Chemiluminescence determination of citrate and pyruvate and their mixtures by the stopped-flow mixing technique

A novel kinetic chemiluminescent method using the stopped-flow mixing technique has been investigated for the rapid and sensitive determination of citrate and pyruvate. The method is based on a tris(2,2′-bipyridiyl)ruthenium(III) (Ru(bpy)₃³⁺) chemiluminescence (CL) reaction. Ru(bpy)₃³⁺ was generated in the mixing chamber by oxidising tris(2,2′-bipyridyl)ruthenium(II) with cerium(IV). After selecting the best operating parameters, calibration graphs were obtained over the concentration ranges 0.38-38 μg ml⁻¹ and 8.7-1300 ng ml⁻¹ for citrate and pyruvate, respectively. The limits of detection were 0.1 μg ml⁻¹ for citrate and 0.3 ng ml⁻¹ for pyruvate. Based on the differential rate of the chemiluminescent reaction corresponding to citrate and pyruvate, a very simple kinetic procedure was developed for the simultaneous determination of both compounds. Mixtures of citrate and pyruvate in ratios between 15:1 and 1.5:1 were satisfactorily resolved. The proposed method was successfully applied to the determination of citrate in pharmaceutical formulations, pyruvate in animal blood serum and both compounds in human urine.     

A novel chemiluminescence assay of organophosphorous pesticide quinalphos residue in vegetable with luminol detection

Background  

Organophosphorous pesticides are the most popular pesticides used in agriculture. As acetylcholinesterase inhibitors, organophosphorous pesticides are toxic organic chemicals. The control and detection of organophosphorous pesticide residue in food, water, and environment therefore plays a very important role in maintaining physical health. A sensitive, rapid, simple chemiluminescence(CL) method has been developed for the determination of quinalphos based on the reaction of quinalphos with luminol-H₂O₂ in an alkaline medium. The method has been applied to detection of quinalphos in vegetable samples with satisfactory results.     

Multivariate calibration applied to the time-resolved chemiluminescence for the simultaneous determination of morphine and its antagonist naloxone

A novel alternative for the simultaneous determination of compounds with similar structure is described, using the whole chemiluminescence-time profiles, acquired by the stopped-flow technique, in combination with mathematical treatments of multivariate calibration. The proposed method is based on the chemiluminescent oxidation of morphine and naloxone by their reaction with potassium permanganate in an acidic medium, using formaldehyde as co-factor. The whole chemiluminescence-time profiles, acquired using the stopped-flow technique in a continuous-flow system, allowed the use of the time-resolved chemiluminescence (CL) data in combination with multivariate calibration techniques, as partial least squares (PLS), for the quantitative determination of both opiate narcotics in binary mixtures.In order to achieve overcoat the additivity of the CL profiles and beside to obtain CL profiles for each drug the most separated as possible in the time, the optimum chemical conditions for the CL emission were investigated. The effect of common emission enhancers on the CL emission obtained in the oxidation reaction of these compounds in different acidic media was studied. The parameters selected were sulphuric acid 1.0 mol L⁻¹, permanganate 0.2 mmol L⁻¹ and formaldehyde 0.8 mol L⁻¹. A calibration set of standard samples was designed by combination of a factorial design, with three levels for each factor and a central composite design. Finally, with the aim of validating the chemometric proposed method, a prediction set of binary samples was prepared. Using the multivariate calibration method proposed, the analytes were determined in synthetic samples, obtaining recoveries of 97-109%.     

Long-wavelength homogeneous enzyme immunoassay for the determination of amikacin in water samples

A simple and rapid homogeneous enzyme immunoassay involving the use of the malic dehydrogenase enzyme and a long-wavelength fluorophor, the oxazine Cresyl Violet, is proposed for the determination of the antibiotic amikacin in water samples. An enzymatic tracer has been synthesized by covalent binding of amikacin to malic dehydrogenase via a carbodiimide derivative. Free tracer catalyses the reaction between Cresyl Violet and malic acid giving rise to a decrease in the fluorescence of the fluorophor. Kinetic curves for this reaction have been monitored at λ_ex 585 and λ_em 624 nm using the stopped-flow mixing technique, being the initial rate measured in only 2-3 s. The dynamic range of the method is 1-15 ng mL⁻¹ and the detection limit is 0.3 ng mL⁻¹, using aqueous standard solutions or water samples. The precision, obtained at 1 and 5 ng mL⁻¹ and expressed as relative standard deviation, was 6.0 and 9.6%, respectively. The method has been applied to the analysis of drinking, river and wastewater samples. The sample pre-treatment involved a solid-phase extraction step for the clean-up of the samples. A recovery study was carried out to validate the method, being the values obtained in the range 80-114%, with a mean value of 96.7%.     

Highly sensitive catalytic determination of molybdenum

A novel, highly sensitive, selective, and simple kinetic method was developed for the determination of Mo(VI) based on its catalytic effect on the oxidation of 1-amino-2-naphthol-4-sulfonic acid (ANSA) with H₂O₂. The reaction was followed spectrophotometrically by tracing the oxidized product at 465 nm after 30 min of mixing the reagents. The optimum reaction conditions were: 10 mmol l⁻¹ ANSA, 50 mmol l⁻¹ H₂O₂, 100 mmol l⁻¹ acetate buffer of pH 5.0 ± 0.05 and at 40 °C. Addition of 200 μg ml⁻¹ diethylenetriaminepentaacetic acid (DTPA) conferred high selectivity for the proposed method. Following the recommended procedure, Mo(VI) could be determined with a linear calibration graph up to 2.5 ng ml⁻¹ and a detection limit, based on the 3S_b-criterion, of 0.027 ng ml⁻¹. The unique sensitivity and selectivity of the implemented method allowed its direct application to the determination of Mo(VI) in natural and industrial waste water. The method was validated by comparison with the standard ETAAS method. Moreover, published catalytic-spectrophotometric methods for the determination of molybdenum were reviewed.     

Determination of trace amounts of bromide by flow injection/stopped-flow detection technique using kinetic-spectrophotometric method

A simple, sensitive and selective method for the determination of bromide in seawater by using a flow injection/stopped-flow detection technique was examined. The detection system was developed for a new kinetic-spectrophotometric determination of bromide in the presence of chloride matrix without any extraction and/or separation. The detection was based on the kinetic effect of bromide on the oxidation of methylene blue (MB) with hydrogen peroxide in a strongly acidic solution. Large amounts of chloride could enhance the sensitivity of the method as an activator. The decolorisation of the blue color of MB was used for the spectrophotometric determination of bromide at 746 nm. A stopped-flow approach was used to improve the sensitivity of the measurement and provide good linearity of the calibration over the range of 0-3.2 μg ml⁻¹ of bromide. The relative standard deviation was 0.74% for the determination of 2.4 μg ml⁻¹ bromide (n = 5). The detection limit (3σ) was 0.1 μg ml⁻¹ with a sampling frequency of 12 h⁻¹. The influence of potential interfering ions was studied. The proposed method was applied to the determination of bromide in seawater samples and provided satisfactory results.     

A new kinetic method for quantification phenoxyl free radicals

In this work, a new kinetic method was proposed for quantification phenoxyl radicals generated in enzyme reaction. Instead of direct detecting the spectral signals of phenoxyl radicals, a molecular probe, the reduced form of nicotinamide adenine dinucleotide (NADH), was employed to indicate the formation of phenoxyl free radicals. It was found that the reactions of NADH and phenoxyl radicals are very fast, but can be followed by using stopped-flow fast scanning spectrophotometric technique. The initial rate of accelerated-oxidation of NADH represents the reactivity of phenoxyl free radical, which is proportional in a certain range to the initial concentration of the parent chlorophenols of the radicals. With this method, the phenoxyl radicals generated in oxidation reaction of chlorophenols (2-CP; 4-CP; 2,4-DCP; 2,4,6-TCP and 2,3,4,6-Tetra-CP) with hydrogen peroxide, catalyzed by horseradish peroxidase, were investigated. The method is highly sensitive. Phenoxyl radicals generated from as low as 1 × 10⁻⁸ M 2,4-DCP, for example, can be readily detected with the proposed method. The results show that the reactivity of various phenoxyl radicals are in the following order: 2,4-DCP > 4-CP > 2-CP > 2,4,6-TCP > 2,3,4,6-Tetra-CP. A mechanism is proposed to explain the possible pathway of the probe reaction. The feasibility of this method was assessed by the determination of enzymatic generation of phenoxyl radicals in lake water samples.     

A high throughput chemiluminescence method for determination of chemical oxygen demand in waters

A novel and high throughput chemiluminescence (CL) method for determination of chemical oxygen demand (COD) in water sample was originally developed based on potassium permanganate-glutaraldehyde CL system. With this method, dissolved organic matter in water samples was digested by excess acid potassium permanganate, the reacted mixture solutions containing surplus KMnO₄ were added in wells of a 96-well plate, followed by injection of glutaraldehyde in the wells, and CL was then produced along with the reaction of the added glutaraldehyde with the surplus KMnO₄ and detected by a photomultiplier tube (PMT). The difference (ΔI) between the CL intensity for distilled water and that for sample water was proportional to the COD value of water sample. The calibration graph was linear in the range of 0.16-19.24 mg L⁻¹ with a detection limit of 0.1 mg L⁻¹. A complete analysis could be performed in 40 min including digestion and detection, giving a very high throughput of 3 × 96 samples in about 60 min. Compared with the conventional methods, this method is simple and sensitive and consumes very limited and cheap reagents. Owing to its rapid, automatic, high throughput and low cost characteristics, the presented CL method has been applied successfully to the determination of COD in real water samples (n = 32) with satisfactory results.     

Stopped-flow kinetic method for the fluorimetric determination of DNA traces in biological samples based on the interaction long-wavelength fluorophor, surfactant and nucleic acid

A simple and rapid method for the determination of DNA, involving the interaction between a surfactant, a long-wavelength fluorophor (LWF) and the nucleic acid, is presented. Different chemical systems based on the local effective charge of the surfactant/LWF system with DNA were tested, choosing cetyltrimethyl ammonium bromide (CTAB) and indocyanine green (ICG) for the development of the method. The fluorescence of ICG increases in the presence of CTAB, but it rapidly decreases in the presence of deoxyribonucleic acid. The initial reaction-rate (v₀) and signal at a prefixed-time (ΔIF₂₀) are monitored at 780 and 802 nm as excitation and emission wavelengths, respectively, using stopped-flow mixing technique, which makes the method applicable to automate routine analysis. Each measurement was obtained in about 30 s, being the integration time 0.1 s. The dynamic range of the calibration graph was 10-1500 ng mL⁻¹, with a detection limit of 5 ng mL⁻¹. The precision of the method, expressed as relative standard deviation, ranged between 2.1% and 4.5%. After a sample treatment consisting on a conventional extraction, the method was applied to the determination of DNA in several samples from different biological materials.     

Sensitive determination of captopril by time-resolved chemiluminescence using the stopped-flow analysis based on potassium permanganate oxidation

The chemiluminescent behaviour of captopril when reacted with a common oxidant, potassium permanganate in different acidic media is described, using the stopped-flow technique in a continuous-flow system. A 22 bit analogue-to-digital converter that acquires analogue signals at −10 and +10 V and allows the power supply to the peristaltic pump to be interrupted is used in the time-resolved chemiluminescence manifold to ensure rapid, efficient mixing of chemiluminescent reagent and sample immediately before reaching the detector; this results in high precision and detectability, particularly with fast, short-lived emissions.The optimum chemical conditions for the chemiluminescence emission were investigated. It was found that a weak CL emission was emitted during the oxidation of this drug with potassium permanganate in acidic solution. The effect of common emission enhancers such as formic acid, formaldehyde, glutaraldehyde, acetaldehyde, quinine, fluorescein, rhodamine B and rhodamine 6G was studied. The parameters selected were 4.0 mol L⁻¹ sulphuric acid, 0.25 mmol L⁻¹ permanganate and 0.75 mol L⁻¹ formaldehyde.Four quantitative parameters adjustable via software settings, two of them typically kinetic parameters, such as rate of the light-development reaction and rate of the light-decay reaction, and the other conventional parameters, such as maximum emission intensity and total emission area, were used to obtain linear calibration graphs with each measurement parameter. The detection limits ranged from 0.011 to 0.026 μg mL⁻¹ and R.S.D. values (n = 10) of 1.21-3.93 at a 0.50 μg mL⁻¹ and 2.01-3.41 at a 1.60 μg mL⁻¹ concentration levels were obtained. The method was satisfactorily applied to the determination of captopril in pharmaceutical preparations.     

Determination of nitrate in environmental water samples by conversion into nitrophenols and solid phase extraction−spectrophotometry, liquid chromatography or gas chromatography-mass spectrometry

Conversion of nitrate into a nitro-phenol derivative by reaction with 2-methylphenol or 2,6-dimethylphenol allowed at least 100-fold enrichment of the derivative on Lichrolut EN polymeric cartridge, and it is stable for up to 1 month on the cartridge. The derivative could be eluted with ammonia-methanol mixture. This reaction for nitrate determination has permitted a choice of final measurement by UV-Vis spectrophotometry, liquid chromatography or gas chromatography-mass spectrometry when the limits of detection were 10, 6 and 3 μg l⁻¹, respectively, and the calibration range 20 μg to 10 mg l⁻¹ nitrate. The method has been validated by spiking natural water samples, when the recovery of nitrate was 98.5-108.4% (relative standard deviation 2.5-6.1%).     

A novel stopped flow injection-amperometric procedure for the determination of chlorate

A novel stopped flow injection—amperometric (sFI-Amp) procedure for determination of chlorate has been developed. The reaction of chlorate with excess potassium iodide and hydrochloric acid, forming iodine/triiodide that is further electrochemically reduced at a glassy carbon electrode at +200 mV versus Ag/AgCl electrode is employed. In order to increase sensitivity without using of too high acid concentration, promoting of the reaction by increasing reaction time and temperature can be carried out. This can be done without increase of dispersion of the product zone by stopping the flow while the injected zone is being in a mixing coil which is immersed in a water bath of 55 ± 0.5 °C. In a closed system of FIA, a side reaction of oxygen with iodide is also minimized. Under a set of conditions, linear calibration graphs were in the ranges of 1.2 × 10⁻⁶-6.0 × 10⁻⁵ mol l⁻¹and 6.0 × 10⁻⁵-6.0 × 10⁻⁴ mol l⁻¹. A sample throughput of 25 h⁻¹ was accomplished. Relative standard deviation was 2% (n = 21, 1.2 × 10⁻⁴ mol l⁻¹ chlorate). The proposed sFI-Amp procedure was successfully applied to the determination of chlorate in soil samples from longan plantation area.     

Homogeneous stopped-flow fluorimmunoassay for gliadin determination in food samples

A homogeneous competitive immunoassay for gliadin determination is described for the first time. Gliadins are labeled with a terbium(III) chelate to form the tracer which competes with unlabeled gliadins for the active sites of anti-gliadin antibodies. The terbium(III) is released from the tracer by means of an “acidic enhancement solution” and the initial rate of the dissociation reaction is monitored using the stopped-flow mixing technique. Initial rate values obtained are different depending upon whether tracer is free or bound to antibody which can be correlated with the analyte concentration. The use of the fixed-time method has been also assayed, but it is not suitable as the results depend on the sample matrix. The detection limit of the assay using standards is 4 ng ml⁻¹, which is equivalent to 4 μg g⁻¹ in food samples, and the dynamic range of the method is 0.008-0.36 μg ml⁻¹. The precision, expressed as the percentage of relative standard deviation, was 5-6.2%. The method has been applied to the analysis of gluten-containing and gluten-free food samples, with recoveries ranging from 83.8 to 115.1%.     

Interference modelling, experimental design and pre-concentration steps in validation of the Fenton's reagent for pesticides determination

The determination of atrazine in real samples (commercial pesticide preparations and water matrices) shows how the Fenton's reagent can be used with analytical purposes when kinetic methodology and multivariate calibration methods are applied. Also, binary mixtures of atrazine-alachlor and atrazine-bentazone in pesticide preparations have been resolved. The work shows the way in which interferences and the matrix effect can be modelled. Experimental design has been used to optimize experimental conditions, including the effect of solvent (methanol) used for extraction of atrazine from the sample. The determination of pesticides in commercial preparations was accomplished without any pre-treatment of sample apart from evaporation of solvent; the calibration model was developed for concentration ranges between 0.46 and 11.6 × 10⁻⁵ mol L⁻¹ with mean relative errors under 4%. Solid-phase extraction is used for pre-concentration of atrazine in water samples through C₁₈ disks, and the concentration range for determination was established between 4 and 115 μg L⁻¹approximately. Satisfactory results for recuperation of atrazine were always obtained.     

On-line sorptive preconcentration platform incorporating a readily exchangeable Oasis HLB extraction micro-cartridge for trace cadmium and lead determination by flow injection-flame atomic absorption spectrometry

A simple, sensitive and inexpensive flow injection solid phase extraction (SPE) system was developed for automatic determination of trace level concentrations of metals. The potentials of this novel scheme, coupled to flame atomic absorption spectrometry (FAAS), were demonstrated for trace cadmium and lead determination in environmental water samples. The method was based on on-line chelate complex formation of target species with ammonium diethyldithiophosphate (DDTP), retention onto the surface of reversed-phase poly(divinylbenzene-N-vinylpyrrolidone) co-polymeric beads (Oasis HLB) and elution with methanol prior to atomization. A special PVC adapter was designed for easy and rapid replacement of the commercially available SPE cartridge. All main chemical and hydrodynamic parameters affecting the complex formation, sorption and elution of the analyte were optimized thoroughly. Moreover, the effect of potential interfering species occurring in environmental samples was also explored.For 90 s preconcentration time, enhancement factors of 155 and 180, detection limits (3s) of 0.09 μg L^− 1 and 0.9 μg L^− 1 and relative standard deviations (R.S.D.) of 2.9% (at 4.0 μg L^− 1) and 2.6% (at 20.0 μg L^− 1) were obtained for cadmium and lead, respectively, with a sample throughput of 24 h^− 1. The measurement trueness of the developed method was evaluated by analyzing a certified reference material and spiked environmental water samples. The proposed method is well suited to detect the target elements at concentration levels below the maximum allowed concentrations endorsed by the European Framework Directive (2008/105/EC) in inland and coastal waters.     

Selective spectrofluorimetric method for paracetamol determination through coumarinic compound formation

A spectrofluorimetrical selective method was designed for determination of paracetamol in tablets. This important technique can be characterized by its sensitivity, simplicity, celerity and cheaper cost than current official methods. The employed methodology involves coumarinic compound formation obtained by reaction between paracetamol and ethylacetoacetate (EAA) in the presence of sulphuric acid as catalyst. The reaction product is highly fluorescent at 478 nm, being excited at 446 nm.The linear concentration range of the application was 0.1-0.4 μg/ml of paracetamol and the detection limit was 57 ng/ml.The influence of different variables was studied and optimized through chemometric techniques. Applying the above-mentioned method good results were obtained with regard to pharmaceutical formulations containing paracetamol. Therefore, it is relevant to suggest this profitable technique for medicament control analysis.     

A green analytical procedure for flow-injection determination of nitrate in natural waters

Nitrate determination in waters is generally carried out with cadmium filings and carcinogenic reagents or by reaction with phenolic compounds in highly concentrated sulfuric acid medium. In this work, it was developed a green analytical procedure for nitrate determination in natural waters based on direct spectrophotometric measurements in ultraviolet, using a flow-injection system with an anion-exchange column for separation of nitrate from interfering species. The proposed method employs only one reagent (HClO₄) in a minimum amount (equivalent to 18 μL concentrated acid per determination), and allowed nitrate determination within 0.50-25.0 mg L⁻¹, without interference of up to 200.0 mg L⁻¹ humic acid; 1.0 mg L⁻¹ NO₂⁻; 200.0 mg L⁻¹ PO₄³⁻; 75.0 mg L⁻¹ Cl⁻; 50.0 mg L⁻¹ SO₄²⁻ and 15.0 mg L⁻¹ Fe³⁺. The detection limit (99.7% confidence level) and the coefficient of variation (n = 20) were estimated as 0.1 mg L⁻¹ and 0.7%, respectively. The results obtained for natural water samples were in agreement with those achieved by the reference method based on nitrate reduction with copperized cadmium at the 95% confidence level.     

Quality control of Metamitron in agrochemicals using Fourier transform infrared spectroscopy in the middle and near range

Two vibrational spectrometry-based methodologies were developed for Metamitron determination in pesticide formulations. Fourier transform-middle infrared (FT-MIR) procedure was based on the extraction of Metamitron by CHCl₃ and latter determination by peak area measurement between 1556 and 1533 cm⁻¹, corrected with a two points baseline established from 1572 to 1514 cm⁻¹. Fourier transform-near infrared (FT-NIR) determination was made after the extraction of Metamitron in acetonitrile and measuring the peak area between 6434 and 6394 cm⁻¹ corrected using a two points baseline defined between 6555 and 6228 cm⁻¹. Repeatability, as relative standard deviation, of 5 independent measurements at mg g⁻¹ concentration level, of 0.16% and 0.07% for MIR and NIR and a limit of detection of 0.03 and 0.004 mg g⁻¹ were obtained for MIR and NIR, respectively.NIR determination provides a sample frequency of 120 h⁻¹, higher than that found by MIR and liquid chromatographic methods (60 and 15 h⁻¹, respectively). On the other hand, the NIR method reduces the solvent consumption and waste generation, to only 1 ml acetonitrile per sample as compared with 3.4 ml chloroform required for the MIR determination and 60 ml acetonitrile used in the chromatographic reference procedure. So, vibrational procedures can be considered serious alternatives to long and time consuming chromatographic methods usually recommended for quality control of commercially available pesticide formulations.     

Fluorometric determination of fluoride ion by reagent tablets containing 3-hydroxy-2'-sulfoflavone and zirconium(IV) ethylenediamine tetraacetate

A reagent tablet for determination of fluoride ion has been prepared using ethylenediamine-N,N,N′,N′-tetraacetate complex of zirconium (Zr-EDTA), 3-hydroxy-2′-flavone (FS) and an appropriate pH buffer. Dissolving of the tablet into water exhibits an intense blue fluorescence (λ_max = 460 nm) upon excitation at 377 nm and the fluorescence intensity decreases with the presence of fluoride ion. Hence, a simple fluorescent detection procedure for fluoride ion in aqueous media was successfully constructed with this tablet. The principle of this detection system is the ligand exchange reaction of FS bound to Zr-EDTA with fluoride ion. The present system provides an easy, rapid and selective determination method of fluoride ion ranging from 5 × 10⁻⁶ to 1 × 10⁻³ mol dm⁻³. The measurement of real samples with this tablet showed the similar results as those by the common method with the Alfusone reagent.     

Chemiluminometric photo-induced determination of Strychnine in a Multicommutation flow assembly

This paper deals on the determination of Strychnine, a potent and dangerous pesticide and the analytical procedure is based on the photo-induced chemiluminescence of the pesticide by means of the Multicommutation continuous-flow methodology. Small segments of the pesticide solution were sequentially alternated with segments of the solution for adjusting the suitable medium for the photodegradation. The required time of UV irradiation was obtained by stopped-flow during 150 s; then, the resulting solution formed alternated segments with the oxidizing solution containing 5 × 10⁻³ mol l⁻¹ Ce(IV) in 0.6 mol l⁻¹ nitric acid. The calibration range, from 2 μg l⁻¹ to 50 mg l⁻¹, resulted in a linear behaviour over the range 25 μg l⁻¹ to 20 mg l⁻¹ and fitting the equation: I = 4706x + 624 with a correlation coefficient of 0.9955. The limit of detection was 2 μg l⁻¹ and the sample throughput 15 h⁻¹. After testing the influence of a large series of potential interferents, the method was applied to different kinds of samples.