Conventional and First Derivative Synchronous Fluorometric Determination of Ethamsylate in Pharmaceutical Preparations and Biological Fluids. Application to Stability Studies

Two simple, accurate and highly sensitive spectrofluorometric methods were developed for the determination of ethamsylate (ETM). Method I is based on measuring the native fluorescence of ethamsylate in water at 354 nm after excitation at 302 nm. The calibration plot was rectilinear over the range of 0.05–1 μg/mL for ETM with limits of detection and quantitation of 7.9 and 26 ng/mL, respectively. Method II involved synchronous and first derivative synchronous fluorometric methods for the simultaneous determination of ethamsylate (ETM) and hydroquinone (HQ) which is considered as an impurity and/or acidic degradation product. The synchronous fluorescence of both the drug and its impurity were measured in methanol at Δ λ of 40 nm. The peak amplitudes (¹D) were estimated at 293.85 or 334.17 nm for ETM and at 309.05 nm for HQ. Good linearity was obtained for ETM over the ranges 0.1–1.4 μg/mL and 0.1–1.0 μg/mL at 293.85 and 334.17 nm, respectively. For HQ, the calibration plot was rectilinear over the range of 0.01–0.14 μg/mL at 309.05 nm. Limits of detection were 20, 2.01 ng/mL and limits of quantitation were 60, 6.7 ng/mL for ETM and HQ by method II, respectively. Both methods were successfully applied to commercial ampoules and tablets. The results were in good agreement with those obtained by the reference method. Method I was utilized to study the stability of ETM and its degradation kinetics using peroxide. The apparent first-order rate constant, half-life times and activation energy of the degradation process were calculated. Method I was further extended to the in-vitro and in-vivo determination of ETM in spiked and real plasma samples. The mean% recoveries were 99.57 ± 3.85 and 89.39 ± 5.93 for spiked and real human plasma, respectively.     

Spectrofluorimetric Determination of Fluvoxamine in Dosage Forms and Plasma Via Derivatization with 4-Chloro-7-Nitrobenzo-2-Oxa-1,3-Diazole

A highly sensitive and simple spectrofluorimetric method has been developed and validated for the determination of the antidepressant fluvoxamine (FXM) in its dosage forms and plasma. The method was based on nucleophilic substitution reaction of FXM with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole in an alkaline medium (pH 8) to form a highly fluorescent derivative that was measured at 535 nm after excitation at 470 nm. The factors affecting the reaction was carefully studied and optimized. The kinetics of the reaction was investigated, and the reaction mechanism was presented. Under the optimized conditions, linear relationship with good correlation coefficient (0.9995) was found between the fluorescence intensity and FXM concentration in the range of 65–800 ng ml⁻¹. The limits of detection and quantitation for the method were 21 and 64 ng ml⁻¹, respectively. The precision of the method was satisfactory; the values of relative standard deviations did not exceed 2.17%. The proposed method was successfully applied to the determination of FXM in its pharmaceutical tablets with good accuracy; the recovery values were 97.8–101.4 ± 1.08–2.75%. The results obtained by the proposed method were comparable with those obtained by the official method. The high sensitivity of the method allowed its successful application to the analysis of FXM in spiked human plasma. The proposed method is superior to the previously reported spectrofluorimetric method for determination of FXM in terms of its simplicity. The proposed method is practical and valuable for its routine application in quality control and clinical laboratories for analysis of FXM.     

Validated Spectrofluorimetric Method for the Determination of Lamotrigine in Tablets and Human Plasma Through Derivatization with <Emphasis Type="BoldItalic">o</Emphasis>-phthalaldehyde

A sensitive, simple and selective spectrofluorimetric method was developed for the determination of Lamotrigine (LMT) in pharmaceutical formulations and biological fluids. The method is based on reaction of LMT with o-phthalaldehyde in presence of 2-mercaptoethanol in borate buffer of pH 9.8 to yield a highly fluorescent derivative that is measured at 448 nm after excitation at 337 nm. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. The fluorescence-concentration plot was rectilinear over the range of 0.1–1.0 μg ml⁻¹ with lower limit of detection (LOD) 0.02 μg ml⁻¹ and limit of quantification (LOQ) 0.06 μg ml⁻¹ respectively. The proposed method was successfully applied to the the analysis of commercial tablets. Statistical comparison of the results obtained by the proposed and reference method revealed no significant difference in the performance of the two methods regarding the accuracy and precision respectively. The proposed method was further extended to the in-vitro and in-vivo determination of the drug in spiked and real human plasma. The mean percentage recoveries in spiked and real human plasma (n = 3) were 95.78 ± 1.37 and 90.93 ± 2.34 respectively. Interference arising from co-administered drugs was also studied. A proposal for the reaction pathway with o-phthalaldehyde was postulated.     

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.     

Spectrofluorimetric Determination of Oxamniquine in Dosage Forms and Spiked Human Plasma through Derivatization with 1-dimethylaminonaphthalene-5-sulphonyl chloride

A sensitive, simple and selective spectrofluorimetric method was developed for the determination of oxamniquine (OXM) in pharmaceutical formulations and biological fluids. The method is based on the reaction between the drug and 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl chloride) in presence of 0.5 M sodium carbonate (pH 10) to yield a highly fluorescent derivative that is measured at 445 nm after excitation at 335 nm. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. The fluorescence concentration plot was rectilinear over the range of 0.02–0.2 μg ml⁻¹ with a lower detection limit (LOD) of 0.007 μg ml⁻¹ and limit of quantitation (LOQ) of 0.02 μg ml⁻¹. The proposed method was successfully applied to the analysis of commercial capsules. The results obtained were in good agreement with those obtained using the official spectrophotometric method. Furthermore, the method was applied for the determination of oxamniquine in spiked human plasma, the mean % recovery (n = 4) is 97.77 ± 1.19. A proposal of the reaction pathway was presented.     

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.     

Photochemically-Induced Fluorescence Dosage of Non-Fluorescent Pyrethroid (Etofenprox) in Natural Water Using a Cationic Micellar Medium

An analytical method based on the use of UV-irradiation to produce fluorescent derivatives from Etofenprox a non-fluorescent pyrethroid insecticide is described. The impact of cetyltrimethylammonium chloride (CTAC) micellar medium on the Etofenprox photochemically-induced fluorescence (PIF) is reported. Parameters influencing the sensitivity and repeatability of the PIF method have been optimized. The alkaline medium (NaOH 6 × 10⁻² M) + CTAC surfactant molecules (3.84 mg/ml) in acetonitrile is found to be very suitable for this pyrethroid insecticide analysis in environment matrices. Linear dynamic range is established over more than two orders of magnitude. The limit of detection is lower than 5 ng/ml. The method seems to be suitable for environmental matrices quality control. Application to the analysis of spiked natural waters gave recoveries rate ranged from 94 to 104% and 107 to 115% respectively for river and pound water.     

First Derivative Synchronous Fluorescence Spectroscopy for the Simultaneous Determination of Sulpiride and Mebeverine Hydrochloride in Their Combined Tablets and Application to Real Human Plasma

A rapid, simple and highly sensitive first derivative synchronous fluorometric method has been developed for the simultaneous analysis of binary mixture of sulpiride (SUL) and mebeverine hydrochloride (MEB). The method is based upon measurement of the synchronous fluorescence intensity of these drugs at ∆λ = 100 nm in water. The different experimental parameters affecting the fluorescence of the two drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.05–1 μg/mL and 0.2–3.2 μg/mL for SUL and MEB respectively with lower detection limits (LOD) of 0.006 and 0.01 μg/mL and quantification limits (LOQ) of 0.0.02 and 0.05 μg/mL for SUL and MEB, respectively. The proposed method was successfully applied for the determination of the two compounds in synthetic mixtures and in commercial tablets. The high sensitivity attained by the proposed method allowed the determination of both of SUL and MEB metabolite (veratic acid) in real human plasma samples applying second derivative synchronous fluorometric technique. The mean% recoveries (n = 3) for both MEB metabolite (veratic acid) and SUL were 99.82 ± 2.53 and 98.84 ± 6.20 for spiked human plasma respectively, while for real human plasma, the mean% recoveries (n = 3) were 91.49 ± 4.25 and 91.36 ± 8.46 respectively.     

Stability-Indicating Spectrofluorimetric Method for the Assay of Ziprasidone in Capsules

A simple, rapid and highly sensitive spectrofluorimetric method was developed for determination of ziprasidone hydrochloride (ZPS) in capsules. The method is based on measuring the native fluorescence of ZPS in acetate buffer of pH 4.5 at 398 nm after excitation at 315 nm. The fluorescence-concentration plot was rectilinear over the range of 0.05–0.80 μg mL⁻¹ with a lower detection limit (LOD) of 6.0 ng mL⁻¹ and quantification limit (LOQ) of 20.0 ng mL⁻¹. The method was fully validated and successfully applied to the determination of ZPS in its capsules with average percentage recovery of 99.7 ± 1.4. The method was extended to stability study of ZPS. 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 Method for Determination and Validation of Cefixime in Pharmaceutical Preparations Through Derivatization with 2-Cyanoacetamide

A simple, sensitive and accurate method has been developed for spectrofluorimetric determination of cefixime in pure form and pharmaceutical preparations. The method is based on the reaction of cefixime with 2-cyanoacetamide in the presence of 21% ammonia at 100 °C. The fluorescent reaction product showed maximum fluorescence intensity at λ 378 nm after excitation at λ 330 nm. The factors affecting the derivatization reaction were carefully studied and optimized. The fluorescence intensity versus concentration plot was rectilinear over the range of 0.02 to 4 μg mL⁻¹ with correlation coefficient of 0.99036. The limit of detection (LOD) and limit of quantification (LOQ) was found to be 2.95 ng mL⁻¹ and 9.84 ng mL⁻¹, respectively. The proposed method was validated statistically and through recovery studies. The method was successfully applied for the determination of cefixime in pure and dosage form with percent recoveries from 98.117% to 100.38%. The results obtained from the proposed method have been compared with the official HPLC method and good agreement was found between them.     

Spectrofluorimetric Protocol for Ceftriaxone in Commercial Formulation and Human Plasma After Condensation with Formaldehyde and Ethyl Acetoacetate

A new spectrofluorimetric method has been developed and validated for the quantification of ceftriaxone in bulk powder, pharmaceutical formulations and spiked human plasma. The developed method is reproducible, accurate, sensitive and cost effective. In this method, ceftriaxone was converted into a fluorescent compound by reacting with 0.8 M ethyl acetoacetate and 25% formaldehyde in a buffered medium (pH = 4.2) at 90 °C. The excitation and emission wavelengths of the fluorescent reaction product are 316 nm and 388 nm respectively. Optimization of the experimental conditions affecting the condensation reaction were carefully carried out and the optimum experimental conditions were incorporated in the procedure. The developed method has a broad linear range (0.2–20 μg mL⁻¹) with a correlation coefficient of 0.9992. The limit of detection (LOD) and limit of quantification (LOQ) was found to be 1.94 × 10⁻² μg mL⁻¹ and 6.47 × 10⁻² μg mL⁻¹ respectively. The common excipients and co-administered drugs were investigated for their interferences effect in the assay. The developed method was validated statistically through recovery studies and successfully applied to ceftriaxone determination in bulk powder, pharmaceutical formulations and spiked human plasma samples. The percent recoveries were found to be in the range of 99.04–100.26% for bulk powder, 98.88–99.92% for pharmaceutical formulations and 94.22–98.48% for spiked human plasma. The results were verified by comparing with reference literature HPLC method and were found in good agreement.     

Second-derivative Synchronous Fluorometric Method for the Simultaneous Determination of Cinnarizine and Domperidone in Pharmaceutical Preparations. Application to Biological Fluids

A rapid, simple and highly sensitive second derivative synchronous fluorometric method has been developed for the simultaneous analysis of binary mixture of cinnarizine (CN) and domperidone (DOM). The method is based upon measurement of the native fluorescence of these drugs at Δλ = 80 nm in aqueous methanol (50% V/V). The different experimental parameters affecting the native fluorescence of the studied drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.1 to 1.3 μg mL⁻¹ and 0.1–3.0 μg mL⁻¹ for CN and DOM, respectively with lower detection limits of 0.017 and 5.77 × 10⁻³ μg mL⁻¹ and quantification limits of 0.058 and 0.02 μg mL⁻¹ for CN and DOM. The proposed method was successfully applied for the determination of the studied compounds in synthetic mixtures and in commercial tablets. The results obtained were in good agreement with those obtained with reference methods. The high sensitivity attained by the synchronous fluorometric method allowed the determination of CN in real and spiked human plasma. The mean % recoveries in case of spiked human plasma (n = 3) were 96.39 ± 1.18 while that in real human plasma (n = 3) was 104.67 ± 4.16.     

Micelle-Enhanced Spectrofluorimetric Method for Determination of Cholesterol-Reducing Drug Ezetimibe in Dosage Forms

A simple and sensitive spectrofluorimetric method was developed for the determination of ezetimibe in its pharmaceutical formulations. The proposed method is based on investigation of the fluorescence spectral behavior of ezetimibe in sodium dodecyl sulfate (SDS) micellar system. In aqueous solution of acetate buffer pH 5.0, the fluorescence intensity of ezetimibe was greatly enhanced, 200% enhancement, in the presence of SDS. The fluorescence intensity of ezetimibe was measured at 380 nm after excitation at 268 nm. The fluorescence-concentration plot was rectilinear over the range of 0.03–3.0 μg/mL with lower detection limit of 3.08 × 10⁻³ μg/mL. The method was successfully applied to the analysis of ezetimibe in its commercial tablets; the results were in good agreement with those obtained with the reported method. The application of the proposed method was extended to the stability studies of ezetimibe after exposure to different forced degradation conditions, such as acidic, alkaline, photo and oxidative conditions, according to ICH guidelines.     

Spectrofluorimetric Determination of Aliskiren in Tablets and Spiked Human Plasma through Derivatization with Dansyl Chloride

A simple and sensitive method has been developed and validated for the determination of aliskiren (ALS) in its dosage forms and spiked plasma. The method was based on the reaction of the drug with dansyl chloride in the presence of bicarbonate solution of pH 10.5 to give a highly fluorescent derivative which was measured at 501 nm with excitition at 378 nm in dichloromethane. Different experimental parameters affecting the development of the method and stability were carefully studied and optimized. The calibration curves were linear over the concentration ranges of 100–700 and 50–150 ng/mL for standard solution and plasma, respectively. The limits of detection were 27.52 ng/mL in standard solution, 4.91 ng/mL in plasma. The developed method was successfully applied to the analysis the drug in the commercial tablets and spiked plasma samples. The mean recovery of ALS from tablets and plasma was 100.10 and 97.81%, respectively. A proposal of the reaction pathway was presented.     

Sensitive Spectrofluorimetric Method of Analysis for Venlafaxine in Spiked Rat Plasma and Formulations

A simple, sensitive, accurate and affordable spectrofluorimetric method was developed and validated for the determination of venlafaxine, both in marketed preparations as well as in spiked rat plasma. Venlafaxine depicted strong native fluorescence property in freshly prepared 0.05 M sulphuric acid. The excitation and emission wavelengths were found to be 237.0 nm and 301.0 respectively. Effect of variations in pH, temperature, concentration, change in molarities of different solvents, and effect of excipients were studied. The calibration graph in case of dosage forms and in spiked plasma was found to be rectilinear in the concentrations of 15–600 ng/ml and 20–650 ng/ml respectively. The intra- day and inter-day accuracy measurements of VEN in formulations ranged from 0.29 to 0.44% and 0.27 to 0.49%, respectively. The intra-day and inter-day accuracy in measurement of VEN in plasma ranged from 0.062 to 2.26% and 0.52 to 2.32%, respectively. The limit of detection (LOD) was found to be 6.0 ng/mL and 4.0 ng/mL in plasma and formulations respectively. The mean recovery of VEN from plasma was 97.46.     

Spectrofluorimetric Methods for the Determination of Gemifloxacin in Tablets and Spiked Plasma Samples

Two new, sensitive and selective spectrofluorimetric methods have been developed for the determination of gemifloxacin (GFX) in tablets and spiked plasma samples. Gemifloxacin, as a primary amine compound, reacts with 7-chloro-4-nitrobenzofurazon (NBD-Cl) (for method A) and fluorescamine (for method B) which are a highly sensitive fluorogenic reagents used in many investigations. For method A, the reaction product was measured spectrofluorimetrically at 516 nm with excitation at 451 nm. The reaction proceeded quantitatively at pH 8.5, 80 °C in 7 min. For method B, the method was based on the reaction between GFX and fluorescamine in borate buffer solution of pH 8.5 to give highly fluorescent derivatives that were measured at 481 nm using an excitation wavelength of 351 nm. The fluorescence intensity was directly proportional to the concentration over the range 40–200 ng mL⁻¹ and 100–1,200 ng mL⁻¹ for method A and B, respectively. Successful applications of the developed methods, for the drug determination in pharmaceutical preparations and spiked plasma samples, were performed.     

Fluorescence Quenching Reaction of Polyvinylpyrrolidone-Eosin Y System for the Determination of Polyvinylpyrrolidone

In pH 1.8 ∼ 2.8 weak acid medium, polyvinylpyrrolidone (PVP) and Eosin Y reacted to form complex that could result in Eosin Y (EY) fluorescence quenching. The maximum quenching wavelength was at 542 nm. The fluorescence quenching (ΔF) was proportional to the concentration of polyvinylpyrrolidone in a certain range. The linear range, the correlation coefficient and the detection limit were 0.33 ∼ 2.0 μg•mL⁻¹, 0.9994 and 99.6 ng•mL⁻¹, respectively. The influences of the coexistence substances were tested and the results showed that the method had good selectivity. Therefore, a new method based on fluorescence quenching of eosin Y by PVP for the determination of trace PVP was developed. The method was sensitive, simple and rapid, which was applied to the determination of trace PVP in the beer with satisfactory results. The reaction mechanism was also discussed.     

Synthesis of Functionalized ZnSe Nanoparticles and Their Applications in the Determination of Bovine Serum Albumin

Luminescent quantum dots (QDs)-semiconductor nanocrystals were promising alternative to organic dyes for fluorescence-based applications. In this paper, we developed procedures to use mercaptoacetic acid (MAA) to modify ZnSe nanoparticles and made the nanoparticles to be soluble for the quantitative and selective determination of bovine serum albumin (BSA). Maximum fluorescence intensity was produced at pH 7.0, with excitation and emission wavelengths at 242 and 348 nm, respectively. Under optimal conditions, the straight line equation: ^△ F = 0.38 + 0.34 C (μg/ml) was found between the relative fluorescence intensity and the concentration of BSA in the range of 9.6–124.8 μg/ml, and the limit of detection was 2 μg/ml.     

Fluorescence enhancement effect for the determination of adenosine 5'-monophosphate with 9-anthracene carboxylic acid-cetyl trimethyl ammonium bromide system

A fluorimetric method based on fluorescence enhancement effect was developed for the determination of adenosine 5′-monophosphate (AMP) with 9-anthracene carboxylic acid (9-ANCA)–cetyl trimethyl ammonium bromide (CTAB) system. Fluorescence intensity of 9-ANCA was decreased by the addition of CTAB but addition of AMP again rose the intensity of 9-ANCA gradually. The observed fluorescence enhancement is attributed to the competitive binding reaction of 9-ANCA and adenosine to CTAB. The enhancement in the fluorescence intensity was found proportional to the concentration of AMP over the range 2.0 × 10⁻⁴ to 1.2 × 10⁻³ mol dm⁻³. The ion pair complex is formed spontaneously between 9-ANCA and CTAB. Since the binding interaction is larger for the adenosine–CTAB pair, the fluorophore 9-ANCA will be released. The quantum yield of free 9-ANCA is higher therefore its fluorescence observed at 417 nm wavelength is enhanced. This mechanism of competitive molecular interaction is further confirmed by conductometric measurements. The method was applied successfully for the determination of AMP from pharmaceutical sample. The method is more selective, sensitive and relatively free from interferences.     

Preparation and Characterization of CdHgTe Nanoparticles and Their Application on the Determination of Proteins

CdHgTe nanoparticles (NPs) with the emission in the near-infrared regions were prepared in aqueous solution, and were characterized by transmission electron microscopy, X-ray diffraction spectrometry, spectrofluorometry and ultraviolet-visible spectrometry. Based on the fluorescence quenching of CdHgTe NPs in the presence of proteins, a novel method for the determination of proteins with CdHgTe NPs as a near-infrared fluorescence probe was developed. Maximum fluorescence quenching was observed with the excitation and emission wavelengths of 500 and 693 nm, respectively. Under the optimal conditions, the calibration graphs were linear in the range of 0.04 × 10⁻⁶–5.6 × 10⁻⁶ g ml⁻¹ for lysozyme (Lyz) and 0.06 × 10⁻⁶–6.1 × 10⁻⁶ g ml⁻¹ for bovine hemoglobin (BHb), respectively. The limits of detection were 13 ng ml⁻¹ for Lyz and 27 ng ml⁻¹ for BHb, respectively. Four synthetic samples were determined and the results were satisfied.