Determination of cimetidine in human plasma by use of coupled-flow injection, solid-phase extraction, and capillary zone electrophoresis

Summary An on-line flow injection-solid-phase extraction-capillary zone electrophoresis (FI-SPE-CZE) method has been developed for determination of cimetidine in human plasma. Sodium dodecylsulfate (SDS) was used as dynamic chemical modifier for elimination of capillary contamination by biological macromolecules. FI on-line preconcentration and cleaning of the analyte by means of a C₁₈ microcolumn was performed automatically and CZE separation was performed consecutively without interruption of the applied voltage and between-run-washing of the capillary. A detection limit of 8 μgL⁻¹ (3×σ) was achieved at a sample throughput of 12h⁻¹. The approach was successfully used for a pharmacokinetic study of cimetidine.     

A novel competitive fluorescence immunoassay for the determination of dibutyl phthalate

A novel, sensitive, and specific competitive fluorescence immunoassay has been developed for the quantitative determination of dibutyl phthalate (DBP) using an antibody-coated plate format. Hapten was synthesized in order to produce polyclonal antibodies against dibutyl phthalate. Polyclonal antisera to dibutyl phthalate were generated in rabbits and used to construct the fluorescence immunoassay for measurement of dibutylphthalate. The assay had a detection limit of about 0.02 μg L⁻¹, a dynamic range of approximately 0.1–300 μg L⁻¹. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique, and the cross-reactivity rates were less than 10%. The study demonstrated that the developed antiserum and fluorescence immunoassay procedure can be used to detect dibutyl phthalate in environmental samples such as tap water, river water, drinking water, and leachate from plastic drinking water bottles.     

Preconcentration technique for manganese by adsorption onto a tantalum wire for tungsten tube atomizer electrothermal atomization atomic absorption spectrometry

A multi-pumping flow system for the spectrophotometric determination of nitrite and nitrate is described. The determination of nitrite is based on the Griess-Ilosvay reaction. Nitrate can be determined after its on-line reduction to nitrite using hydrazine sulphate in alkaline medium. Calibration was linear up to 3 mg NO₂ ⁻ L⁻¹ with a limit of detection (3s_b/S) of 0.013 mg NO₂ ⁻ L⁻¹ an injection throughput of 55 injections h⁻¹ and a repeatability (RSD) of 0.5% for the direct determination of nitrite. Two calibration graphs within the ranges 0.039–7 mg NO₃ ⁻ L⁻¹ and 0.026–5 mg NO₂ ⁻ L⁻¹ were run for the determination of nitrate and nitrite under reducing conditions, respectively. A limit of detection of 0.039 mg NO₃ ⁻ L⁻¹ was obtained. An injection throughput of 27 injections h⁻¹ and an RSD lower than 1.5% were achieved. The method was successfully applied to the determination of nitrite and nitrate in water samples. Correspondence: Víctor Cerdà, Department of Chemistry, University of the Balearic Islands, Carretera de Valldemossa Km7.5, 07122 Palma de Mallorca, Spain     

Automatic determination of phylloquinone in vegetables and fruits using on-line photochemical reduction and fluorescence detection via solid phase extraction and flow injection

A very simple, rapid and highly sensitive flow injection fluorimetric method was developed for the determination of phylloquinone. The assay was based on the on-line reduction of phylloquinone in dodecylsulfate micelles after irradiation with UV light. The micellar medium enhanced the fluorescence and stability of the reduced phylloquinone. Under optimum experimental conditions, the range of application of the technique was between 0.09 and 45.0 μg mL⁻¹ and the detection limit was 0.05 μg mL⁻¹. The sample throughput was 90 injections per hour. The reliability of the method for the routine analysis of phylloquinone in vegetables and fruits is demonstrated. Extractions were made with hexane, and an automated solid phase extraction system was used to purify the sample extracts prior to injection into the flow injection manifold.     

Analysis of ofloxacin in plasma samples by high-performance liquid chromatography

Summary A sensitive HPLC method has been developed for determination of ofloxacin (OFL) in biological fluids. Sample preparation was performed by adding phosphate buffer (pH 7.4, 0.1m) then extraction with trichloromethane. OFL and the internal standard, sarafloxacin (SAR), were separated on a reversed-phase column with aqueous phosphate solution-acetonitrile, 80∶20, as mobile phase. The fluorescence of the column effluent was monitored at λ_ex 338 and λ_em 425 nm. The retention times were 2.66 and 4.24 min for OFL and SAR, respectively, and the detection and quantitation limits were 8 and 15 ng mL⁻¹, respectively. Plots of response against ofloxacin concentration were linear in the range 8 to 2000 ng mL⁻¹. Recovery was 92.9% for OFL.     

Fluorescence enhancement assay for trace iron(II) using Pyr-tempo as a spin label fluorescent probe

Pyrene-tetramethylpiperidinyl (Pyr-Tempo) as a spin label fluorescent probe for iron(II) was synthesized. It exhibited weak fluorescence (λ_exc/λ_em = 346/399 nm) in aqueous solution due to an intramolecular quenching pathway. A method for determination of iron(II) was proposed based on the fluorescence enhancement of the probe in the presence of iron(II) in acidic medium. Under optimum conditions, the fluorescence enhancement of Pyr-Tempo is linearly proportional to the iron(II) concentration range of 6.0 × 10⁻⁸ to 9.6 × 10⁻⁶ mol L⁻¹ with a detection limit of 8.0 × 10⁻⁹ mol L⁻¹. The relative standard deviation (RSD) of six replicate measurements is 1.95% for 3.0 × 10⁻⁷ mol L⁻¹ iron(II). The developed spin label fluorescence probe is found to be rapidly and sensitively responsive to iron(II) with high selectivity compared to existing fluorescence methods. The proposed method was successfully applied to iron(II) detection in five real samples with satisfactory results obtained by manual UV/Vis spectrophotometry (standard method) with 1,10-phenanthroline.     

Multisyringe flow injection analysis hyphenated with liquid core waveguides for the development of cleaner spectroscopic analytical methods: improved determination of chloride in waters

In this work, the hyphenation of the multisyringe flow injection analysis technique with a 100-cm-long pathlength liquid core waveguide has been accomplished. The Cl⁻/Hg(SCN)₂/Fe³⁺ reaction system for the spectrophotometric determination of chloride (Cl⁻) in waters was used as chemical model. As a result, this classic analytical methodology has been improved, minimizing dramatically the consumption of reagents, in particular, that of the highly biotoxic chemical Hg(SCN)₂. The proposed method features a linear dynamic range composed of two steps between (1) 0.2–2 and (2) 2–8 mg Cl⁻ L⁻¹, thus extended applicability due to on-line sample dilution (up to 400 mg Cl⁻ L⁻¹). It also presents improved limits of detection and quantification of 0.06 and 0.20 mg Cl⁻ L⁻¹, respectively. The coefficient of variation and the injection throughput were 1.3% (n = 10, 2 mg Cl⁻ L⁻¹) and 21 h⁻¹. Furthermore, a very low consumption of reagents per Cl⁻ determination of 0.2 μg Hg(II) and 28 μg Fe³⁺ has been achieved. The method was successfully applied to the determination of Cl⁻ in different types of water samples. Finally, the proposed system is critically compared from a green analytical chemistry point of view against other flow systems for the same purpose.     

Completely automated in-syringe dispersive liquid–liquid microextraction using solvents lighter than water

This paper describes the development of a new multisyringe flow injection analysis set-up that enables the complete automation of the dispersive liquid–liquid microextraction (DLLME) technique using solvents lighter than water. Its hyphenation with a liquid chromatographic separation is implemented using a single multisyringe pump obtaining a compact, simple, easy to operate, and fast instrument. DLLME is carried out with a throughput of 42 h⁻¹ and DLLME for the extraction of benzo(a)pyrene and its subsequent chromatographic determination can be carried out with an analysis throughput of 7 h⁻¹.     

Binding properties of a monoclonal antibody against the Cry1Ab from <Emphasis Type="Italic">Bacillus Thuringensis</Emphasis> for the development of a capillary electrophoresis competitive immunoassay

Experimental work performed was aimed at the assessment of a competitive capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF) detection for the determination of the Cry1Ab endotoxin from Bacillus thuringensis. The binding constant of a monoclonal antibody, raised against the insecticide protein Cry1Ab, was determined on a microplate by indirect enzyme-linked immunosorbent assay (ELISA) and compared with that obtained in-capillary under nonequilibrium separation conditions. The two binding constants appear comparable—(5.0 ± 1.2) × 10⁶ M⁻¹ and (9.06 ± 5.7) × 10⁶ M⁻¹—reflecting good preservation of the antibody binding behavior in the capillary electrophoresis format. These results allow use of a calibration curve possible between 0.2 and 150 nM of endotoxin protein, with a limit of detection of 0.5 nM (33 μg L⁻¹). Preliminary recovery experiments on maize extracts spiked with known amounts of Cry1Ab endotoxin also showed promising results in detecting the toxin in complex real matrices.     

An ab initio Molecular Orbital Study of the Conformational Properties of all-( Z )-Cyclododeca-1,4,7,10-tetraene

 Ab initio HF/6-31G^* and MP2/6-31G^*//HF/6-31G^* methods were used to calculate the structure optimization and conformational interconversion pathways for all-(Z )-cyclododeca-1,4,7,10-tetraene. This compound adopts the symmetrical crown (C _4v) conformation. Ring inversion takes place via symmetrical intermediates, such as boat-chair (BC, C _s) and twist (C _2h) conformers and requires about 22.3 kJ · mol⁻¹. The calculated strain energies for BC and twist conformers are 5.9 and 13.5 kJ · mol⁻¹. The results of semiempirical AM1 calculations for structural parameters and relative energies of the important geometries of the title compound are in good agreement with the results of ab initio methods.     

A rapid fluorescence polarization immunoassay for the determination of T-2 and HT-2 toxins in wheat

A rapid fluorescence polarization (FP) immunoassay has been developed for the simultaneous determination of T-2 and HT-2 toxins in naturally contaminated wheat samples. Syntheses of four fluorescein-labelled T-2 or HT-2 toxin tracers were carried out and their binding response with seven monoclonal antibodies was evaluated. The most sensitive antibody-tracer combination was obtained by using an HT-2-specific antibody and a fluorescein-HT-2 tracer. The developed competitive FP immunoassay in solution showed high cross-reactivity for T-2 toxin (CR% = 100%) while a very low CR% for neosolaniol (0.12%) and no cross-reactivity with other mycotoxins frequently occurring in wheat. A rapid extraction procedure using 90% methanol was applied to wheat samples prior to FP immunoassay. The average recovery from spiked wheat samples (50 to 200 μg kg⁻¹) was 96% with relative standard deviation generally lower than 8%. A limit of detection of 8 μg kg⁻¹ for the combined toxins was determined. Comparative analyses of 45 naturally contaminated and spiked wheat samples by both the FP immunoassay and high-performance liquid chromatography/immunoaffinity clean-up showed a good correlation (r = 0.964). These results, combined with the rapidity (10 min) and simplicity of the assay, show that this method is suitable for high throughput screening as well as for quantitative determination of T-2 and HT-2 toxins in wheat.     

Preparation of antibodies and development of a sensitive immunoassay with fluorescence detection for triazine herbicides

Specific polyclonal antibodies against s-triazine herbicides were obtained by preparing immunogens coupling home-synthesized haptens derivatives of simazine (6-chloro-N-ethyl-N′-ethyl-1,3,5-triazine-2,4-diamine) to lysine groups of hemocyanin from keyhole limpets and bovine serum albumin carrier proteins. Three highly sensitive rabbit antisera were obtained and evaluated with a battery of six enzyme tracers derived from triazine structures in an optimized ELISA format. The antiserum As8 and the HRP-2f tracer, which yield the best assay sensitivity for simazine (detection limit 0.11 ± 0.02 μg L⁻¹, IC₅₀ 0.88 ± 0.04 μg L⁻¹), were applied to the development of a sensitive flow-through immunoassay for the analysis of this herbicide. The automated assay was based on a direct competitive immunosorbent assay and fluorescence detection. The optimized method presents an IC₅₀ value of 0.35 ± 0.04 μg L⁻¹ with a detection limit of 1.3 ± 0.9 ng L⁻¹ and a dynamic range from 0.010 to 7.5 μg L⁻¹ simazine. The generic nature of the antiserum was shown by good relative cross-reactivities with other triazines such as atrazine (420%) or propazine (130%) and a lower response to terbutylazine (6.4%) and desethyl-atrazine (2.2%). No cross-reactivity was obtained for nonrelated pesticides such as 2,4-dichlorophenoxyacetic acid or linuron and the assay could be applied as a screening method for triazine herbicides. The total analysis time was 30 min per determination and the immunosensor could be reused for more than 150 cycles without significant loss of activity. The immunosensor has been successfully applied to the direct analysis of simazine in surface water samples at the nanogram per liter level. The results obtained by comparative analysis of the immunosensor with a chromatographic procedure for triazines showed a close correspondence.     

Fluorescence enhancement of the protein–curcumin–sodium dodecyl benzene sulfonate system and protein determination

Protein can greatly enhance the fluorescence of curcumin (CU) in the presence of sodium dodecyl benzene sulfonate (SDBS). Experiments indicate that under the optimum conditions, the enhanced intensity of fluorescence is proportional to the concentration of proteins in the range of 0.0050–20.0 μg mL⁻¹ for bovine serum albumin (BSA), 0.080–20.0 μg mL⁻¹ for human serum albumin (HSA), and 0.040–28.0 μg mL⁻¹ for egg albumin (EA). Their detection limits (S/N=3) are 1.4 ng mL⁻¹, 20 ng mL⁻¹, and 16 ng mL⁻¹, respectively. The method has been satisfactorily used for the determination of proteins in actual samples. In comparison with most of fluorimetric methods, this method is quick and simple, has high sensitivity and good stability. The interaction mechanism is also studied.     

CdS quantum dots as fluorescence probes for the sensitive and selective detection of highly reactive HSe<Superscript>−</Superscript> ions in aqueous solution

Water-soluble cadmium sulfide (CdS) quantum dots (QDs) capped by mercaptoacetic acid were synthesized by aqueous-phase arrested precipitation, and characterized by transmission electron microscopy, spectrofluorometry, and UV-Vis spectrophotometry. The prepared luminescent water-soluble CdS QDs were evaluated as fluorescence probes for the detection of highly reactive hydrogen selenide ions (HSe⁻ ions). The quenching of the fluorescence emission of CdS QDs with the addition of HSe⁻ ions is due to the elimination of the S²⁻ vacancies which are luminescence centers. Quantitative analysis based on chemical interaction between HSe⁻ ions and the surface of CdS QDs is very simple, easy to develop, and has demonstrated very high sensitivity and selectivity features. The effect of foreign ions (common anions and biologically relevant cations) on the fluorescence of the CdS QDs was examined to evaluate the selectivity. Only Cu²⁺ and S²⁻ ions exhibit significant effects on the fluorescence of CdS QDs. With the developed method, we are able to determine the concentration of HSe⁻ ions in the range from 0.10 to 4.80 μmol L⁻¹, and the limit of detection is 0.087 μmol L⁻¹. The proposed method was successfully applied to monitor the obtained HSe⁻ ions from the reaction of glutathione with selenite. To the best of our knowledge, this is the first report on fluorescence analysis of HSe⁻ ions in aqueous solution. Figure CdS quantum dots as fluorescence probes for the sensitive and selective detection of highly reactive HSe- ions in aqueous solution     

Determination of sub-ppb reserpine by an optosensing device based on photochemically induced fluorescence

A flow injection–solid-phase spectroscopy (FI-SPS) system implemented with photochemically induced fluorescence (PIF) is described for the rapid and very sensitive determination of reserpine in biological fluids and pharmaceutical formulations. An intensively fluorescent photoproduct is in-line generated, retained on C₁₈ silica gel in the detection area and monitored at 394/489 nm (λ _ex/λ _em). After the establishment of the appropriate working variables, the system is calibrated at two different injection volumes, 100 and 800 μL, achieving detection limits of 0.33 and 0.05 ng mL⁻¹, respectively. The RSD for reserpine at 2 ng mL⁻¹ (800 μL) was 1.5% (n = 10). The sampling rates were 46 and 43 h⁻¹ for each injection volume, respectively. The potential interference of some common species coexisting with reserpine in the analysed samples was also studied. The procedure was successfully applied to commercial formulations, urine and serum without any previous treatment of samples. Recoveries ranged from 94.9 to 100.2%.     

Determination of paralytic shellfish poisoning toxins in cultured microalgae by high-performance liquid chromatography with fluorescence detection

A novel method for the determination of paralytic shellfish poisoning (PSP) toxins using high-performance liquid chromatography with fluorescence detection was developed. The fluorescent derivates of neosaxitoxin (neoSTX), saxitoxin (STX), gonyautoxins 1 and 4 (GTX1+4), and gonyautoxins 2 and 3 (GTX2+3) were separated on a μBondapak NH₂ column (300 mm × 3.9 mm, 10 μm) using water and acetate buffer (pH 6.5) as the mobile phase (1.00 mL min⁻¹) in gradient mode with fluorescence detection at 390 nm (excitation at 330 nm). The linear ranges of neoSTX, STX, GTX1+4 and GTX2+3 were 3.31–331, 0.952–95.2, 3.78–378 and 0.124–12.4 ng mL⁻¹, respectively. The detection limits of neoSTX, STX, GTX1+4 and GTX2+3 were 1.10, 0.32, 1.26 and 0.041 ng mL⁻¹, respectively. The method was successfully applied to the determination of PSP toxins in microalgae. The recoveries ranged from 88±2% to 107±4% and the relative standard deviations were 0.16% to 4.4%. The procedure is also environmentally friendly because no organic solvent is used in the mobile phase.     

Flow injection spectrofluorimetric determination of iron(III) in water using salicylic acid

A simple and fast flow injection fluorescence quenching method for the determination of iron in water has been developed. Fluorimetric determination is based on the measurement of the quenching effect of iron on salicylic acid fluorescence. An emission peak of salicylic acid in aqueous solution occurs at 409 nm with excitation at 299 nm. The carrier solution used was 2 × 10⁻⁶ mol L⁻¹ salicylic acid in 0.1 mol L⁻¹ NH₄⁺/NH₃ buffer solution at pH 8.5. Linear calibration was obtained for 5–100 μg L⁻¹ iron(III) and the relative standard deviation was 1.25 % (n = 5) for a 20 μL injection volume iron(III). The limit of detection was 0.3 μg L⁻¹ and the sampling rate was 60 h⁻¹. The effect of interferences from various metals and anions commonly present in water was also studied. The method was successfully applied to the determination of low levels of iron in real samples (river, sea, and spring waters).     

An ab initio Molecular Orbital Study of the Conformational Properties of all-(Z)-Cyclododeca-1,4,7,10-tetraene

Summary.  Ab initio HF/6-31G^* and MP2/6-31G^*//HF/6-31G^* methods were used to calculate the structure optimization and conformational interconversion pathways for all-(Z )-cyclododeca-1,4,7,10-tetraene. This compound adopts the symmetrical crown (C _4v) conformation. Ring inversion takes place via symmetrical intermediates, such as boat-chair (BC, C _s) and twist (C _2h) conformers and requires about 22.3 kJ · mol⁻¹. The calculated strain energies for BC and twist conformers are 5.9 and 13.5 kJ · mol⁻¹. The results of semiempirical AM1 calculations for structural parameters and relative energies of the important geometries of the title compound are in good agreement with the results of ab initio methods. Received July 9, 2001. Accepted September 26, 2001     

A compact miniaturized continuous flow system for the determination of urea content in milk

A multicommutation-based flow system with photometric detection was developed, employing an analytical microsystem constructed with low temperature co-fired ceramics (LTCC) technology, a solid-phase reactor containing particles of Canavalia ensiformis DC (urease source) immobilized with glutaraldehyde, and a mini-photometer coupled directly to the microsystem which monolithically integrates a continuous flow cell. The determination of urea in milk was based on the hydrolysis of urea in the solid-phase reactor and the ammonium ions produced were monitored using the Berthelot reaction. The analytical curve was linear in the urea concentration range from 1.0 × 10⁻⁴ to 5.0 × 10⁻³ mol L⁻¹ with a limit of detection of 8.0 × 10⁻⁶ mol L⁻¹. The relative standard deviation (RSD) for a 2.0 × 10⁻³ mol L⁻¹ urea solution was lower than 0.4% (n = 10) and the sample throughput was 13 h⁻¹. To check the reproducibility of the flow system, calibration curves were obtained with freshly prepared solutions on different days and the RSD obtained was 4.7% (n = 6). Accuracy was assessed by comparing the results of the proposed method with those from the official procedure and the data are in close agreement, at a 95% confidence level.     

Preparation of a novel composite particles and its application in the fluorescent detection of proteins

A new fluorescence method for the detection of proteins with novel composite nanoparticles (CdS/PPA) has been developed. The composite nanoparticles have been prepared through an in-situ polymerization method under ultrasonic irradiation. The surface of the composite nanoparticles was covered with functional groups (-COOH). These groups may play a major role in the improving the water solubility and biocompatibility of the nanoparticles. The composite particles is combined with proteins in NaAc-HCl buffer solution (pH=1.99), which can result in strong fluorescence, and the response is linearly proportional to the concentration of proteins. In λem/λex=650 nm/365 nm place (the stoke’ shift is 285 nm), its fluorescent strength reaches the maximum. Under the optimum conditions, the linear range is 0.10–20.0 μg·ml⁻¹ with the detection limit of 41 ng·ml⁻¹ for HSA, and 0.10–15.0 μg·ml⁻¹ with the detection limit of 35 ng·ml⁻¹ for Human γ-IgG . The method has been applied to the determination of the total protein in human serum samples collected from the hospital and the results are satisfactory.