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.     

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.     

Second‐derivative synchronous spectrofluorimetric assay of dapagliflozin: Application to stability study and pharmaceutical preparation

A highly accurate, simple and sensitive spectrofluorimetric analytical method for dapagliflozin (DGF) quantitation was developed. The proposed method was successively applied to DGF analysis in both its pure and pharmaceutical dosage forms. This method was developed to investigate DGF stability in its degradation products, as laid out in International Council for Harmonisation (ICH) rules. Kinetics of alkaline degradation of DGF was also calculated. The half‐life time (t_1/2) of the reaction was 75.32 min. An alkaline degradation pathway was described. The present study involved measurement of the second‐derivative synchronous fluorescence intensity of DGF at Δλ = 30 nm. Peak amplitude was measured at 322 nm. Linear range of the calibration curve was 0.1–1.0 μg ml^?1. Lower detection and quantitation limits were 0.023 and 0.071 μg ml^?1, respectively, and indicated good sensitivity of the proposed method. Mean per cent recovery was 99.78 ± 1.78%. The proposed analytical approach was successfully applied to DGF in the quality control laboratory and would be suitable as a stability‐indicating assay.     

Utility of the fluorogenic characters of benzofurazan for analysis of tigecycline using spectrometric technique; application to pharmacokinetic study,urine and pharmaceutical formulations

Tigecycline (TIGE) is the newest tetracycline derivative antibiotic with low toxicity, it is used for management of infectious diseases caused by Gram‐positive and Gram‐negative bacteria. Hence, an efficient, selective and sensitive method was developed for analysis of TIGE in commercial formulations, human plasma and urine. The spectrofluorimetric technique based on the reaction of secondary amine moiety in TIGE with 4‐chloro‐7‐nitrobenzofurazan (NBD‐Cl) in slightly alkaline medium producing a highly fluorescent product measured at 540 nm (λ_ex at 470 nm) after heating for 15 min at 75°C. The proposed strategy was upgraded and approved by ICH rules and bio‐analytical validated using US‐FDA recommendations. A linear relationship between fluorescence intensity and TIGE concentration was observed over the concentration range 40–500 ng mL^?1 with limit of quantification (LOQ) 21.09 ng mL^?1 and limit of detection (LOD) 6.96 ng mL^?1.The ultra‐affectability and high selectivity of the proposed strategy permits analysis of TIGE in dosage form, human plasma and urine samples with good recovery ranged from 97.23% to 98.72% and from 99.36% to 99.80% respectively, without any interfering from matrix components. Also, the developed strategy was used to examine the stability of TIGE in human plasma and applied for pharmacokinetic investigation of TIGE.     

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.     

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.     

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.     

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.     

Utility of Hantzsch reaction for development of highly sensitive spectrofluorimetric method for determination of alfuzosin and terazosin in bulk,dosage forms and human plasma

A highly sensitive, cheap, simple and accurate spectrofluorimetric method has been developed and validated for the determination of alfuzosin hydrochloride and terazosin hydrochloride in their pharmaceutical dosage forms and in human plasma. The developed method is based on the reaction of the primary amine moiety in the studied drugs with acetylacetone and formaldehyde according to the Hantzsch reaction, producing yellow fluorescent products that can be measured spectrofluorimetrically at 480 nm after excitation at 415 nm. Different experimental parameters affecting the development and stability of the reaction products were carefully studied and optimized. The fluorescence–concentration plots of alfuzosin and terazosin were rectilinear over a concentration range of 70–900 ng ml^?1, with quantitation limits 27.1 and 32.2 ng ml^?1 for alfuzosin and terazosin, respectively. The proposed method was validated according to ICH guidelines and successfully applied to the analysis of the investigated drugs in dosage forms, content uniformity test and spiked human plasma with high accuracy.     

Pharmacokinetic evaluation of daclatasvir and ledipasvir in healthy volunteers using a validated highly sensitive spectrofluorimetric method

A simple, highly sensitive and selective spectrofluorimetric method has been developed and fully validated for the determination of daclatasvir (DAC) and ledipasvir (LED) in tablets and human plasma. The method is based on measurement of the native fluorescence in methanol at λ_em 384 nm after excitation at λ_ex 318 nm for DAC and in acetonitrile at λ_em 402 nm after excitation at λ_ex 340 nm for LED. The fluorescence intensity (FI) concentration plot was rectilinear over the ranges 1.2–12, 0.1–18 ng ml^?1 and 9–90, 1–100 ng ml^?1 with a good correlation of r = 0.9994 to r = 0.9997 in standard solution and human plasma for DAC and LED, respectively. The extraction of analytes from plasma was performed using methanol and acetonitrile as a precipitating agent with lower limit of quantification (LLOQ) of 0.1 and 1.0 ng ml^?1 for DAC and LED; respectively. The proposed method was validated according to the US Food and Drug Administration (FDA) guidelines and successfully applied for estimating the pharmacokinetic parameters of DAC and LED following oral administrations of their tablets.     

Utilization of erythrosine B in spectrofluorimetric quenching analysis of amlodipine and perindopril in pharmaceutical and biological matrices

A spectrofluorimetric approach that is sensitive, simple, validated, and cost-effective has been proposed for the estimation of amlodipine (AML) and perindopril (PER) in their bulk powders, pharmaceutical formulations, and spiked human plasma. The recommended approach utilized the quantitative quenching effect of the two cited drugs on the fluorescence intensity of erythrosine B, as a result of complex binary reactions among each drug with erythrosine B at pH 3.5 (Teorell and Stenhagen buffer). The quenching of erythrosine B fluorescence was recorded at 554 nm after excitation at 527 nm. The calibration curve was detected in the range 0.25–3.0 μg ml⁻¹, with a correlation coefficient of 0.9996 for AML, and 0.1–1.5 μg ml⁻¹, with a correlation coefficient of 0.9996 for PER. The established spectrofluorimetric approach was validated for the estimation of the cited drugs with high sensitivity regarding International Council on Harmonization guidelines. Therefore, the established approach could be utilized for quality control of the cited drugs in their pharmaceutical formulations.     

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.     

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).     

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.     

Approved spectrofluorimetric strategies for assurance of three modern antineoplastic drugs: tepotinib,sotorasib, and darolutamide in their dose forms and biological liquids utilizing mercurochrome

An approved, straightforward, fast, and delicate spectrofluorimetric strategy was developed for the estimation of tepotinib (TEPO), sotorasib (SOTO), and darolutamide (DARO) as new antineoplastic drugs. The spectrofluorimetric strategy was based on quantitative fluorescence quenching of MER at 538 nm after being excited at 350 nm by the addition of the cited drugs in the presence of acetate buffer (pH 3.5). The degree of fluorescence quenching was directly proportional to the concentrations of the cited drugs within the concentration range of 0.5–10.0, 0.2–10, and 0.4–10.0 μg ml⁻¹ for TEPO, SOTO, and DARO, respectively. Mean ± standard deviation (SD) were calculated for the studied drugs as follows; 99.9 ± 0.87, 99.72 ± 1.08, and 100.21 ± 1.44, for TEPO, SOTO, and DARO, respectively. Limit of detection (LOD) values were 0.16, 0.05, and 0.11 μg ml⁻¹, whereas limit of quantitation (LOQ) values were 0.5, 0.15, and 0.36 μg ml⁻¹ for TEPO, SOTO, and DARO, respectively. Statistical comparison through detailed strategies produced greater understanding and found that there were no noteworthy contrasts in exactness and exactness between strategies. The proposed strategy was used effectively to analyze the measurement of different forms of the examined drugs. Moreover, the recommended fluorimetric strategy was used for examination of TEPO, SOTO, and DARO in human plasma and urine tests.     

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.     

Utility of green chemistry for sustainable fluorescence derivatization approach for spectrofluorimetric quantification of Darolutamide as antineoplastic drug in pharmaceutical formulation and spiked human plasma

Darolutamide is an oral nonsteroidal androgen receptor antagonist used to delay the process of prostate cancer to metastatic disease and to increase the quality of life for people with advanced prostate cancer. Here, a second spectrofluorimetric method was advanced for quantifying Darolutamide in pharmaceutical formulation and spiked human plasma. This method depends on the fluorescence derivatization of Darolutamide with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) at 75°C in a (pH 9) of borate buffer to produce a fluorescent derivative that can be detected at 520 nm after excitation at 460 nm. The method has been validated using ICH criteria, and it demonstrated linearity in the range 5–200 ng ml⁻¹_. The limit of detection (LOD) and limit of quantitation (LOQ) were 1.15 and 3.84 nm, respectively. The proposed method was applied precisely and accurately for quantifying Darolutamide within the pharmaceutical formulation and spiking human plasma without any interferences. Moreover, the method's sustainability was evaluated and compared with the published method using two greenness assessment tools termed analytical eco-scale and Analytical GREEnness (AGREE). These findings suggest that the method is more sustainable than the published method.     

Determination of sodium artesunate in plasma using ion-pairing high-performance liquid chromatography

A chromatographic method is described for the determination of sodium artesunate in plasma. This includes cetyltrimethylammonium bromide as a cationic pairing ion in a reversed-phase system using an octadecylsilica 100×4.6 mm I.D. 3 μm analytical column with a mobile phase of acetonitrile/acetate buffer at pH7. Column switching incorporating a 5 μm octadecylsilica 100×4.6 mm I.D. precolumn is used in addition to off-line solid-phase extraction for pretreatment of plasma samples in order to eliminate interference from endogenous components. Detection is by post-column derivatisation with 1.0 M methanolic KOH followed by UV detection at 289 nm. Calibration is linear over the range 100–1600 ng ml⁻¹ and the limit of detection is estimated as 20 ng ml⁻¹. Illustrative results are shown of the artesunate plasma levels determined by the proposed method following the administration of artesunate as tablets and as suppositories to healthy volunteers.     

A novel spectrofluorimetric method for determination of lomefloxacin adopting on zinc(II) chelation strategy: Application in human plasma

A new sensitive and instantaneous spectrofluorimetric method for efficient determination of lomefloxacin (LMX) in its pure, dosage form and human plasma was designed. The developed method depends on formation of a metal-chelation compound of LMX as a ligand with zinc(II) in a buffer of acetate (pH 5.5). The following parameters; type of metal, concentration of metal, pH, type of buffer and diluting solvent were optimized. After carefully investigation; 0.2 mM zinc, 2.0 ml acetate buffer (pH 5.5) and water as diluting solvent were set as optimum reaction conditions. Under these conditions, a large increase in the intensity of the fluorescence of LMX was attained at 450 after excitation at 284 nm. The limits of detection and quantification were 5.8 and 1.9 ng ml⁻¹, respectively, with linearity range of 10.0 to 500.0 ng ml⁻¹. The binding mode of LMX and zinc(II) ion (Zn²⁺) was found to be 2:1, respectively, and confirmed by Job's plot method. Furthermore, it extended to the analysis of LMX in the spiked plasma of humans with percentage recovery (98.70 ± 0.97 to 100.30 ± 1.69%, n = 3).