Spectrofluorimetric Protocol for Estimation of Commonly Used Antispasmodic Drotaverine. Fluorescence Quenching Study.

A fast, simple, accurate, precise, and cheap fluorimetric protocol has been proposed for analysis of a phosphodiesterase-IV inhibitor, namely drotaverine hydrochloride. Fluorimetric protocol is based on estimating the decrease in the eosin Y fluorescence intensity by quantitative addition of drotaverine at pH 3.1 (acetate buffer). An ion pair complex is formed, which leads to quenching in the fluorescence intensity of the dye without need of prior extraction at 534 nm (λex. 339 nm). Different reaction perimeters which influence the production of complex (ion pair between drotaverine and eosin) were deeply investigated and optimized. The developed fluorimetric protocol is capable for quantitative estimation of drotaverine in linear range of 0.4 to 2.5 µg mL-1. After method validation in respect to ICH guidelines, it was applied to determine drotaverine in its commercial preparation. By comparing with other reported method, the developed and validated fluorimetric protocol is capable for estimation of drotaverine in commercial preparation with good accuracy and excellent precision.

Sensitive spectrofluorimetric determination of the prokinetic drug prucalopride succinate based on supramolecular aggregation approach with evaluation of method greenness: application to content uniformity test.

The prokinetic drug, prucalopride (PCP) succinate, was determined using a new spectrofluorimetric approach with a highly sensitive, rapid, and simple procedure. The method exploited the enhancement of the inherent native fluorescence of PCP by micellar aggregation with sodium lauryl sulfate (SLS) as an anionic surfactant. Different factors that could affect the fluorescence intensity were carefully studied in order to achieve the maximal fluorescence signal. Measurement of the enhanced fluorescence was done at 354 nm after the excitation at 276 nm. The fluorescence intensity-concentration plot was rectilinear in the concentration range of 50-600 ng/ml with detection and quantitation limits of 13.9 and 42.1 ng/ml, respectively. The method underwent validation according to the International Council for Harmonisation criteria in order to assess its analytical performance, and promising results were achieved that proved the validity and reliability of the method. Furthermore, the method was employed effectively for the analysis of the cited drug in commercial pharmaceutical tablets.

An eco-friendly matrix-augmented fluorescence spectroscopic approach for the analysis of mitoxantrone, an oncogenic therapy; application to the dosage form and biological matrices.

Mitoxantrone (MXN) is a synthetic anthracenedione oncogenic therapy. It is often prescribed as an anticancer agent to manage a variety of cancers. A green, fast, and easy fluorimetric technique for the assay of MXN as a topoisomerase type II enzyme suppressor. An investigation of MXN's fluorescence behavior in various media and solvents constituted the basis for this new technique. Methanol was shown to enhance the intrinsic fluorescence considerably. After excitation at 610 nm, the highest fluorescence intensity was found at 675 nm. Various experimental parameters, such as media, solvents, and pH levels, were tested and adjusted. ICH (International Conference on Harmonization) guidelines were followed when validating procedures. It was possible to achieve linearity in the 0.02-1.50 µg ml-1 with the method. The sensitivity (in terms of limit of detection and limit of quantification) was 0.003 and 0.008 µg ml-1 , indicating low toxicity. As a result, the current technology has a remarkable recovery for detecting residues in diverse bodily fluids. Also, the quantum yield was estimated for the designed system. Finally, the method was rated by eco-scale scoring.

Resonance Rayleigh scattering approach based on association complex formation with erythrosine B for determination of venlafaxine, application to the dosage form and spiked human plasma.

The interaction of venlafaxine hydrochloride (VLX) with erythrosine B was investigated using a resonance Rayleigh scattering (RRS) spectroscopic technique. In acetate buffer (pH 3.4), erythrosine B reacted with VLX to form a 1:1 ion-pair complex with concomitant enhancement in RRS intensity that was measured at 330 nm. In addition, the stability constant and the change in free energy of the reaction were estimated. Based on this interaction a new method was developed for a sensitive VLX analysis using erythrosine B as a probe. The results indicated that this method had good selectivity in the presence of coexisting compounds. The scattering intensity (ΔIRRS ) was linearly dependent on VLX concentration over the range 0.04-1.0 µg ml-1 with a determination coefficient (r) of 0.9998. The limit of detection and limit of quantitation were 0.01 and 0.03 µg ml-1 , respectively. This method could be suitably used for analysis of VLX in pharmaceutical capsules and human plasma.

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.

Facile spectrofluorimetric quantitation of octreotide, a synthetic peptide, in its pure form and pharmaceutical formulation; evaluation of the method's greenness.

A new, rapid, highly sensitive, and affordable spectrofluorimetric approach has been constructed and validated for the determination of octreotide in its authentic form and pharmaceutical dosage form. Octreotide is an important synthetic analog of the naturally occurring somatostatin hormone. The developed spectrofluorimetric approach is actually dependent on the measurement of octreotide native fluorescence at emission wavelength of 342 nm after excitation at 218 nm. At optimal reaction conditions, the calibration curve has been constructed over the concentration range 200-2000 ng ml-1 , with excellent linearity. The limits of detection and quantitation values were found to be 55 and 169 ng ml-1 , respectively. The developed approach has been effectively used to determine octreotide in its pharmaceutical ampoules, without interference from the excipients in the dosage form. The developed approach is simple, time-saving, and does not require multiple pretreatment steps for samples, costly apparatus, or dangerous materials. As a result, it can be used to detect and quantify octreotide acetate in quality control laboratories.

Simple TLC-spectrodensitometric method for studying lipophilicity and quantitative analysis of hypoglycemic drugs in their binary mixture.

A selective and simple salting-out-assisted thin-layer chromatographic methodology was developed for the simultaneous determination of two oral hypoglycemic drugs, dapagliflozin (DAPA) and metformin (MET) in their pure forms, tablets and spiked human plasma samples. Silica gel 60 F254 plates were used in the separation of the two drugs using a mobile phase consisting of 0.5 m (NH4 )2 SO4 and methanol (3:7, v/v). The plates were scanned in the reflectance mode at λmax = 237 nm. The obtained retardation factor (Rf ) values for DAPA and MET were 0.77 ± 0.02 and 0.25 ± 0.02, respectively. The thin-layer chromatography method was validated according to International Conference on Harmonization guidelines. The peak areas were linearly increased with the increases in concentrations of 45-1,000 and 50-1,500 ng/band for DAPA and MET, respectively. Moreover, the method was applied to estimate the molecular lipophilicity parameters of DAPA and MET via retention data. The suggested method was efficiently utilized for the analysis of DAPA and MET in pharmaceutical tablets and plasma samples with recoveries 98.4-100.4 and RSDs in the ranges of 1.4-2.6 and 2.2-3.0% for DAPA and MET, respectively.

Investigating the interaction of mitoxantrone with anionic surfactants by spectrofluorimetry and its application for the feasible analysis of pharmaceutical preparation and biological fluids.

The behaviour of mitoxantrone (MTX), an anthracenedione antineoplastic agent, in different types of organized medium was explored using molecular spectrofluorometry. The original fluorescence and quantum yield of MTX were augmented by about five-fold in the aqueous buffered solution (Britton-Robinson, pH 3.0) by the addition of sodium dodecyl sulfate. Enhancement in the fluorescence intensity did not come from the boost in the ultraviolet (UV) light absorbance of the drug in the presence of micelles but due to shielding of the lowest excited singlet state of the drug from a radiationless process inside the cavity of the micelle. Accordingly, a versatile, sensitive, and feasible spectrofluorimetric method was constructed and evaluated for MTX determination. Fluorescence measurements were performed at 675 nm (λex 610 nm). A linear relationship was shown between fluorescence intensity and drug concentration within the range 0.01-2.0 µg ml-1 of MTX with a correlation coefficient of 0.9999 and a detection limit of 2 ng ml-1 . The developed method was effectively used for analysis of MTX in biological samples and dosage forms. In addition, the method was expanded to study the stability of MTX exposed to different drastic degradations and the kinetic parameters of the degradation were calculated.

Investigation of the association complex formed between dapoxetine and erythrosine-B for facile dapoxetine assay in pharmaceutical formulation using resonance Rayleigh scattering and spectrofluorimetric techniques.

Premature ejaculation is a male sexual problem that is marked by rapid ejaculation and quick attainment of orgasm. Dapoxetine belongs to the antidepressant category and modulates its action by preventing the reuptake of serotonin by neurons. Dapoxetine is marketed as an off-label therapy for premature ejaculation. Here, two facile, sensitive, and green compatible approaches were established for dapoxetine assay. The approaches chemically rely on association complex formed between erythrosine-B and dapoxetine in an acidic buffered medium. The quenching effect of the formed complex on the native erythrosine fluorescence at emission 553.5 nm is simply the main idea of spectrofluorimetric assay, while resonance Rayleigh scattering methodology uses augmentation of resonance Rayleigh scattering spectrum at 345 nm by the added dapoxetine. The current approaches exhibit linearity between response and dapoxetine concentration over 0.2-2.5 µg/ml and 0.3-3.0 µg/ml for spectrofluorimetric and resonance Rayleigh scattering (RRS) methods, respectively. All variables affecting methods and complex formation were studied and precisely optimized. The criteria of validation were performed by the directives provided by International Conference on Harmonization's (ICH) Guidelines and limits of detection were 0.06 and 0.05 µg/ml for spectrofluorimetric and RRS techniques, respectively. Finally, the approaches were applied with acceptable results for pharmaceutical formulation analysis.

Use of Hantzsch reaction for quantitation of milnacipran as a magic treatment for fibromyalgia syndrome in human plasma and urine.

A new fluorimetric procedure is described for analysis of milnacipran in its bulk, tablet dosage forms, as well as in biological human samples such as plasma and urine. The suggested method relies on the construction of a derivative with strong fluorescence called dihydropyridine derivative. This derivative resulted from the interaction of the primary amino group in the studied drug and acetylacetone/formaldehyde in McIlvaine buffer (pH 5). The fluorescent dihydropyridine derivative was measured at 470 nm. Influences of experimental variables namely pH, reagent concentration and temperature were examined and optimized. The calibration curve showed linearity over the range of 0.15-1.25 µg ml-1 of milnacipran with an R2 value of 0.9998. The detection limit was 0.02 µg ml-1 and the determination limit was 0.07 µg ml-1 . The developed procedure was successfully used in the assay of the studied drug in Avermilan® tablets with excellent selectivity. In addition, the reaction was applied to estimate the drug in spiked human plasma and urine with mean percentage recoveries of 100.04 ± 1.61 and 99.78 ± 0.81% for urine and plasma, respectively.

A new feasible approach based on utility of ninhydrin for selective fluorimetric analysis of baclofen. Application to content uniformity evaluation.

In the current proposed analysis, a new, feasible, and selective fluorimetric approach was designed for baclofen assay. Baclofen is a medication prescribed as a therapy for muscle spasticity that originated from multiple sclerosis or a spinal cord injury, and other cases such as hiccups. The analytical approach relies on the use of ninhydrin to form a fluorescent derivative that was monitored at λex 386 nm or λem 480 nm. Under suitable reaction conditions, the primary amino moiety in baclofen was condensed with ninhydrin and phenylacetaldehyde in the presence of Teorel buffer as a buffered medium. The method exhibited linearity when baclofen concentration was plotted against response in the range 1-10 µg ml-1 . Adjustment of the reaction variables and study of validation parameters according to ICH directives were performed correctly. An interference study was implemented to ensure that no discrepancy from the excipient had occurred. Finally, the proposed method was applied successfully for baclofen assay in dosage form and extended to test Mylobac content uniformity.

Resonance Rayleigh scattering and spectrofluorimetric approaches for the selective determination of rupatadine using erythrosin B as a probe: application to content uniformity test.

In this study, spectrofluorimetric and resonance Rayleigh scattering techniques were applied for the first time for determination of rupatadine through two validated methods. The proposed methods were based on a facile association complex formation between rupatadine and erythrosin B reagent in acidic medium. Spectrofluorimetric determination relied on the quenching effect of rupatadine on the fluorescence intensity of erythrosin B at 556 nm (excitation = 530 nm). Conversely, the resonance Rayleigh scattering (RRS) method relied on enhancement in the resonance Rayleigh scattering spectrum of erythrosin B at 344 nm after the addition of rupatadine. The developed methods produced linear results over ranges 0.15-2.0 µg/ml and 0.1-1.5 µg/ml, with detection limits of 0.030 µg/ml and 0.018 µg/ml for the spectrofluorimetric method and the RRS method, respectively. All reaction conditions for rupatadine-erythrosin B formation were optimized experimentally and both methods were validated according to International Council for Harmonisation guidelines. The developed methods were applied to estimate rupatadine content in its pharmaceutical tablet dosage form with acceptable recoveries. Furthermore, a content uniformity test for the commercial rupatadine tablets was successfully applied by the suggested spectroscopic methods according to United States Pharmacopeia guidelines.

Development and validation of stability indicating chromatographic methods for simultaneous determination of citicoline and piracetam.

Citicoline and piracetam were subjected separately to different stress conditions as recommended by the international conference on harmonization. In addition, new stability indicating thin layer chromatographic and ultra high performance liquid chromatographic methods have been developed and validated for simultaneous determination of citicoline and piracetam in presence of their degradation products. Separation on the proposed thin layer chromatographic method was carried out using a developing system containing methanol:chloroform:ammonium chloride buffer (9:1:2, v/v/v) on silica gel plates at 230 nm. On the other hand, the mobile phase in the ultra high performance liquid chromatographic method was composed of water (containing 0.1% triethylamine):ethanol (92:8, v/v). The flow rate was 1 mL/min and ultraviolet detection was at 230 nm. Moreover, results of the developed methods were statistically compared to those obtained by the reported high-performance liquid chromatography method and no significant difference between them was found. The greenness profile of ultra high performance liquid chromatographic method was assessed and compared with those of the previously published high-performance liquid chromatography methods, it was noticed that the proposed ultra high performance liquid chromatographic method more environmentally friendly and greener than other methods.

Impact of single nucleotide polymorphisms (R132Q and W120R) on the binding affinity and metabolic activity of CYP2C19 toward some therapeutically important substrates.

Although CYP2C19 is minor human liver enzyme, it is responsible for the metabolism of many clinically important drugs. In this work, CYP2C19 wild type and its SNP mutants (R132Q and W120R) were prepared using over-expression system in E. coli, purified by column chromatography and their biological activities were compared. The enzyme activity toward certain drugs (amitriptyline, imipramine, lansoprazole and omeprazole) was investigated. Resonance Raman and UV-VIS spectroscopies revealed a minimal effect of SNP mutations on the heme structure. However, the mutation greatly affected the drug metabolism activities of CYP2C19. The degree of these effects was dependent on both the mutation and the chemical structure of the substrate. Surprisingly, the affected amino acid residue is located remotely from the heme center. Therefore, the direct effect of the mutation on the metabolic center is excluded. Alternatively, the significant impairment in the drug metabolism of these mutants could be attributed to a decrease in the electron flow to the iron center. Accordingly, understanding the effect of SNPs of CYP2C19, and the extents in which they participate in the drug metabolism, are important pillars that can enhance the therapeutic drugs efficacy and improve the patient outcomes toward the development of patient's tailored medicine.

New approach for stability study and determination of fluvoxamine in raw materials and pharmaceuticals through condensation with 2,2-dihydroxyindane-1,3-dione.

The present study describes the validation of a selective spectroscopic method for assay of fluvoxamine maleate (FXM). The validated method relies on condensation of FXM with 2,2-dihydroxyindane-1,3-dione and phenylacetaldehyde using Teorell-Stenhagen buffer (pH 6.6) to give coloured fluorescent product measured at 482 nm using 386 nm as the excitation wavelength. The parameters influencing the reaction were studied precisely and adjusted accurately. The constructed calibration graph appeared rectilinear over the following range (0.8-14 µg ml-1 ) and the estimated limit of detection was 0.25 µg ml-1 . Two pharmaceutical products from the Egyptian market were assayed using the suggested method and the final results agreed with measurements from other reported methods. Moreover, the drug was subjected to diverse stress conditions including acidic, alkaline, thermal, and photolytic degradation to examine the FXM stability. Directives from the International Conference on Harmonisation guidelines were applied to establish the validity of the work.

Thin-layer chromatography/fluorescence detection approach for sensitive and selective determination of hepatitis C virus antiviral (velpatasvir): application to human plasma.

A validated thin-layer chromatography (TLC) method combined with fluorescence detection mode was developed for the selective determination of a recently approved anti-hepatitis C virus (HCV) drug (velpatasvir). The separation was performed on silica gel 60 F254 plates using ethylacetate:methanol:triethylamine (48:1.5:1.0, v/v/v) as a mobile phase. Plates were scanned in the fluorescence mode after excitation at 335 nm. This method provided an excellent separation of velpatasvir from sofosbuvir with RF values of 0.22 and 0.46 for velpatasvir and sofosbuvir, respectively, after scanning the developed plates in the ultraviolet detection mode at 335 nm. The calibration curve was linear over the range 4-40 ng/band with a correlation coefficient of 0.9994. The developed procedure was validated according to ICH guidelines with a detection limit of 1.30 ng/band and quantitation limit of 3.95 ng/band. The suggested method could selectively determine velpatasvir with high sensitivity in a synthetic tablet powder containing a co-formulated anti-HCV drug (sofosbuvir) without any interference from excipients or sofosbuvir. In addition, the method was successfully applied for determination of velpatasvir in spiked human plasma with adequate % recovery.

Use of acetylacetone for nano-level assay of fluvoxamine maleate in pure form and pharmaceutical formulation.

The present study developed and validated a selective spectrofluorimetric method designed to assay fluvoxamine maleate (FLX). The validated method relied on the condensation reaction between FLX and acetylacetone/formaldehyde using acetate buffer (pH 4.2). The formed fluorescent product was measured at an emission wavelength of 479 nm after excitation at 419 nm. Parameters that influenced the reaction were studied and adjusted accurately. The constructed calibration graph was rectilinear over the range 200-2000 ng ml-1 and the estimated limit of detection was 60 ng ml-1 . Two products from an Egyptian market were assayed using the suggested method and the final results agreed with the measurements of the reported method. The selectivity of the proposed approach was evaluated using the standard addition method and results were acceptable and confirmed the reliability of this approach. Finally, directives from the International Council for Harmonisation guidelines were applied to establish the validity of the study.

Microenvironment improvement protocol for the sensitive spectrofluorimetric determination of an hepatitis C virus antiviral (Simeprevir): application to human plasma.

Simeprevir (SPV) is a powerful antihepatitis C virus agent that was newly introduced into the pharmaceutical market. We here established and validated an easy, simple, and sensitive spectrofluorimetric method for its estimation at λem 427 nm (λex 337 nm). The suggested procedure was based on two times enhancement in the original emission of SPV through modifying its microenvironment in buffered aqueous solution by adding Triton X-100. The relationship between the concentration of SPV and the observed fluorescence intensity was linear in the range 0.06-1.0 µg ml-1 with a correlation coefficient of 0.9997. The limits of detection and quantitation were 21 and 64 ng ml-1 , respectively. The present method was effectively applied to quantify SPV content in pharmaceutical tablets and human plasma spiked with the drug with no interference from tablet excipients or plasma components.

Micellar-based spectrofluorimetric method for the selective determination of ledipasvir in the presence of sofosbuvir: application to dosage forms and human plasma.

A fast, low-cost, sensitive, and selective spectrofluorimetric method for the determination of ledipasvir was developed and validated. The method is based on an enhancement in the native fluorescence intensity of ledipasvir by 500% of its original value by the formation of hydrogen bonds between the cited drug and Tween-20 in the micellar system (pH = 5.0). All fluorescence measurements were carried out at 425 nm and 340 nm for emission and excitation wavelengths, respectively. A linear relationship between the concentration of ledipasvir and the observed fluorescence intensity was achieved in the range of 0.1-2.0 µg ml-1 with 0.028, 0.084 µg ml-1 , for detection and quantitation limits, respectively. The acquired selectivity and sensitivity using the proposed method facilitate the analysis of ledipasvir in spiked human plasma with sufficient percentage recovery (95.36-99.30%). The proposed method was developed and validated according to International Council for Harmonisation (ICH) guidelines. Moreover, the cited drug was successfully determined in its pharmaceutical dosage form using the proposed method. In addition, the validity of the proposed results was statistically confirmed using Student's t-test, variance ratio F-test, and interval hypothesis test.

Spectrofluorometric determination of alogliptin an antidiabetic drug in pure and tablet form using fluorescamine, a fluorogenic agent: application to content uniformity test.

Alogliptin is an antidiabetic drug that belongs to a group called dipeptidyl peptidase-4 enzyme inhibitors. As the drug contains a primary amino group in its structure, it readily reacts with fluorescamine in slightly alkaline medium (borate buffer, pH 8.8) to form a highly fluorescent product. Emission of this product was measured at 477 nm (λex = 387 nm). The linear range between the fluorescence intensity and the drug concentration was 0.1-0.5 µg ml-1 with a good correlation coefficient (0.9986). Limits of detection and quantitation were 22 and 72 ng ml-1 , respectively. Guidelines of the International Conference for Harmonisation were followed to validate the developed method with acceptable results. Alogliptin content was determined successfully in its commercial dosage form using the fluorescamine method with good recovery (98.60-101.26%). The method has excellent levels of accuracy and precision compared with the reported method as assessed using Student's t-test and Fisher's exact test. The method was applied successfully for the content uniformity test with high recovery and low relative standard deviation.