Fluorescence Intensity-concentration Plot Research Articles

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.

Spectrofluorimetric determination of labetalol in pharmaceutical preparations and biological fluids

A simple and highly sensitive spectrofluorimetric method has been developed for the determination of labetalol (LBT) in pharmaceutical preparations and biological fluids. The method is based on the reaction between the nitroso-derivative of LBT and 2-cyanoacetamide (2-CAA) in the presence of ammonia to give fluorescent product with excitation wavelength of 335 nm and emission wavelength of 420 nm. The reaction conditions were studied and optimized. The fluorescence intensity-concentration plot is rectilinear over the concentration range of 0.025-0.250 μg/mL with minimum detectability of 1.40 ng/mL (3.6x10-9 M). The proposed method was successfully applied to commercial tablets containing LBT; the percentage recoveries agreed well with those obtained using the reference method. The method was further extended to the in-vitro determination of LBT in spiked human urine and plasma samples. The percentage recovery was 100.10 ± 3.44 and 101.27 ± 4.97, respectively. A proposed reaction pathway was postulated.

New spectrofluorimetric analysis of empagliflozin in its tablets and human plasma using two level full factorial design.

An efficient, accurate and sensitive spectrofluorimetric method was developed for analysis of empagliflozin (EGF) in pure form, dosage form and human plasma. The proposed procedure was based on formation of yellow fluorescent product between benzofurazan reagent and empagliflozin in slightly alkaline medium that is measured at 521nm, when excitation at 455nm. The present study was validated according to ICH guidelines and bioanalytical validated according to US-FDA guidance. The fluorescence intensity-concentration plot was linear over the range of 50-1000ngml-1 with limit of detection (LOD) and quantitation (LOQ) of 15.55 and 46.63ngml-1, respectively. The correlation (r) and determination (r2) coefficient was 0.9998 and 0.9997, respectively. Due to high sensitivity and selectivity of the proposed method, it is successfully used for analysis of empagliflozin in its dosage form and human plasma with good recoveries of 98.89% and 98.70%, respectively, without any interfering from matrix components. The corresponding regression equation, Y=0.756X+141.93, (r2=0.9994) for spiked plasma sample. Two level full factorial designs were used to study different experimental parameters that affect the reaction product and to get the optimum method conditions. The suggested method can be used in quality control lab as well as in pharmacokinetic studies of empagliflozin.

Selectivity Improvement for Spectrofluorimetric Determination of Oseltamivir Phosphate in Human Plasma and in the Presence of Its Degradation Product.

A simple and sensitive spectrofluorimetric method has been developed and validated for determination of oseltamivir phosphate (OSP). The proposed method is based on condensation reaction of the primary amino group of OSP with ninhydrin and phenylacetaldehyde in buffered medium (pH6.5). The formed yellow fluorescent product exhibits excitation and emission maxima at 390 and 460nm, respectively. The selectivity improvement of our proposed method is based on the water insolubility of the oseltamivir carboxylic acid (OSC) the active metabolite of OSP, which contains the same primary amino group as OSP but cannot, condensed with ninhydrin and phenylacetaldehyde reagents. The different experimental parameters affecting the formation and stability of the reaction product were carefully studied and optimized. The fluorescence intensity concentration plot is rectilinear in the range of 2-15μg ml-1 with detection and quantitation limits of 0.32 and 0.98μg ml-1, respectively. The proposed method was successfully applied for determination of OSP in commercial capsules, suspension and spiked human plasma with good percentage recovery. In addition, the developed procedure was extended to study the stability of OSP under different stress conditions; including acid and alkali hydrolysis, oxidation, photolysis, and thermal degradation. Furthermore, the kinetic of alkaline and acidic degradation of the cited drug were investigated. The apparent first order degradation rate constants were 0.258 and 0.318K h-1 with half times of 2.68 and 2.17h, for acidic and alkaline degradation, respectively.

Validated spectrofluorimetric methods for the determination of apixaban and tirofiban hydrochloride in pharmaceutical formulations

Apixaban and Tirofiban Hydrochloride are low molecular weight anticoagulants. The two drugs exhibit native fluorescence that allow the development of simple and valid spectrofluorimetric methods for the determination of Apixaban at λ ex/λ em=284/450nm and tirofiban HCl at λ ex/λ em=227/300nm in aqueous media. Different experimental parameters affecting fluorescence intensities were carefully studied and optimized. The fluorescence intensity-concentration plots were linear over the ranges of 0.2–6μgml−1 for apixaban and 0.2–5μgml−1 for tirofiban HCl. The limits of detection were 0.017 and 0.019μgml−1 and quantification limits were 0.057 and 0.066μgml−1 for apixaban and tirofiban HCl, respectively. The fluorescence quantum yield of apixaban and tirofiban were calculated with values of 0.43 and 0.49. Method validation was evaluated for linearity, specificity, accuracy, precision and robustness as per ICH guidelines. The proposed spectrofluorimetric methods were successfully applied for the determination of apixaban in Eliquis tablets and tirofiban HCl in Aggrastat intravenous infusion. Tolerance ratio was tested to study the effect of foreign interferences from dosage forms excipients. Using Student's t and F tests, revealed no statistically difference between the developed spectrofluorimetric methods and the comparison methods regarding the accuracy and precision, so can be contributed to the analysis of apixaban and tirofiban HCl in QC laboratories as an alternative method.

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

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

Spectrofluorimetric and micelle-enhanced spectrofluorimetric methods for determination of Felodipine and Nimodipine in pharmaceutical preparations and human plasma

Rapid, simple and sensitive two spectrofluorimetric methods have been developed for determination of Felodipine (FLD) and Nimodipine (NDP). The first method is based on measuring the native fluorescence of either Felodipine or Nimodipine at 426nm after excitation at 385nm. The fluorescence intensity-concentration plots of Felodipine and Nimodipine were rectilinear over the concentration ranges (0.2–3.0) and (0.5–4.0) μgml−1, respectively. The second method is based on measuring the fluorescence intensity of the studied drugs in micellar media (0.3% Tween-80) at λex=385nm and λem=423nm. In the presence of 0.3% Tween-80, about 1.6-fold and 2.1-fold enhancement can be achieved in the relative fluorescence intensity (RFI) of Felodipine and Nimodipine, respectively. The fluorescence intensity-concentration plots of Felodipine and Nimodipine with Tween-80 were rectilinear over the concentration ranges (0.05–4.0) and (0.1–4.0) μgml−1, respectively with determination coefficients (r2) of 0.9981 and 0.9990, and limit of quantitation of 0.05 and 0.027μgml−1 for FLD and NDP, respectively. The proposed methods were validated according to ICH guidelines and have been successfully applied to the analysis of these drugs in their commercial tablets with high accuracy (97.6–98.8±0.50–1.42%, n=5). The high sensitivity of micellar method permits its application for determination of the cited drugs in spiked human plasma with % recovery (91.9–106.6±0.66–1.7%, n=6).

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

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

Application of new spectrofluorometric techniques for determination of atorvastatin and ezetimibe in combined tablet dosage form.

Two accurate, reliable, and highly sensitive spectrofluorometric methods were developed for simultaneous determination of the binary mixture of Atorvastatin and Ezetimibe without prior separation steps. The first method is based on double scan synchronous fluorescence spectrometry. Each of Atorvastatin and Ezetimibe can be determined independent of the other when scanned at Δλ=100 nm and 40 nm, respectively. The relative fluorescence intensity-concentration plots at two wavelengths, 272 (Δλ=100 nm) and 266 nm (Δλ=40 nm) were rectilinear over the range of 0.4-8 µg/mL (for Atorvastatin) and 0.6-8 µg/mL (for Ezetimibe), respectively. The second method is based on the technique of simultaneous equations (Vierodt's method), in which two equations are solved simultaneously after using a single excitation wavelength of 273 nm and λEm1=380 nm of Atorvastatin and λEm2=301 nm of Ezetimibe. Under the optimum conditions, linear relationships were found between the relative fluorescence intensity and the concentrations of the investigated drugs in the range of 0.4-8 µg/mL (for Atorvastatin) 0.6-8 µg/mL (for Ezetimibe). The different experimental parameters affecting the fluorescence intensities of the two drugs were carefully studied and optimized. The proposed methods were successfully applied for the determination of the investigated drugs in pure form, dosage form and in synthetic mixtures with good recovery and the results obtained were favorably compared to those obtained with a reference method.

Stability-Indicating Spectrofluorimetric Method for Determination of Itopride Hydrochloride in Raw Material and Pharmaceutical Formulations

A simple, sensitive and rapid spectrofluorimetric method for determination of itopride hydrochloride in raw material and tablets has been developed. The proposed method is based on the measurement of the native fluorescence of the drug in water at 363nm after excitation at 255nm. The relative fluorescence intensity-concentration plot was rectilinear over the range of 0.1-2μg/mL (2.5 × 10(-7)-5.06 × 10(-6) mole/L), with good correlation (r = 0.9999), limit of detection of 0.015μg/mL and a lower limit of quantification of 0.045 μg/mL. The described method was successfully applied for the determination of itopride hydrochloride in its commercial tablets with average percentage recovery of 100.11 ± 0.32 without interference from common excipients. Additionally, the proposed method can be applied for determination of itopride in combined tablets with rabeprazole or pantoprazole without prior separation. The method was extended to stability study of itopride. The drug was exposed to acidic, alkaline, oxidative and photolytic degradation according to ICH guidelines. Moreover, the method was utilized to investigate the kinetics of the alkaline, acidic and oxidative degradation of the drug. A proposal for the degradation pathways was postulated.

Spectrofluorimetric determination of oseltamivir phosphate through derivatization with o‐phthalaldehyde. Application to pharmaceutical preparations with a preliminary study on spiked plasma samples

A simple and sensitive spectrofluorimetric method has been developed and validated for the determination of oseltamivir phosphate (OST) in pharmaceutical preparations. The method is based on the reaction between oseltamivir phosphate and o-phthalaldehyde in presence of 2-mercapto-ethanol in borate buffer, pH 10.8, to give a highly fluorescent product measured at 450 nm after excitation at 336 nm. The different experimental parameters affecting the development and stability of the reaction product were studied and optimized. The fluorescence intensity-concentration plot is rectilinear over the range 0.05-1.0 µg/mL, with a lower detection limit of 5 ng/mL and limit of quantitation of 16 ng/mL. The developed method was successfully applied to the analysis of the drug in its commercial capsules and suspension, mean recoveries of OST were 99.97 ± 1.67% and 100.17 ± 1.18%, respectively (n = 3). Statistical comparison of the results obtained by the proposed and comparison method revealed no significant difference in the performance of the two methods regarding accuracy and precision. The proposed method was further extended to in vitro determination of the studied drug in spiked human plasma as a preliminary investigation; the mean recovery (n = 3) was 98.68 ± 5.8%. A reaction pathway was postulated.

Spectrofluorimetric determination of aliskiren in dosage forms and urine

A new, simple and sensitive spectrofluorimetric method has been developed for the determination of aliskiren (ALS) in dosage forms and human urine. The method is based on the reaction between ALS and fluorescamine in borate buffer solution, pH 9, to give a highly fluorescent derivative which is measured at 482 nm after excitation at 382 nm. The factors affecting the reaction were carefully studied. The fluorescence intensity concentration plots were rectilinear over the range 140-1400 ng/mL with a limit of detection 13.47 ng/mL and limit of quantitation 40.81 ng/mL. The developed method was successfully applied to the analysis of the drug in tablets and human urine; the average recoveries (n = 6) were 99.88 ± 0.38% and 99.57 ± 0.44%, respectively. The analytical performance of the method was fully validated and the results were satisfactory. The stability of the drug was studied by subjecting it to acidic, basic, oxidative and thermal degradation.

Synchronous fluorescence spectrofluorimetric method for the simultaneous determination of metoprolol and felodipine in combined pharmaceutical preparation

A rapid, simple and sensitive synchronous specrtofluorimetric method has been developed for the simultaneous analysis of binary mixture of metoprolol (MTP) and felodipine (FDP). The method is based upon measurement of the synchronous fluorescence intensity of the two drugs at Δλ of 70 nm in aqueous solution. The different experimental parameters affecting the synchronous fluorescence intensities of the two drugs were carefully studied and optimized. The fluorescence intensity-concentration plots were rectilinear over the ranges of 0.5-10 μg/mL and 0.2-2 μg/mL for MTP and FDP, respectively. The limits of detection were 0.11 and 0.02 μg/mL and quantification limits were 0.32 and 0.06 μg/mL for MTP and FDP, respectively. The proposed method was successfully applied for the determination of the two compounds in their commercial tablets and the results obtained were favorably compared to those obtained with a comparison method.

Validated Spectroflurimetric Determination of Some H1 Receptor Antagonist Drugs in Pharmaceutical Preparations Through Charge Transfer Complexation

A validated simple, rapid, and selective spectrofluorimetric method was developed for the determination of some antihistaminic H(1) receptor antagonist drugs namely ebastine (EBS), cetirizine dihydrochloride (CTZ), and fexofenadine hydrochloride (FXD). The method is based on the reaction of the cited drugs with some Π acceptors namely p-chloranilic acid (CLA), tetracyanoethylene (TCNE), and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) to give highly fluorescent derivatives. The fluorescence intensity-concentration plots were rectilinear over the concentration ranges of 0.2-3.0, 0.2-2.5 and 0.15-2.0μg/ml for EBS with CLA, DDQ, and TCNE respectively; 0.5-7.0, 0.5-6.0, and 0.2-4.0μg/ml for CTZ with the previously mentioned reagents, and 0.2-3.5, 0.5-6.0, and 0.2-3.5μg/ml for FXD. The factors affecting the formation of the reaction products were carefully studied and optimized. The method was applied for the determination of the studied drugs in their dosage forms. The results obtained were in good agreement with those obtained by the comparison methods. Reactions Stoichiometries of the complexes formed between the studied drugs and Π acceptors were defined by the Job's method of the continuous variation and found in 1:1 in all cases.

Spectrofluorometric Determination of Methocarbamol in Pharmaceutical Preparations and Human Plasma

A simple, sensitive and rapid spectrofluorometric method for determination of methocarbamol in pharmaceutical formulations and spiked human plasma has been developed. The proposed method is based on the measurement of the native fluorescence of methocarbamol in methanol at 313 nm after excitation at 277 nm. The relative fluorescence intensity-concentration plot was rectilinear over the range of 0.05-2.0 μg/mL, with good correlation (r=0.9999), limit of detection of 0.007 μg/ mL and a lower limit of quantification of 0.022 μg/ mL. The described method was successfully applied for the determination of methocarbamol in its tablets without interference from co-formulated drugs, such as aspirin, diclofenac, paracetamol and ibuprofen, The results obtained were in good agreement with those obtained using the official method (USP 30).The high sensitivity of the method allowed the determination of the studied drug in spiked human plasma with average percentage recovery of 99.42 ± 3.84.

Simple and sensitive spectrofluorimetric method for the determination of pregabalin in capsules through derivatization with fluorescamine

A new, simple and sensitive spectrofluorimetric method has been developed for the determination of pregabalin (PG) in capsules. The method is based on the reaction between pregabalin and fluorescamine in borate buffer solution of pH 10 to give a highly fluorescent derivative that is measured at 487 nm after excitation at 390 nm. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. The fluorescence intensity concentration plot was rectilinear over the range of 0.01-0.3 µg mL⁻¹ with a lower detection limit of 0.0017 µg mL⁻¹ and limit of quantitation of 0.005 µg mL⁻¹. The developed method was successfully applied to the analysis of the drug in its commercial capsules. The mean percentage recovery of PG in its capsule was 99.93±1.24 (n = 3). Statistical comparison of the results with those of the comparison method revealed good agreement and proved that there was no significant difference in the accuracy and precision of the two methods. A proposed reaction pathway was postulated.

Simple and Sensitive Spectrofluorimetric Method for the Determination of Oseltamivir Phosphate in Capsules Through Derivatization with Fluorescamine

A new, simple and sensitive spectrofluorimetric method has been developed for the determination of oseltamivir phosphate (OSP) in capsules. The method is based on the reaction between oseltamivir and fluorescamine in borate buffer solution of pH 8.50 to give highly fluorescent derivatives that are measured at 483 nm using an excitation wavelength of 381. The different experimental parameters effecting the development and stability of the reaction product were carefully studied and optimized. The fluorescence intensity concentration plot is rectilinear over the range 50-450 ng mL(-1) with a lower detection limit (LOD) of 1.219 ng mL(-1) and limit of quantitation (LOQ) of 4.064 ng mL(-1). Selectivity was validated by subjecting stock solution of OSP to acidic, basic, oxidative, and thermal degradation. No interference was observed from excipients present in formulations. The developed method was successfully applied to determination of the drug in capsules. The mean % recovery (n = 6) was 100.08. The results obtained were in good agreement with those obtained using a reported spectrophotometric method.

Spectrofluorimetric Determination of Famotidine in Pharmaceutical Preparations and Biological Fluids. Application to Stability Studies

A simple, economic, selective, and stability indicating spectrofluorimetric method was developed for the determination of famotidine (FMT); is based on its reaction with 9, 10-phenanthraquinone in alkaline medium to give a highly fluorescent derivative measured at 560 nm after excitation at 283 nm. The fluorescence intensity-concentration plot was rectilinear over the concentration range of 50-600 ng/ml with minimum quantification limit (LOQ) of 13.0 ng/ml and minimum detection limit (LOD) of 4.3 ng/ml. The factors affecting the development of the fluorescence intensity of the reaction product were carefully studied and optimized. The method was applied for the determination of FMT in its dosage forms. The stability of the compound was studied, and the proposed method was found to be stability indicating one. The results obtained were in good agreement with those obtained by the official method. Furthermore, the method was applied for the determination of FMT in spiked and real human plasma. The mean % recovery (n = 4) was found to be 99.94 +/- 0.24, and 105.13 +/- 0.64 for spiked and real human plasma, respectively. The composition of the reaction product as well as its stability constant was also investigated. Moreover, the method was utilized to investigate the kinetics of both alkaline and oxidative induced degradation of the drug. The apparent first order rate constant and half life time of the degradation product was calculated. A proposal of the reaction pathway was postulated.

Spectrofluorometric Determination of Drotaverine Hydrochloride in Pharmaceutical Preparations

A highly sensitive, spectrofluorometric method was developed for the determination of drotaverine HCl in pharmaceutical preparations. The proposed method is based on the measurement of the native fluorescence of the drug in 0.1 M H2SO4 at emission 465 nm after excitation at 295 nm. The relative fluorescence intensity-concentration plot was rectilinear over the range of 0.16–4 µg·ml−1, with good correlation (r = 0.9999) and a lower limit of detection (LOD) of 0.032 µg·ml−1. The proposed method was successfully applied for the determination of drotaverine HCl in tablets and ampoules with a recovery percentage of 100.42 ± 0.601,99.83 ± 0.82 and 99.43 ± 1.08, respectively, which were in accordance with those obtained by a compendial method.

Spectrofluorometric determination of nicardipine, nifedipine and isradipine in pharmaceutical preparations and biological fluids

Abstract A simple and highly sensitive spectrofluorometric method was developed for the determination of some 1,4-dihydropyridine compounds namely, nicardipine, nifedipine and isradipine in pharmaceutical preparations and biological fluids. The method is based on the reduction of nicardipine, nifedipine and isradipine with Zn/HCl and measuring the fluorescence intensity obtained (λem/λex) at 460/364, 450/393 and 446/360 nm, respectively. The factors affecting the development of the fluorophore and its stability were studied and optimized. The effect of some surfactants such as β-cyclodextrin (βCD), carboxymethylcelullose (CMC), sodium dodecyl sulphate (SDS) and triton X-100, on the fluorescence intensity was studied. The fluorescence intensity-concentration plots of nicardipine, nifedipine and isradipine were rectilinear over the ranges 0.4–6.0, 0.2–4.0 and 0.1–9.0 μg ml−1 with detection limits of 0.0028, 0.017 and 0.016 μg ml−1, respectively. The proposed method was successfully applied to commercial tablets containing the compounds; the percentage recovery agreed well with those obtained using the official methods. The method was further extended to the in vitro determination of the compounds in spiked human plasma and urine samples. A proposal of the reduction reaction pathway was postulated.