الفهرس 
Quantitative determination of Furosemide, Spironolactone and Canrenone; the major related substance of SpironolactoneOnly 14 pages are availabe for public view
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Abstract
This Thesis Consists Of Three Parts; Each Part Contains An Introduction, Literature Review And Explained Experimental Work For The Studied Components. It Ends With References And A Summary In Arabic.
Part I: Quantitative Determination Of Furosemide, Spironolactone And Canrenone, The Major Related Substance Of Spironolactone
This Part Includes:
Section A: Introduction And Literature Review
This Introduction Describes The Pharmacological Action Of The Studied Drugs, Their Chemical Structures, Physical Properties And Review Of The Published Methods Developed For Their Analysis.
Section B: Quantitative Determination Of Furosemide, Spironolactone And Canrenone By Successive Ratio Subtraction Method (SRS)
Successive Ratio Subtraction (SRS) Starts With Successive Extension Of (RS) And (ERS). The Introduced Novel Method Was Developed For Resolving Ternary Mixture Consisting Of Three Components (X, Y And Z) Represented As CN, FR And SP, Respectively. The Unextended Component (X), Showing A Wavelength Region Of No Interference from (Z) Could Be Estimated By RS, While The Extended One (Y) Could Be Estimated By (ERS). For Estimation Of The Most Unextended One In The Mixture (Z), A New Spectrophotometric Method Named (SRS) Is Newly Introduced In The Presented Work where The Interference from (X) And (Y) Could Be Removed By (RS) And (ERS), Respectively., For Measuring CN Content (Component X) In The Mixtures Using (RS) Method, The Spectra Of The Laboratory Prepared Mixtures Were Divided By The Standard Spectrum Of 12 µg Ml-1 Of FR. The Value Of The Absorbance In The Plateau Region (315-365 Nm) Was Subtracted And The Obtained Curves Were Multiplied By The Standard Spectrum Of 12 µg Ml-1 Of FR. The Obtained Curves Were Used For Determination Of CN Using The Regression Equation Previously Computed At 285 Nm. For Determination Of FR Content (Component Y) In The Mixture, (ERS) Method Was Applied By Dividing The Obtained CN Spectra By 4 µg Ml-1 Of CN As A Divisor, So A Constant Value In The Plateau Region (275-305 Nm.) Was Obtained. The Laboratory Prepared Mixtures Were Then Divided By The Same Divisor, (4 µg Ml-1 CN), Then The Obtained Constant Values Were Subtracted Producing New Spectra Which Were Then Multiplied By The Divisor, (4 µg Ml-1 CN). The Obtained Curves Were Used For Determination Of FR Using The Regression Equation Previously Calculated At 273.6 Nm. Finally, SP Content (Component Z) In The Mixture Was Successfully Determined By (SRS) Method, Overcoming The Severe Interference from FR And CN. The Spectrum Of The Mixture Was Divided By (12 µg Ml-1 Of FR) Followed By Subtraction Of The Plateau Region And Then Multiplied By The Same Divisor (12 µg Ml-1 Of FR). Complementing The Previous Steps, The Obtained Spectrum Was Divided By 4 µg Ml-1 Of CN, Followed By Subtraction Of The Constant Value And Then Multiplied By The Same Divisor (4 µg Ml-1 Of CN). The Severe Overlapped Spectra Of The Three Components Over The Range (200-270 Nm) Were Eliminated By The Newly Developed Method And The Third Component (SP) Was Accurately Determined At 240 M.
Section C: Quantitative Determination Of Furosemide, Spironolactone And Canrenone By Double Divisor Ratio Derivative Spectrophotometric Method (DDRD)
In This Section, The (DDRD) Spectrophotometric Method Was Applied For Determination Of SP, FR And CN. Regarding FR, Each Absorption Spectrum Was Divided By The Spectra Of Mixture Containing Equal Concentrations Of Both Of SP And CN (4 µg Ml-1 Of Each). The 1D Spectra Of FR Were Recorded At Δλ= 4 Nm And Scaling Factor= 10. The Peak Amplitudes At 236.8 Nm Were Recorded Then Calibration Graph Was Constructed. The Regression Equation Was Then Computed For The Studied Drug At The Specified Wavelength And Used For Determination Of Unknown Samples Containing FR.
Concerning SP, Each Absorption Spectrum Was Divided By The Spectra Of Mixture Containing Equal Concentrations Of Both Of FR And CN (2 µg Ml-1 Of Each). The 1D Spectra Of SP Were Recorded At Δλ= 4 Nm And Scaling Factor= 10. The Peak Amplitudes At 249.2 Nm Were Recorded Then Calibration Graph Was Constructed. The Regression Equation Was Then Computed For The Studied Drug At The Specified Wavelength And Used For Determination Of Unknown Samples Containing SP.
According To CN, Each Absorption Spectrum Was Divided By The Spectra Of Mixture Containing Equal Concentrations Of Both Of FR And SP (2 µg Ml-1 Of Each). The 1D Spectra Of SP Were Recorded At Δλ= 4 Nm And Scaling Factor= 10. The Peak Amplitudes At 292.2 Nm Were Recorded Then Calibration Graph Was Constructed. The Regression Equation Was Then Computed For The Studied Drug At The Specified Wavelength And Used For Determination Of Unknown Samples Containing CN.
Section D: Quantitative Determination Of Furosemide, Spironolactone And Canrenone By HPTLC Method In Pure Form, Tablets And Spiked Human Plasma.
Application Of HPTLC Samples Was Automatically Done By A Camag Linomat IV Applicator To HPTLC Plates (20 × 10 Cm Pre-Washed Using Methanol And Dried At 105°C For 30 Min Before Sample Application) In The Form Of Bands. The Bands Were Applied With 6 Mm Width, Spaced 5 Mm from Each Other And 10 Mm Apart from The Bottom End Of The Plate. The chromatographic Chamber Was Saturated With Ethyl Acetate: Triethylamine: Acetic Acid (9: 0.7: 0.5, By Volume) For 30 Min Before Applying Linear Ascending Technique For chromatographic Separation To Provide Peaks That Are Sharp And Well Resolved. The Developed Plates Were Left To Dry In Air And Scanned At 254 Nm.
Section E: Quantitative Determination Of Furosemide, Spironolactone And Canrenone By Green RP-HPLC Method In Pure Form, Tablets And Spiked Human Plasma.
Isocratic Elution Was Implemented On ZOBRAX Eclipse Plus C18 (4.6 X 100 Mm) Column With 3.5 µm Particle Size Using Mobile Phase Consisting Of Ethanol: Deionized Water (45: 55, V/V) And Adjusted To Ph 3.5 Using Glacial Acetic Acid. Flow Rate Of The Mobile Phase Was Controlled To Be 1.0 Ml/Min. The Injection Volume Was Adjusted To Be 20 µl And UV Scanning At 254 Nm At Room Temperature Was Achieved. Elution With Good Separation Of The Three Compounds Was Achieved Within 5 Min.
Part II: Quantitative Determination Of Pyridostigmine Bromide And Its’ Related Substances
This Part Includes:
Section A: Introduction And Literature Review
This Introduction Represented A Concise View About The Structure, Properties And Different Published Methods For The Assay Of The Drug Of Interest In Pure Form, Pharmaceutical Formulation And Also In Plasma.
Section B: Quantitative Determination Of Pyridostigmine Bromide And Its’ Related Substances By Induced Ratio Difference (IRD) Spectrophotometric Method.
To Run IRD Method, One Of The Three Components Must Be Extended Over The Other Two As Summarized Before. For PR, The Stored Spectra Of PR Were Divided By, 20 Μg Ml-1 Of IMP B. The Resulted Constant Was Subtracted from The Ratio Spectra And The Equality Factor Of IMP A At 229 And 276 Nm Was Accurately Determined And Found To Be 1.9. A Calibration Curve Was Immediately Constructed By Plotting The Amplitudes’ Differences Of The Obtained Ratio Spectra At 229 And 276 Nm Multiplied By The Determined Equality Factor Of IMP A (1.9) Versus The Corresponding Concentrations Of PR. As Per IMP A, The Stored Spectra Of IMP A Were Divided By The Same Divisor (20 Μg Ml-1 Of IMP B) And The Equality Factor Of PR At 272 And 286 Nm Was Calculated And Found To Be 2.02. A Calibration Curve Was Directly Constructed Between The Amplitudes’ Differences Of The Gained Ratio Spectra At 272 And 286 Nm Multiplied By The Determined Equality Factor Of PR (2.02) And The Corresponding Concentrations Of IMP A. Regarding IMP B, The Stored Zero order Absorption Spectra Were Simply Used To Set A Calibration Curve Against Its Corresponding Concentrations.
Section C: Quantitative Determination Of Pyridostigmine Bromide And Its’ Related Substances By Derivative Ratio Difference (DRD) Spectrophotometric Method.
In order To Improve The Sensitivity Of The Studied Components, DRD Was Developed. The Absorption Spectra Of PR And IMP A Were Divided By 20 µg Ml-1 Of IMP B. 1DD Curves Of The Obtained Ratio Spectra Of PR And IMP A Were Recorded At Δλ= 2 Nm And Scaling Factor = 10. For Determination Of PR, The Difference In Amplitude Values Of The Derivative Ratio Spectra Of PR At 230 And 270 Nm (Corresponding To Zero Difference In Case Of IMP A And IMP B), Was Plotted Versus The Corresponding Concentration Of PR To Construct The Calibration Curve. Regarding IMP A, The Difference In Amplitude Values Of The Derivative Ratio Spectra Of IMP A Between 231.2 And 275.4 Nm (Corresponding To Zero Difference In Case Of IMP B And PR), Was Directly Proportional To The Corresponding Concentration Of IMP A Then The Calibration Curve Was Directly Constructed. As Per IMP B, 20 µg Ml-1 Of IMP A Was Carefully selected As A Divisor. The Stored Absorption Spectra Of PR, IMP A And IMP B Were Divided By The selected Divisor, Then 1DD Curves Of Their Corresponding Ratio Spectra Were Recorded Using Δλ= 2 Nm And Scaling Factor = 10. Amplitudes’ Differences Of The Obtained Derivative Ratio Spectra Of IMP B Between 228.8 And 246 Nm (Corresponding To Zero Difference In Case Of IMP A And PR) Were Recorded And The Calibration Curve Was Constructed By Plotting The Values Of The Amplitude Difference Between The selected Wavelengths Against The Corresponding Concentrations. Section D: Quantitative Determination Of Pyridostigmine Bromide And Its’ Related Substances By Ratio Subtraction Ratio Difference (RSRD) Spectrophotometric Method.
Owing To The Determination Of The Studied Components In Their Laboratory Prepared Mixtures Without Any Derivatization Step As In The Previous Method (DRD), RSRD Was Newly Developed. The Spectra Of The Laboratory Prepared Mixtures Were Divided By The Standard Spectrum Of 30 µg Ml-1of IMP B. Values Of The Absorbance In The Plateau Region (320-340 Nm) Were Subtracted, And The Obtained Curves After The Subtraction Were Multiplied By The Standard Spectrum Of 30 µg Ml-1 Of IMP B Eliminating The Interference Of IMP B. Moreover, Direct Determination Of IMP B At 329 Nm, where No Interference from PR And IMP A, Was Done. The Concentrations Were Calculated Using The Corresponding Regression Equations. Linear Relationships Were Founds Between The Values Of The Amplitudes’ Differences At The Chosen Wavelengths And Their Respective Concentrations.
Section E: Determination Of Pyridostigmine Bromide In Presence Of Related Impurities By Four Modified Classical Least Square Based Models: A Comparative Study
Novel Manipulations Of The Well-Known Classical Least Square Multivariate Calibration Model Are Explained As A Comparative Study In The Presented Research Study. Prior To Modeling With CLS, First Derivatization And Orthogonal Projection To Latent Structures (OPLS) Are Implemented As Preprocessing Methods To Produce Two Novel Manipulations Of Classical Least Square Based Model. Moreover, Spectral Residual Augmented Classical Least Squares (SRACLS) Model Is Included In The Presented Comparative Study. Quantitative Determination Of Pyidostigmine Bromide (PR) In Presence Of Its Two Related Substances Impurity A (IMP A) And Impurity B (IMP B) Was Considered As A Case Study For Constructing The Comparison. A 3 Factor 4 Level Experimental Design Was Used To Achieve A Training Set Of 16 Mixtures With Different Percentages Of The Studied Components. An Independent Test Set With Of 9 Mixtures Was Applied To Confirm The Predictive Ability Of The Proposed Models. Coupling Of CLS Model With OPLS Preprocessing Method Produces Significant Improvement Of The Predictive Ability Of It. The Root Mean Square Error Of Prediction RMSEP For The Test Set Mixtures Was Used As A Key Comparison Tool. RMSEP Was Found 1.367 With No Preprocessing Method, 1.352 When First Derivative Data Was Applied, 0.2100 When OPLS Preprocessing Method Was Applied And 0.2747 When SRACLS Was Achieved.
Section F: Green Simultaneous chromatographic Separation Of Pyridostigmine Bromide And Its Related Substances By HPTLC In Pure Form, Tablets And Spiked Human Plasma.
Application Of HPTLC Samples Was Automatically Done By A Camag Linomat IV Applicator To HPTLC Plates (20 × 10 Cm Pre-Washed Using Methanol And Dried At 105°C For 30 Min Before Sample Application) In The Form Of Bands. The Bands Were Applied With 6 Mm Width, Spaced 5 Mm from Each Other And 10 Mm Apart from The Bottom End Of The Plate. Saturation Of The chromatographic Chamber With Acetone: Acetic Acid (80:20, V/V) For 30 Min Was Necessarily Performed Before Linear Ascending Development. The Developed Plates Were Left To Completely Dry In The Air. Under The Specified Instrumental Conditions, The Developed Plates Were Scanned At 260 Nm.
Part III: Quantitative Determination Of Amiloride Hydrochloride, Hydrchlorothiazide And Their Related Substances
This Part Includes:
Section A: Introduction And Literature Review
This Introduction Contain A Brief Ideas About Pharmacological Actions, Structures, Related Substances And Physical Properties Of Amiloride Hydrochloride And Hydrochlorothiazide And Literature Survey Of The Published Methods Developed For Them Either Alone Or In Their Mixture.
Section B: Quantitative Determination Of Amiloride Hydrochloride And Hydrochlorothiazide In Presence Of Their Related Substances By Quadruple Divisor Ratio Derivative Spectrophotometric Method (QDRD)
Quadruple Divisor Ratio Derivative Spectrophotometric (QDRD) Method Is A Novel Manipulation Of Double Divisor Ratio Derivative Spectrophotometric Method And Newly Applied To Determine The Studied Drugs In Presence Of Their Related Substances. Regarding AM, Each Absorption Spectrum Was Divided By The Spectra Of Quaternary Mixture Containing Equal Concentrations Of HCZ, CL, SA And CLP (2 µg Ml-1 Of Each). The 1D Spectra Of AM Were Recorded At Δλ= 4 Nm And Scaling Factor= 10. The Peak Amplitudes At 239.6 Nm Were Recorded Then Calibration Graph Was Constructed. The Regression Equation Was Then Computed For The Studied Drug At The Specified Wavelength And Used For Determination Of Unknown Samples Containing AM. Concerning HCZ, Each Absorption Spectrum Was Divided By The Spectra Of Quaternary Mixture Containing Equal Concentrations Of AM, CL, SA And CLP (2 µg Ml-1 Of Each). The 1D Spectra Of SP Were Recorded At Δλ= 4 Nm And Scaling Factor= 10. The Peak Amplitudes At 232.2 Nm Were Recorded Then Calibration Graph Was Constructed. The Regression Equation Was Then Computed For The Studied Drug At The Specified Wavelength And Used For Determination Of Unknown Samples Containing HCZ.
Section C: Quantitative Determination Of Amiloride Hydrochloride And Hydrochlorothiazide In Presence Of Their Related Substances By HPTLC Method
Application Of The Prepared Samples To HPTLC Plates (20 × 11 Cm Pre-Washed Utilizing Methanol And Dried For 30 Min At 105°C Prior To Sample Application) Was Automatically Performed Using A Camag Linomat IV Applicator In The Form Of Bands. The Applied Bands Were Of Space Of 5 Mm Apart from Each Other, Width Of 6 Mm And 10 Mm from The Bottom Border Of The Plate. To Achieve Optimum Separation Of The Studied Components With Sharp Peaks, The chromatographic Chamber Was Allowed To Be Saturated With The Mobile Phase Which Consisting Of Ethyl Acetate: Ethanol: Ammonia Solution (8: 2: 0.2, By Volume) For Consecutive 30 Minutes Just Before Applying The Linear Ascending Technique Of The chromatographic Separation. Before Scanning Of The Plates At 273 Nm, They Were Left To Dry In Air.
Section D: Quantitative Determination Of Amiloride Hydrochloride And Hydrochlorothiazide In Presence Of Their Related Substances By RP-HPLC Method
ZOBRAX Eclipse Plus C18 (4.6 X 100 Mm) Column Of 3.5 µm Particle Size, Mobile Phase Containing 0.1% Phosphoric Acid Solution: Acetonitrile (90: 10, V/V) With Adjusted Ph Of 3.6 Were Used To Achieve The Optimum Isocratic Elution. The Flow Rate Of The Mobile Phase Was Adjusted To Be 1.0 Ml/Min. The Injection Volume Was Also Controlled To Be 20 µl. UV Scanning Was Accomplished At 273 Nm At Room Temperature. Rapid Elution Rapid And Optimum Separation Of The Five Compounds Was Completed Within 5 Min.
This Thesis Contains 207 References, 108 Figures, 71 Tables And Ends With A Summary In Arabic.