Characterisation and optimisation of an immunoprobe for triazines

The characterisation and optimisation of an optical immunoassay with label free detection based on Reflectometric Interference Spectroscopy (RIfS) is presented. The immunoprobe is operated in a sequential scheme, where Fab-fragments react with analyte molecules in a first step. In a second step the optical transducer is used to quantify the amount of unoccupied Fab- fragments in the reaction mixture binding to the hapten-modified transducer surface. For optimisation of the test, the Fab-fragment concentration was varied between 2×10^-8 mol/l and 2.5× 10^–9 mol/l. Down to a concentration of 5×10^-9 mol/l a reduction in the limit of detection has been observed. At the lowest concentration investigated no further improvement has been found due to a reduced binding of the analyte and a strong decrease of antibody binding at the transducer surface. This finding could be explained by the thermodynamics of the antigen-antibody reaction and the performance of the optical transducer used. The limit of detection obtained is discussed with respect to thermodynamics, transducer characteristics and immunoprobe test format.     

Electrochemical determination of hydrogen peroxide using o -dianisidine as substrate and hemoglobin as catalyst

A new electrochemical method for the determination of microamounts of hydrogen peroxide utilizing o-dianisidine (ODA) as substrate and hemoglobin (Hb) as catalyst is described in this paper. Hb can be used as mimetic peroxidase and it can catalyse the reduction of hydrogen peroxide with the subsequent oxidation of ODA. The oxidative reaction product is an azo compound, which is an electroactive substance and has a sensitive second-order derivative polarographic reductive peak at the potential of -0.58 V (vs. SCE) in pH 80 Britton-Robinson (B-R) buffer solution. The conditions of Hb-catalytic reaction and polarographic detection of the reaction product were carefully studied. By using this polarographic peak and under optimal conditions, the calibration curve for the H₂O₂ was constructed in the linear range of 2.0 x 10^-7 ∼ 10 x 10^-4 mol/l with the detection limit of 5.0 x 10^-8 mol/l. This method can also be used to the determination of Hb content in the range of 20 x 10^-9 ∼ 30 x 10^-7 mol/l with a detection limit of 10 x 10^-9 mol/l. The proposed method was further applied to the determination of the content of H₂O₂ in fresh rainwater with satisfactory results. The catalytic reaction mechanism and the electrode reductive process of the reaction product were carefully studied.     

Chemiluminescence flow injection analysis of 1,3-dichloro-5,5-dimethylhydantoin in swimming pool water

A new chemiluminescence (CL) flow-injection method is proposed for the determination of 1,3-dichloro-5,5-dimethylhydantoin (DDH). The method is based on the chemiluminescent reaction of DDH and luminol-H(2)O(2) in an alkaline medium (pH 12.0-12.5). The concentration of the analyte shows a good linear relationship with the produced luminescence intensity in the range of 3.0x10(-8) to 8.0x10(-6) mol l(-1). The detection limit of the proposed method is 1.0x10(-8) mol l(-1) and the relative standard deviation (R.S.D.) is 4.7% (n=5) at 5.0x10(-7) mol l(-1). This method was successfully applied to the determination of trace amounts of this disinfectant in water samples obtained from five different swimming pools. Satisfying recovery values were also obtained.     

Porphyrin hybrid complex ([2Fe-2S]2TPPS) as a catalyst for determination of hydrogen peroxide.

A new promising mimetic enzyme, a [2Fe-2S] cluster-porphyrin hybrid complex ([2Fe-2S]2TPPS), has been synthesized and applied to the determination of hydrogen peroxide. Under the optimum condition, the calibration graph has a linear range of 8.0 x 10(-8) - 1.0 x 10(6) mol/l H2O2 with a detection limit (3sigma, N = 9) of 5.3 x 10(-9) mol/l.     

Constructing a new optical sensor for monitoring ammonia in water samples using bis(acetylacetoneethylendiamine)-tributylphosphin cobalt(III) tetraphenylborate complex-coated triacetylcellulose.

A new ammonia optical sensor was designed using bis(acetylacetoneethylendiamine)tributylphosphin cobalt(III) tetraphenylborate complex, coated on transparent triacetylcellulose film as membrane. The change in the absorbance of the optode at the maximum wavelength of 408 nm was related to ammonia concentration in aqueous samples. A buffer solution with a pH of 9 (sodium borate-HCl) was used. The optode was fully regenerated in pH 2. The linear dynamic range for determination of ammonia was 3.3 x 10(-4) to 6.9 x 10(-3) mol l(-1) with a detection limit of 5.0 x 10(-5) mol l(-1) and a response time range of 4 - 6 min. This membrane was successfully applied for determination of ammonia in drinking water.     

The synthesis and membrane transport characteristics of macrocyclic polyether ligands composed of 1,10-phenanthroline as carriers for primary amine species

Four macrocyclic polyether derivatives of o-phenanthroline were synthesized and used as neutral carriers for preparing poly(vinyl chloride) (PVC) membrane electrodes to sense primary amine species. The potentiometric response characteristics of the electrodes prepared were investigated. The electrode sensitive to benzyl amine as model analyte showed a linear response range of 8.0 x 10(-6)-0.1 mol/l with a detection limit of 8.9 x 10(-7) mol/l and a slope of 56.5 mV/decade. The linear potentiometric response of the mexiletine-sensitive electrode was 4.7 x 10(-6)-0.1 mol/l, and the detection limit was 5.0 x 10(-7)1 mol/l with a slope of 59.0 mV/decade. The transfer behavior of amines and ammonium ions through an organic phase was investigated by means of the bulk liquid membrane transport experiment. The effects of pH, counter anions and other factors on the transfer of the amine and ammonium species were studied. The mass transfer rates of the test species facilitated by macrocyclic polyether derivatives of o-phenanthroline were determined and the following sequence was found: benzyl amine > ethyl amine > tetramethyl ammonium > triethyl amine > diethyl amine > K(+) > ammonium > Na(+) > Ca(2+) > Mg(2+). This was exactly the potentiometric selectivity sequence of the membrane electrodes prepared by using these carriers. The mechanism of transfer of benzyl amine through a membrane phase induced by the carriers has been elucidated on the basis of experimental observations.     

Spectrophotometric flow injection determination of formetanate and m-aminophenol in water after reaction with p-aminophenol

An automated procedure has been developed for the determination of formetanate and its metabolite m-aminophenol (MAP) in water samples. MAP can be selectively determined in the presence of formetanate by direct on-line reaction with p-aminophenol and spectrophotometric measurement of the absorbance at 576 nm in the presence of KIO(4), as oxidizing agent. The method has a limit of detection of 5 x 10(-7)M, it provides a recovery percentage from 95 to 104% and permits one to carry out 120 measurements/hr. The spectrophotometric determination of formetanate must be carried out after a previous hydrolysis to MAP. To determine formetanate in the presence of MAP, two steps are necessary. Firstly, the MAP content is selectively determined as has been mentioned above. After that, the sample is treated with 0.05M NaOH at 90' degrees C, to hydrolyze the formetanate to MAP, and then the sum of both is determined spectrophotometrically. The difference between the results obtained in each step gives the formetanate concentration. The developed procedure for the determination of formetanate provides a sensitivity of 1070 absorbance units mol(-1) l and a limit of detection of 1.9 x 10(-7)M, which corresponds to 50 mug/l of formetanate hydrochloride. The method has been applied to the analysis of natural water samples fortified with formetanate and MAP, and formetanate has also been quantitatively recovered in irrigation waters at a concentration level of 1.9 x 10(-6)M which corresponds to 500 mug/l. On the other hand, working in the stopped-flow mode, for a reaction time of 100 sec, the sensitivity of the formetanate determination can be increased to 4642 absorbance units mol(-1) l but the limit of detection remains of the order of 44 mug/l.     

Quantification of dibromodimethylhydantoin disinfectants in water by chemiluminescent method.

Quantification of 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) was studied by its chemiluminescence (CL) reaction with luminol in an alkaline medium. The stability of DBDMH, 1,3-dichloro-5,5-dimethylhydantoin (DCDMH) and 1-bromo-3-chloro-5,5-dimethylhydantoin (BCDMH) in water was initially assessed by its CL reaction capability. The results indicated that the hydrolysis process was critically dependent on the types of reagents and their pHs. Capillary electrophoresis (CE) separation with CL detection procedure was applied to the DBDMH solution. It was found that at least 3 species in the aqueous DBDMH solution could oxidize luminol to give luminescence: one of them was confirmed to be hypobromite and the others could be the unhydrolyzed or active oxygen produced in the hydrolysis reaction. Finally, a flow-injection chemiluminescent method was proposed for the determination of DBDMH. The concentration of the analyte showed a linear relationship with the CL intensity in the range of 1.2x10(-10) to 1.0x10(-6) mol dm-3 and the detection limit was as low as 6.2x10(-11) mol dm-3. The relative standard deviation (RSD) is 1.7% (n=9) for 2.8x10(-7) mol dm-3 DBDMH.     

Flow injection turbidimetric determination of thiamine in pharmaceutical formulations using silicotungstic acid as precipitant reagent

A simple flow injection system is proposed for the determination of thiamine in pharmaceutical formulations. The determination is based on the precipitation reaction of thiamine with silicotungstic acid in acidic medium to form a thiamine silicotungstate suspension that is measured at 420 nm. Adding 0.05% (w/v) poly(ethyleneglycol) in the carrier solution (0.5 mol l(-1) hydrochloric acid), an improvement in the sensitivity, repeatability and baseline stability of the flow injection system was obtained. The calibration graph was linear in the thiamine concentration range from 5.0x10(-5) to 3.0x10(-4) mol l(-1) with a detection limit of 1.0x10(-5) mol l(-1). The relative standard deviations for ten successive measurements of 1.0x10(-4) mol l(-1) and 2.5x10(-4) mol l(-1) thiamine were less than 1% and an analytical frequency of 90 h(-1) was obtained.     

Determination of morphine in process streams by sequential injection analysis with chemiluminescence detection

A procedure for the determination of morphine in process streams by sequential injection analysis based on the chemiluminescence reaction of morphine with acidic potassium permanganate in the presence of sodium hexametaphosphate is presented. The chemiluminescence emission has been monitored using an in-house detection system which consisted of a fibre optic flowthrough cell and a sensitive, low dark current, photomultiplier tube. The calibration graph (range 2 x 10(-8) to 1 x 10(-4) mol/l) was not linear over the entire range of concentration, with a polynomial equation of best fit of y = 1.0 x 10(15) x(3) - 2.2 x 10(11) x(2) + 1.3 x 10(7) x - 8.3. The calibration function approximates linearity over the concentration range 2.5 x 10(-6) to 3.0 x 10(-5) mol/l where the slope of the log-log plot is 1.09 +/- 0.16. The detection limit was estimated at about 10(-8) mol/l from the response of the lowest calibration standard (2.5 x 10(-8) mol/l) which gave a signal to noise ratio of 3 : 1. Although the structurally related codeine did not interfere significantly the results suggest that this method may be susceptible to matrix effects, dependent on the location of sampling from the process stream.     

Selective determination of hydrogen peroxide by adduct formation with a dinuclear iron(III) complex and flow injection analysis/tandem mass spectrometry

A highly selective method for the determination of hydrogen peroxide is presented. In a flow injection analysis (FIA) instrument, the analyte is brought into contact with a dinuclear heptadentate iron(III) complex. The formation of the peroxide adduct is quantified using electrospray tandem mass spectrometry (ESI-MS/MS). Selected reaction monitoring (SRM) based on the transition from the triply charged peroxide adduct with m/z = 251.2 to the triply charged fragment ion of m/z = 240.5 is performed. The limit of detection for hydrogen peroxide is 10(-7) mol dm(-3), limit of quantification is 3 x 10(-7) mol dm(-3), and a linear range of 2.5 decades starting at the limit of quantification is observed.     

Spectrophotometric determination of fluoxetine by batch and flow injection methods

A rapid, simple, and accurate spectrophotometric method is presented for the determination of fluoxetine by batch and flow injection analysis methods. The method is based on fluoxetine competitive complexation reaction with phenolphthalein-beta-cyclodextrin (PHP-beta-CD) inclusion complex. The increase in the absorbance of the solution at 554 nm by the addition of fluoxetine was measured. The formation constant for fluoxetin-beta-CD was calculated by non-linear least squares fitting. Fluoxetine can be determined in the range 7.0 x 10(-6)-2.4 x 10(-4) mol l(-1) and 5.0 x 10(-5)-1.0 x 10(-2) mol l(-1) by batch and flow methods, respectively. The limit of detection and limit of quantification were respectively 4.13 x 10(-6) mol l(-1) and 1.38 x 10(-5) mol l(-1) for batch and 2.46 x 10(-5) mol l(-1) and 8.22 x 10(-5) mol l(-1) for flow method. The sampling rate in flow injection analysis method was 80+/-5 samples h(-1). The method was applied to the determination of fluoxetine in pharmaceutical formulations and after addition to human urine samples.     

An investigation of electroosmotic flow and pressure pumped luminol chemiluminescence detection for cobalt analysis in a miniaturised total analytical system

The luminol chemiluminescence reaction has been investigated for the determination of cobalt in a planar glass micro reactor. Reagents were mobilised within an etched glass chip with 200 microm wide channels using either electrically driven flow with voltages below 200 V cm(-1) or negative pressure pumping. Surfactants were used to assist mobilisation of the luminol for electrically driven flow and this also increased the chemiluminescence intensity in that system. A calibration over seven orders of magnitude was achieved for cobalt(II) nitrate with the negative pressure pumping system, and the limit of detection at the 95% confidence level was 3 x 10(-11) mol l(-1). The limit of detection for the electrokinetic flow was 4 x 10(-11) mol l(-1) and the linear range was between 10(-10) to 10(-6) mol l(-1). The advantages and disadvantages of both mobilisation methods are discussed for this reaction.     

Flow injection spectrophotometric determination of isoproterenol using an avocado (Persea americana) crude extract immobilized on controlled-pore silica reactor

An enzymatic reactor was constructed by the immobilization of polyphenol oxidase (PPO) from avocado (Persea americana) crude extract in an inorganic support of controlled pore silica (CPS), after a previous step of silanization. This inorganic support has been used as an excellent carrier to immobilize this enzyme and the enzymatic reactor was used in a flow injection system for the determination of isoproterenol in pharmaceutical products. The procedure is based on the oxidation reaction of this drug with immobilized PPO and the product obtained was monitored at 492 nm. This system presented an analytical curve from 1.23x10(-4) to 7.38x10(-4) mol l(-1) isoproterenol with a detection limit of 6.25x10(-5) mol l(-1). Recoveries of isoproterenol between 98.5 and 103.1%, a relative standard deviation (R.S.D.) less than 1% (n=10) and 36 determinations per h were obtained.     

Stopped-flow spectrophotometric determination of hydrogen peroxide with hemoglobin as catalyst

A rapid and sensitive method was proposed for the determination of hydrogen peroxide based on the catalytic effect of hemoglobin using o-phenylenediamine as the substrate. Stopped-flow spectrophotometric method was used to study the kinetic behavior of the oxidation reaction. The catalytic effectiveness of hemoglobin was compared with other four kinds of catalysts. The initial rate of the formation of the reaction product 2,3-diaminophenazine at the wavelength of 425 nm was monitored, permitting a detection limit of 9.2x10(-9) mol/l H(2)O(2). A linear calibration graph was obtained over the H(2)O(2) concentration range 5.0x10(-8)-3.5x10(-6) mol/l, and the relative standard deviation at a H(2)O(2) concentration of 5.0x10(-7) mol/l was 2.08%. Satisfied results were obtained in the determination of H(2)O(2) in real samples by this method.     

A fluorescence quenching method for the determination of nitrite with Rhodamine 110

A simple and sensitive fluorescence quenching method for the determination of trace nitrite has been developed. The method is based on the reaction of Rhodamine 110 with nitrite in acidic medium to form a new compound, which has much lower fluorescence. The optimum experimental conditions were studied. The linear range was obtained at a nitrite concentration of 1.0 x 10(-8)-3.0 x 10(-7)mol l(-1) with a detection limit of 7.0 x 10(-10) mol l(-1) (S/N=3). The proposed method has been successfully applied to the determination of nitrite in tap water and lake water without extraction.     

Polarographic determination of total iron content using a Fe(2+/3+)-Methylthymol Blue-NO2- system.

In the buffer solution of NH3-NH4Cl (pH = 8.5, 0.04 mol l(-1)), iron-Methylthymol Blue (MTB) can produce a sensitive polarographic wave at -1.10 V (vs. SCE) in the NaNO2. The peak current is linear with the concentration of the iron in the range of 3 x 10(-8)-5 x 10(-6) mol l(-1), and the detection limit is 1 x 10(-8) mol l(-1). By studying the characteristics of the wave and the electrode reaction mechanism, we can prove that the catalytic wave is an adsorption wave and that the peak current comes from the reduction of Fe(II). The molar ratio of iron to ligand was found to be 1:1. Adsorption particles are neutral molecules, the saturated adsorption quantity of the complex on the mercury electrode is 1.92 x 10(-9) mol cm(-2), according with the Frumkin isothermal formula. In the experiments, the adsorption coefficient (beta) is 4.05 x 10(5); the adsorption factor (gamma) is 0.70: the electron transfer number (n) is 2; the free energy (deltaG(o)) is 31.99 kJ mol(-1); the transfer coefficient of the irreversible adsorption is 0.42-0.45; and the reaction velocity constant (Ks) is 1.35 s(-1). This method, whose result is satisfying, can be applied to the detection of trace total iron contents in medicinal products.     

Chemiluminescence determination of sulfite in sugar and sulfur dioxide in air using Tris(2,2'-bipyridyl)ruthenium(II)-permanganate system

A chemiluminescence (CL) detection for the determination of sulfite using the reaction of Ru(bipy)(3)(2+) (bipy=2,2'-bipyridyl) -SO(3)(2-)-KMnO(4) is described. The concentration of sulfite is proportional to the CL intensity from 5.0x10(-8) to 1.25x10(-4) mol l(-1). The limit of detection is 2.5x10(-8) mol l(-1) and the relative standard deviation is 4.9% for the 2x10(-5) mol l(-1) sulfite solution in six repeated measurements. This method has been successfully applied to the determination of sulfite in sugar and sulfur dioxide in air by using triethanolamine (TEA) as the absorbent material.     

The effect of cetyltrimethyl ammonium bromide on the electrochemical determination of thyroxine

Electrochemical behavior of thyroxine at a polyvinylpyrrolidone modified carbon paste electrode in the presence of cetyltrimethyl ammonium bromide was described. Thyroxine underwent totally irreversible oxidation at this system and a well-defined peak at 0.42 V was obtained. The influence of various surfactants on the oxidation of thyroxine was examined by cyclic voltammetry. Chronocoulometry was also used to investigate the electrode process. In the range 2 x 10(-7) to 9 x 10(-6)mol/l, the thyroxine concentration was linear with the oxidation peak current and a low detection limit of 8 x 10(-8) mol/l was obtained for 5 min accumulation.     

Terbium-sensitized fluorescence method for the determination of pazufloxacin mesilate and its application.

A simple, rapid and highly sensitive fluorometric method for the determination of pazufloxacin mesilate (PZFX) is described. It is based on the formation of the complex [Tb(PZFX)2](3+), which shows the intensive characteristic bands of Tb3+. Optimum conditions for the determination were investigated. Under the optimum experimental condition, the fluorescence intensity responds linearly to the PZFX concentration in the range of 2.0 x 10(-8) - 5.0 x 10(-6) mol/l with a detection limit of 6.2 x 10(-9) mol/l. The method has been successfully applied to the determination of PZFX in urine and serum samples.