the isoporphyrin complexes 2a and 2b exhibit an intense peak at near IR region . Electrochemical measurements of the synthesized isoporphyrins showed a typical irreversible reduction peak at lower potential. S-containing nucleophiles, which work as reducing agents, convert the zinc isoporphyrins to their parent porphyrins, which supports the electrochemical observations. Their structural properties have been studied using powder X-ray diffraction. The luminescence properties of isoporphyrins were compared with the parent zinc porphyrins.     

Preconcentration of trace metal ions by combined complexation-anion exchange : Part 2. Cobalt,zinc, cadmium and lead with 8-hydroxyquinoline-5-sulfonic acid [1]

The ligand, 8-hydroxyquinoline-5-sulfonic acid, forms anionic complexes with cobalt(II), zinc(II), cadmium(II), and lead(II), each resulting complex showing a high affinity for anion-exchange resins. The effect of pH, ligand/metal ratio, volume, and concentration on percent retention of the anionic complexes by an anion-exchange resin are reported. At optimum conditions, all four metals are quantitatively retained by the column. Zinc, cadmium and lead(II) ions are completely eluted with 11 ml or less of 2 M HN0₃; cobalt(II) is totally removed by 12 M HCl and 2 M HNO₃. Concentration enhancements of 100-fold are easily achieved. All four anionic complexes can be left on the column for 7 days and still be quantitatively (99%) recovered. A ligand-loaded resin column can also remove all four metals quantitatively. Distribution coefficients for the metal complexes and their ligand/metal ratios were determined by using batch methods that may also serve as the isolation procedure.     

Liquid Chromatographic Analysis of Tryptophan and Serotonin Metabolites. Comparison of UV,Electrochemical and Spectrofluorimetric Detection

Abstract

Tryptophan and five of its indolic metabolites have been separated by reversed-phase high performance liquid chromatography. The isocratic chromatographic system consisted of a column (20 × 0.46 cm i.d.) packed with RSil C18 HL5 and a methanol/NaClO₄ O.2 M-HClO₄ (pH 1.4) mobile phase.

Three detection methods were tested : UV detection, fluorometric detection and electrochemical detection.

The limits of detection were found to be 4 picomoles (serotonin, 5 HTrp) and 20 pmoles (Trp, 5 HIAA, IAA, N-acetyl Trp) in the case of UV detection; 1 pmole (serotonin), 4 pmoles (5H-Trp) and 10 pmoles (5 HIAA, Trp, IAA, N-acetyl Trp) for fluorometric detection; and 1 pmole for electrochemical detection.

Analysis of human plasma were carried out using the above three detection methods to compare their relative specificity.