الفهرس 
Part I: General introductionOnly 14 pages are availabe for public view
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Abstract
The present thesis focused on the development of new cost effective and environmentally safe analytical methods for the analysis of some newly approved direct acting hepatic antivirals, namely; SMV, LDS and SOF. These methods are easy to operate, have high accuracy and precision and enjoy good sensitivity while maintaining the required selectivity.
The developed methods in the present study include spectrofluorimetric, spectrophotometric and chromatographic (TLC) methods.The presented thesis contains three parts.
Part I: General Introduction
This part provides a general introduction about the investigated compounds such as, chemical structure, pharmacological aspect and some physical properties. In addition, this part also includes previously reported analytical review for the determination of the studied drugs in pure forms, dosage forms and biological fluids. The aim of the suggested work was described at the end of this part.
Part II: Spectroscopic techniques
This part falls in five chapters:
Chapter I: Sensitive spectrofluorimetric assay based on micelle enhanced protocol for the determination of hepatitis C antiviral agent (simeprevir): Application to dosage form and human plasma.
In the present chapter, we hereby introduce a validated rapid, sensitive and straight forward fluorimetric method for its estimation at (ex 290 nm, em 428 nm). The fluorescence spectra of SMV were inspected in both aqueous and surfactant systems. It was found that; about 2.2 folds enhancements in the fluorescence intensity of SMV was occurred by the addition of Tween-80 in presence of Teorell buffer (pH 5.0) compared with the aqueous system. There was a linear relationship between SMV concentration and corresponding fluorescence intensity between 0.05 –1.0 µg mL-1 with determination coefficient of 0.9996. The detection and quantitation limits were 0.016 and 0.048 µg mL-1, respectively. The proposed method was effectively used to quantify SMV in dosage form and spiked human plasma without significant interference from matrix.
Chapter II: Micelle sensitized synchronous spectrofluorimetric approaches for the simultaneous determination of simeprevir and ledipasvir: application to pharmaceutical formulations and human plasma.
Two spectrofluorimetric approaches were combined together in this chapter for the development of highly sensitive, rapid, simple and accurate method for simultaneous quantification of SMV and LDS. The native fluorescence intensity values of SMV and LDS were enhanced by the addition of Tween-80 micellar system, while second derivative of the synchronous fluorescence intensity of the drugs at Δλ = 120 nm enabled the determination of both drug concomitantly. Different experimental parameters affecting the synchronous fluorescence of the cited drugs were carefully evaluated for their optimization. The peak amplitudes of the second derivative synchronous fluorimetry were measured at 429 nm for SMV and at 417 nm for LDS. The calibration plots were rectilinear over concentration ranges of 60–1500 and 36–540 ng mL-1 for SMV and LDS with good linearity. The limits of detection were 9 and 6 ng mL-1 for SMV and LDS, respectively. The method was successfully applied for the determination of both drugs in their pure forms, their pharmaceutical products and human plasma without any significant interference. Statistical comparison with the reported method revealed excellent accuracy and precision of the proposed method.
Chapter III: Development and validation of spectrofluorimetric method for the determination of hepatitis C antiviral ledipasvir through fluorescence quenching of eosin Y: Application to content uniformity test.
This chapter describes the development and validation of a simple, rapid, sensitive, and non-extractive spectrofluorometric method for the estimation of LDS in pharmaceutical preparation based on an ion-pair complex formation with eosin Y. The method was based on measuring the quenching effect of LDS on the native fluorescence of eosin Y at pH 3.2 by using acetate buffer. It was found that the formation of binary complex of eosin Y with the studied drug LDS have a quenching effect on the native fluorescence of the dye at λex/ λem 301 / 539 nm. The relationship was linear over the range of 0.05–0.7 µg ml-1 for LDS. The coefficients of correlation (r) and determination (r2) were 0.9998 and 0.9996, respectively, indicating the excellent linearity of the proposed method. The method was validated according to ICH guidelines and successfully applied for the analysis of the dosage form and for testing the content uniformity of these dosage forms.
Chapter IV: Resonance Rayleigh scattering technique using erythrosine B, as novel spectrofluorimetric method for determination of hepatitis C antiviral agent ledipasvir: Application for dosage form and human plasma.
This chapter demonstrates the development of a new, simple, and rapid spectrofluorimetric method for a sensitive and selective estimation of LDS in the presence of SOF by applying the Rayleigh resonance scattering (RRS) technique. RRS is a special elastic scattering that can be measured using a special synchronous fluorimetry equal to zero (i.e. when the excitation wavelength equals the emission wavelength). The method is based on the formation of a binary-complex between LDS and one of xanthene dyes, erythrosine B, in the aqueous medium (pH 3.4) by electrostatic attraction and hydrogen bonding. The formation of this complex leads to a significant improvement in the Rayleigh resonance scatter spectrum, and leads to the emergence of a new RRS peak measured at 340 nm for both excitation and emission. The relationship between the intensity of the Rayleigh resonance scattering and the LDS concentration was linear in the range of 0.05-0.8 µg ml-1. The detection and quantitative limits were 0.01 and 0.03 µg ml-1, respectively. Due to the sensitivity and selectivity of the proposed method, the method provided a good estimate of the cited drug in its pure forms, as well as human plasma and pharmaceutical formulations, without any significant interference from the co-formulated drug (SOF) which is present in the same pharmaceutical tablets. The proposed method was validated in accordance with the guidelines for the ICH.
Chapter V: Spectrophotometric determination of the new antiviral drug ledipasvir in presence of sofosbuvir by using surface plasmon
resonance of silver nanoparticles: Application to pharmaceutical table. This chapter refers to the use of silver nanoparticles as a simple and selective colorimetric probe for estimation of hepatitis C antiviral agent LDS in pure form and pharmaceutical tablets. Upon the addition of LDS to AgNPs solution in buffered aqueous solution (Teorell – Stenhagen buffer pH 3.0), the color changed to red and the absorbance at 395 nm decreased with simultaneous appearance of a new absorption band at 505. The change in spectrum based on AgNPs aggregation was adopted to develop a simple and rapid colorimetric methodology for the determination of LDS. The proposed method was validated in accordance with the guidelines for the ICH. By using the optimal reaction conditions, good linear relationship was obtained between the absorbance and LDS concentration in the range of 4-30 µg mL-1. The detection and quantitation limits were 0.6 and 1.82 µg mL-1, respectively. This method was successfully applied for estimation of LDS in their tablet formulation without any significant interference from tablet excipients or the co-formulated drug (SOF) which is present in the same pharmaceutical tablets.
Part III: chromatographic technique
This part falls in two chapters:
Chapter I: Stress stability study of simeprevir, a hepatitis C virus inhibitor, using feasible TLC- spectro-densitometry: application to pharmaceutical dosage form and human plasma.
This chapter refers to the use of a simple, highly selective and stability-indicating, thin-layer chromatography (TLC) method for the assay of SMV in pharmaceutical dosage form and spiked human plasma. The method used silica gel 60 F254 coated TLC aluminum plates as the stationary phase. The mobile phase system was ethyl acetate ‒ hexane‒methanol (5: 4: 1, v/v/v). The wavelength was set at 288 nm. This system was found to give a compact spot of SMV having a retardation factor of 0.67 ± 0.02. The guidelines of the International Council on Harmonization were followed to validate the proposed analytical method, and the results were acceptable. The calibration curve was linear over the range of 80–1000 ng /band. The limit of detection was 19 ng/ band, and the limit of quantitation was 57 ng/ band. The proposed method was employed successfully for the accurate and reproducible analysis of the pharmaceutical preparation and human plasma containing the drug with precision and accuracy similar to those of a reported method. The method was extended to study the stability of the drug under various stress conditions. The drug was subjected to hydrolytic, oxidative and UV-induced degradation. The results showed that the proposed method could efficiently separate the degradation products from the intact drug and allow its satisfactory quantitation.
Chapter II: Feasible TLC- spectro-densitometry technique for Simultaneous determination of two hepatitis C antiviral drugs; sofosbuvir and simeprevir: application to pharmaceutical dosage forms and human plasma.
This chapter is developing and validating, a high-performance thin-layer chromatographic method was developed and validated for the concurrent determination of SMV and SOF. The chromatographic separation was attained on silica gel 60 F254 as stationary phase and ethyl acetate‒hexane‒methanol (5: 4: 1, v/v/v) as developing solvent with UV detection at 273 nm. The retardation factor values were 0.67 and 0.43 for SMV and SOF; respectively. The method has been validated in respect to the ICH guidelines. Linearity was maintained between 60–1000 and 70–1200 ng/ band for SMV and SOF; respectively with good correlation coefficients (0.9993- 0.9997) for both drugs. The suggested method was highly sensitive as the calculated detection limits were 15 and 22 ng/ band while the quantitation limits were 44 and 66 ng/band for SMV and SOF; respectively. The suggested methodology has been effectively employed for the determination of the mentioned drugs in their pure forms and their pharmaceutical dosage forms as well as human plasma without significant interference from pharmaceutical excipients or plasma components.
In addition, the thesis includes a list of (155) References, (55) tables, (56) figures and (4) schemes, as well as summary in English and another in Arabic.