Room-temperature luminescence optosensings based on immobilized active principles actives: Application to nafronyl and naproxen determination in pharmaceutical preparations and biological fluids

This paper presents two easy and selective methods for determining the active principles nafronyl (NFL) and naproxen (NAP), using a flow-through fluorescence optosensor based on the on-line immobilization on a nonionic-exchanger (Silica Gel, Davisil™ and Amberlite XAD 7, respectively) solid support. The determination was performed in 5×10⁻³ M HAc/NaAc buffer solution at pH 5 for NFL and 15×10⁻³ M glycine/HCl buffer solution at pH 2.5 for NAP at a working temperature of 20 °C. The fluorescence intensities were measured at λ_ex/em=294/336 nm and λ_ex/em=332/354 nm for NFL and NAP, respectively. The response time for these optosensors were practically instant, obtaining a linear concentration range between 0 and 700.0 ng ml⁻¹ with a detection limit of 20.8 ng ml⁻¹, an analytical sensitivity of 10.1 ng ml⁻¹ and a standard deviation of 1.27% at a 500 ng ml⁻¹ concentration level for NFL and a linear concentration range between 0 and 200.0 ng ml⁻¹ with the detection limit of 13.3 ng ml⁻¹, an analytical sensitivity of 6.0 ng ml⁻¹ and a standard deviation of 3.52% at a 100 ng ml⁻¹ concentration level for NAP. The proposed methods were satisfactorily applied to real samples (three commercial formulations and urine samples). The effects of the possible interferences were evaluated in all cases.     

Quantification of sex pheromone from Anocentor nitens by gas chromatography-mass spectrometry-selected ion monitoring

Two highly sensitive and selective methods based on gas chromatography coupled to mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode have been developed for the quantification of 2,6-dichlorophenol (2,6-DCP), a sex pheromone of the tick females of Anocentor nitens. Standard addition method and calibration curve techniques using 5-bromine-4-hydroxy-3-methoxybenzaldehyde (5-BrV) as internal standard (IS) afforded detection limit of 0.1 ng ml⁻¹. The calibration curve was linear over the concentration range from 0.5 to 500 ng ml⁻¹ for 2,6-DCP. Results show that the concentration range of sex pheromone in the extracts samples was 1.08-10.35 ng ml⁻¹. The methods developed provided reliable procedures to determine amounts of 2,6-DCP present in ticks.     

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.     

Partial least squares multicomponent fluorimetric determination of fluoroquinolones in human urine samples

Ternary mixtures of fluoroquinolones, with a 7-piperazinyl substituted group have been simultaneously determined in human urine samples by application of a multivariate calibration partial least squares (PLS) model. The calibration set was designed with 15 urine samples containing different concentrations of the three fluoroquinolones and 16 blank urine samples. The concentration range for the fluoroquinolones were up to 25 ng ml⁻¹ for norfloxacin (NOR), 80 ng ml⁻¹ for ofloxacin (OFLO) and 300 ng ml⁻¹ for enoxacin (ENO). The method is based on the native fluorescence emission of these compounds in sodium dodecyl sulfate (SDS) medium, at pH 4.0, when exciting at 277 nm. A selection of the emission wavelength range used for the analysis was made for each component. Intraday and interday precision values were determined. Figures of merit as selectivity, sensitivity, limit of detection (LOD) and analytical sensitivity were also calculated. Using the standard addition methodology, five urine samples from five different persons, fortified with three concentration levels of the fluoroquinolones, were analyzed. The limits of detection in urine were 10.0, 0.5 and 0.8 ng ml⁻¹ for ENO, NOR and OFLO, respectively.     

Determination of cadmium by flame atomic absorption spectrometry after preconcentration on naphthalene-methyltrioctylammonium chloride adsorbent as tetraiodocadmate (II) ions

A sensitive and simple solid-phase preconcentration procedure for enrichment of cadmium prior to analysis by flame atomic absorption spectrometry (FAAS) is described. The method is based on the adsorption of cadmium as CdI₄²⁻ on naphthalene-methyltrioctylammonium chloride adsorbent, elution by nitric acid and subsequent determination by FAAS. The effect of pH, iodide concentration, sample flow rate, volume of the sample and diverse ions on the recovery of the analyte was investigated and optimum conditions were established. A preconcentration factor of 40 was achieved using the optimum conditions. The calibration graph was linear in the range 1-100 ng ml⁻¹ cadmium in the initial solution. The detection limit based on the 3S_b criterion was 0.6 ng ml⁻¹ and the relative standard deviations (RSD) were 3.9 and 1.05% for 5 and 40 ng ml⁻¹, respectively (n=8). The method was successfully applied to the determination of cadmium added to river, tap and Persian Gulf water samples.     

Near-infrared fluorimetric determination of nucleic acids by shifting the ion-association equilibrium between heptamethylene cyanine and Alcian blue 8GX

A new method based on near-infrared (near-IR) fluorescence recovery, employing a two-reagent system which is composed of an anionic heptamethylene cyanine (HMC) and a polycationic phthalocyanine dye, Alcian blue 8GX, is presented for the determination of nucleic acids. With a maximum excitation wavelength at 766 nm and a maximum emission wavelength at 796 nm, the fluorescence recovery is linear with the concentration of nucleic acids added. Factors including the acidity of the medium, the reaction time, the optimal ratio of the two reagents, as well as the influence of foreign substance were all investigated. Meanwhile, the mechanism of fluorescence recovery was also studied. Under the optimal conditions, the linear ranges of the calibration curves were 10-250 ng ml⁻¹ for calf thymus DNA (CT DNA) and 10-200 ng ml⁻¹ for yeast RNA. The detection limits were 6.8 ng ml⁻¹ for CT DNA and 6.3 ng ml⁻¹ for yeast RNA, respectively. The method has been applied to the analysis of practical samples and the recovery results were satisfactory.     

Determination of carbendazim, fuberidazole and thiabendazole by three-dimensional excitation-emission matrix fluorescence and parallel factor analysis

We simultaneously determined carbendazim, fuberidazole and thiabendazole by excitation-emission matrix (EEM) fluorescence in combination with parallel factor analysis (PARAFAC). Three-way deconvolution provided the pure analyte spectra from which we estimated the selectivity and sensitivity of the pesticides, and the relative concentration in the mixtures from which we established a linear calibration. Special attention was given calculating such figures of merit as precision, sensitivity and limit of detection (LOD), derived from the univariate calibration curve. The method, which had a relative precision of around 2-3% for the three pesticides, provided limits of detection of 20 ng ml⁻¹ for carbendazim, 4.7 ng ml⁻¹ for thiabendazole and 0.15 ng ml⁻¹ for fuberidazole. The accuracy of the method, evaluated through the root mean square error of prediction (RMSEP), was 27.5, 1.4, and 0.03 ng ml⁻¹, respectively, for each of the pesticides.     

Potassium permanganate-glyoxal chemiluminescence system for flow injection analysis of cephalosporin antibiotics: cefalexin, cefadroxil, and cefazolin sodium in pharmaceutical preparations

A sensitive flow injection chemiluminescence (FL-CL) method for the determination of cephalosporin antibiotics, was developed. The method was based on that cephalosporin antibiotics could enhance the CL reaction of glyoxal and KMnO₄ in sulfuric acid. Method development included the optimization of reagent concentrations and flow-rate. Under the optimized conditions, three cephalosporin antibiotics: cefalexin, cefadroxil, and cefazolin sodium, were determined. The detection limits of the method are 10 ng ml⁻¹ cefalexin, 2 ng ml⁻¹ cefadroxil, and 2 ng ml⁻¹ cefazolin sodium. The method was successfully applied to the determination of three cephalosporin antibiotics in pharmaceutical preparations.     

Direct spectrophotometric determination of calcium in clinical samples with carboxyazo-p-CH3

A highly sensitive and relatively interference-free spectrophotometric method for determination of calcium is described. The method is based on the reaction between calcium ions and carboxyazo-p-CH₃ in aqueous citrate medium of pH 7, to form a blue complex with maximum absorption at 716 nm. The calibration is linear up to 0.12 μg ml⁻¹ calcium with a repeatability (R.S.D.) of 1.0% at a concentration of 0.04 μg ml⁻¹ (n=5). The molar absorptivity of the complex and Sandell’s sensitivity are 3.5×10⁵ l mol⁻¹ cm⁻¹ and 0.11 ng cm⁻², its 10σ limit of quantification and the 3σ limit of detection were found to be 0.3 ng ml⁻¹ and 0.09 ng ml⁻¹ respectively. The influence of reaction variables and the effect of interfering ions are studied; no interference was observed in clinical samples. The proposed method has been applied directly to the determination of calcium in clinical samples without the need for pre-concentration, masking metal ions and digesting samples.     

Kinetic method for determination of nanogram amounts of copper(II) by its catalytic effect on hexacynoferrate(III)-citric acid indicator reaction

This paper describes a highly sensitive, selective catalytic-kinetic-spectrophotometric method for the determination of copper(II) concentration as low as 6 ng ml⁻¹. The method is based on the catalytic effect of copper(II) on the oxidation of citric acid by alkaline hexacyanoferrate(III). The reaction was followed by measuring the decrease in absorbance of hexacyanoferrate(III) at 420 nm (λ_max of [Fe(CN)₆]³⁻, ∈ = 1020 dm³ mol⁻¹ cm⁻¹). The dependence of rate of the indicator reaction on the reaction variables has been studied and discussed to propose a suitable mechanism to get a relation between the reaction rate and [Cu²⁺]. A fixed time procedure has been used to obtain a linear calibration curve between the initial rate and lower [Cu²⁺] or log[Cu²⁺] in the range 1 × 10⁻⁷ to 4 × 10⁻⁴ mol l⁻¹ (6.35-25,400 ng ml⁻¹). The detection limit has been calculated to be 4 ng ml⁻¹. The maximum average error is 3.5%. The effect of the presence of various cations, commonly associated with copper(II) and some anions has also been investigated and discussed. The proposed method is sensitive, accurate, rapid and inexpensive compared to other techniques available for determination of copper(II) in such a large range of concentration. The new method has been successfully applied for the determination of copper(II) in various samples.     

Flow-injection chemiluminescent determination of metoclopramide hydrochloride in pharmaceutical formulations and biological fluids using the [Ru(dipy)(3)(2+)]-permanganate system

A flow-injection (FI) methodology using (2,2′-dipyridyl) ruthenium(II) [Ru(dipy)₃²⁺] chemiluminescence (CL) was developed for the rapid and sensitive determination of metoclopramide hydrochloride. The method is based on the CL reaction of metoclopramide with Ru(dipy)₃²⁺ and KMnO₄ in a sulfuric acid medium. Under the optimum conditions, a calibration graph was obtained over the concentration range 0.005-3.5 μg ml⁻¹ with a limit of detection (S/N=2) of 1 ng ml⁻¹. The correlation coefficient was 0.99993 (n=8) with a relative standard deviation of 0.48% for 10 determinations of 1 μg ml⁻¹ of drug. The method was successfully applied to the determination of metoclopramide in pharmaceutical preparations and biological fluids after IP administration of 25 mg kg⁻¹ dose to rats. The elimination half-life was 2.5±0.4 h.     

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.     

Synthesis of a new biacridine and its use as the chemiluminescent probe for immunoassay of carcinoembryonic antigen

A new biacridine compound, 10,10′-dimethyl-3,3′-disulfo-9,9′-biacridine (DMDSBA) was synthesized, and its chemiluminescent characteristics were investigated in detail. DMDSBA was used to label anti-CEA antibody. The labeling ratio was estimated to be 1.15-1.32, and the average labeling ratio was 1.25. The results show that there are no obvious changes in the immunoreactivity of the labeled anti-CEA antibody and the quantum efficiency of DMDSBA after attaching to the anti-CEA antibody. In addition, the conditions of labeling reaction, sandwich immunoassay and chemiluminescent reaction have been studied. A new sandwich chemiluminescent immunoassay method was firstly established, and used to determine CEA in human serum, the calibration range is 1.0-100 ng ml⁻¹ and the minimal detectable concentration of CEA is 0.53 ng ml⁻¹, the relative standard deviation is 6.5% for 20 ng ml⁻¹ CEA. This method was well-matched with radio immunoassay.     

Solid phase preconcentration of iron as methylthymol blue complex on naphthalene-tetraoctylammonium bromide adsorbent with subsequent flame atomic absorption determination

A sensitive and selective preconcentration method for the determination of trace amounts of iron by atomic absorption spectrometry has been developed. Iron forms a complex with methylthymol blue at pH=3. This complex is retained by naphthalene tetraoctylammonium bromide adsorbent in a column with a height of about 2 cm. The adsorbed metal complex is then eluted from the column with nitric acid and its iron content is determined by flame atomic absorption spectrometry (FAAS). The effect of different variables such as pH, reagent concentration, flow rate and interfering ions on the recovery of the analyte was investigated. The calibration graph was linear in the range 25-350 ng ml⁻¹ of iron in the initial solution with r=0.9994. The limit of detection based on 3S_b criterion was 12 ng ml⁻¹ and the relative standard deviation for eight replicate measurements of 150 and 300 ng ml⁻¹ of iron was 3.1 and 1.8%, respectively. This procedure was successfully applied to the determination of iron in tap and sewage water samples.     

Group-specific enzyme-linked immunosorbent assay for fenitrooxon and 3-methyl-4-nitrophenol in water samples based on a polyclonal antibody

Fenitrooxon [O,O-dimethyl-O-(4-nitro-m-tolyl)phosphate] is the major metabolite of the organophosphorus insecticide fenitrothion, and 3-methyl-4-nitrophenol is its major degradation product. In the present study, we describe the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of these compounds in water samples based on a group-specific polyclonal antiserum generated with a “bifunctional hapten”, which has two functions: the conventional function of producing an antibody against an antigen and a unique function of promoting the production of the antibodies in rabbit. For application to water samples, the influence of several factors such as organic solvent, pH, and detergent was studied. Under optimized conditions, the quantitative working range of the fenitrooxon ELISA was 0.71-27 ng ml⁻¹ with a limit of detection (LOD) of 0.32 ng ml⁻¹, and the fenitrooxon concentration giving 50% reduction of the maximum signal (IC₅₀) was 4.2 ng ml⁻¹. The quantitative working range of the 3-methyl-4-nitrophenol ELISA was 0.67-27 ng ml⁻¹ with a LOD of 0.38 ng ml⁻¹ and an IC₅₀ of 3.7 ng ml⁻¹. No significant matrix effect originating from the water sample (river water, tap water, purified water, and bottled water) was shown by addition of Tween 20 to the assay buffer. Water samples spiked with each of these compounds at 1, 5, 10, and 20 ng ml⁻¹ were directly analyzed without extraction and clean-up by the proposed ELISA. The mean recovery was 100.9%, and the mean coefficient of variation (CV) was 7.7% for the fenitrooxon ELISA and for the 3-methyl-4-nitrophenol ELISA, the mean recovery was 97.6%, and the mean CV was 7.2%. The proposed ELISA allows precise and accurate determination of these compounds in water at such low levels.     

Determination of proteins with Alizarin Red S by Rayleigh light scattering technique

A new protein determination method by enhanced Rayleigh light scattering (RLS) technique has been developed. In acid condition (pH=3.60), RLS of 1,2-dihydroxyanthraquinone-3-sulfonate (Alizarin Red S) can be greatly enhanced by addition of proteins, resulting in two characteristic peaks, 360 and 505 nm, respectively. The new protein assay is based on the RLS enhancement and spectrum change. The optimum condition for the reaction was investigated. The linear range is 0.20-24.9 μg ml⁻¹ for BSA and 0.20-15.5 μg ml⁻¹ for HSA. The detection limits (S/N=3) are 9.59 ng ml⁻¹ for BSA and 9.51 ng ml⁻¹ for HSA. The results of determination for human serum samples were comparable to those obtained by Bradford method. The binding stoichiometry was determined.     

A rapid determination of propiverine and its N-oxide metabolite in human plasma by high performance liquid chromatography-electrospray ionization tandem mass spectrometry

A simple, fast and sensitive high-performance liquid chromatography (HPLC)-electrospray ionization (ESI) tandem mass spectrometric method (LC-MS/MS) has been developed for determination of propiverine and propiverine N-oxide metabolite in human plasma using oxybutynin as internal standard. Instead of extracting propiverine from plasma using organic solvents, which should be separated from the aqueous phase and evaporated before injecting the sample into the chromatograph, plasma sample containing propiverine and N-oxide was directly injected after precipitating proteins with acetonitrile. Numerous compounds in the plasma did not interfere with the highly specific multiple reaction monitoring in tandem mass spectrometric detection following C₈ reversed-phase chromatographic separation under conditions that eluted propiverine, N-oxide and oxybutynin within 2 min (0.1% formic acid in water/acetonitrile, 25:75, v/v). The LC-MS/MS method and an alternative LC-MS method, using methyl-t-butyl ether extraction and selected ion monitoring, were validated over 1-250 ng ml⁻¹ of propiverine and 2 to 500 ng ml⁻¹ of N-oxide, and successfully applied in a pharmacokinetic study. The lower limit of quantitation was 1 ng ml⁻¹ for propiverine and 2 ng ml⁻¹ for N-oxide in both methods.     

An immunoassay for bisphenol A based on direct hapten conjugation to the polystyrene surface of microtiter plates

Based on direct hapten coated format a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for bisphenol A (BPA) was developed. Polystyrene surface was modified by 3-Aminopropyltriethoxysilane (APTES) to produce amino groups after H₂SO₄/HNO₃-pre-treatment. 4,4-bis (4-hydroxyphenyl) valeric acid (BVA) which is analogue of BPA, was successfully immobilized on the surface of microtiter plates by N,N′-dicyclohexylcarbodiimide (DCC) method. The essential steps of the assay were optimized, especially blocking procedure which is key step to prevent unspecific binding of antibody. The results indicated that compared with hapten-protein coated format (IC₅₀ = 176.67 ng ml⁻¹, LOD = 15.90 ng ml⁻¹), the direct hapten coated format (IC₅₀ = 23.50 ng ml⁻¹, LOD = 0.27 ng ml⁻¹) could improve assay sensitivity and the detection ranges were 2.30 ng∼157.60 ng ml⁻¹ with good signal reproducibility (P value > 0.05) after careful optimization of assay conditions. Tap water samples and seawater samples were spiked with a known amount of BPA and measured by ciELISA. The average recoveries were between 70 and 142%. As far as we are aware this is the most sensitive ELISA for BPA yet reported.     

Determination of naproxen with solid substrate room temperature phosphorimetry based on an orthogonal array design

A solid substrate room temperature phosphorimetric method (SSRTP) for the determination of naproxen in pharmaceutical products was developed. The experimental conditions were optimized by a L₂₅ (5⁶) orthogonal array design (OAD) with five factors at five levels using statistical analysis. The five factors contained pH value of the sample solution, drying time (t_d) and drying temperature (T_d) of solid substrate paper in the oven, concentration (c_I⁻) of heavy atom (I⁻), and exposure time (t_e) of solid substrate paper after being dried. The pH value, t_d, c_I⁻ and t_e had significant influences on the measurement of phosphorescence intensity. The optimization for sample preparation improved greatly the analytical performance of SSRTP. Under the optimal conditions, naproxen can be determined in a linear range from 10 to 400 ng ml⁻¹ with a detection limit of 2.7 ng ml⁻¹ at 3σ. The method has been applied satisfactorily to the determination of naproxen in a commercial product.     

Resonance Rayleigh scattering, frequency doubling scattering and second-order scattering spectra of the heparin-crystal violet system and their analytical application

In pH 6.0-11.2 Britton-Robinson buffer solution, binding of heparin with crystal violet (CV) can result in a significant enhancement of resonance Rayleigh scattering (RRS) and resonance non-linear scattering, such as frequency doubling scattering (FDS) and second-order scattering (SOS). Their maximum scattering wavelengths, λ_ex/λ_em, appear at 492 nm/492 nm for RRS, 984 nm/492 nm for FDS and 492 nm/984 nm for SOS, respectively. The optimum conditions of the reaction, the influencing factors and the relationship between the three scattering intensities and the concentration of heparin have been investigated. New methods for the determination of trace amounts of heparin based on the RRS, FDS and SOS methods have been developed. The methods exhibit high sensitivities, the detection limit for heparin is 2.9 ng ml⁻¹ for the RRS method, 3.5 ng ml⁻¹ for the FDS method and 3.3 ng ml⁻¹ for the SOS method. The methods have good selectivity and were applied to the determination of heparin in heparin sodium injection samples with satisfactory results.