Novel spectrofluorimetric quantification of alogliptin benzoate in biofluids exploiting its interaction with 4‐chloro‐7‐nitrobenzofurazan

The direct determination of alogliptin benzoate (ALO) using fluorescence has not yet been accomplished because ALO cannot fluoresce naturally. Accordingly, it should be derivatized first with a fluorogenic reagent to enhance the sensitivity required for its bioanalysis. This method is the first spectrofluorimetric assay for ALO quantification exploiting the nucleophilic nature of its amino group to react with 4‐chloro‐7‐nitrobenzofurazan (NBD‐Cl) in borate buffer at pH 8.5 to produce a strong fluorescent compound that is excited at and emits at wavelengths 470 and 527 nm, respectively. Experimental variables concerning the conditions of reaction and fluorogenic intensity were carefully investigated and optimized. Linearity was from 1–250 ng ml^?1 with a lower detection limit of 0.29 ng ml^?1 and a lower quantification limit of 0.88 ng ml^?1. Validation of the current study was accomplished with mean per cent recovery of 100.62 ± 1.59 in tablets and 99.86 ± 0.82 in human plasma. Furthermore, the current method has been utilized in the bioanalysis of ALO in real rat plasma after oral administration with a simple specimen preparation. The developed method has proven to be a promising alternative method for ALO analysis in bioequivalence studies.     

Ultrafast liquid chromatographic analysis of erdosteine in human plasma based on fluorimetric detection and precolumn derivatization with 4‐bromomethyl‐7‐methoxycoumarin: Application to pharmacokinetic studies

In this study, a new analytical method for erdosteine (ERD) in plasma based on high‐performance liquid chromatography and a fluorimetric detector, is presented. Precolumn derivatization of ERD with 4‐bromomethyl‐7‐methoxy coumarin (BrMmC) and dibenzo‐18‐crown‐6‐ether as a reaction catalyst led to the production of a fluorescent compound. ERD was monitored by fluorescence with an excitation wavelength λ_ext. = 325 nm and emission wavelength λ_em. = 390 nm. Optimum reaction conditions were carefully studied and optimized. A chromatographic procedure was performed using a C18 column of 150 × 4.6 mm and 3 μm particle size and a mobile phase consisting of methanol:acetonitrile:water (30:30:40, v/v/v) under a flow rate of 0.5 ml min^?1. A calibration plot was established covering analyte concentration range 0.2–3.0 μg ml^?1; the detection limit was 0.015 μg ml^?1 and quantification limit was 0.05 μg ml^?1. Mean recovery was 87.33% and relative standard deviation was calculated to be less than 4.4%. The developed method was successfully used to determine pharmacokinetic preparations of ERD subsequent to administration of a 900 mg dose capsule to a healthy 40‐year‐old woman volunteer.     

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.     

Spectrofluorometric determination of clonazepam in dosage forms: Application to content uniformity testing and human plasma

The present paper describes a developed and validated simple, highly sensitive and cost‐effective spectrofluorometric method for determination of clonazepam (CNP). The proposed method depends on forming a highly fluorescent product through the reduction of CNP with Zn/HCl. The produced fluorophore exhibits a strong fluorescence at λ_em 350 nm after excitation at λ_ex 250 nm. The use of carboxymethylcellulose (CMC) greatly enhanced the fluorescence intensity of the produced fluorophore to the extent of about 100%. Calibration curve showed good linear regression (r ² > 0.9998) within test ranges of 20–400 ng ml^?1 with a lower detection limit of 0.67 ng ml^?1 and lower quantification limit of 2.22 ng ml^?1 upon using CMC. The method was successfully applied to the analysis of CNP in its pharmaceutical formulations and the results were in agreement with those obtained using a reference method. Furthermore, the content uniformity testing of the tablets was also performed. The application of the proposed method was extended to determine CNP in spiked human plasma sample as a preliminary investigation and the results were satisfactory.     

Aggregation‐Induced Emission of 1,8‐Naphthalimide?Casein Micelle: Investigation by Synchronous Spectrographic Method

A novel 1,8‐naphthalimide probe 1 , bearing two acetic‐acid moieties was synthesized. The acetic‐acid groups, docked into the sub‐domains of casein micelle and bound with tryptophan residues, and the 1,8‐naphthalimide chromophore adsorbed on the surface of casein micelle, forming a supermolecule, 1 ? casein micelle, which exhibited the aggregation‐induced synchronous emission (AISE) characters. The effect of pH on the intensity of supermolecule was investigated, and the result indicated that the emission enhancement was mainly due to the 1,8‐naphthalimide chromophore aggregated onto the casein micelle. Based on AISE, a novel casein quantification method was developed, which exhibited a good linear range of 0.05–10.0 μg ml^?1 and 0.07–9.5 μg ml^?1 with the detection limits of 2.8 and 3.0 ng ml^?1. The effects of metal ions and pH on the system of 1 ? casein micelle were investigated. The proposed method was applied to determine casein in milk samples, and the results were in good agreement with the result of the Biuret method.     

A novel spectrofluorimetric method for determination of imatinib in pure,pharmaceutical preparation,human plasma,and human urine

The following paper represents a simple, highly sensitive, responsive validated and developed spectrofluorimetric method for estimation of imatinib (IMB) in its pure, commercial preparation, human urine and human blood plasma. The calibration curve was in the range 4–900 ng ml^?1 for pure form and urine and 8–900 ng ml^?1 for plasma in a medium contains carboxymethyl cellulose (CMC) and acetate buffer (pH 5) with excitation wavelength (λ_ex) 230 nm and emission wavelength (λ_em) 307 nm. The limit of detection (LOD) was 0.37 ng ml^?1 for the pure form, 0.64 ng ml^?1 for human urine, and 0.70 ng ml^?1 for human plasma, while the limit of quantitation (LOQ) was 1.2 for pure form, 1.91 for urine and 2.1 for plasma. The suggested method was successfully applied for evaluation of IMB in tablets within 99% mean percentage recovery. The excipients that are usually used as additives in pharmaceutical dosage form did not interfere with the suggested method. The method was efficiently used for estimation of IMB in human urine and human plasma. The effect of some cations that might be present in urine and plasma was also studied. The method was also focused on human volunteers and in vitro drug release.     

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.     

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 λ_em 427 nm (λ_ex 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.     

Spectrofluorimetric determination of the antineoplastic agent lomustine based on the sensitizing effect of β‐cyclodextrin

The effects of solvent dipolarity/polarizability and solvent–solute hydrogen bonding on the photophysical properties of the antineoplastic drug lomustine were analysed by means of the linear solvation energy relationship (LSER) concept proposed by Kamlet and Taft. The LSER method enabled the overall solvent effects to be quantitatively estimated and separated into specific and non‐specific contributions. The molecular encapsulation of lomustine by β‐cyclodextrin (β‐CD) has been studied using fluorescence spectroscopy. The results are discussed in terms of the binding parameter and the effect of the solvent used. It was concluded that β‐CD forms a 1:1 inclusional complex with lomustine in acetonitrile solution and its association constant was calculated to be 500 M^?1. In addition, and for the first time, a simple, rapid and high sensitive fluorimetric method for the determination of lomustine was developed based upon the enhancement effect produced through complex formation with β‐CD. The new approach for the quantification of lomustine in the presence of β‐CD was described in aqueous and organic solutions. Better limits of detection (0.31 µg ml^?1) and quantification (1.05 µg ml^?1) were obtained in aqueous solution with respect to those obtained in organic solvent. Copyright © 2015 John Wiley & Sons, Ltd.     

Highly luminescent nitrogen‐doped carbon dots for simultaneous determination of chlortetracycline and sulfasalazine

Here, we have presented a green and facile strategy to fabricate nitrogen‐doped carbon dots (N‐CDs) and their applications for determination of chlortetracycline (CTC) and sulfasalazine (SSZ). The fluorescent N‐CDs, prepared by one‐step hydrothermal reaction of citric acid and l ‐arginine, manifested numerous excellent features containing strong blue fluorescence, good water‐solubility, narrow size distribution, and a high fluorescence quantum yield (QY) of 38.8%. Based on the fluorescence quenching effects, the as‐synthesized N‐CDs as a fluorescent nanosensor exhibited superior analytical performances for quantifying CTC and SSZ. The linear range for CTC was calculated to be from 0.85 to 20.38 μg ml^?1 with a low detection limit of 0.078 μg ml^?1. Meanwhile, the linear range for SSZ was estimated to be from 0.34 to 6.76 μg ml^?1 with a low detection limit of 0.032 μg ml^?1. Therefore, the N‐CDs hold admirable application potential for constructing a fluorescent sensor for pharmaceutical analysis.     

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.     

Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry

Carvedilol is an antihypertensive drug available as a racemic mixture. (?)‐(S)‐carvedilol is responsible for the nonselective β‐blocker activity but both enantiomers present similar activity on α₁‐adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T® (Teicoplanin) column. Protonated ions [M + H]⁺ and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml^?1 for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)‐(R)‐carvedilol (AUC^0–∞ 205.52 vs. 82.61 (ng h) ml^?1), with an enantiomer ratio of 2.48. Chirality, 2012. © 2012 Wiley Periodicals, Inc.     

Enantiomeric assay of escitalopram S(+)‐enantiomer and its “in‐process impurities” using two different techniques

The enantiomeric purity of escitalopram oxalate ESC and its “in‐process impurities,” namely, ESC‐N‐oxide, ESC‐citadiol, and R(?)‐enantiomer were studied in drug substance and products using high‐performance liquid chromatography (HPLC)‐UV (Method I), synchronous fluorescence spectroscopy (SFS) (Method IIA), and first derivative SFS (Method IIB). Method I describes as an isocratic HPLC‐UV for the direct resolution and determination of enantiomeric purity of ESC and its “in‐process impurities.” The proposed method involved the use of α_l‐acid glycoprotein (AGP) chiral stationary phase. The regression plots revealed good linear relationships of concentration range of 0.25 to 100 and 0.25 to 10 μg mL^?1 for ESC and its impurities. The limits of detection and quantifications for ESC were 0.075 and 0.235 μg mL^?1, respectively. Method II involves the significant enhancement of the fluorescence intensities of ESC and its impurities through inclusion complexes formation with hydroxyl propyl‐β‐cyclodextrin as a chiral selector in Micliavain buffer. Method IIA describes SFS technique for assay of ESC at 225 nm in presence of its impurities: R(?)‐enantiomer, citadiol, and N‐oxide at ?λ of 100 nm. This method was extended to (Method IIB) to apply first derivative SFS for the simultaneous determination of ESC at 236 nm and its impurities: the R(?)‐enantiomer, citadiol, and N‐oxide at 308, 275, and 280 nm, respectively. Linearity ranges were found to be 0.01 to 1.0 μg mL^?1 for ESC and its impurities with lower detection and quantification limits of 0.033/0.011 and 0.038/0.013 μg mL^?1 for SFS and first derivative synchronous fluorescence spectra (FDSFS), respectively. The methods were used to investigate the enantiomeric purity of escitalopram.     

Copper nanocluster‐based fluorescence enhanced determination of d‐penicillamine

Taking advantage of the compelling properties of d ‐penicillamine (d ‐PA) combined with copper, a method for the sensitive and selective determination of d ‐PA was established using copper nanocluster (Cu NC)‐based fluorescence enhancement. d ‐PA molecules containing a thiol compound showed a strong tendency to combine with the surface of Cu NCs, causing the re‐dispersion of nanoclusters and therefore fluorescence intensity was enhanced. Fluorescence enhancement efficiency of Cu NCs induced by d ‐PA was linear, with the d ‐PA concentration varying from 0.6–30 μg ml^?1 (R² = 0.9952) and with a detection limit of 0.54 μg ml^?1. d ‐PA content in human urine samples was detected with recoveries of 104.8–112.99%. Fluorescence‐enhanced determination of d ‐PA using Cu NCs was established for the first time and this rapid, easy and sensitive method should attract much attention for this application.     

Seasonal changes in the concentration of estrogens and testosterone in the plasma of the stallion

A blood sample was taken from each of 15 stallions at monthly intervals for 14 consecutive months. Plasma concentrations of estrogens and testosterone were measured by radioimmunoassay methods. Estrogens in peripheral blood were present in much higher amounts than testosterone and were principally in a water-soluble, solvolyzable form (> 98%). The major component in the solvolyzed extracts behaved chromatographically as estrone. The mean plasma level (± S.E.) of estrogens averaged across months was 52.9 ± 4.5 ng ml^?1. Individual stallions showed considerable month-to-month variation; for example, single monthly samples ranged from 29.5 to 160.6 ng ml^?1 for the stallion with the highest single value.The highest mean monthly concentration was 69 ± ng ml^?1 in May, and plasma levels were < 40 ng ml^?1 during November and December. For the 11 Thoroughbred stallions in the study, the mean concentrations of estrogens were 73 ± 5.8 ng ml^?1 for May to July and 45 ± 4.1 ng ml^?1 for November to January (P > 0.001).The mean monthly concentrations (± S.E.) of testosterone ranged from 0.22 ± 0.05 to 0.90 ± 0.14 ng ml^?1, and individual samples ranged from < 0.02 to 2.8 ng ml^?1 of plasma. While the highest mean level of testosterone was seen in September, there was a significant difference (P < 0.01) between the values in the breeding season (May–July, 0.73 ± 0.07 ng ml^?1) and the non-breeding season (November–January, 0.38 ± 0.08 ng ml^?1). No marked seasonal changes were observed, however, in testosterone levels in several stallions. It was concluded that plasma estrogen levels may provide a more sensitive index of endocrine function of the testes in the stallion.     

Utility of Von Pechman synthesis of coumarin reaction for development of spectrofluorimetric method for quantitation of salmeterol xinafoate in pharmaceutical preparations and human plasma

Simple, precise and selective spectrofluorimetric technique was evolved for quantitation of selective β₂ agonist drug namely salmeterol xinafoate (SAL). Utilizing its phenolic nature, a method was described based on the reaction of the studied drug with ethyl acetoacetate (EAA) to yield extremely fluorescent coumarin product which can be detected at 480 nm (λ_ex = 420 nm). The procedure obeys Beer's law with a correlation coefficient of r = 0.9999 in the concentration range between 500 and 5000 ng ml^?1 with and 177 ng ml^?1 for limit of detection (LOD) and limit of quantification (LOQ), respectively. Diverse reaction variables influencing the firmness and formation of the coumarin product were accurately examined and modified to ensure greatest sensitivity of the procedure. The proposed technique was performed and examined according to the US Food and Drug Administration (FDA) guidelines for bio‐analytical methods and was efficiently applied for quantitation of SAL in both pharmaceutical preparations (% recovery = 100.06 ± 1.07) and spiked human plasma (% recovery = 96.64–97.14 ± 1.01–1.52).     

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.     

Isoquinoline-based intrinsic fluorescence assessment of erythropoiesis-stimulating agent,Roxadustat (FG-4592), in tablets: applications to content uniformity and human plasma evaluation

In the present study, a first validated and green spectrofluorimetric approach for its assessment and evaluation in different matrices was investigated. After using an excitation wavelength of 345 nm, Roxadustat (ROX) demonstrates a highly native fluorescence at an emission of 410 nm. The influences of experimental factors such as pH, diluting solvents, and different organized media were tested, and the most appropriate solvent choice was ethanol. It was confirmed that there was a linear relationship between the concentration of ROX and the relative fluorescence intensity in the range 60.0–1000.0 ng ml^−1, with the limit of detection and limit of quantitation, respectively, being 17.0 and 53.0 ng ml⁻¹. The mean recoveries % [±standard deviation (SD), n = 5] for pharmaceutical preparations were 100.11% ± 2.24%, whereas for plasma samples, they were 100.08 ± 1.08% (±SD, n = 5). The results obtained after the application of four greenness criteria, Analytical Eco-Scale metric, NEMI, GAPI, and AGREE metric, confirmed its eco-friendliness. In addition, the whiteness meter (RGB12) confirmed its level of sustainability. The International Council for Harmonisation (ICH) criteria were used to verify the developed method through the study in both spiked plasma samples and content uniformity evaluation. An appropriate standard for various applications in industry and quality control laboratories was developed.     

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.     

Utility of Cremophor RH 40 as a micellar improvement for spectrofluorimetric estimation of sorafenib in pure form,commercial preparation,and human plasma

An easy, quick, simple and accurate spectrofluorimetric method was recognized and validated for evaluation of sorafenib (SOR) in pure form and biologically in plasma. Cremophor RH 40 (Cr RH 40) used for enhancing the fluorescence activity of SOR in phosphate buffer (pH 7). Cr RH 40 improved the native fluorescence of SOR remarkably in water. The fluorescence spectrum of SOR was observed at 405 nm after excitation at 265 nm. The linearity appeared to be in the range of 5 to 600 ng ml^?1 for pure and from 9 to 500 ng ml^?1 for plasma using the protein precipitation (ppt) method while from 10 to 500 ng ml^?1 for plasma using liquid–liquid extraction method. The precisions and the accuracy of the estimated method gave satisfactory results. The recommended method was effectively applied for determination of SOR in human plasma with high recovery values. The results of some compounds that are possibly found in plasma were studied. The proposed method was also focused on real volunteers and a drug dissolution test.