Photolysis of formylmethylflavin in aqueous and organic solvents.

The photolysis of formylmethylflavin (FMF), a major intermediate in the photodegradation sequence of riboflavin, has been carried out in water (pH 7.0) and in several organic solvents. FMF produces lumichrome (LC) in organic solvents and LC and lumiflavin (LF) in aqueous solution. FMF and its photoproducts have been analysed using a specific multicomponent spectrophotometric method. FMF undergoes a bimolecular redox reaction on photolysis. The second-order rate constants for the reaction range from 0.66 (chloroform) to 2.44 M(-1) s(-1) (water) and are a linear function of the solvent dielectric constant. A plot of ln k against 1/epsilon is linear for the reactions in 1-butanol, 1-propanol, ethanol, methanol, acetonitrile and water (epsilon approximately 17-79) and non-linear in chloroform and dichloroethane (epsilon approximately 5-10) suggesting a change in reaction mechanism in the two regions. This may be explained on the basis of the existence of a dipolar intermediate along the reaction pathway. The rate of photolysis is governed by the solvation of the intermediate and is thus influenced by the dielectric constant of the medium. The solvent effect on the rate of photolysis of FMF has been expressed in terms of the solvent acceptor number. A linear relationship has been found between ln k and the solvent acceptor number.     

Micelle‐enhanced spectrofluorimetric determination of amlexanox in bioadhesive buccal tablets: application to content uniformity testing

A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of Amlexanox (AMX) in its bioadhesive buccal tablets. The proposed method is based on measuring the native fluorescence of the methanolic solution of AMX at 400 nm after excitation at 242 nm in 0.2 M borate buffer (pH 10) and 0.5% w/v sodium dodecyl sulfate (SDS) solution. The interaction of AMX with SDS was studied, and the enhanced fluorescence intensity was exploited to develop an assay method for the determination of AMX. The relative fluorescence intensity–concentration plot was rectilinear over the range 5.0–80.0 ng/mL, with a lower detection limit of 0.57 ng/mL and a lower quantification limit of 1.74 ng/mL. The proposed method was successfully applied to the analysis of AMX in its commercial tablets. Moreover, content uniformity testing was conducted by applying official USP guidelines. Statistical evaluation and comparison of the data obtained using the proposed and comparison methods revealed good accuracy and precision for the proposed method. Copyright © 2015 John Wiley & Sons, Ltd.     

New spectrofluorimetric analysis of dapagliflozin after derivatization with NBD‐Cl in human plasma using factorial design experiments

A new validated spectrofluorimetric method was proposed for dapagliflozin (DGF) analysis in bulk, plexin its commercially available tablets and in spiked human plasma. The proposed spectrofluorimetric method depended on the formation of a fluorescent complex soluble in organic liquids by a substitution reaction between 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl) reagent and DGF in aqueous buffered solution at pH 7. The fluorescence intensity was measured at 522 nm after excitation at 453 nm. The high selectivity of the proposed method allowed analysis of DGF in dosage form and human plasma samples with average recovery values of 99.84 ± 1.38% and 98.71 ± 1.80%, respectively, without any interference from matrix components. The calibration range was 50–1000 ng ml^?1. The limit of detection (LOD) and limit of quantitation (LOQ) were 14.24 ng ml^?1 and 43.14 ng ml^?1, respectively. The estimated relative standard deviation values were lower than 2.0%, this showed the excellent precision at both levels. Factorial design was used to get the optimum method conditions for the analysis of the resulting DGF fluorescence complex in different matrices. The proposed method could be used in routine analysis of DGF in quality control laboratories. Also, it could be used to assay DGF in human plasma and be applied for pharmacokinetic investigation of DGF.     

Spectrofluorimetric investigation with green analytical procedures for estimation of bambuterol and terbutaline: Application to pharmaceutical dosage forms and content uniformity testing

Bambuterol (BAM) and terbultaline (TER) are well known and effective bronchodilators. In this article highly sensitive, green and cost‐effective spectrofluorimetric methods are designed to determine low concentrations of such drugs. The proposed methods are based on an investigation of the native fluorescence properties of aqueous solutions of BAM at 298 nm after excitation at 263 nm and of TER at 313 nm after excitation at 275 nm. Under optimum conditions, the plots of the relative fluorescence intensity versus concentration were rectilinear over the range 0.1–1.2 μg/mL for BAM and 0.05–0.5 μg/mL for TER with a limit of quantitation of 0.067 μg/mL for BAM and 0.018 μg/mL for TER. The methods are simple and hence suitable for application to the quantification of BAM and TER in syrups and tablets without interference from common excipients. Furthermore, based on United States Pharmacopeia (USP) guidelines, the application was extended to determine the content uniformity of the cited drugs in low dose tablets. The developed methods were fully validated according to the guidelines of the International Conference on Harmonization (ICH).     

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.     

Two green and sensitive spectrofluorimetric approaches for determination of Ambroxol and guaifenesin in their single and combined pharmaceutical formulations

Ambroxol hydrochloride (AMX) and guaifenesin (GFN) are approved drugs utilized to treat coughs through their potent mucolytic and expectorant properties. Due to their massive, combined administration in many illnesses, there is a persistent need for their concurrent estimation in different pharmaceutical formulations. Two sensitive, environmentally friendly spectrofluorimetric methods were developed. AMX was determined using the first method (I) without interference from GFN. This method depends on the quenching of Erythrosine B (EB) native fluorescence at 552 nm after excitation at 527 nm due to the formation of a non-fluorescent AMX-EB ion-pair complex in Britton–Robinson buffer (BRB) solution pH (3.5). The concentration plot is linear over the 0.25–5.0 μg/mL range, with a mean percent found value of 99.74%. Method (II) depends on measuring the native fluorescence of aqueous GFN solution at two analytical wavelengths, either 300 or 600 nm, after excitation at 274 nm. Relative fluorescence intensity (RFI)–concentration plots are linear over the ranges of 0.02–0.5 and 0.1–2.0 μg/ml, with mean percent found at 99.96% and 99.91% at dual wavelengths, respectively. The proposed methods were successfully applied to assay both drugs in raw materials and different single and combined pharmaceutical formulations. These methods have been thoroughly validated following International Committee on Harmonisation (ICH) guidelines. National Environmental Methods Index, Analytical Eco-Scale, and Green Analytical Procedure Index were used to prove greenness, thereby enhancing their applicability. The proposed techniques provide straightforward, precise, and cost-effective solutions for routine formulation analysis in quality control laboratories.     

Ultraviolet radiation-induced photodegradation and 1O2, O2-. production by riboflavin, lumichrome and lumiflavin

Although UVA (320-400 nm) is considered less harmful to skin as compared to UVB (290-320 nm) and UVC (200-290 nm) radiation, certain endogenous chromophores may enhance UVA-induced cutaneous reactions by largely O2-dependent photodynamic reactions. Photodegradation pattern and singlet oxygen (1O2), superoxide anion radical (O2-.) producing capacity of riboflavin (RF), lumiflavin (LF) and lumichrome (LC) were examined to assess their phototoxic potential under UVA. Photolysis of RF upon exposure to UVA, UVB or UVC revealed considerable degradation to LF and LC with a near identical spectral pattern of photodegradation between 250-500 nm. Both LF and LC were stable to UVA (3 J/cm2) and UVB (400 mJ/cm2), whereas RF was photodegraded by 30 and 20%, respectively, under similar irradiation conditions. UVA-sensitized LF and LC respectively, produced nearly 15% higher and 60% lower yield of 1O2 in comparison to RF, whereas, O2-. was generated predominently by RF. Both RF and LF thus appeared to be potential chromophores for evoking deleterious effects of UVA in normal human skin.     

Determination of omeprazole via fluorescence-quenching methodology; application to content uniformity testing

A new, proven, economical spectrofluorimetric approach has been used to determine the proton pump inhibitor omeprazole (OMP). This innovative technique is based on the ability of OMP to quench the native fluorescence of the mercurochrome dye in an acidic (pH 3.6) solution. Because it was discovered that quenching is proportional to the drug concentration, this dye was used as a sensor for OMP detection. The fluorescence intensity was measured at 518/540 nm, and its linear response ranged from 0.2–10.0 μg/mL with a linear coefficient of 0.9999. The computation yielded a limit of quantification (LOQ) of 0.20 μg/mL and a limit of detection (LOD) of 0.07 μg/mL. Every circumstance and element impacting the reaction product was examined in detail. Pharmacopeial standards carried out the validation. The approved method investigated several commercial preparations and formulations, and the results were favorably compared with those provided by a reference method. According to United States Pharmacopeia (USP) rules, content consistency for two distinct formulations was evaluated.     

Orthophthaldehyde as a fluorescent probe for the feasible and sensitive assay of heptaminol: application to dosage forms and real human plasma

The antihypotensive drug heptaminol was determined using a spectrofluorimetric method and ortho-phthaladehyde as a fluorescence probe. The drug was mixed with the reagent in the presence of 2-mercaptoethanol and the reaction was carried out in slightly alkaline aqueous solution containing 0.1 M sodium hydroxide. The resulting product exhibited high fluorescence activity that was measured at 451 nm after excitation at 334 nm. The linearity range of the method was 5–100 ng ml⁻¹ with a lower detection limit of 1.8 ng ml⁻¹. The procedure was evaluated according to the International Council of Harmonization guidelines. The proposed method was applied to analyze the drug in pharmaceutical tablets and oral drops. In addition, the present study represents the first spectrofluorimetric method for the determination of the cited drug in real human plasma. The method provided high recovery percentages without any interference from coexisting pharmaceutical excipients or the components of human plasma.     

Micelle‐enhanced spectrofluorimetric method for determination of sitagliptin and identification of potential alkaline degradation products using LC‐MS

A novel, quick, simple and highly sensitive spectrofluorimetric method was developed and validated for the determination of sitagliptin (SG) in its pharmaceutical formulations. The proposed method is based on investigation of the fluorescence spectral behavior of sitagliptin in an SDS micellar system. In an aqueous solution of phosphate buffer pH 4.0, the fluorescence intensity of SG in the presence of SDS was greatly enhanced, by 200%, i.e. twofold enhancement. The fluorescence intensity of SG was measured at 300 nm after excitation at 270 nm. The method showed good linearity in the range 0.03–10.0 µg/mL with a good correlation coefficient (r = 0.9998). The limits of detection and quantitation values were 5.31 and 16.1 ng/mL, respectively. The proposed method was successfully applied to the analysis of SG in its single and co‐formulated commercial tablets; the results were in good agreement with those obtained using a reference method. Application of the proposed method was extended to stability studies of SG after exposure to different forced degradation conditions according to the ICH guidelines, such as acidic, alkaline, thermal, photo‐ and oxidative stress. The chemical structure of certain potential degradation products (DPs) were investigated using LC‐MS. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of sensitive benzofurazan‐based spectrometric methods for analysis of spectinomycin in vials and human biological samples

Spectinomycin hydrochloride (SPEC) is an aminoglycoside antibiotic that has a broad spectrum against several bacterial strains of humans and veterinary infections. However, SPEC lacks a fluorophore or chromophore and this lack makes its analysis a challenge. Our study provides a simple, sensitive and low‐cost spectrofluorimetric/spectrophotometric method based on the reaction between secondary amine groups and a benzofurazan reagent using borate buffer (pH 9.2). The yielding compound was measured fluorimetrically at 530 nm (excitation at 460 nm) with colorimetry at 410 nm. The calibration curves ranged from 30 to 400 ng ml^?1 and from 0.2 to 6 μg ml^?1 for spectrofluorimetric and spectrophotometric analyses, respectively. The limits of detection were calculated to be 4.15 ng ml^?1 and 0.05 μg ml^?1 for spectrofluorimetric and spectrophotometric processes, respectively. The ultra‐affectability and high selectivity of the proposed method permitted analysis of SPEC in the dosage form and in human plasma samples, with good recoveries of about 101.19 and 97.11%, respectively, without any matrix interference. The proposed strategy was validated using International Conference on Harmonization standards and validated bioanalytically using USFDA recommendations.     

Spectrofluorimetric Method for the Determination of Doxepin Hydrochloride in Commercial Dosage Forms

A novel spectrofluorimetric method has been developed for the determination of doxepin hydrochloride in commercial dosage forms. The method is based on the fluorescent ion pair complex formation of the drug with eosin Y in the presence of sodium acetate–acetic acid buffer solution of pH 4.52 which is extractable in dichloromethane. The extracted complex showed fluorescence intensity at λ_em = 567 nm after excitation at 464 nm. The calibration curve was linear over the working range of 0.1–0.8 μg ml⁻¹. Under the optimized experimental conditions, present method is validated as per International Conference on Harmonization guidelines. The limit of detection for the developed method is 2.95 ng ml⁻¹. The method has been successfully applied to the determination of doxepin hydrochloride in commercial dosage forms. The results are compared with the reference spectrofluorimetric method.     

Spectrofluorimetric determination of certain adrenergic agonist drugs in their pure forms and pharmaceutical formulations: Content uniformity test application

A new, simple, sensitive and rapid spectrofluorimetric method has been developed for determination of certain adrenergic agonists such as isoxsuprine hydrochloride, ritodrine hydrochloride and etilefrine hydrochloride in their pure forms and pharmaceutical dosage forms. The method depends on micellar enhancement of the native fluorescence of investigated drugs by using 2% w /v sodium dodecyl sulfate (SDS) as an anionic surfactant. The enhanced fluorescence intensity of investigated drugs was measured at 305 nm after excitation at 278 nm. The interaction of studied drugs with SDS was studied, and the enhanced fluorescence intensity was exploited to develop an assay method for the determination of investigated drugs. The relative fluorescence intensity–concentration plots were rectilinear over the range 0.15–3.00 μg ml^?1, with low quantification limits of 0.132, 0.123 and 0.118 μg mL^?1 for isoxsuprine, ritodrine and etilefrine, respectively. The proposed method was successfully applied for determination of studied drugs in their pharmaceutical formulations. Moreover, the high sensitivity of the proposed method allows performing the content uniformity testing of the studied drugs in their tablets by using the official United States Pharmacopeia (USP) guidelines. Statistical comparisons of the results with those of the reported methods revealed excellent agreement and indicated no significant difference in accuracy and precision.     

Factorial design optimization of micelle enhanced synchronous spectrofluorimetric assay of Omarigliptin: Applied to content uniformity testing and in vitro drug release

A micelle enhanced spectrofluorimetric method was developed for determination of Omarigliptin (OMG) based on its native fluorescence behavior. The interaction of OMG with surfactants and macromolecules was studied. In aqueous solution, the relative fluorescence intensity (RFI) of OMG was enhanced by 24% in the presence of Tween 80 at pH 3.5. The optimal conditions for the micelle enhanced fluorescence were attained by Minitab® program using Plackett–Burman factorial design. Pareto chart, contour plots and surface plots were used to exclude the insignificant variables and optimize the significant factors. The spectrofluorimeter was operated under synchronous mode using ?λ = 30 nm and recording the RFI of the intense narrow band at 267 nm for OMG in 0.5% w/v Tween 80 + 0.2 M acetate buffer (pH 3.5) system using water as diluent. Using synchronous scan mode offered many advantages including considerable reduction of spectral overlap and enhanced linearity of the calibrators. Validation parameters were satisfied over the concentration range 0.1–2 μg/ml. The developed method was the first analytical procedure for OMG assay in Marizev® tablets. Moreover, content uniformity testing and in vitro drug release of tablets were performed.     

Validated spectrofluorimetric assay of two co-administered drug mixtures containing hydroxychloroquine with either moxifloxacin or ofloxacin as a drug regimen for hospital-acquired pneumonia in patients with COVID-19

Moxifloxacin and ofloxacin are two broad-spectrum quinolone antibiotics. They are among the most widely used antibiotics, at this time, applied to control the COVID-19 pandemic. Hydroxychloroquine is an FDA-approved drug for the treatment of COVID-19. This work describes a simple, green, selective, and sensitive spectrofluorimetric method for the assay of moxifloxacin and ofloxacin in the presence of hydroxychloroquine, two co-administered mixtures used in the treatment of hospital-acquired pneumonia in patients with COVID-19. Simultaneous assay of hydroxychloroquine and moxifloxacin was carried out in methanol using a direct spectrofluorimetric method (method I) at 375 and 550 nm, respectively, after excitation at 300 nm. The direct spectrofluorimetric assay was rectilinear over concentration ranges 50.0–400.0 and 300.0–2500.0 ng/ml for hydroxychloroquine and moxifloxacin, respectively, with limits of detection (LOD) of 6.4 and 33.64 ng/ml and limits of quantitation (LOQ) of 19.4 and 102.6 ng/ml, respectively, for the two drugs. The assay for hydroxychloroquine and ofloxacin was carried out by measuring the first derivative synchronous amplitude for hydroxychloroquine at the zero crossing point of ofloxacin and vice versa at Δλ = 140 nm (method II). Hydroxychloroquine was measured at 266 nm, while ofloxacin was measured at 340 nm over the concentration range 4–40 ng/ml for hydroxychloroquine and 200–2000 ng/ml for ofloxacin with LOD of 0.467 and 25.3 ng/ml and LOQ of 1.42 and 76.6 ng/ml, respectively, for the two drugs. The two methods were validated following International Conference on Harmonization guidelines and were applied to the analysis of the two drugs in plasma with good percentage recoveries (109.73–93.17%).     

Spectrofluorimetric determination of dipyridamole in serum — a comparison of two methods

Two spectrofluorimetric methods for the determination of dipyridamole in plasma are described. The thin-layer chromatographic—fluoridensitometric method utilizes 1 ml of plasma which is extracted at pH 10 with diethyl ether—dichloromethane (80:20). The organic phase is evaporated to dryness, reconstituted in 250 μl dichloromethane and 5 μl are spotted on a silica gel 60 plate. The plate is developed in ethyl acetate—methanol—ammonia (85:10:5), dried, dipped in a paraffin wax solution, dried, and scanned using 380 nm as excitation wavelength, a 430 nm cut-off filter, and collecting all emitted light on the photomultiplier. Quantitation was done by the external standard method, peak heights being measured and a calibration graph constructed. For the spectrofluorimetric method 1 ml of plasma is extracted at pH 10 with 8 ml of hexane—isoamyl alcohol (95:5) and the organic phase used directly for the measurement of the fluorescence intensity (excitation 405 nm, emission 495 nm). Quantitation was done by measuring the fluorescence of standards that were treated as above and constructing a calibration graph of concentration versus fluorescence intensity. Concentrations of unknowns were found by interpolation from this graph. The two methods were found to exhibit good correlation but the spectrofluorimetric method proved to be more amenable to the analysis of a large number of samples.     

Micelle‐enhanced direct spectrofluorimetric method for the determination of linifanib: Application to stability studies

A new simple stability‐indicating spectrofluorimetric method has been developed and validated for the determination of the tyrosine kinase inhibitor, linifanib (LNF). The proposed method makes use of the native fluorescence characteristics of LNF in a micellar system. Compared with aqueous solutions, the fluorescence intensity of LNF was greatly enhanced upon the addition of Tween‐80. The relative fluorescence intensity of LNF was measured in a diluting solvent composed of 2% Tween‐80: phosphate buffer pH 8.0 (20: 80, v/v) using excitation and emission wavelengths of 290 and 450 nm, respectively. The proposed method was fully validated as per the ICH guidelines. The recorded fluorescence intensity of LNF was rectilinear over a concentration range of 0.3–2 μg/ml with a high correlation coefficient (r = 0.9990) and low limits of detection (0.091 μg/ml) and quantitation (0.275 μg/ml). The applicability of the method was extended to study the inherent stability of LNF under different stress degradation conditions including, alkaline, acidic, oxidative, photolytic and thermal degradation. Moreover, the method was utilized to study the kinetics of the alkaline and oxidative degradation of LNF. The pseudo‐first order rate constants and half‐lives were calculated.     

Photochemical properties of Yt base in aqueous solution.

Photoreactivity of Yt base [I] has been studied in aqueous solution [pH approximately 6] saturated with oxygen. Two photoproducts (II,III], resulting from irradiation at lambda = 253.7 nm and lambda greater than or equal to 290 nm, were isolated and their structures determined. The quantum yield for Yt base disappearance [zeta dis] is 0.002 [lambda = 313 nm]. It was shown that dye-sensitized photooxidation of Yt base in aqueous solution occurs according to a Type I mechanism, as well as with participation of singlet state oxygen. Quantum yields, fluorescence decay times and phosphorescence of Yt base have been also determined.     

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.     

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.