Spectrophotometric and spectrofluorimetric determination of indacaterol maleate in pure form and pharmaceutical preparations: application to content uniformity

Two simple, rapid, sensitive and precise spectrophotometric and spectrofluorimetric methods were developed for the determination of indacaterol maleate in bulk powder and capsules. Both methods were based on the direct measurement of the drug in methanol. In the spectrophotometric merthod (Method I) the absorbance was measured at 259 nm. The absorbance‐concentration plot was rectilinear over the range 1.0–10.0 µg mL^?1 with a lower detection limit (LOD) of 0.078 µg mL^?1 and lower quantification limit (LOQ) of 0.238 µg mL^?1. Meanwhile in the spectrofluorimetric method (Method II) the native fluorescence was measured at 358 nm after excitation at 258 nm. The fluorescence‐concentration plot was rectilinear over the range of 1.0–40.0 ng mL^?1 with an LOD of 0.075 ng mL^?1and an LOQ of 0.226 ng mL^?1. The proposed methods were successfully applied to the determination of indacaterol maleate in capsules with average percent recoveries ± RSD% of 99.94 ± 0.96 for Method I and 99.97 ± 0.81 for Method II. In addition, the proposed methods were extended to a content uniformity test according to the United States Pharmacopoeia (USP) guidelines and were accurate, precise for the capsules studied with acceptance value 3.98 for Method I and 2.616 for Method II. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of sensitive spectrofluorimetric and spectrophotometric methods for the determination of duloxetine in capsule and spiked human plasma

A new, sensitive and selective spectrofluorimetric method has been developed for the determination of duloxetine (DLX) in capsule and spiked human plasma. DLX, as a secondary amine compound, reacts with 7‐chloro‐4‐nitrobenzofurazon (NBD‐Cl), a highly sensitive fluorogenic and chromogenic reagent used in many investigations. The method is based on the reaction between the drug and NBD‐Cl in borate buffer at pH 8.5 to yield a highly fluorescent derivative that is measured at 523 nm after excitation at 478 nm. The fluorescence intensity was directly proportional to the concentration over the range 50–250 ng/mL. The reaction product was also measured spectrophotometrically. The relation between the absorbance at 478 nm and the concentration is rectilinear over the range 1.0–12.0 µg/mL. The methods were successfully applied for the determination of this drug in pharmaceutical dosage form. The spectrofluorimetric method was also successfully applied to the determination of duloxetine in spiked human plasma. The suggested procedures could be used for the determination of DLX in pure form, capsules and human plasma being sensitive, simple and selective. Copyright © 2014 John Wiley & Sons, Ltd.     

Two spectroscopic methods for estimation of anti-migraine medications: Sumatriptan and Zolmitriptan based on nucleophilic substitution reaction

Sensitive and selective spectrophotometric and spectrofluorimetric methods have been developed for the estimation of two anti-migraine drugs, namely sumatriptan succinate (SUM) and zolmitriptan (ZOL). These methods depend on producing a yellow-coloured product after the reaction of the two drugs with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). The reaction products exhibited maximum absorbance at 481 nm in borate buffer of pH 9 and fluorescence emission peak at 540 nm after excitation at 470 nm for the two drugs. The linear ranges were 5–60 μg/ml for SUM and 5–50 μg/ml for ZOL in the spectrophotometric method (Method I), whereas this was 0.4–4 μg/ml for SUM and 0.5–5 μg/ml for ZOL in the spectrofluorimetric method (Method II). The method validity was assessed according to International Council for Harmonisation (ICH) guidelines. Statistical analysis of the results obtained from the proposed and comparison methods confirmed that the proposed methods were highly accurate and precise. The suggested methods could be used for the determination of the mentioned drugs in both pure form and in tablets.     

On the limits of applicability of spectrophotometric and spectrofluorimetric methods for the determination of chlorophyll a/b ratio

The concentration limits for spectrophotometric and spectrofluorimetric determinations of the chlorophyll (Chl) a/b ratio in barley leaves were studied using 80% acetone extracts at room temperature. The optimum sample absorbances (at 663.2 nm – maximum of the Q_Y) band of Chl a) for the Chl a/b determination were determined. For given spectrometers and sample positions, these absorbances ranged between 0.2 and 1.0 and 0.008–0.1 for the absorption and fluorescence methods, respectively. Precision of the measurements and the distorting effects are discussed. The lower limits of both absorption and fluorescence methods depend on sensitivity of the spectrometers for the Chl b detection. The spectrophotometric determination of Chl a/b ratio at higher Chl concentrations can be distorted by the chlorophyll fluorescence signal. The extent of this distortion depends on sample-detector geometry in any given type of the spectrometer. The effect of inner filter of Chl molecules and the detection instrumental function affect the value of the upper limit for the spectrofluorimetric method. Both methods were applied to estimate the Chl a/b ratio in pigment extracts from greening barley leaves, which are characterized by a low Chl concentration and a high Chl a/b ratio at the beginning of greening process.     

Application of π acceptors to the spectrophotometric and spectrofluorimetric determination of vincamine and naftidrofuryl oxalate in their pharmaceutical preparations

Three different spectrophotometric and two spectrofluorimetric methods have been developed and validated for the determination of vincamine (VN) and naftidrofuryl oxalate (NF) in tablets. The spectrophotometric methods depend on charge transfer complex formation between each of VN and NF with 7,7,8,8‐tetracyano‐quinodimethane (TCNQ), 2,6‐dichloroquinone‐4‐chloroimide (DCQ) and 2,3‐dichloro‐5,6‐dicyano‐1,4‐benzoquinone (DDQ) at 843, 580 and 588 nm, respectively. The spectrofluorimetric methods are based on the formation of charge transfer complex between each of the two drugs and TCNQ, with measurement of the fluorophore formed at 312/375 and 284/612 nm, respectively, or with DDQ at 400/475 and 284/396 nm, respectively. In the spectrophotometric measurements, Beer's law was obeyed at concentration ranges of 1.5–16, 10–180 and 12–140 μg/ml for VN with TCNQ, DCQ, and DDQ, respectively. For NF, the corresponding concentrations were 2–28, 5–75 and 25–150 μg/ml with TCNQ, DCQ, and DDQ, respectively. In the spectrofluorimetric measurements, the ranges for VN were 0.05–0.9 and 0.3–4 μg/ml with TCNQ and DDQ, respectively, whereas for NF the ranges were 0.05–0.85 and 0.5–8 μg/ml with TCNQ and DDQ, respectively. The different experimental parameters affecting the development and stability of the formed color or fluorophore were studied and optimized and the molar ratios of the complexes were calculated. The proposed methods were validated according to ICH guidelines and were successfully applied for the determination of VN and NF in their tablet dosage forms.     

Synchronous fluorescence determination of ferulic acid with Ce(IV) and sodium tripolyphosphate

In this study, a synchronous fluorescence detection method for ferulic acid (FA) is proposed based on a redox reaction between FA and Ce(IV) sulfate in dilute sulfuric acid medium at room temperature. It was found that FA could reduce Ce(IV) to Ce(III) in acidic medium, and sodium tripolyphosphate could further enhance the intrinsic fluorescence of the Ce(III) produced. The enhanced extent of synchronous fluorescence intensity was in proportion to the concentration of FA over the range 3.0 × 10^‐8 to 1.0 × 10^‐5 mol/L. The corresponding limit of determination (S/N = 3) was 1.3 × 10^‐8 mol/L. The proposed method was applied to the determination of sodium ferulate for injection sample with satisfactory results. Copyright © 2013 John Wiley & Sons, Ltd.     

Spectroscopic determination of succinylcholine in dosage forms using eosin Y

Two simple and sensitive analytical assay methods using spectrophotometry and spectrofluorimetry techniques were developed for the estimation of succinylcholine chloride (SUC) in pharmaceutical preparations. The suggested methods are based on the formation of an ion pair complex formed between the drug and eosin Y spectrophotometrically (Method I), or the suppressive effect of succinylcholine on the native fluorescence property of eosin Y (Method II). The spectrophotometric method (Method I) involves measuring the absorbance of the complex between succinylcholine and eosin Y at 550 nm in Britton Robinson buffer of pH 3. However, the spectrofluorimetric method (Method II) involves measuring the quenching effect of the studied drug on the native fluorescence property of eosin Y at the same pH at 550 nm after excitation at 480 nm. The absorbance versus concentration of the drug is rectilinear over the range of 0.5 to 15 μg/ml. The formation constant was 3.5 × 10⁴ and the Gibb's free energy change was ?2.5 × 10⁴ J/mol. In Method II, the relative fluorescence intensity was directly proportional to SUC concentration over the range of 0.05 to 1 μg/ml. The proposed methods allowed a successful application to the estimation of succinylcholine ampoules. An explanation of the reaction pathway was postulated.     

Green spectrophotometric and spectrofluorimetric determination of biperiden hydrochloride using erythrosine B sensing probe

Erythrosine B (EB) is a food colorant antiviral xanthene dye that has many applications as a color additive in pharmaceuticals and cosmetics. Its use as a sensor for spectrofluorimetric and spectrophotometric analysis of amine-based pharmaceuticals renders many advantages because of its availability, low cost, rapid labeling, and high sensitivity. Herein, two fast and sensitive spectrofluorimetric and spectrophotometric methods were established for the estimation of the anti-Parkinson drug, biperiden (BIP) hydrochloride (HCl), in its raw material and tablet forms. The proposed methods depended on the interaction between the phenolic group of EB and the tertiary amino group of the studied analyte to form an ion-pair complex at pH 4 using the Britton Robinson buffer. The spectrofluorimetric method is based on the measurement of the quenching power of BIP HCl on the fluorescence intensity of EB at λ_ex/em = 527.0/550.9 nm. This method was rectilinear over the concentration range of 0.1–1.0 μg/mL with a limit of detection (LOD) = 0.017 μg/mL and a limit of quantification (LOQ) = 0.05 μg/mL. Meanwhile, the colorimetric method involved monitoring the absorbance of the formed ion-pair complex at 555 nm, showing a linearity range of 0.4–5.0 μg/mL with LOD = 0.106 μg/mL and LOQ = 0.322 μg/mL. The proposed methods were assessed for the greenness, indicating the greenness of the developed methods.     

Optimizing the spectrofluorimetric determination of cefdinir through a Taguchi experimental design approach

The aim of this work is to optimize a spectrofluorimetric method for the determination of cefdinir (CFN) using the Taguchi method. The proposed method is based on the oxidative coupling reaction of CFN and cerium(IV) sulfate. The quenching effect of CFN on the fluorescence of the produced cerous ions is measured at an emission wavelength (λ_em) of 358 nm after excitation (λ_ex) at 301 nm. The Taguchi orthogonal array L₉ (3⁴) was designed to determine the optimum reaction conditions. The results were analyzed using the signal‐to‐noise (S/N) ratio and analysis of variance (ANOVA). The optimal experimental conditions obtained from this study were 1 mL of 0.2% MBTH, 0.4 mL of 0.25% Ce(IV), a reaction time of 10 min and methanol as the diluting solvent. The calibration plot displayed a good linear relationship over a range of 0.5–10.0 µg/mL. The proposed method was successfully applied to the determination of CFN in bulk powder and pharmaceutical dosage forms. The results are in good agreement with those obtained using the comparison method. Finally, the Taguchi method provided a systematic and efficient methodology for this optimization, with considerably less effort than would be required for other optimizations techniques. Copyright © 2015 John Wiley & Sons, Ltd.     

Prostaglandin H synthetase as a multisubstrate enzyme. Fluorimetric study of enzyme kinetics

The kinetics of a multisubstrate enzymatic reaction catalyzed by prostaglandin H synthase (PGH-synthase, EC 1.14.99.1) was studied, using homovanillic acid, a new electron donor for the given system. Homovanillic acid was shown to be a participant in a reaction with arachidonic acid/O2 stoichiometric ratios and is oxidized to a readily fluorescing product with an absorbance maximum (excitation) at 315 nm and fluorescence maximum at 425 nm. This allows for determination of the rate of enzymatic reaction with the sensitivity exceeding by one order of magnitude that of polarographic or spectrophotometric assays. Using fluorescent techniques, the dependence of the rate of PGH-synthase reaction on substrate (arachidonic acid, O2 and homovanillic acid) concentrations was studied, and the corresponding Km values were determined. The effect of Tween-20 and Lubrol PX concentrations on the reaction rate were examined. It was shown that with a decrease in the surfactant concentration the reaction rate increases.     

Synthesis,characterization and fluorescent properties of cerium(III) glutathione complex

The cerium (III) glutathione complex was synthesized by the redox reaction of cerium (IV) with glutathione reduced (GSH) in aqueous solution. The Job‐plots indicate an ML (L = GSSG) stoichiometry of the complex. The fluorescent properties of the compound were investigated. The as‐prepared complex showed the characteristic maximum emission spectra of Ce(III) at 350 nm (λ_ex = 255 nm). The fluorescence results show that the Ce(IV) ions are first reduced to Ce(III), and then form Ce(III) complex after reacting with GSH. The complex was characterized by element analysis and FT‐IR spectra; the stability of the complex was analyzed by cyclic voltammeters and DSC‐TG as well. Finally, Ce(IV) was successfully employed to determine the concentrations of GSH in the presence of GSSG, in which the fluorescence intensities are proportional to the concentrations of GSH in the range of 1–100 nM with the detection limit of 0.05 nM of GSH, without interference from the presence of GSSG. Copyright © 2009 John Wiley & Sons, Ltd.     

Liquid chromatographic and spectrofluorimetric assays of empagliflozin: Applied to degradation kinetic study and content uniformity testing

Stability‐indicating high‐performance liquid chromatography (HPLC) and spectrofluorimetric methods were developed for determination of empagliflozin (EGF). EGF was subjected to oxidation, wet heat, photo‐degradation, acid hydrolysis and alkali hydrolysis. The alkaline degradation pathway was subjected to a kinetics study as the major product obtained after stress conditions. Arrhenius plots were constructed and the activation energies of the degradation process were calculated. HPLC was used for the kinetic study as it enabled simultaneous determination of EGF and the degradation product while the spectrofluorimetric assay was applied to content uniformity testing due to its higher sensitivity and lower limit of detection (LOD). Isocratic chromatographic elution was attained for HPLC on a Intersil® C₁₈ column (150 mm × 4 mm, 5 μm), using a mobile phase of acetonitrile–potassium dihydrogen phosphate buffer pH 4, (50:50, v/v) at a flow rate of 1 ml/min with ultraviolet (UV) detection at 225 nm. The relative fluorescence intensity was recorded by spectrofluorimeter applying synchronous mode using ?λ = 70 nm at 297.6 nm. Linearity ranges were found to be 5–50 μg/ml and 50–1000 ng/ml for HPLC and spectrofluorimetric methods, respectively.     

Sensitive spectrofluorometric method for the determination of ascorbic acid in pharmaceutical nutritional supplements using acriflavine as a fluorescence reagent

An easily performed, specific, sensitive, rapid, reliable and inexpensive procedure for the spectrofluorometric quantitation of ascorbic acid was proposed using acriflavine as a fluorescence quenching reagent. The procedure was based on the determined quenching effect of ascorbic acid on the natural fluorescence signal of acriflavine and the reaction between ascorbic acid and acriflavine in Britton–Robinson buffer solution (pH 6) to produce an ion‐associated complex. The reduction in acriflavine fluorescence intensity was detected at 505 nm, while excitation occurred at 265 nm. The relationship between quenching fluorescence intensity (?F) and concentration of ascorbic acid was linear (R² = 0.9967) within the range 2–10 μg/ml and with a detection limit of 0.08 μg/ml. No significant interference was detected from other materials often found in pharmaceutical nutritional tablets. The obtained results were compared with those from high‐performance liquid chromatography and appeared in good agreement, with no important differences in precision or accuracy. The proposed spectrofluorimetric method was used to determine the amount of ascorbic acid in a number of commercial pharmaceutical nutritional supplement tablets with a 95% confidence performance.     

Application of quinone‐based fluorophore and native fluorescence for the spectrofluorimetric determination of agomelatine in dosage form: Identification of acidic and alkaline‐ induced degradation products by LC–MS/TOF

Three spectrofluorimetric methods were developed for agomelatine (AGM) determination in commercial tablets. Method A is based on measuring the native fluorescence of AGM aqueous solution at 230/360 nm. Methods B and C are based on the formation of a charge transfer complex between AGM and 2,3‐dichloro‐5,6‐dicyano‐1,4‐benzoquinone (DDQ) and 7,7,8,8‐tetracyanoquinodimethane (TCNQ) with measurement of the formed fluorophore at 365/475 nm and 250/304 nm, respectively. The relative fluorescence intensity (RFI) of AGM–DDQ complex was greatly enhanced in the presence of methyl‐β‐cyclodextrin (CD). The methods were linear over the concentration ranges of 0.015–0.5, 0.5–8.0, 0.09–6.0 and 0.05–0.2 μg/ml for AGM‐native fluorescence, AGM–DDQ, AGM–DDQ–CD and AGM–TCNQ complexes, respectively with excellent correlation coefficients (r = 0.9999). The methods were validated as per the International Conference on Harmonization (ICH) guidelines and all validation requirements were satisfied. The developed methods were extended to the analysis of AGM in commercial tablets. Furthermore, the stability of AGM was studied under different stress conditions (alkaline, acidic, oxidative and photolytic). The potential alkaline and acidic degradation products were identified by LC–MS/TOF.     

A novel solid‐phase extraction–spectrofluorimetric method for the direct determination of atenolol in human urine

A novel, simple, sensitive and selective solid‐phase extraction (SPE)–spectrofluorimetric method has been developed for the determination of atenolol (ATE) in human urine. Because an extraction procedure is required to isolate ATE or eliminate the interfering molecules present in complex human urine for the direct spectrofluorimetric determination, a pH‐sensitive poly(acrylic acid‐ethylene glycol dimethacrylate) [poly(AA‐EGDMA)] hydrogel was developed and used as a SPE adsorbent. Some factors affecting the ATE extraction efficiency, such as washing solvent type and volume, and the volume of elution solvent were optimized. Eluates from SPE cartridges were analyzed using a spectrofluorimeter (λ_ex = 277 nm and λ_em = 300 nm). The calibration graph was linear over the concentration range 0.15–4.0 µg/mL. Limit of detection (LOD) and limit of quantification (LOQ) values were found to be 0.03 and 0.10 µg/mL, respectively. Relatively high intraday [2.06%, mean relative standard deviation (RSD)] and interday (2.6%, mean RSD) precisions were achieved. High mean recovery (95.4%) and low RSD values (3.8%) were obtained for spiked ATE in human urine. The spectrofluorimetric method presented here can be easily applied to assay trace amounts of ATE in pharmaceuticals and biological samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Esterases as a marker for growth of BY-2 tobacco cells and early somatic embryos of the Norway spruce

Growth is one of the basic properties of biological systems. The methods which are commonly used for the determination of growth are usually difficult and not very accurate. In the present work we decided to use esterase activity as a growth marker in tobacco suspension culture (BY-2 line) and in early somatic embryos of Norway spruce (clone 2/32) grown on a semi-solid medium. Esterase activity correlates well with the classical growth characteristics of BY-2 and spruce early somatic embryos. Determination of esterase activity is based on spectrophotometric and spectrofluorimetric detection of reaction products, which arise from the enzymatic hydrolysis of two substrates (p -nitrophenyl acetate and fluorescein diacetate) by esterase. The spectrophotometric method enabled us to detect approximately 10⁴ BY-2 cells and 25 spruce embryos whereas the more sensitive spectrofluorimetric method allowed us to detect approximately 800 BY-2 cells and 5 early somatic embryos of Norway spruce.     

Spectrofluorimetric determination of certain biologically active phenothiazines in commercial dosage forms and human plasma

A validated simple and sensitive spectrofluorimetric method was developed for the determination of chlorpromazine hydrochloride, promethazine hydrochloride, trifluperazine hydrochloride, thioridazine hydrochloride, perazine maleate and oxomemazine. The method was based on condensation of malonic acid/acetic anhydride (MAA) under the catalytic effect of the tertiary amine moiety of the studied phenothiazines to provide a deep yellow to brown colour with green florescence. Relative fluorescence intensity of the products was measured at λ_exc 398 nm and λ_em 432 nm. Different variables affecting the reaction were studied and optimized. The method was successfully applied for the determination of the studied drugs in commercial dosage forms. The lower detection limits allowed the application of this method for the determination of the compounds in plasma as an example of a biological fluid. In addition, the method was considered specific for the determination of tertiary amines in the presence of primary and secondary amines; as a result, it was deemed suitable for the determination of the cited drugs in the presence of their degradation products resulting from N‐dealkylation or oxidation of the corresponding sulphoxides or sulphones. Copyright © 2012 John Wiley & Sons, Ltd.     

Use of eosin for green spectroscopic determination of mebendazole

New, sensitive, and reliable spectroscopic methods were constructed for the fast determination of the anthelmintic drug mebendazole. The methods depended on the reaction of the amino group in mebendazole with eosin in acidic medium forming an ion pair complex. The first method, Method I, relied on quenching of the native fluorescence of eosin after reaction with mebendazole at pH 3.7 using acetate buffer. Fluorescence quenching was measured at 538 nm after excitation at 518 nm. This method showed a linear response over the concentration range 5.0–20.0 μg/ml. The second method, Method II, was based on measuring the absorbance of the formed complex at 554 nm; the method showed good linearity from 7.0 to 22.0 μg/ml. Different parameters that influenced the formation of the reaction product were carefully investigated to reach the optimized conditions. A comparison between the proposed methods and a previous spectrophotometric method was carried out and there was no significant difference between them. The methods could be applied successfully to determine mebendazole in its tablet form. Moreover, the methods used water as diluting solvent, which made them compatible with the ‘green’ analytical chemistry principles. No organic solvents were used throughout the study.     

Validated spectrofluorimetric method for the determination of carbamazepine in pharmaceutical dosage forms after reaction with 4‐chloro‐7‐‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl)

A sensitive and simple spectrofluorimetric method has been developed and validated for the determination of the anti‐epileptic drug carbamazepine (CBZ) in its dosage forms. The method was based on a nucleophilic substitution reaction of CBZ with 4‐chloro‐7‐nitrobenzo‐2‐ oxa‐1,3‐diazole (NBD‐Cl) in borate buffer (pH 9) to form a highly fluorescent derivative that was measured at 530 nm after excitation at 460 nm. Factors affecting the formation of the reaction product were studied and optimized, and the reaction mechanism was postulated. The fluorescence–concentration plot is rectilinear over the range of 0.6–8 µg/mL with limit of detection of 0.06 µg/mL and limit of quantitation of 0.19 µg/mL. The method was applied to the analysis of commercial tablets and the results were in good agreement with those obtained using the reference method. Validation of the analytical procedures was evaluated according to ICH guidelines. Copyright © 2015 John Wiley & Sons, Ltd.     

Spectrofluorimetric determination of folic acid in tablets and urine samples using 1,10‐phenanthroline‐terbium probe

A new spectrofluorimetric method was reported for the determination of folic acid (FA), based on its quenching effect on the fluorescence intensity of Tb³⁺–1,10‐phenanthroline complex as a fluorescent probe. The quenched fluorescence intensity at an emission wavelength of 545 nm was proportional to the concentration of FA in Tris–HCl buffer solution of pH 6.2. The effects of pH, time, order of addition of reagents, temperature and concentrations of Tb³⁺, buffer and 1,10‐phenanthroline were investigated and optimized. The linear range for the determination of FA was 0.01–1.1 mg/L. The detection limit was 0.003 mg/L and the relative standard deviation for replicated determination of 1 mg/L of folic acid was 1.2%. This method was simple, practical and relatively free from interference effects. It was successfully applied to assess FA in pharmaceutical tablets and urine samples. Copyright © 2010 John Wiley & Sons, Ltd.