Solid-phase fluorescence spectroscopy for the determination of acetylsalicylic acid in powdered pharmaceutical samples

A rapid, simple and rugged procedure without requiring any prior sample treatment was developed for the determination of acetylsalicylic acid (ASA) in tablets formulations by solid-phase fluorescence spectroscopy. The method was carried out on powdered samples, consisting of an active substance dispersed in lactose, maize starch, talc and magnesium stearate. Previous knowledge of the sample bulk composition is needed for proper application of the method. Wavelengths for maximum excitation and emission were 288 and 318 nm, respectively, and the fluorescence intensity was linear with ASA concentration within the 50-170 mg g⁻¹ range. Detection and quantification limits were 2.2 and 7.3 mg g⁻¹, and the analytical frequency was 200 h⁻¹. For a typical sample, the relative standard deviation of results was estimated as 2.3% (n = 10). Accuracy was assessed by comparing the analytical results obtained with the proposed method with those related to a reference method recommended by British Pharmacopoeia: no differences between the methods were found at the 95% confidence level.     

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.     

Fluorescence resonance energy transfer between acridine orange and rhodamine 6G and its analytical application for vitamin B12 with flow-injection laser-induced fluorescence detection

By coupling flow-injection with laser-induced fluorescence detection, a setup was developed and a novel method combining fluorescence resonance energy transfer (FRET) and flow-injection analysis (FIA) was proposed for the determination of vitamin B₁₂ (VB₁₂) based on its fluorescence quenching on the system of acridine orange (AO)/rhodamine 6G (R6G). The effective energy transfer could occur between AO and R6G in the dodecyl benzene sodium sulfonate (DBS) while 454 nm argon laser was used as the excitation source, and as a result, the fluorescence emission of R6G has been increased significantly. It was found that the fluorescence of the above system could be sharply diminished by VB₁₂. By using the mixed solution AO-R6G-DBS and the same solution containing VB₁₂ as the carrier and sample, respectively, a series of negative peaks which could be applied for the quantification of VB₁₂ were obtained. The detection limit for VB₁₂ was 1.65 × 10⁻⁶ mol/L. The linear range for determining VB₁₂ was 4 × 10⁻⁴ to 2 × 10⁻⁶ mol/L (correlation coefficient, r = 0.9923). The method was applied to measure VB₁₂ injections with satisfactory results.     

Use of a solid sensing zone implemented with unsegmented flow analysis for simultaneous determination of thiabendazole and warfarin

For the first time, a solid sensing zone implemented with unsegmented flow analysis is described for the simultaneous determination of two pesticides, thiabendazole and warfarin. The system works as a simple and rapid spectrofluorimetric biparameter sensor. The sensor is based on the retention of the analytes on the sensing solid zone (octadecyl silane C₁₈ gel) placed in the detection zone itself into a quartz flow-cell. A temporary sequentiation in the arrival of the analytes to the sensing zone is achieved by on line separation using a pre-column of the same gel placed just before the flow cell. Thiabendazole is determined the first (using methanol 30% (v/v) as carrier/elution solution) because it passes through the pre-column while warfarin is strongly retained in it. Then, warfarin is conveniently eluted from the pre-column (using methanol 50% (v/v) as carrier/elution solution) the intrinsic fluorescence peak height measured at an excitation wavelength of 309 nm and an emission wavelength of 368 nm is used as analytical signal. Using a low sample volume (40 μl), the analytical signal showed a very good linearity in the range 10-800 ng ml⁻¹ and 2-40 μg ml⁻¹ with detection limits of 2.35 ng ml⁻¹ and 0.54 μg ml⁻¹ for thiabendazole and warfarin, respectively. The sensor was satisfactorily applied to the determination of these two analytes in pesticides and pharmaceutical preparations.     

CoFe2O4 nano-particles functionalized with 8-hydroxyquinoline for dispersive solid-phase micro-extraction and direct fluorometric monitoring of aluminum in human serum and water samples

A simple dispersive solid-phase micro-extraction method based on CoFe₂O₄ nano-particles (NPs) functionalized with 8-hydroxyquinoline (8-HQ) with the aid of sodium dodecyl sulfate (SDS) was developed for separation of Al(III) ions from aqueous solutions. Al(III) ions are separated at pH 7 via complex formation with 8-HQ using the functionalized CoFe₂O₄ nano-particles sol solution as a dispersed solid-phase extractor. The separated analyte is directly quantified by a spectrofluorometric method at 370 nm excitation and 506 nm emission wavelengths. A comparison of the fluorescence of Al(III)–8-HQ complex in bulk solution and that of Al(III) ion interacted with 8-HQ/SDS/CoFe₂O₄ NPs revealed a nearly 5-fold improvement in intensity. The experimental factors influencing the separation and in situ monitoring of the analyte were optimized. Under these conditions, the calibration graph was linear in the range of 0.1–300 ng mL⁻¹ with a correlation coefficient of 0.9986. The limit of detection and limit of quantification were 0.03 ng mL⁻¹ and 0.10 ng mL⁻¹, respectively. The inter-day and intra-day relative standard deviations for six replicate determinations of 150 ng mL⁻¹ Al(III) ion were 2.8% and 1.7%, respectively. The method was successfully applied to direct determine Al(III) ion in various human serum and water samples.     

Fluorometric quantification of total d-gluconate by a flow-injection system using an immobilized-enzyme reactor

A method for the quantification of total d-gluconate by flow-injection analysis was developed using an immobilized-enzyme reactor and fluorescence detection. d-Gluconate was quantified using co-immobilized gluconate kinase (GK) and 6-phosphogluconate dehydrogenase (PGDH) reactor. d-Gluconate was phosphorylated to 6-phospho-d-gluconate by GK in the presence of ATP, and then the 6-phospho-d-gluconate produced was oxidized by PGDH with NADP⁺. The NADPH produced by the GK-PGDH reactor was monitored fluorometrically at 455 nm (excitation at 340 nm). A linear relationship between the responses and concentrations of d-gluconate was obtained in the ranges of 1.0 × 10⁻⁶-1.6 × 10⁻⁴ M. The relative standard deviation for 10 successive injections was 0.57% at the 0.1 mM level. This analytical method was applied to the quantification of d-gluconate in honeys, vinegars and noble rot wines, and the results showed good agreement with those obtained using the conventional F-kit method.     

Flow-injection fluorimetric determination of menadione using on-line photo-reduction in micellar media

A very sensitive fluorimetric method for the determination of menadione using a flow injection system is proposed. The method is based on the on-line reduction of menadione in dodecylsulphate micelles upon irradiation with UV light. The strong fluorescence of the reduced menadione in micellar medium is measured at 410 nm with excitation at 340 nm. The method shows a linear range between 2.42 and 245 ng ml⁻¹ and a limit of detection of 0.18 ng ml⁻¹. The sample throughput was 90 injections per hour. The applicability of the assay was demonstrated by analysing this vitamin in commercial pharmaceutical preparations.     

Spectroscopic and HPLC methods for the determination of alendronate in tablets and urine

Two methods (spectrophotometric and HPLC) have been developed and validated for the analysis of alendronate sodium in tablet dosage form. Both methods depend on the ability of alendronate sodium to react with o-phthalaldehyde (OPA) at basic pH to produce a light-absorbing derivative. The derivative was found to possess absorption maximum at 330 nm where neither the derivatizing agent nor the analyte had any absorption. Thus, spectroscopic method was based on the derivatization-induced absorption of alendronate sodium at 333 nm. The HPLC method was based on separation of the formed derivative from other ingredients in tablets with detection at 333 nm. Both methods were satisfactory with regard to accuracy, prescion and linearity. Moreover, a HPLC method with fluorescence detection (HPLC-FD) was developed for the quantification of alendronate sodium in urine. The method was also based on the derivatization of alendronate with OPA, but fluorescence detection was employed. Linearity, recovery, selectivity, prescision and sensitivity were satisfactory for the proposed HPLC-FD method. Yet a new quantification limit (0.6 ng ml⁻¹) for alendronate in urine was achieved.     

Detection and quantification of plasma amyloid-β by selected reaction monitoring mass spectrometry

Amyloid-β (Aβ) in human plasma was detected and quantified by an antibody-free method, selected reaction monitoring mass spectrometry (SRM-MS) in the current study. Due to its low abundance, SRM-based quantification in 10 μL plasma was a challenge. Prior to SRM analysis, human plasma proteins as a whole were digested by trypsin and high pH reversed-phase liquid chromatography (RPLC) was used to fractionate the tryptic digests and to collect peptides, Aβ_1–5, Aβ_6–16, Aβ_17–28 and Aβ_29–40(42) of either Aβ_1–40 or Aβ_1–42. Among those peptides, Aβ_17–28 was selected as a surrogate to measure the total Aβ level. Human plasma samples obtained from triplicate sample preparations were analyzed, obtaining 4.20 ng mL⁻¹ with a CV of 25.3%. Triplicate measurements for each sample preparation showed CV of <5%. Limit of quantification was obtained as 132 pM, which corresponded to 570 pg mL⁻¹ of Aβ_1–40. Until now, most quantitative measurements of Aβ in plasma or cerebrospinal fluid have required antibody-based immunoassays. Since quantification of Aβ by immunoassays is highly dependent on the extent of epitope exposure due to aggregation or plasma protein binding, it is difficult to accurately measure the actual concentration of Aβ in plasma. Our diagnostic method based on SRM using a surrogate peptide of Aβ is promising in that actual amounts of total Aβ can be measured regardless of the conformational status of the biomarker.     

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%.     

Determination of various alcohols based on a new immobilized enzyme fluorescence capillary analysis

A novel method for the determination of ethanol in tequila based on the immobilized enzyme fluorescence capillary analysis (IE-EFCA) has been proposed. Alcohol dehydrogenase (ADH) was immobilized in inner surface of a capillary and an immobilized enzyme capillary bioreactor (IE-ECBR) was formed. After nicotinamide adenine dinucleotide (NAD⁺) as an oxidizer is mixed with alcohol sample solution, it was sucked into the IE-ECBR. The fluorescence intensity of the mixed solution in the IE-ECBR was detected at λ_ex = 350 nm and λ_em = 459 nm. The experimental conditions are as follows: The reaction time is 20 min; temperature is 40 °C; the concentrations of phosphate buffer solution (pH 7.5) and NAD⁺ are 0.1 mol L⁻¹ and 5 mmol L⁻¹, respectively; immobilization concentration of ADH is 10 U L⁻¹. The determination range of ethanol is 2.0-15.0 g L⁻¹ (F = 10.44C + 6.6002, r > 0.9958); its detection limit is 1.11 g L⁻¹; and relative standard deviation is 1.9%. IE-EFCA method is applicable for the determination of the samples containing alcohol in medicine, industry and environment.     

Alternative spectrophotometric method for standardization of chlorite aqueous solutions

The chlorite ion is the principal by-product of the treatment of drinking water by chlorine dioxide. In function of the chlorite salt instability, standard solutions of this ion need standardization by iodometric titration, which is a reliable method although labor intensive and time consuming. An alternative method to standardization of aqueous chlorite solutions, based on its direct UV absorption measurement, was presented. Besides the maximum absorption (260 nm) generally used in other studies, the minimum (239 nm) and isosbestic (248 nm) wavelengths were proposed as supplementary points to chlorite quantification and their molar absorptivity coefficients were estimated (155.2 ± 0.6, 104.5 ± 1.0 and 69.0 ± 1.2 L cm⁻¹ mol⁻¹, respectively). The direct spectrophotometric determination of chlorite could be made selectively even in the presence of high concentration of major contaminants (chorine dioxide, chloride and chlorate), being a simple and rapid method, consuming very low volume of sample and generating low quantities of laboratory wastes.     

Selective determination of bisphenol A (BPA) in water by a reversible fluorescence sensor using pyrene/dimethyl β-cyclodextrin complex

A bifurcated optical fiber chemical sensor for continuous monitoring of bisphenol A (BPA) has been proposed based on the fluorescence quenching (λ_ex/λ_em = 286/390 nm) of pyrene/dimethyl β-cyclodextrin (HDM-β-CD) supramolecular complex immobilized in a plasticized poly(vinyl chloride) (PVC) membrane, in which pyrene served as a sensitive fluorescence indicator probe. The decrease of the fluorescence intensity of pyrene/HDM-β-CD complex upon the addition of BPA was attributed to the displacement of pyrene by BPA, which has been utilized as the basis of the fabrication of a BPA-sensitive fluorescence sensor. The response mechanism of the sensor was discussed in detail. The sensor exhibited a dynamic detection range from 7.90 × 10⁻⁸ to 1.66 × 10⁻⁵ mol L⁻¹ with a detection limit of 7.00 × 10⁻⁸ mol L⁻¹, and showed excellent reproducibility, reversibility, selectivity, and lifetime. The proposed sensor was successfully used for the determination of BPA in water samples and landfill leachate.     

Gel-surface enhanced fluorescence sensing system coupled to a continuous-flow assembly for simultaneous monitoring of benomyl and carbendazim

A novel, versatile and sensitive continuous-flow on-line solid phase fluorescence based system is proposed for the simultaneous determination of benomyl and carbendazim. The continuous-flow system is based on the on-line preconcentration and resolution of the pesticides on a solid sensing zone, followed by the sequential measure of their native fluorescence, monitored at 235/306 and 293/398 nm (λ_exc/λ_em for carbendazim and benomyl, respectively), and later desorption of these analytes (from the flow-through cell filled with C₁₈ silica gel) using aqueous methanol mixtures as carrier and eluent solutions.A double discrimination is used for the simultaneous monitoring of these analytes: (1) the usage of two pair of excitation/emision wavelengths, performed by the use of a multiwavelength fluorescence detection mode and (2) a temporary sequentiation in the arrival of the analytes to the sensing system by on-line separation due to the different kinetics showed by the analytes in the sorption-desorption process performed just in the solid support placed in the flow-through cell. Carbendazim is determined the first, because it shows a weaker retention in the C₁₈ bonded phase silica beads, while benomyl is strongly fixed. Then, benomyl is conveniently eluted from the flow-through sensing zone and its native fluorescence signal is measured (at 398 nm). The sensor was calibrated for two different injection volumes: 400 and 2000 μl. Using a 2000 μl sample volume, the analytical signal showed linearity in the range 0.050-1.0 and 0.020-0.50 μg ml⁻¹ with detection limits of 3.0 and 7.5 ng ml⁻¹ for carbendazim and benomyl, respectively, and R.S.D. values smaller than 2% for both analytes. A recovery study was performed on four different spiked environmental water samples at concentration levels from 0.05 to 0.35 μg ml⁻¹. The recovery percentage ranged from 97 to 104%, and from 98 to 104%, for benomyl and carbendazim, respectively.     

Determination of picloram in natural waters employing sequential injection square wave voltammetry using the hanging mercury drop electrode

This paper describes the development of a sequential injection analysis method to automate the determination of picloram by square wave voltammetry exploiting the concept of monosegmented flow analysis to perform in-line sample conditioning and standard addition. To perform these tasks, an 800 μL monosegment is formed, composed by 400 μL of sample and 400 μL of conditioning/standard solution, in medium of 0.10 mol L⁻¹ H₂SO₄. Homogenization of the monosegment is achieved by three flow reversals. After homogenization the mixture zone is injected toward the flow cell, which is adapted to the capillary of a hanging drop mercury electrode, at a flow rate of 50 μL s⁻¹. After a suitable delay time, the potential is scanned from −0.5 to −1.0 V versus Ag/AgCl at frequency of 300 Hz and pulse height of 25 mV. The linear dynamic range is observed for picloram concentrations between 0.10 and 2.50 mg L⁻¹ fitting to the linear equation I_p = (−2.19 ± 0.03)C_picloram + (0.096 ± 0.039), with R² = 0.9996, for which the slope is given in μA L mg⁻¹. The detection and quantification limits are 0.036 and 0.12 mg L⁻¹, respectively. The sampling frequency is 37 h⁻¹ when the standard addition protocol is followed, but can be increased to 41 h⁻¹ if the protocol to obtain in-line external calibration curve is used for quantification. The method was applied for determination of picloram in spiked water samples and the accuracy was evaluated by comparison with high performance liquid chromatography using molecular absorption at 220 nm for detection. No evidences of statistically significant differences between the two methods were observed.     

A green method for the determination of hypochlorite in bleaching products based on its native absorbance

A new, green method for the determination of hypochloride in bleaching products is here presented. The method, based on a flow injection system and measurement of the native absorbance of hypochlorite at 292 nm, allows the determination of hypochlorite in the range 0.07-0.42 g Cl₂ l⁻¹. In order to achieve high selectivity a mini-column containing cobalt oxide, which effectively catalyses hypochlorite decomposition to chloride and oxygen, was inserted in the flow system. The difference in absorbance of samples no circulated and circulated through the mini-column was selected as analytical signal; thus, the method only requires 20 mg of solid, reusable catalyst, and a NaOH solution of pH 10.4; providing a sample throughput of 12 samples h⁻¹ in triplicate injection. Its usefulness for analysis of bleaching products is demonstrated.     

A new resonance scattering spectral method for the determination of trace amounts of iodate with rhodamine 6G

Under the conditions of 0.04 mol L⁻¹ HCl-8.0 × 10⁻⁴ mol L⁻¹ KI, there is a fluorescence peak at 540 nm and a synchronous fluorescence peak at 540 nm for rhodamine 6G (RhG). When there is IO₃⁻, it reacts with exceed I⁻ to form I₃⁻. And I₃⁻ and RhG combine into ion association particles. The particles exhibit three resonance scattering peaks at 320, 400 and 595 nm. And there is fluorescence quenching at 540 nm. Iodine concentration is proportional to the intensity of the resonance scattering intensity at 400 nm in the range of 1.0-20 × 10⁻⁷ mol L⁻¹. And a new resonance scattering spectral (RSS) method has been described for the determination of IO₃⁻ in salt samples. The spectral results have been verified that the formation of (RhG-I₃)_n association particles and solid-liquid interfaces are the main factor that cause the fluorescence quenching and resonance scattering effects.     

Photophysical properties of gatifloxacin in aqueous solution by laser flash photolysis and pulse radiolysis

GFX in water, at pH 7.0, shows intense absorption bands with peaks at 284 and 333 nm, (ε=24,670 and 12,670 M⁻¹ cm⁻¹). Both the absorption and emission properties of GFX were pH-dependent; the pK_a values for the protonation equilibria of the ground state (5.7 and 8.9) and excited singlet state (3.6 and 7.5) of GFX were determined spectroscopically. GFX fluoresces weakly, with a maximum quantum yield for fluorescence emission (0.06) at pH 4.7. A series of experiments were performed to characterize the transient species of GFX in aqueous solution using laser flash photolysis and pulse radiolysis. GFX undergoes monophotonic photoionization with a quantum yield of 0.16 on a 355 nm laser excitation. This process leads to the formation of a long-lived cation radical with a maximum absorption at 380 nm. Triplet-triplet absorption had maximum absorption at 510 nm. The reaction of GFX with one-electron oxidant N₃• was investigated and the bimolecular rate constant was determined to be 3.1×10⁹ M⁻¹ s⁻¹.     

A simple route to nanocrystalline silicon carbide

Nanocrystalline silicon carbide has been prepared via reacting magnesium silicide (Mg₂Si) with carbon tetrachloride (CCl₄) in an autoclave at 450-600°C. X-ray diffraction patterns of the products can be indexed as the cubic cell of SiC with the lattice constant, a=4.352 Å, in good agreement with a=4.349 Å (JCPDS card No. 75-0254). The transmission electron microscopy images show that the sample mainly consists of nanoparticles with an average size from 30 to 80 nm co-existing with a small fraction of nanorods and nanowires. Typically the nanorods range from 20 to 40 nm in diameter and the nanowires have diameters of 20 nm and lengths up to 10 μm. The Raman spectrum shows a characteristic sharp peak at 790 cm⁻¹. X-ray photoelectron spectra (XPS) gives an atomic ratio of Si to C as 1.08:1.00 from the quantification of the peak intensities. Photoluminescence spectrum reveals that the SiC sample emits ultraviolet light of 328 nm. A possible mechanism and the influence of temperature on the formation of crystalline SiC are proposed.     

Ultrasensitive determination of silver ion based on synchronous fluorescence spectroscopy with nanoparticles

Based on the characteristics of synchronous fluorescence spectroscopy (SFS), a new method with high sensitivity and selectivity was developed for rapid determination of silver ion with functional cadmium sulphide (CdS) nanoparticles as a fluorescence probe. When Δλ (λ_em − λ_ex) = 215 nm, maximum synchronous fluorescence is produced at 304 nm. Under optimal conditions, functional cadmium sulphide displayed a calibration response for silver ion over a wide concentration range from 0.8 × 10⁻¹⁰ to 1.5 × 10⁻⁸ mol L⁻¹. The limit of detection was 0.4 × 10⁻¹⁰ mol L⁻¹ and the relative standard deviation of seven replicate measurements for the lowest concentration (0.8 × 10⁻¹⁰ mol L⁻¹) was 2.8%. Compared with several fluorescence methods, the proposed method had a wider linear range and improved the sensitivity. Furthermore, the concentration dependence of the synchronous fluorescence intensity is effectively described by a Langmuir-type binding isotherm.