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Abstract This thesis is concerned with the development of different simple, sensitive and selective methods for determination of anti-viral drugs; namely valaciclovir HCl, oseltamivir phosphate and zanamivir either alone or in presence of their acid and/or alkaline degradates. Furthermore, the work aimed to determine aciclovir, the active metabolite of valaciclovir HCl, in human plasma which could be used for bioequivalence studies. The establishment of a method that quantify the pure drug in presence of its different degradates is of great pharmaceutical value, because any degradation occurring at normal storage conditions can be determined. This thesis includes five parts: Part one: Introduction This part is divided into two sections Section A: General Introduction This section comprises a brief data about anti-viral drugs, their classes, mode of action and drug stability. Section B: Literature Review In this section, the structures, properties and review of the published analytical procedures developed for their analysis, are stated. XIX Part two: chromatographic Methods This part is divided into nine sections Section A: Introduction to Ultra High Pressure Liquid chromatography. Section B: Stability Indicating Rapid Resolution Liquid chromatography (RRLC) Method for Determination of Valaciclovir HCl: This section describes a new stability indicating RRLC method for determination of valaciclovir HCl in presence of its alkaline- and acid- degradates . This method based on the separation of intact drug using ZORBAX SB phenyl 50.0 mm L x 4.6 mm id x 1.8 μm particle size column as stationary phase , Binary mixture of 0.01M of n- tetra butyl ammonium hydrogen sulfate and methanol in the ratio of (95:5, v/v) as mobile phase, with flow rate of 0.3 ml min -1and UV detection at 254 nm .Linear correlation was obtained in the range of( 0.40-40.0 μg ml-1) without interference from the alkaline and acid degradates. The proposed method was applied for the analysis of valaciclovir HCl in pure form, laboratory prepared mixtures containing different percentages of its degradates and in pharmaceutical preparation. The validity was assessed by applying the standard addition technique. The obtained results were statistically compared with those of the reference method. Satisfactory results were obtained upon applying the suggested method in pharmaceutical formulation which encourages the use of the proposed method in quality control laboratories. XX Section C: Bio-analytical RRLC Method for Determination of Aciclovir, the Active Metabolite of Valaciclovir, in Spiked Human Plasma: This section is an application of proposed RRLC method describes in section B for determination of aciclovir, the active metabolite of valaciclovir HCl, in human plasma. Section D: Stability Indicating Reversed Phase Rapid Resolution Liquid Chromatography (RP-RRLC) Method for Determination of Oseltamivir Phosphate: This section describes a new stability indicating RP- RRLC method for determination of oseltamivir phosphate in presence of its alkaline-degradate. This method based on the separation of the intact drug using Zorbax XDB C18 50.0 mm L x 4.6 mm id x 1.8μm particle size column as stationary phase ,water (pH 3.0): methanol (50:50,v/v) as mobile phase with flow rate of 1.0 ml min-1 and UV detection at 215 nm. Linear correlations obtained in the range of (4.0-240.0 μg ml-1) without interference of the alkaline degradate. The proposed method was successively applied for the analysis of oseltamivir phosphate in pure form, laboratory prepared mixtures containing different percentages of its alkaline-degradate and in pharmaceutical preparation. The validity was assessed by applying the standard addition technique. The results obtained were statistically compared with those obtained with reference method and no significant difference was observed regarding accuracy and precision. The results indicate that the proposed method could be used for routine analysis of oseltamivir phosphate in presence of its alkalinedegradate. XXI Section E: Stability Indicating Reversed Phase High Performance Liquid Chromatography (RP-HPLC) Method for Determination of Valaciclovir HCl : This section describes a new stability indicating RP- HPLC method for determination of valaciclovir HCl in presence of its alkaline- and acid- degradates, based on the separation of the intact drug using Hypersil BDS C18 250.0 mm L x 4.6 mm id x 5.0 μm particle size column as stationary phase, Phosphate buffer (pH 4.6): Methanol (90:10,v/v) as mobile phase with flow rate of 1.0 ml min-1 and UV detection at 252 nm. Linear correlations obtained in the range of (0.5-20.0 μg ml-1) without interference of the alkaline- and acid -degradates . The suggested method was applied for the analysis of valaciclovir HCl in pure form, laboratory prepared mixtures containing different ratios of its degradates and in pharmaceutical preparation. The validity was assessed by applying the standard addition technique. Statistical analysis of the results showed no significant difference when compared with those obtained with reference method regarding accuracy and precision. Section F: Stability Indicating Reversed Phase High Performance Liquid Chromatography (RP-HPLC) Method for Determination of Oseltamivir Phosphate: This section describes a new stability indicating RP- HPLC method for determination of oseltamivir phosphate in presence of its alkaline-degradate. This method based on the separation of the intact drug using Symmetry C8 150.0 mm L x 4.6 mm id x 5.0μm particle size column as stationary phase ,phosphate buffer (pH 3.0): methanol (60:40,v/v) as mobile phase with flow rate of 1.5 ml min-1 and UV detection at 215 nm. Linear XXII correlations obtained in the range of (1.0-100.0 μg ml-1) without interference of the alkaline -degradate. The proposed method was applied for the analysis of oseltamivir phosphate in pure form, laboratory prepared mixtures containing different ratios of its alkaline-degradate and also in pharmaceutical preparation. The validity was assessed by applying the standard addition technique. The obtained results were statistically compared with those of the reference method. Satisfactory results were obtained upon applying the suggested method in pharmaceutical formulation so it can be used in quality control laboratories. Section G: Stability Indicating Reversed Phase High Performance Liquid Chromatography (RP-HPLC) Method for Determination of Zanamivir: This section describes a new stability indicating RP- HPLC method for determination of zanamivir in presence of its acid-degradate. This method based on the separation of the intact drug using Xterra C18 250.0 mm L x 4.6 mm id x 5.0μm particle size column as stationary phase ,0.01M octane sulfonic acid sodium salt( pH 6.0) as mobile phase with flow rate of 1.0 ml min-1 and UV detection at 235 nm. Linear correlations obtained in the range of (2.0-100.0 μg ml-1) without interference of the acid -degradate. Laboratory prepared mixtures containing zanamivir and different ratios of the aciddegradate were analyzed by the proposed method and satisfactory results were obtained. The proposed method was also used for determination of zanamivir in pure form and in XXIII pharmaceutical preparation and good results were obtained. The validity of the suggested method was assessed by applying standard addition technique. The obtained results were statistically compared with those obtained by the reference method. The calculated t and F values were less than the theoretical ones, indicating that both methods are equally accurate and precise. Section H: Stability Indicating Spectrodensitometric Thin Layer chromatography (TLC) Method for Determination of Valaciclovir HCl: This section describes a new stability indicating densitometric TLC for determination of valaciclovir HCl in the presence of its alkaline-degradate . This method based on spotting methanolic solution of valaciclovir HCl on a silica gel F254 plate, using methanol: ethyl acetate: ammonia in ratio of (50:50:10, by volume) as mobile phase. Scanning of the chromatogram was carried out at 254 nm by reflection mode. Linear correlation was obtained in the range of (0.4-4.0μg/spot). Application of the proposed method to the analysis of valaciclovir HCl in pure form, laboratory prepared mixtures containing different ratios of its alkaline- degradate and also in pharmaceutical preparation showed that neither the excipients usually formulated in the market preparation nor the alkaline –degradate interfere with the determination . The validity was assessed by applying the standard addition technique. The reliability of the proposed method was determined through statistical comparison with the reference method. The calculated t and F values were found to be less than the theoretical ones, XXIV indicating that there is no significant difference between the two methods in respect to accuracy and precision which encourages the use of the proposed method in quality control laboratories. Section I: Stability Indicating Spectrodensitometric Thin Layer chromatography (TLC) Method for Determination of Oseltamivir Phosphate: This section describes a new stability indicating densitometric TLC for determination of oseltamivir phosphate in the presence of its alkaline-degradate . This method based on spotting methanolic solution of oseltamivir on a silica gel F254 plate, using methanol: ethyl acetate: diethyl ether: ammonia (80:60:20:5, by volume) as mobile phase. Scanning of the chromatogram was carried out at 215 nm by reflection mode. Linear correlation was obtained in the range of (5.0-50.0μg/spot). The proposed method was applied for the analysis of oseltamivir phosphate in pure form, laboratory prepared mixtures containing different ratios of its alkaline- degradate and also in pharmaceutical preparation. The validity was assessed by applying the standard addition technique. Statistical comparison of the results obtained by the proposed densitometric method and the reference method was done and no significant difference was observed regarding accuracy and precision. XXV Part three: Spectrophotometric methods This part is divided into two sections Section A: Stability Indicating First Derivative of Ratio Spectra Spectrophotometric Method for determination of Oseltamivir Phosphate in Presence of its Alkaline- Degradate: This section describes stability indicating method used for the determination of oseltamivir phosphate in the presence of its alkaline-degradates. This method based on the use of first derivative ratio spectrophotometric method (1DD). Oseltamivir could be determined in presence of its alkaline-degradates by dividing the spectra of the intact drug by the spectrum of its alkaline-degradate (10.0 μg ml-1) as divisor .The peak amplitudes of the first derivative ratio spectra were measured at 223nm .A linear relationship was obtained in concentration range of (5.0-45.0μg ml-1)for determination of oseltamivir phosphate without interference from the alkaline-degradate. The proposed derivative ratio method was used for the determination of oseltamivir phosphate in laboratory prepared mixtures and in the available pharmaceutical dosage form where good results were obtained. The validity of the suggested procedure was proved by applying the standard addition technique. Statistical comparison of the results obtained by applying the proposed method and the reference method permits one to conclude with 95% of confidence, that there is no significant difference between the proposed method and the reference method. XXVI The proposed analytical procedure is simple, rapid, specific and could be applied for quality and stability monitoring of oseltamivir phosphate. Section B: Stability Indicating First Derivative of Ratio Spectra Spectrophotometric Method for determination of Zanamivir in Presence of its acidic- Degradate: This section describes stability indicating method used for the determination of zanamivir in the presence of its acid-degradate. This based on the use of first derivative ratio spectrophotometric method (1DD). Zanamivir could be determined in presence of its acid-degradates by dividing the spectra of the intact drug by the spectrum of its aciddegradate (12.0 μg ml-1) as divisor .The peak amplitudes of the first derivative ratio spectra were measured at 222 nm .A linear relationship was obtained in concentration range of (4.0-36.0μg ml-1) for determination of zanamivir without interference from the acid-degradate. The proposed method was applied for the analysis of zanamivir in pure form, laboratory prepared mixtures containing different ratios of its acid-degradate and also in pharmaceutical preparation. The validity was assessed by applying the standard addition technique. The obtained results were statistically compared with those of the reference method. Satisfactory results were obtained upon applying the suggested method in pharmaceutical formulation which encourages the use of the proposed method in quality control laboratories. XXVII Part four: General Discussion This part shows a short summary of the developed methods. A comparison between the studied methods, choosing the most sensitive method for each drug, is also stated. Part five: References This thesis refers to 115 references, contains 64tables and 24 figures and ends with an Arabic summary. |