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.

Green micellar spectrofluorimetric determination of α-mangostin found in herbal products as antioxidant and other biological activities beneficial to human health.

Pharmaceutical product quality control (QC) needs quick, sensitive and economical procedures to deliver high throughput at low cost, which is the key factor considered by such economic facilities. To lessen the risky effects of research laboratories, researchers must take into account the ecological impacts. α-Mangostin (MAG) exhibit anti-inflammatory, antioxidant, anticancer, anti-allergic, antibacterial, antifungal, antiviral and antimalarial activities. Based on the spectrofluorimetric approach, a novel straightforward, sensitive and environmentally friendly method for MAG determination was developed and validated. Many variables were investigated to improve MAG native fluorescence, including solvent type, buffers, pH and additional surfactants. The best MAG fluorescence sensitivity was found in Britton-Robinson buffer (pH 4) at 450 nm after irradiation at 350 nm in the concentration range of 5-50 ng ml-1 . The technique was successfully used to determine the presence of MAG in both its approved dose forms and in samples of spiked human plasma, as per FDA standards for validation. According to their evaluation on two recent greenness criteria (GAPI [Green Analytical Procedure Index] and AGREE [Analytical GREEnness]), the suggested approach has been shown to be environmentally beneficial because it normally uses biodegradable chemicals in solvent-free aqueous phases.

Sensitivity improvement for spectrofluorimetric determination of commonly used antifungal drug 'nystatin': application for oral suspension.

In this paper, two simple, rapid and highly sensitive spectrofluorimetric methods were developed and validated for nystatin determination in its pure form and pharmaceutical dosage form (oral suspension). The first method was based on measuring the nystatin native fluorescence after dilution with isopropyl alcohol at 407 nm (excitation 303 nm). The fluoresence intensity was linearly dependant on the nystatin concentration within the specified range 50-500 ng ml-1 . The second was based on micellar enhancement of nystatin fluorescence using sodium dodecyl sulphate (SDS). In the presence of 2% w/v SDS, an ~1.9-fold enhancement could be achieved in the relative fluorescence intensity of nystatin. The linear range for the second method was 20-100 ng ml-1 . The limits of quantification and detection were found to be 43.23 ng ml-1 and 14.27 ng ml-1 (Method I), 6.08 ng ml-1 and 2.0 ng ml-1 (Method II). According to percentage recoveries and relative standard deviations (RSDs) obtained, the proposed methods were precise (RSDs were less than 2%), reproducible, and accurate and could be successfully applied for quantitative estimation of nystatin in its dosage form. The statistical results of this method were compared with that of the reported method and showed excellent agreement with respect to accuracy and precision.

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.

Green micellar solvent-free HPLC and spectrofluorimetric determination of favipiravir as one of COVID-19 antiviral regimens.

Quality control (QC) of pharmaceutical products requires fast, sensitive as well as economic methodologies in order to provide high through output at low cost which are the main aspects considered by such economic facilities. Meanwhile, the ecological impacts must be considered by researchers to minimize the hazardous effects of research laboratories. Favipiravir (FAV) is an antiviral agent recently approved for treatment of COVID-19 infections during 2020 pandemic crisis, so the size of its production by international pharmaceutical corporations evolved dramatically within the past few months. Two novel simple, sensitive, and green methods were developed and validated for FAV determination based on solvent-free micellar LC and spectrofluorimetry techniques. To improve FAV native fluorescence, several factors were studied including solvent type, buffering, pH and added surfactants. The best sensitivity for FAV fluorescence was obtained in Britton-Robinson buffer (pH 4) at 436 nm after excitation at 323 nm within concentration range of 20-350 ng mL-1. Another HPLC method was validated using C18-RP (5 µm, 250 × 4.6 mm) stationary phase and solvent-free mobile phase consisting of (0.02 M Brij-35, 0.15 M SDS, and 0.02 M disodium hydrogen phosphate, pH 5.0) isocratically eluted at a flow rate of 1 mL min-1 and detection wavelength of 323 nm. LC method was validated across concentration range of 10-100 µg mL-1 and FAV eluted in 3.8 min. The methods were validated according to the FDA guidelines and were applied successfully for determination of FAV in its marketed tablet dosage forms and in spiked human plasma samples. The proposed methods are eco-friendly since they are typically based on biodegradable reagents in aqueous solvent-free phases, which was proven by their assessment on two recent greenness metrics (GAPI and AGREE) to prove their eco-friendly properties.

Green spectrofluorimetric determination of salmeterol xinafoate in its pure forms, medicinal commercial formula, and human plasma: Application for stability studies.

A natural, accurate, extremely rapid, and precise spectrofluorometric method has been developed and validated for determination of salmeterol (SAL) xinafoate in its medicinal commercial form and spiked human plasma. The native SAL fluorescence has been measured at 415 nm (after 340 nm as excitation) in distilled water. Different factors affecting the native fluorescence consistency of SAL were surveyed and optimized. The suggested procedure was capable of SAL determination over concentrations ranging 200-2000 ng.mL-1 with excellent correlation coefficient of 0.9995. The limits of detection and quantification were estimated as 44.44 ng mL-1 and 134.66 ng mL-1 , respectively. The method has good accuracy and precision. The green spectrofluorometric method was used for determination of SAL in its commercial preparations and the results were in accordance with other reported methods regarding accuracy and precision. Moreover, the proposed procedure was enforced for stability indicating assay of SAL and for SAL determination in spiked human plasma. Nearly no cost, high sensitivity, and wide application make the proposed method ideally suited for analysis of SAL in quality control laboratories.

Elucidating the Anomalous Binding Enhancement of Isoquinoline Boronic Acid for Sialic Acid Under Acidic Conditions: Expanding Biorecognition Beyond Vicinal Diols.

A zwitterionic heterocyclic boronic acid based on 4-isoquinolineboronic acid (IQBA) exhibits the highest reported binding affinity for sialic acid or N-acetylneuraminic acid (Neu5Ac, K=5390±190 m-1 ) through the formation of a cyclic boronate ester complex under acidic conditions (pH 3). This anomalous pH-dependent binding enhancement does not occur with common neutral saccharides (e.g., glucose, fructose, sorbitiol), because it is mediated via selective complexation to a α-hydroxycarboxylate moiety forming a stable ion pair and ternary complex with Neu5Ac in phosphate buffer. IQBA expands biorecognition beyond classical vicinal diols under neutral or alkaline buffer conditions, which enables the direct analysis of Neu5Ac by native fluorescence with sub-micromolar detection limits.

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).

Early detection of colorectal adenocarcinoma: a clinical decision support tool based on plasma porphyrin accumulation and risk factors.

BACKGROUND: An increase in naturally-occurring porphyrins has been described in the blood of subjects bearing different kinds of tumors, including colorectal, and this is probably related to a systemic alteration of heme metabolism induced by tumor cells. The aim of our study was to develop an artificial neural network (ANN) classifier for early detection of colorectal adenocarcinoma based on plasma porphyrin accumulation and risk factors. METHODS: We measured the endogenous fluorescence of blood plasma in 100 colorectal adenocarcinoma patients and 112 controls using a conventional spectrofluorometer. Height, weight, personal and family medical history, use of alcohol, red meat, vegetables and tobacco were all recorded. An ANN model was built up from demographic data and from the integral of the fluorescence emission peak in the range 610-650 nm. We used the Receiver Operating Characteristic (ROC) curve to assess performance in distinguishing colorectal adenocarcinoma patients and controls. A liquid chromatography-high resolution mass spectrometry (LC-HRMS) analytical method was employed to identify the agents responsible for native fluorescence. RESULTS: The fluorescence analysis indicated that the integral of the fluorescence emission peak in the range 610-650 nm was significantly higher in colorectal adenocarcinoma patients than controls (p < 0.0001) and was weakly correlated with the TNM staging (Spearman's rho = 0.224, p = 0.011). LC-HRMS measurements showed that the agents responsible for the fluorescence emission were mainly protoporphyrin-IX (PpIX) and coproporphyrin-I (CpI). The overall accuracy of our ANN model was 88% (87% sensitivity and 90% specificity) with an area under the ROC curve of 0.83. CONCLUSIONS: These results confirm that tumor cells accumulate a diagnostic level of endogenous porphyrin compounds and suggest that plasma porphyrin concentrations, indirectly measured through fluorescence analysis, may be useful, together with risk factors, as a clinical decision support tool for the early detection of colorectal adenocarcinoma. Our future efforts will be aimed at examining how plasma porphyrin accumulation correlates with survival and response to therapy.

Validated sensitive spectrofluorimetric method for determination of antihistaminic drug azelastine HCl in pure form and in pharmaceutical dosage forms: application to stability study.

A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of azelastine HCl (AZL) in either its pure state or pharmaceutical dosage form. The proposed method was based on measuring the native fluorescence of the studied drug in 0.2 M H2 SO4 at λem  = 364 nm after excitation at λex  = 275 nm. Different experimental parameters were studied and optimized carefully to obtain the highest fluorescence intensity. The proposed method showed a linear dependence of the fluorescence intensity on drug concentration over a concentration range of 10-250 ng/mL, with a limit of detection of 1.52 ng/mL and limit of quantitation of 4.61 ng/mL. Moreover, the method was successfully applied to pharmaceutical preparations, with percent recovery values (± SD) of 99.96 (± 0.4) and 100.1 (± 0.52) for nasal spray and eye drops, respectively. The results were in good agreement with those obtained by the comparison method, as revealed by Student's t-test and the variance ratio F-test. The method was extended to study the stability of AZL under stress conditions, where the drug was exposed to neutral, acidic, alkaline, oxidative and photolytic degradation according to International Conference on Harmonization (ICH) guidelines. Copyright © 2016 John Wiley & Sons, Ltd.

Validated spectrofluorimetric method for determination of two phosphodiesterase inhibitors tadalafil and vardenafil in pharmaceutical preparations and spiked human plasma.

A valid, sensitive and rapid spectrofluorimetric method has been developed and validated for determination of both tadalafil (TAD) and vardenafil (VAR) either in their pure form, in their tablet dosage forms or spiked in human plasma. This method is based on measurement of the native fluorescence of both drugs in acetonitrile at λem 330 and 470 nm after excitation at 280 and 275 nm for tadalafil and vardenafil, respectively. Linear relationships were obtained over the concentration range 4-40 and 10-250 ng/mL with a minimum detection of 1 and 3 ng/mL for tadalafil and vardenafil, respectively. Various experimental parameters affecting the fluorescence intensity were carefully studied and optimized. The developed method was applied successfully for the determination of tadalafil and vardenafil in bulk drugs and tablet dosage forms. Moreover, the high sensitivity of the proposed method permitted their determination in spiked human plasma. The developed method was validated in terms of specificity, linearity, lower limit of quantification (LOQ), lower limit of detection (LOD), precision and accuracy. The mean recoveries of the analytes in pharmaceutical preparations were in agreement with those obtained from the comparison methods, as revealed by statistical analysis of the obtained results using Student's t-test and the variance ratio F-test.

Determination of Some Non-sedating Antihistamines via Their Native Fluorescence and Derivation of Some Quantitative Fluorescence Intensity - Structure Relationships.

A validated simple, novel, and rapid spectrofluorimetric method was developed for the determination of some non-sedating antihistamines (NSAs); namely cetirizine (CTZ), ebastine (EBS), fexofenadine (FXD), and loratadine (LOR). The method is based on measuring the native fluorescence of the cited drugs after protonation in acidic media and studying their quantitative fluorescence intensity - structure relationships. There was a linear relationship between the relative fluorescence intensity and the concentration of the investigated drug. Under the optimal conditions, the linear ranges of calibration curves for the determination of the studied NSAs were 0.10-2.0, 0.20-6.0, and 0.02-1.0 [Formula: see text] for (CTZ, FXD), (EBS), and (LOR); respectively. The factors affecting the protonation of the studied drugs were carefully studied and optimized. The method was validated according to ICH guidelines. The suggested method is applicable for the determination of the four investigated drugs in bulk and pharmaceutical dosage forms with excellent recoveries (97.67-103.80%). Quantitative relationships were found between the relative fluorescence intensities of the protonated drugs and their physicochemical parameters namely: the pKa, log P, connectivity indexes (χ(v)) and their squares. Regression equations (76) were obtained and not previously reported. Six of these equations were highly significant and used for the prediction of RFI of the studied NSAs.

Characterization and classification of pathogenic bacteria using native fluorescence and spectral deconvolution.

Identification and classification of pathogenic bacterial strains is of current interest for the early treatment of diseases. In this work, protein fluorescence from eight different pathogenic bacterial strains were characterized using steady state and time resolved fluorescence spectroscopy. The spectral deconvolution method was also employed to decompose the emission contribution from different intrinsic fluorophores and extracted various key parameters, such as intensity, emission maxima, emission line width of the fluorophores, and optical redox ratio. The change in average lifetime values across different bacterial strains exhibits good statistical significance (p ≤ 0.01). The variations in the photophysical characteristics of bacterial strains are due to the different conformational states of the proteins. The stepwise multiple linear discriminate analysis of fluorescence emission spectra at 280 nm excitation across eight different bacterial strains classifies the original groups and cross validated group with 100% and 99.5% accuracy, respectively.

Spectrofluorometric determination of cinacalcet hydrochloride: greenness assessment and application to biological fluids andin-vitrodissolution testing.

A facile, simple, green and sensitive spectrofluorometric method was developed for determination of the calcimimetic drug cinacalcet hydrochloride. It is used for the treatment of hyperparathyroidism. The drug showed high native fluorescence intensity at 320 nm after excitation at 280 nm. The method was linear over the range of 5.0-400.0 ng ml-1with excellent correlation (R2= 0.9999). Limit of detection (LOD) and limit of quantitation (LOQ) values were 1.19 and 3.62 ng ml-1, respectively. The percentage recovery was found to be 100.42% ± 1.39 (n=8). The proposed method was successfully applied for determination of cinacalcet in spiked human plasma samples with % recoveries of (87.23 to 109.69%). Two recent greenness metrics (GAPI and Analytical Eco-Scale) were chosen to prove the eco-friendly nature of the method. Furthermore, the proposed method was successfully applied to dissolution study of commercial cinacalcet tablets. The interference likely to be introduced by some commonly co-administrated drugs such as metoprolol and itraconazole was studied; the tolerance limits were calculated.

Sodium dodecyl sulfate-capillary gel electrophoresis with native fluorescence detection for analysis of therapeutic proteins.

A 280 nm light-emitting diode (LED) was used as the excitation source for native fluorescence detection (NFD) of proteins in capillary electrophoresis. The NFD scheme was evaluated in sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) for monoclonal antibody (mAb) characterization. Utilizing a technique by which we filtered the LED emission through a 280 nm bandpass filter, we were able to increase overall concentration sensitivity of SDS-CGE-NFD ~2.3-fold. Under the optimized conditions, the assay linear dynamic range was > 4 orders of magnitude with a correlation coefficient (r2) of 0.9999, and the limit of detection was 8.3 ng/mL. The SDS-CGE-NFD assay was applied to quantitation and purity analysis of Etanercept, a therapeutic protein. Over the range of 50 - 150% of the target concentration, 200 µg/mL, recoveries were in the range of 97.02 - 101.6%. The SDS-CGE-NFD assay allowed for simultaneous quantitation of high- and low-molecular-weight species in Etanercept.

Green quantitative spectrofluorometric analysis of rupatadine and montelukast at nanogram scale using direct and synchronous techniques.

Two green-sensitive spectrofluorometric methods were investigated for assay of rupatadine (RUP) [method I] and its binary mixture with montelukast (MKT) [method II]. Method I depends on measuring native fluorescence of RUP in the presence of 0.10 M H2SO4 and 0.10%w/v sodium dodecyl sulfate at 455 nm after excitation at 277 nm. The range of the first method was 0.20-2.00 µg ml-1 with detection and quantitation limits of 59.00 and 179.00 ng ml-1, respectively. Method II depends on the first derivative synchronous spectrofluorometry. The derivative intensities were measured for the two drugs in an aqueous solution containing Mcllvaine's buffer pH 2.60 at fixed Δλ of 140 nm. Each drug was estimated at zero-contribution of the other. The intensity was measured at 261 and 371 nm for RUP and MKT, respectively. The method was linear over 0.10-4.00 and 0.20-1.60 µg ml-1 with limits of detection 31.00 and 66.00 ng ml-1 and limits of quantitation 94.00 and 200.00 ng ml-1 for RUP and MKT, respectively. The method was extended to determine this mixture in laboratory-prepared mixtures and combined tablets. Method validation was performed according to ICH guidelines. Statistical interpretation of data revealed good agreement with the comparison method. Method greenness was confirmed by applying three different assessment tools.

Capillary isoelectric focusing with UV fluorescence imaging detection enables direct charge heterogeneity characterization of erythropoietin drug products.

An imaged capillary isoelectric focusing (icIEF) - UV fluorescence imaging detection method is described for the direct charge heterogeneity characterization of recombinant human erythropoietin (rhEPO) drug products (DPs). rhEPO is one of the most important protein therapeutics for biopharmaceutical industry worldwide. As a heavily glycosylated protein therapeutic, its charge heterogeneity must be carefully monitored in each step of manufacturing and storage. Current charge characterization methods suffer from challenges to characterize rhEPO DPs, due to low sensitivity of the method and potential for interference from the DP's formulation. The method described herein leverages the separation power of imaged cIEF separation combined with the increased sensitivity afforded by UV fluorescence imaging detection and requires no pre-treatment of the DP sample prior to analysis. The method was evaluated initially using a simulated DP, and subsequently a mini method validation was performed using a commercial rhEPO DP sample according to the guideline set by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). The limit of quantitation (LOQ) of the method is validated to be 20.3 IU/mL (or 0.10 µg/mL), which is approximately 100 times more sensitive than CZE - UV absorption detection method. To demonstrate the applicability of the method for use, 8 different commercial rhEPO DPs with concentrations ranging from 2000 IU/mL - 10,000 IU/mL were successfully evaluated. This method allows for sensitive, rapid analysis of low concentration rhEPO drug products without sample pre-treatment to provide critical charge heterogeneity information.

After enjoying curry: urine metabolism analysis of curcuminoids by microemulsion electrokinetic chromatography with laser-induced native fluorescence detection.

Considering pH-dependent fluorescence of curcuminoids, a microemulsion electrokinetic chromatographic (MEEKC) method was developed under acidic conditions for their separation and detection using laser-induced native fluorescence (LINF), so as to solve the analysis of urine metabolism for curcuminoids. The microemulsion composition was optimized by response surface methodology (RSM), and the effects of buffer pH and organic modifiers were systematically investigated. The optimal buffer for the separation of curcuminoids was chosen as follows: 2.8% (v/v) ethyl acetate, 80 mM SDS and 2.8% (v/v) n-butanol to form microemulsion, 28% (v/v) ethanol as organic modifier, and 20 mM phosphoric acid as electrolyte at pH 3.0. Under these conditions, four curcuminoids including curcumin, demethoxy curcumin (DMC), bisdemethoxy curcumin (BDMC) and demethyl curcumin (DEC) could be well separated within 18 min, and the detection limits (LOD, based on S/N=3) were calculated to be 71, 60, 22, and 147 pg mL-1, respectively. Combined with solid-phase extraction (SPE), the developed MEEKC-LINF method has been successfully applied to continuously monitor the curcuminoids and related metabolites in human urine collected from a healthy volunteer after oral administration of curry, testifying that this method has potential for evaluating the pharmacological activity of curcuminoids.

Native fluorescence detection with a laser driven light source for protein analysis in capillary electrophoresis.

While ultraviolet light (UV) absorbance detection is the most widely used detection mode in capillary electrophoresis (CE), it can yield poor concentration sensitivity and has tendencies to exhibit baseline fluctuations. In order to overcome these challenges, alternative detection strategies, including the use of dedicated wavelength lasers, have been applied, resulting in enhancements of concentration sensitivity as well as decreased baseline disturbance. In this work, using a laser driven light source for excitation, we reported a native fluorescence detection (NFD) scheme for use in a commercial CE platform, PA 800 Plus Pharmaceutical Analysis System, for protein analysis. The CE-NFD system was characterized using tryptophan and a reduced IgG. We compared NFD with UV absorbance detection as applied to sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and capillary isoelectric focusing (cIEF). In SDS-CGE, with the reported NFD a non-reduced IgG standard sample yielded a signal-to-noise ratio which was 14.6 times higher than with UV absorbance detection at 214 nm. In cIEF analysis of NISTmAb, Humanized IgG1k, with NFD ∼170 times less sample mass was needed to obtain similar profile quality to that with UV absorbance detection at 280 nm. NFD also eliminated baseline anomalies observed with UV absorbance detection and showed less interference by other absorbing species. These results suggest that CE-NFD is a practical and powerful tool for protein characterization in the biopharmaceutical industry.

Native and synchronous fluorescence spectroscopy for determination of avanafil in presence of its co-formulated drug (dapoxetine hydrochloride): Application to pharmaceutical product, biological fluid and content uniformity.

The native and synchronous fluorescence spectroscopy procedures have been established and validated for the simultaneous determination of a binary mixture of dapoxetine hydrochloride (DAP) and avanafil (AVA). The first procedure is based on measurement of native fluorescence intensity of both drugs at λEm 337 nm and 370 nm using λEx 290 nm and 314 nm for DAP and AVA in methanol respectively. The second procedure describes a measurement of synchronous fluorescence intensity of these drugs at 232 nm for DAP, and 267 nm for AVA, using Δλ of 90nm. In the first procedure the fluorescence concentration were 0.1-4.0 µg/mL for DAP and 0.5-16 µg/mL for AVA. For the second procedure fluorescence concentrations were 0.025-1.0 µg/mL and 0.5-16 µg/mL for DAP and AVA respectively, with lower detection limit and quantification limits. The processes were successfully used for the limitation of DAP and AVA in their drug product without pre-separation. Then, the techniques were utilized for the determination of DAP and AVA in biological fluids. There is a good agreement between these results and the results obtained using a reference method.