الفهرس 
Part I: General IntroductionOnly 14 pages are availabe for public view
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Abstract
The present thesis is devoted for the development and validation of cost effective and environmentally safe analytical methods for the determination of some newly approved anti-HCV drugs namely; daclatasvir dihydrochloride, ledipasvir, sofosbuvir, and velpatasvir. The developed methods in the present study include spectrofluorimetric, spectrophotometric, FTIR spectroscopic and TLC densitometric methods.
The presented thesis falls into three main parts:
Part I: General introduction
This part provides an overview of the pharmaceutical importance, pharmacological effect, mechanism of action and chemical structure of the studied drugs. It also includes the analytical review for the analytical techniques that were previously reported for the determination of the studied drugs in pure forms, dosage forms and biological fluids. At the end of this part, the objective of the suggested work was discussed.
Part II: Spectroscopic techniques
This part falls in six chapters:
Chapter I: Micellar spectrofluorimetric protocol for the innovative determination of HCV antiviral (daclatasvir) with enhanced sensitivity; Application to human plasma and stability study.
This chapter is devoted for the development of a sensitive, simple, fast and specific fluorometric method for the estimation of DAC in the presence of SOF. The method is based on enhancement of the fluorescence intensity of DAC by 170 % of its original value in an aqueous micellar solution of hexadecyl-trimethylammonium bromide (pH 5.5, Teorell and Stenhagen buffer). The fluorescence intensity were measured at 387 nm after excitation at 328 nm. A linear relationship between the DAC concentration and the fluorescence intensity was achieved in the range of 0.05-2.0 µg mL-1 with determination and correlation coefficients of 0.9998 and 0.9999. The detection and quantitation limits were 13.4 and 40.8 ng mL-1, respectively. The excellent sensitivity and specificity of the proposed method allowed the efficient estimation of DAC in real human plasma with adequate recovery (81.78 ± 1.57). In addition, the proposed method was successfully applied for the determination of DAC in its tablets dosage form. Moreover, the proposed method was extended to examine the stability of DAC under different stress conditions such as oxidative, alkaline, acidic, UV, daylight and sunlight degradations according to ICH guidelines. Furthermore, the kinetics study of the alkaline and oxidative degradations of DAC was investigated. The half-life times of the reaction (t1/2) were 1.92 and 1.16 h and the first-order reaction rate constants were 0.359 and 0.598, for the alkaline and oxidative degradations, respectively.
Chapter II: Xanthene based spectroscopic probe for the analysis of HCV antiviral, daclatasvir dihydrochloride, through feasible complexation reaction.
In this chapter two simple and rapid analytical methods were developed and validated utilizing spectroscopic techniques for the estimation of DAC in pharmaceutical preparations based on an ion-pair complex formation with eosin Y. In the first method, the maximum absorbance of the formed complex was measured at 545 nm in 0.1 M acetate buffer (pH 3.2). Beer’s law was obeyed in the range of 1.0–10.0 µg mL-1 for the spectrophotometric method (Method I). The spectrofluorimetric method (Method II) was based on measuring the quenching effect of DAC on the native fluorescence of eosin Y at pH 3.2, and the measurements were carried out at 543 nm after excitation at 301 nm. The rectilinear calibration graph between DAC concentration and the fluorescence quenching values (ΔRFI) was achieved over the range of 0.1-2.0 µg mL-1. The formation constant for the binary complex was 4.4 × 104 and Gibb’s free energy change was −27 kJ mol-1. Both methods were validated according to ICH guidelines and successfully applied for the analysis of the dosage forms with good % recovery. A suggestion for the reaction pathway was presented.
Chapter III: Micellar based spectrofluorimetric method for the selective determination of ledipasvir in the presence of sofosbuvir: Application to dosage forms and human plasma.
This chapter describes a fast, low-cost, sensitive and selective spectrofluorometric method for the estimation of LDP. The method is based on the formation of hydrogen bonding between the hydroxyl groups in both Tween-20 and LDP, producing a remarkable improvement of the native fluorescence intensity of LDP by 500 % of its original value at pH = 5.0. All fluorescence measurements were achieved at 425 and 340 nm for emission and excitation, respectively. A linear relationship between the concentration of LDP and the fluorescence intensity was achieved in the range of 0.1-2.0 µg mL-1 with 0.028 and 0.084 µg mL-1, for detection and quantitation limits, respectively. The good selectivity and sensitivity of the proposed method facilitate the determination of LDP in human plasma with acceptable percentage recovery (95.36-99.30 %). The proposed procedure was validated according to ICH guidelines. Moreover, the proposed method was successfully applied for the determination of LDP in its pharmaceutical dosage form.
Chapter IV: Novel approach for spectrofluorimetric determination of ledipasvir through UV- irradiation: Application to biological fluids, pharmacokinetic study and content uniformity test.
In this chapter, a highly sensitive and specific spectrofluorimetric approach was developed for quantitation of LDP. The innovative approach relies on the enhancement of the fluorescence intensity of LDP upon its exposure to direct UV irradiation. The fluorescence of the resultant solution was measured at 375 nm (321 nm for excitation). The fluorescence properties of the resultant product was carefully investigated and the linear range of the method was 5.0–150.0 ng mL−1 with a detection limit of 0.9 ng mL−1 and a quantitation limit of 2.7 ng mL−1. The excellent analytical features of the suggested method allow the specific and sensitive estimation of LDP either in plasma or in tablets dosage form without any interference from plasma components, pharmaceutical excipients or other co-formulated anti-HCV drugs (SOF). The developed analytical and bio-analytical procedures were validated according to ICH and FDA guidelines, respectively. Since the main elimination route for LDP is via the faecal excretion, the suggested method was applied successfully for the determination of LDP for the first time in rats faecal samples. Moreover, the pharmacokinetic parameters (C_max, t_max, t_(1/2), 〖AUC〗_(0-t), and 〖AUC〗_(0-∞)) for LDP were calculated. Additionally, the suggested methodology was successfully applied for testing the content uniformity of LDP in its pharmaceutical tablets.
Chapter V: Study on fluorescence properties of HCV antiviral (velpatasvir) and its fluorimetric determination in the presence of sofosbuvir; Application to stability study and human plasma.
In this chapter, a highly sensitive, simple, and selective fluorometric method for the determination of VLP in the presence of SOF was developed and validated. The fluorescence behaviour of VLP in different organic solvents was examined. Methanol was found to be the best solvent. The native fluorescence intensity of VLP was measured at 383 nm (339 nm for excitation). The effect of experimental variables included pH and various organized media were examined and optimized. A linear relationship was achieved between VLP concentration and the fluorescence intensity in the range of 0.1-5.0 µg mL-1 with quantitation and detection limits of 0.030 and 0.093 µg mL-1, respectively. The proposed method was utilized for analyzing VLP in human plasma. Furthermore, the proposed method was extended to examine the stability of VLP under various stress conditions, such as oxidative, alkaline, acidic, UV, daylight and sunlight conditions, according to ICH guidelines. Moreover, the kinetics of acidic and oxidative degradations of VLP was examined. The half-life times of the reaction (t1/2) and the first-order reaction rate constants were estimated.
Chapter VI: Employment fourier transform infrared spectroscopy for the determination of second-generation anti-HCV (sofosbuvir, daclatasvir) drugs: Application to pharmaceutical dosage form.
In this chapter, a selective and non-destructive fourier transform infrared spectroscopic method was developed for the direct determination of DAC and SOF in their sold form. The method based on measuring the absorbance of carbonyl group bands at 1695 and 1740 cm-1 for DAC and SOF, respectively. The proposed method was validated according to ICH guidelines. The linear ranges were 0.25 -20.0 and 0.5-20.0 mg/g for DAC and SOF, respectively. The values of LOQ were 0.149 and 0.172 mg/g and the values of LOD were 0.049 and 0.057 mg/g for DAC and SOF, respectively. Also, the correlation coefficients (r) were 0.9995 and 0.9993 for DAC and SOF, respectively. The method was successfully applied for the determination of DAC and SOF in their pharmaceutical tablets.
Part II: chromatographic technique
This part falls in two chapters:
Chapter I: Innovative TLC-fluorescence detection approach for sensitive and specific determination of ledipasvir in rat faeces and pharmaceutical dosage form. Application to content uniformity test
This chapter is devoted to a newly developed and validated TLC method combined with fluorescence detection mode for the specific estimation of LDP. The separation was performed on silica gel 60 F254 plates using ethylacetate : hexane : acetonitrile : triethylamine; (6.0 : 3.5 : 1.5 : 0.5, v/v/v/v) as a mobile phase. The suggested method involved the exposure of the developed TLC plate of LDP to strong UV irradiation, resulting in a great enhancement in the fluorescence properties of LDP. The UV-irradiated plates were scanned in the fluorescence mode after excitation at 315 nm. The method provided a sufficient separation of LDP from SOF with R_F-values of 0.28 and 0.36 for LDP and SOF, respectively. The developed procedure was validated according to ICH and FDA guidelines. The calibration curve was linear over the range of 5.0-50.0 ng/band with a correlation coefficient of 0.9995. The excellent analytical features of the suggested method allow the specific and sensitive determination of LDP in pharmaceutical tablets containing either the drug alone or in combination with its co-formulated anti-HCV drugs (SOF) without any interference from excipients or SOF. As the main elimination route for LDP was via the faecal excretion (86 %), the method was applied for the determination of LDP for the first time ever in faecal specimens with adequate recovery. In addition, the method was successfully applied for testing the content uniformity of LDP in commercial tablets without exhausting extraction steps.
Chapter II: Thin layer chromatographic method coupled with fluorescence detection mode for sensitive and selective determination of velpatasvir: Application to human plasma
A validated TLC method coupled with fluorescence detection mode was developed in this chapter for the selective determination of VLP. The separation was performed on silica gel 60 F254 plates using ethylacetate : methanol : triethylamine; (48 : 1.5 : 1.0, v/v/v) as a mobile phase. The plates were scanned in the fluorescence mode after excitation at 335 nm. The method provided an excellent separation of VLP from SOF with R_F-values of 0.22 and 0.46 for VLP and SOF, respectively. The developed procedure was validated according to ICH guidelines. The calibration curve was linear over the range of 4.0 – 40.0 ng/band with a correlation coefficient of 0.9994. The suggested method could selectively determine VLP with high sensitivity in a synthetic tablet powder containing the co-formulated anti-HCV drugs (SOF) without any interference from excipients or SOF. In addition, the method was successfully applied for the determination of VLP in spiked human plasma with adequate % recovery.
In addition, the thesis includes a list of 147 References, 61 Tables, 49 Figures, a summary in English and another in Arabic.