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Abstract This Thesis Contains Three Separate Parts, Which Contain Different Analytical Methods For Quantitative Determination Some Of Pharmaceutical Compounds Containing Amine group Either In Their Pharmaceutical Formulations Or In Biological Fluids. Part I: Quantitative Determination Of Sofosbuvir, Paracetamol, And DL- Methionine In Rat Plasma By Different chromatographic Methods With Application To Pharmacokinetic Study This Part Is Focused On Investigating Accurate, Sensitive, And Specific Methods For Determination Of Sofosbuvir (SOF), Paracetamol (PAR), And DL- Methionine (MET) In Rat Plasma By Two Different selective chromatographic Methods. These Methods Were The First Developed Ones For Simultaneous Determination Of The Studied Drugs In Rat Plasma With Their Application To Pharmacokinetic Study. This Part Includes Three Sections: Section A: Introduction And Literature Review This Section Includes An Introduction About The Pharmacological Actions Of Sofosbuvir (SOF), Paracetamol (PAR), And DL- Methionine (MET), Their Chemical Structures, Physical Properties And Summary Of The Published Methods Developed For Their Analysis. Section B: Determination Of Sofosbuvir, Paracetamol, And DL- Methionine In Rat Plasma Using Thin-Layer chromatography And Its Application To Pharmacokinetic Study In This Section, selective TLC-Densitometric Method Were Developed And Validated Regarding To FDA Guidelines For The Simultaneous Analysis Of The Three Recommended Drugs With A Developing System Consisted Of Chloroform: Methanol: Glacial Acetic Acid: Formic Acid In The Ratio Of (9.5: 1: 1.5: 0.5, By Volume). The Studied Analytes And The Internal Standard (Naphazoline Hydrochloride) Were Scanned At Wavelength 210 Nm. The Method Showed Linearity In The Concentration Range Of 160- 3000 Ng/Band For Sofosbuvir And Paracetamol, 300- 3000 Ng/Band For DL- Methionine. Furthermore, Application Of The Developed Method Has Been Extended To In Vivo Analysis In Rat Plasma. In Addition, Pharmacokinetic Behaviors Of These Drugs Have Been Studied. Section C: Determination Of Sofosbuvir, Paracetamol, And DL- Methionine In Rat Plasma Using High Performance Liquid chromatography And Its Application To Pharmacokinetic Study In This Section, Rapid And Sensitive HPLC Method Has Been Developed And Validated For Simultaneous Determination Of Sofosbuvir In Rat Plasma With Two Co- Administered Drugs, Paracetamol And DL- Methionine Depended On Using HPLC Method Whereas The Analytes And The Internal Standard (Cinnarizine) Were Separated On An Xterra® HPLC RP C18 Column (250 × 4.6 Mm, 5µm) Using Gradient Mode With A Mobile Phase Consisted Of Methanol And 0.1% Aqueous Triethylamine (TEA) At Ph 3 Adjusted With Orthophosphoric Acid. The UV Detection Was Carried Out At Wavelength 210 Nm. The Method Was Linear And Validated Over A Concentration Range Of 150- 5000 Ng Ml-1 For Sofosbuvir, 300- 5000 Ng Ml-1 For Both Paracetamol And DL- Methionine. All Validation Parameters Met The Acceptance Criteria According To FDA Guidelines. The Developed Method Was Successfully Applied To A Pharmacokinetic Study Of Sofosbuvir, Paracetamol, And DL- Methionine In Rats And The Results Suggested The Availability Of Co- Administration Of These Drugs Without Any Significant Effect On Their Pharmacokinetic Properties. Part II: Stability Study Of Benztropine Mesylate Using Different Analytical Methods This Part Is Focused On Developing Simple, Accurate, Sensitive, And selective Spectrophotometric And chromatographic Stability- Indicating Methods For Determination Of Benztropine Mesylate (BNZ) And Its Hepatotoxic And Carcinogenic Degradation Product; Benzophenone (BPH) In Pure Form And In Pharmaceutical Formulation. The Most Important Advantage Of This Work Is That The Developed Methods Are The First Developed Ones For Determination Of BNZ And Its Carcinogenic Degradation Product. This Part Includes Four Sections: Section A: Introduction And Stability Study Of Benztropine Mesylate This Section Includes An Introduction About The Pharmacological Actions Of Benztropine Mesylate (BNZ), Its Chemical Structure And Physical Properties. Also, It Is Includes Results Of Stability Study Of BNZ, Preparation Of Degradation Product And Summary Of The Published Methods Developed For Analysis Of BNZ. Section B: Stability Indicating Spectrophotometric Methods For Determination Of Benztropine Mesylate And Its Carcinogenic Degradation Product In This Section, Three Simple, Accurate, Sensitive, And selective Spectrophotometric Stability- Indicating Methods For Determination Of Benztropine Mesylate (BNZ) And Its Hepatotoxic And Carcinogenic Degradation Product; Benzophenone (BPH); Were Developed And Validated. The Developed Spectrophotometric Methods Are; First Derivative, First Derivative Of Ratio Spectra And Ratio Difference Methods. Calibration Curves Of These Methods Are Linear Over The Concentration Ranges Of 5- 40 And 1- 20 Μg Ml-1 For BNZ And BPH, Respectively. Method (I); First Derivative Spectrophotometric One where BNZ Was Measured At 228.2 Nm While BPH Was Measured At 244 Nm. Method (II); First Derivative Of Ratio Spectra Spectrophotometric One where The Overlapping Spectra Of BNZ And BPH Were Well Resolved And BNZ Was Measured At 228.8 Nm While BPH Was Measured At 211 Nm. Method (III); Ratio Difference Spectrophotometric Method Which Depended On Measuring The Ratio Difference Between 214 And 224 Nm For Determination Of BNZ And At 227.5 And 237.5 Nm For BPH. These Methods Were Validated According To ICH Guidelines. Section C: Stability Indicating TLC- Densitometric Method For Determination Of Benztropine Mesylate And Its Carcinogenic Degradation Product In This Section, An Accurate, Precise And Highly selective Stability Indicating TLC- Densitometric Method Was Adopted For Simultaneous Determination Of Benztropine Mesylate (BNZ) In Presence Of Its Hepatotoxic And Carcinogenic Degradation Product, Benzophenone (BPH). The Method Depended On Separation Of BNZ from Its Degradate On TLC Aluminum Plates Precoated With Silica Gel 60 F254 As The Stationary Phase Using A Developing System Consisted Of Hexane: Methylene Chloride: Triethylamine (50: 50: 6, By Volume) And Scanning The Separated Bands At 235 Nm. The Method Was Successfully Applied To Available Marketed Dosage Form where No Interference from Excipients Was Found. Section D: Stability Indicating UPLC Method For Determination Of Benztropine Mesylate And Its Carcinogenic Degradation Product In This Section, A selective, Sensitive, And Fast Ultra-Performance Liquid Chromatographic (UPLC) Method Was Developed And Validated To Determine BNZ And Its Oxidative Degradate (BPH) At Which The Mixture Was Separated On A Reversed Phase C8 Analytical Column (50 × 2.1 Mm, 1.9 µm) Using A Mobile Phase Of Acetonitrile: Aqueous Sodium Dodecyl Sulfate (SDS) (50: 50, V/V) Adjusted To Ph =3 With Phosphoric Acid, At A Flow Rate Of 0.5 Ml Min-1. Quantification Was Achieved At 210 Nm Based On Peak Area And Linear Calibration Curves Over Concentration Ranges Of (2- 200 Μg Ml-1) And (0.5- 50 Μg Ml-1) For BNZ And BPH, Respectively, Were Obtained. The Investigated Method Were Successfully Applied To Dosage Form And Method Validation Has Been Carried Out. The Results Obtained By Applying This Method Were Analyzed And Compared With Reported One And No Significant Difference Was Obtained Regarding Both Accuracy And Precision. Part III: Quantitative Determination Of Clotrimazole And Hydrocortisone By Different Analytical Methods This Part Is Focused On Investigating Simple And Accurate Spectrophotometric And Chromatographic Methods For The Determination Of Clotrimazole (CLO) And Hydrocortisone (HDC) In Their Pure Form And In Their Combined Formulation. The Developed Spectrophotometric And chromatographic (UPLC) Methods Were Applied For Determination Of CLO And HDC In The Topical Cream With High selectivity And Sensitivity. This Part Includes Four Sections: Section A: Introduction And Literature Review This Section Includes An Introduction About The Pharmacological Actions Of Clotrimazole (CLO) And Hydrocortisone (HDC), Their Chemical Structures, Physical Properties And Summary Of The Published Methods Developed For Their Analysis In Their Single Forms And In Their Binary Mixture. Section B: Quantitative Determination Of Clotrimazole And Hydrocortisone By Different Spectrophotometric Methods Manipulating Ratio Spectra In This Section, Different Precise, Accurate, And selective Three Spectrophotometric Methods Have Been Developed And Validated For The Determination Of Clotrimazole (CLO) And Hydrocortisone (HDC) In Their Combined Dosage Form. The Developed Spectrophotometric Methods Were First Derivative Spectrophotometry (1D), The Second Derivative Of Ratio Spectra (2DD), And Mean Centering Of Ratio Spectra (MCR) Spectrophotometric Methods. All These Methods Were Tested By Their Application For Determination Of The Studied Drugs In Pure Forms And Laboratory Prepared Mixtures And No Interference from Any Of Them On Determination Of The Other Was Found. Methods Were Then Validated And Applied To Pharmaceutical Dosage Form. Section C: Quantitative Determination Of Clotrimazole And Hydrocortisone By Different Spectrophotometric Methods Manipulating Absorbance Difference In This Section, Two Accurate, Sensitive, And selective Spectrophotometric Methods Have Been Developed For The Determination Of Clotrimazole (CLO) And Hydrocortisone (HDC) In Their Combined Dosage Form. The Developed Methods Were Dual Wavelength (DW) Method where CLO Was Determined Using The Absorbance Difference Between 225.4 And 264 Nm, While HDC Was Determined Using The Absorbance Difference Between 228 And 247 Nm. The Second Method Was Advanced Absorbance Subtraction (AAS) Method At Which A Unified Regression Equation Was Constructed Using The Absorbance At ʎiso = 225.4 Nm And Used For Calculating Concentrations Of CLO And HDC In The Binary Mixture After Simple Mathematical Calculations. Methods Validation Were Carried Out Regarding Linearity, Accuracy, Precision, And selectivity And They Were Found To Be Suitable For Quality Control Analysis Of The Studied Drugs. Section D: Quantitative Determination Of Clotrimazole And Hydrocortisone By UPLC Method In This Section, A Sensitive, And Fast Analytical Method For Simultaneous Quantitative Determination Of Clotrimazole (CLO) And Hydrocortisone (HDC) Was Developed And Validated By Using UPLC Method. The Mixture Was Separated On Hypersil Gold C18 Column (150 × 4.6 Mm, 3 µm) Using An Isocratic Method With Mobile Phase Consisted Of Acetonitrile: Water (50: 50, V/V), At A Flow Rate Of 1 Ml Min-1 At Room Temperature With Analysis Time Of Less Than 2.5 Min. The Drugs Were Detected At 228 Nm Over A Concentration Range Of (5- 35 Μg Ml-1) And (5- 50 Μg Ml-1) For CLO And HDC, Respectively. The Proposed Method Have Been Successfully Applied For Determination Of CLO And HDC In Bulk And In Their Pharmaceutical Formulations. The Results Obtained Were Statistically Analyzed And Compared With The Reported One Using F And Student’s T-Tests And No Significant Differences Were Obtained Regarding Both Accuracy And Precision. |