Validated spectrofluorimetric method for the determination of tamsulosin in spiked human urine,pure and pharmaceutical preparations

A novel, sensitive and selective spectrofluorimetric method was developed for the determination of tamsulosin in spiked human urine and pharmaceutical preparations. The proposed method is based on the reaction of tamsulosin with 1‐dimethylaminonaphthalene‐5‐sulfonyl chloride in carbonate buffer pH 10.5 to yield a highly fluorescent derivative. The described method was validated and the analytical parameters of linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, recovery and robustness were evaluated. The proposed method showed a linear dependence of the fluorescence intensity on drug concentration over the range 1.22 × 10^‐7 to 7.35 × 10^‐6 M. LOD and LOQ were calculated as 1.07 × 10^‐7 and 3.23 × 10^‐7 M, respectively. The proposed method was successfully applied for the determination of tamsulosin in pharmaceutical preparations and the obtained results were in good agreement with those obtained using the reference method. Copyright © 2013 John Wiley & Sons, Ltd.     

Simplex optimization of the variables affecting the micelle-stabilized room temperature phosphorescence of 6-methoxy-2-naphthylacetic acid and its kinetic determination in human urine

This article reports the kinetic determination of 6-methoxy-2-naphthylacetic acid (6-MNA), the major metabolite of nabumetone, from micelle-stabilized room temperature phosphorescence (MS-RTP) measurements made by using the stopped-flow mixing technique. This methodology allows one to determine analytes in complex matrices without the need for a tedious separation process. It also shortens analysis times substantially. The proposed method uses simplex methodology to optimize the chemical and instrumental variables affecting the phosphorescence. It was applied to the determination of 6-MNA in human urine. The maximum phosphorescence signal is obtained within only 10 s after the sample is prepared. The maximum slope of the kinetic curve, which corresponds to the maximum rate of the phosphorescence development, is measured at lambda(ex)=273 nm and lambda(em)=516 nm. Least-squares regression was used to fit experimental data, and the detection limit, repeatability, and standard deviation for replicate samples were determined.     

Validated spectrofluorimetric method for the determination of clonazepam in pharmaceutical preparations

A simple, highly sensitive and validated spectrofluorimetric method was applied in the determination of clonazepam (CLZ). The method is based on reduction of the nitro group of clonazepam with zinc/CaCl₂, and the product is then reacted with 2‐cyanoacetamide (2‐CNA) in the presence of ammonia (25%) yielding a highly fluorescent product. The produced fluorophore exhibits strong fluorescence intensity at ?_em = 383 nm after excitation at ?_ex = 333 nm. The method was rectilinear over a concentration range of 0.1–0.5 ng/mL with a limit of detection (LOD) of 0.0057 ng/mL and a limit of quantification (LOQ) of 0.017 ng/mL. The method was fully validated and successfully applied to the determination of CLZ in its tablets with a mean percentage recovery of 100.10 ± 0.75%. Method validation according to ICH Guidelines was evaluated. Statistical analysis of the results obtained using the proposed method was successfully compared with those obtained using a reference method, and there was no significance difference between the two methods in terms of accuracy and precision. Copyright © 2015 John Wiley & Sons, Ltd.     

Investigation of luminescent banding in solid coral: the contribution of phosphorescence

This study investigates the nature and components of annual luminescent banding in massive Porites coral skeletons, with a view to refining the technique for using this banding to reconstruct past environmental conditions. Three-dimensional excitation-emission-matrix spectroscopy and optical fibre beam delivery have been used to investigate the luminescence properties of the bright and dull bands of solid coral. Six characteristic excitation/emission peaks have been identified: 280/450–600, 340/450, 370/470, 390/485, 420/505 and 450/530 nm. The first peak corresponds to protein-type fluorescence. The others are characteristic of humic acid luminescence. The difference in luminescence intensity between bright and dull bands has been quantified and characterised spectroscopically. The luminescence of the bright bands is up to 25% more intense than their neighbouring dull bands with the greatest increase in relative intensity in the long wavelength emission region, between 500 and 600 nm. The contribution of long-lived phosphorescence to the total luminescence intensity has been determined by time-resolved measurements on the 100 ms timescale. Both bright and dull bands show long-lived phosphorescence with decay times up to 1.5 s. This phosphorescence accounts for about 10% of the total luminescence intensity of bright bands. The difference in phosphorescence intensity between bright and dull bands is substantially greater than the difference in total luminescence intensity: the phosphorescence of bright bands is up to twice as intense as that of dull bands. This suggests that phosphorescence plays an important role in defining luminescent banding in coral. Furthermore, the large observable difference in phosphorescence between bright and dull bands indicates that measurement of phosphorescence profiles across growth bands in corals may prove to be a more sensitive indicator of past environmental conditions than measurements of total luminescence. Received: 18 March 1999 / Accepted: 20 December 1999     

Direct determination of phosphate in urine by sequential-injection analysis with single on-line dilution-calibration method and photometric detection

In this work we report a sequential-injection (SI) analysis method for the direct determination of phosphate in urine with on-line dilution and photometric detection. The developed SI manifold incorporated an additional dilution coil (DC) connected to one of the ports of the selection valve. On-line dilution was performed by aspirating a zone of sample with volume V(S) from the sample line into the holding coil (HC), transferring volume V(T) of that zone into the DC, and, finally, aspirating a portion of V(T) with volume V(A) back into the HC. This dilution introduces an additional dispersion of the initial sample zone represented by the effective dispersion coefficient D(eff) which can be tailored to the desired value by appropriate selection of the volumes V(S), V(T), and V(A). Actual detection of phosphates was performed by the molybdenum blue method with detection at 690 nm. By employing an on-line dilution step with a D(eff)= 100, the calibration curve for phosphate was linear in the range 0.05 x 10(-2) to 3 x 10(-2) mol L(-1) which covers the actual phosphate concentration in urine samples. The regression coefficient was r2 = 0.996, the relative standard deviation was sr = 3.9% at the 1 x 10(-2) mol L(-1) level (n = 10), and the injection frequency was 30 injections h(-1). The method was applied to urine samples with recoveries > 97%.     

Kinetic study on oxygenation of Lingula unguis hemerythrin using the stopped-flow 02-jump method

O₂-jump experiments with an improved stopped-flow apparatus have been used to study oxygenation and deoxygenation processes in Lingula unguis hemerythrin. With an O₂ electrode set in the observation cell, O₂ concentration conld be obtained directly. The reliability of this method has been compared with other conventional methods.O₂-jump (up and down) experiments were carried out with L. unguis hemerythrin at pH 6.8 (non-cooperative pH) and at pH 7.6 (cooperative pH). At pH 6.8, both O₂-jump (up) and O₂-jump (down) experiments showed single exponential processes which were consistent with the following scheme: . The value of k _on was estimated to be (4.4 ± 0.5) × 10⁵ M^–1 s ^–1, and k _off was (15 ± 5) s^–1. These values are consistent with those obtained by the temperature-jump method (Zimmer et al. 1986). At pH 7.6, O₂-jump (up) experiments showed two relaxation processes, whereas O₂-jump (down) experiments showed a single exponential process. The faster process in the O₂-jump (up) experiments could be attributed to the same process as that seen in the temperature-jump experiments (Zimmer et al. 1986). The slower process in the O₂-jump (up) experiments corresponds to the process obtained in the O₂-jump (down) experiments. The results are discussed in terms of a state with intermediate affinity in O₂-binding and with the possible existence of a slow step in O₂-binding.Abbreviations Hr hemerythrin - Hb hemoglobin - CM carboxymethyl - T-jump temperature-jump Offprint requests to: H. Kihara     

Determination of atenolol by the micelle-stabilized room-temperature phosphorescence methodology.

A micellar-stabilized room-temperature phosphorescence (MS-RTP) method for the determination of atenolol has been developed in micellar solutions of sodium dodecylsulphate (SDS) in the presence of thallium(I) as a heavy atom and sodium sulphite as an oxygen scavenger. The effects of thallium(I) nitrate, SDS and sodium sulphite concentrations on atenolol MS-RTP intensity were studied. Optimized conditions to obtain maximum sensitivity were 0.015 mol/L thallium(I) nitrate, 0.1 mol/L SDS and 0.0075 mol/L sodium sulphite. The maximum phosphorescence signal was completely developed in 10 min and the intensity was measured at lambda(ex) = 272 nm and lambda(em) = 412 nm. The linear range of application obtained was 2.01-16.00 microg/mL. The detection limit estimated from the least-squares regression analysis was 0.86 microg/mL and the relative standard deviation of 10 replicates was 1.7%. The proposed method was applied to the determination of atenolol in a pharmaceutical formulation. The quantitation was carried out by means of standard calibration, standard-additions calibration and Youden calibration. These three experiments were necessary to evaluate the presence of constant and proportional errors due to the matrix.     

Determination of 4-methylpropranolol in cerebrospinal fluid,serum, and urine by nonprotected fluid room-temperature phosphorescence using simplex optimization

A direct and simple procedure for the determination of 4-methylpropranolol, a specific beta-adrenergic receptor blocking agent, in biological fluids was developed. The method was based on the measurement of the nonprotected fluid room-temperature phosphorescence of the drug. This technique enables us to determine analytes in complex matrices without the need for a tedious prior separation process. The appropriate experimental conditions to obtain suitable reproducibility and maximum phosphorescence signal, when sodium sulfite is used to eliminate the oxygen from the solution and when potassium iodide is used as heavy atom, were studied. The optimum concentration of KI was 3.2 M. The optimization of Na(2)SO(3) (7.0 x 10(-3) M) and the accurate value of pH (10.88) were determined using a simplex as the method of optimization. A sodium carbonate-hydrogen carbonate buffer solution (5.0 x 10(-2) M) was used to adjust the value of pH. The delay time (124 micros), gate time (206 micros), and time between flashes (5 ms) were also optimized using a simplex. Under the above conditions, the maximum signal of phosphorescence appears instantly once the sample has been prepared, and the intensity was measured at lambda(ex) = 300 nm and lambda(em) = 537 nm, in the concentration range 25-500 ng/ml. Overall least-squares regression was used to find the straight line that fit the experimental data. The detection limit according to the error propagation theory was 6.2 ng/ml and the detection limit calculated as proposed by C. A. Clayton et al. (1987, Anal. Chem. 59, 2506) was 11.7 ng/ml. The repeatability was studied using 10 solutions of 200 ng/ml 4-methylpropranolol; if error propagation theory was assumed, the relative error was 1.78% and the standard deviation for replicate samples was 3.5 ng/ml. This method was successfully applied to the determination of 4-methylpropranolol in urine, serum, and cerebrospinal fluid, with recoveries of 99.3 +/- 0.5% in the case of urine, 99.8 +/- 0.2% for serum, and 101.5 +/- 1.5% for cerebrospinal fluid.     

Sensitive spectrofluorimetric determination of tizanidine in pharmaceutical preparations,human plasma and urine through derivatization with dansyl chloride

A sensitive spectrofluorimetric method was developed for the determination of tizanidine in human plasma, urine and pharmaceutical preparations. The method is based on reaction of tizanidine with 1‐dimethylaminonaphthalene‐5‐sulphonyl chloride (dansyl chloride) in an alkaline medium to form a highly fluorescent derivative that was measured at 511 nm after excitation at 383 nm. The different experimental parameters affecting the fluorescence intensity of tizanidine was carefully studied and optimized. The fluorescence–concentration plots were rectilinear over the ranges 50–500 and 20–300 ng/mL for plasma and urine, respectively, detection limits of 1.81 and 0.54 ng/mL and quantification limits of 5.43 and 1.62 ng/mL for plasma and urine, respectively. The method presents good performance in terms of linearity, detection and quantification limits, precision, accuracy and specificity. The proposed method was successfully applied for the determination of tizanidine in pharmaceutical preparations. The results obtained were compared with a reference method, using t‐ and F‐tests. Copyright © 2011 John Wiley & Sons, Ltd.     

Glucose biosensor based on the room-temperature phosphorescence of TiO2/SiO2 nanocomposite

The direct immobilization of glucose oxidase (GOD) on TiO₂/SiO₂ nanocomposite and its application as glucose biosensor were investigated. The room-temperature phosphorescence of TiO₂/SiO₂ nanocomposite can be quenched by hydrogen peroxide (H₂O₂). The detection of glucose may be accomplished by monitoring the formation of hydrogen peroxide which generated in the oxidation process of glucose with the catalysis of GOD. To our surprise, by using a 96-hole polyporous plate accessory of fluorescence spectrophotometer, the biosensor exhibits excellent linear response to glucose concentrations ranging from 1.0 × 10⁻⁹ to 1.0 × 10⁻² M with a detection limit of 1.2 × 10⁻¹⁰ M. The TiO₂/SiO₂ nanocomposite can be used as both supporting material and signal transducer. The phosphorescence intensity and color of the biosensor change obviously and even could be observed with naked eyes by continuous addition of glucose. Based on the room-temperature phosphorescence of TiO₂/SiO₂ nanocomposite, a new method of solid substrate-room-temperature phosphorimetry (SS-RTP) for glucose determination is proposed. A glucose biosensor was fabricated with wide determination concentration range, low detection limit, high sensitivity, and fast response time. And the biosensor has been successfully applied to the determination of glucose in human blood serum. The coacervation of GOD enzyme and its interaction with TiO₂/SiO₂ nanocomposite enlarge the surface area and enhance the chemical stability of GOD. The nice biocompatibility, large surface area, good chemical stability and nontoxicity of the TiO₂/SiO₂ nanocomposite have made this material suitable for functioning as biosensor.     

Optimization and validation of spectrofluorimetric method for determination of cefadroxile and cefuroxime sodium in pharmaceutical formulations

A simple, accurate, precise and validated spectrofluorimetric method is proposed for the determination of two cephalosporins, namely, cefadroxile (cefa) and cefuroxime sodium (cefu) in pharmaceutical formulations. The method is based on a reaction between cephalosporins with 1,2‐naphthoquinone‐4‐sulfonate in alkaline medium, to form fluorescent derivatives that are extracted with chloroform and subsequently measured at 610 and 605 nm after excitation at 470 and 460 nm for cefa and cefu respectively. The optimum experimental conditions have been studied. Beer's law is obeyed over the concentrations of 20–70 ng/mL and 15–40 ng/mL for cefa and cefu, respectively. The detection limits were 4.46 ng/mL and 3.02 ng/mL with a linear regression correlation coefficient of 0.9984 and 0.998, and recoveries ranging 97.50–109.96% and 95.73–98.89% for cefa and cefu, respectively. The effects of pH, temperature, reaction time, 1,2‐naphthoquinone‐4‐sulfonic concentration and extraction solvent on the determination of cefa and cefu, have been examined. The proposed method can be applied for the determination of cefa and cefu in pharmaceutical formulations in quality control laboratories. Copyright © 2013 John Wiley & Sons, Ltd.     

Spectrofluorimetric determination of tobramycin in human serum and pharmaceutical preparations by derivatization with fluorescamine

A simple, sensitive and selective spectrofluorimetric method has been developed for the determination of tobramycin (TOB) in human serum and pharmaceutical preparations. The method is based on the reaction between the primary amino group of TOB and fluorescamine in borate buffer, pH 8.5, to give a highly fluorescent derivative which is measured at 469 nm after excitation at 388 nm. The fluorescence intensity was directly proportional to the concentration over the range 300–1500 ng/mL, with a limit of detection of 65 ng/mL and limit of quantitation of 215 ng/mL. All variables were investigated to optimize the reaction conditions. The method was validated according to International Conference on Harmonization guidelines in terms of specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. Good recoveries were obtained ranging from 97.4 to 100.64%, indicating that no interference was observed from concomitants usually present in pharmaceutical dosage forms. The method was successfully, applied for the analysis of the drug substance in its pharmaceutical preparations and spiked serum samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Inhibition of calmodulin-activated Ca2+-ATPase by propranolol and nadolol

Propranolol, at concentrations ranging from 0.05 to 0.5 mM, inhibits the calmodulin-activated Ca²⁺-ATPase of human erythrocyte membranes. In the same concentration range it is without effect on the basal Ca²⁺-ATPase. The inhibition is competitive and appears to be due to membrane binding, rather than to combination with cytoplasmic calmodulin as is the case for phenothiazines. This effect of propranolol may explain its ability to open the calcium-gated potassium channel, and could also be related to its action as a β-adrenergic blocker. Nadolol, another β-adrenergic blocker, is also an inhibitor of calmodulin-activated Ca²⁺-ATPase.     

Effect of gelatin on molecular mobility in amorphous sucrose detected by erythrosin B phosphorescence

We have used phosphorescence from the triplet probe erythrosin B (Ery B) to evaluate the effect of gelatin on the molecular mobility of the amorphous sucrose matrix as a function of temperature. Ery B was dispersed in amorphous sucrose and sucrose-gelatin films at ratios of approximately 1:10(4) (probe/sucrose), and delayed emission spectra and emission decay transients were measured over the temperature range from 5 to 100 degrees C. Analysis of spectra using a lognormal function provided the peak energy and bandwidth of the emission. The emission peak frequency decreased at low (0.00022-0.0007) gelatin concentrations and increased at high (above 0.0022) gelatin concentrations, indicating that gelatin increased the extent, and thus the rate, of dipolar relaxation at low gelatin content and decreased the extent at higher gelatin content. Decay transients were well fit to a stretched exponential function at all gelatin contents and temperatures. Analysis of the emission lifetimes provided a measure of the rate of non-radiative decay to the ground state, an indicator of matrix molecular mobility. This rate increased at low (0.00022-0.0022) and decreased at high (>0.0073) gelatin wt ratios. Analysis of the effect of gelatin on the emission bandwidth, the stretching exponent beta, and the variation of lifetime across the emission band indicated that matrix dynamic site heterogeneity increased at low and decreased at high gelatin wt ratios. These results provide a novel insight into the complex dynamic effects of the gelatin polymer on the molecular mobility of the amorphous sucrose matrix.     

Development and validation of a sensitive spectrofluorimetric method for the determination of amoxapine in human plasma and urine

A selective and sensitive spectrofluorimetric method was developed and validated for the determination of amoxapine in human plasma and urine. The developed method is based on labeling with 5‐dimethylaminonaphthalene‐1‐sulfonyl chloride (dansyl chloride) and monitoring at 397 nm (excitation)/514 nm (emission). The method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, recovery and robustness. The calibration curves were linear over a concentration range of 250–2500 and 50–1250 ng/mL for plasma and urine, respectively. The LOD values were calculated to be 13.31 and 13.17 ng/mL for plasma and urine, respectively. The proposed method was applied to study of amoxapine in human plasma and urine. Copyright © 2013 John Wiley & Sons, Ltd.     

Improvement and validation of a liquid chromatographic method for the determination of levosulpiride in human serum and urine

A rapid, selective and highly sensitive reversed-phase high-performance liquid chromatography (HPLC) method was developed for the determination of levosulpiride, 5-(aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxy benzamide, in human serum and urine. The method involved the extraction with a dichloromethane followed by back-extraction into 0.025 M sulfuric acid. HPLC analysis was carried out using reversed-phase isocratic elution with a Luna C(18)(2) 5 microm column, a mobile phase of acetonitrile-0.01 M potassium hydrogen phosphate (30:70, v/v, adjusted to pH 8.5 with triethylamine), and a fluorescence detector with excitation at 300 nm and emission at 365 nm. The chromatograms showed good resolution and sensitivity and no interference of human serum and urine. The calibration curves were linear over the concentration range 0.25-200 ng/ml for serum and 0.2-20 microg/ml for urine with correlation coefficients greater than 0.997. Intra- and inter-day assay precision and accuracy fulfilled the international requirements. The mean absolute recovery for human serum was 89.8+/-3.7%. The lower limits of quantitation in human serum and urine were 0.25 ng/ml and 0.2 microg/ml, respectively, which were sensitive enough for pharmacokinetic studies. Stability studies showed that levosulpiride in human serum and urine was stable during storage, or during the assay procedure. This method was successfully applied to the study of pharmacokinetics of levosulpiride in human volunteers following a single oral administration of levosulpiride (25 mg) tablet.     

Selective spectrofluorimetric method for determination of Lisinopril in pharmaceutical preparations and in presence of hydrochlorothiazide: Application to content uniformity testing

A novel sensitive and cost‐effective spectrofluorimetric method has been developed and validated for determination of lisinopril (an angiotensin converting enzyme inhibitor) in its pure form and pharmaceutical preparations. The method is based on the reaction of the drug with ninhydrin and phenylacetaldehyde in buffered medium (pH 7.0) to form a highly fluorescent product measured at 460 nm after excitation at 390 nm. Different experimental parameters were optimized and calibration curve was constructed. The fluorescence‐concentration relationship was linear in the range of 0.15–4.0 μg mL^?1. The calculated Limit of detection (LOD) and Limit of quantitation (LOQ) were 0.04 and 0.12 μg mL^?1, respectively. The method was successfully applied for the analysis of pharmaceutical preparations containing the studied drug either alone or co‐formulated with hydrochlorothiazide. The obtained results were in agreement with those of the reported method in respect to accuracy and precession. Moreover, the method was applied content uniformity testing according to United States Pharmacopeia (USP) guidelines.