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TESISUMK

TESISUMK

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Published by Sudhanshu Singh

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Published by: Sudhanshu Singh on Jan 04, 2012
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Development and Validation of Bioanalytical Assay Methods forSildenafil in Human Plasma
Michael Kidane TesfuA dissertation submitted in fulfilment of the requirements of:
Master of Science
Department of Chemistry, University of the Free State2004
Supervisor: Prof. S.S. BassonJoint Supervisors: Prof. H.K.L. HundtMr. J.A. Venter
 
 
Table of Contents I
Table of Contents
List of abbreviations...............................................................................................................................IV
 
List of tables............................................................................................................................................VI
 
List of figures.........................................................................................................................................IX
 
INTRODUCTION AND OBJECTIVES..............................................................................................................1
 
1 LITERATURE REVIEW 2
 
1.1 METHOD DEVELOPMENT AND VALIDATION.................................................................................2
 
1.1.1 Method development.....................................................................................................................3
 
1.1.1.1 Limits of detection and quantification (LLOD and LLOQ)...............................................4
 
1.1.1.2 Calibration line...........................................................................................................................5
 
1.1.2 Method validation..........................................................................................................................6
 
1.1.2.1 Selectivity....................................................................................................................................8
 
1.1.2.2 Precision.......................................................................................................................................9
 
1.1.2.2.1 Intra-Assay Precision.............................................................................................................9
 
1.1.2.2.2 Inter-Assay Precision.............................................................................................................9
 
1.1.2.3 Accuracy....................................................................................................................................10
 
1.1.2.4 Recovery....................................................................................................................................10
 
1.1.2.5 Stability......................................................................................................................................11
 
1.1.2.5.1 Long term stability................................................................................................................11
 
1.1.2.5.2 Standard stock solution stability.........................................................................................11
 
1.1.2.5.3 Short term matrix stability...................................................................................................12
 
1.1.2.5.4 On-instrument sample stability...........................................................................................12
 
1.1.2.5.5 Freeze-thaw stability............................................................................................................12
 
1.1.2.6 Sensitivity..................................................................................................................................12
 
1.2 BACKGROUND INFORMATION ON SILDENAFIL..........................................................................13
 
1.2.1 Introduction...................................................................................................................................13
 
1.2.2 Structure and chemical properties of sildenafil......................................................................13
 
1.2.2.1 Pharmacophoric group of sildenafil......................................................................................15
 
1.2.2.2 Mode of action of sildenafil....................................................................................................15
 
1.2.3 Metabolism of sildenafil.............................................................................................................16
 
1.2.4 Pharmacokinetics of sildenafil..................................................................................................16
 
1.2.4.1 Pharmacokinetic interactions of sildenafil...........................................................................17
 
1.2.4.2 Stability of sildenafil................................................................................................................18
 
1.2.5 Side effects of sildenafil.............................................................................................................19
 
1.2.6 Bioanalytical assay methods overview of sildenafil..............................................................20
 
2 EXPERIMENTAL 25
 
2.1 HPLC-UV ASSAY METHOD FOR SILDENAFIL................................................................................25
 
2.1.1 Method development...................................................................................................................25
 
2.1.1.1. Materials...................................................................................................................................25
 
2.1.1.2 Instruments................................................................................................................................25
 
2.1.1.3 Preparation of the stock solutions..........................................................................................26
 
 
Table of Contents II
2.1.1.4 Spectrophotometric Measurement.........................................................................................26
 
2.1.1.5 Optimisation of the mobile phase..........................................................................................28
 
2.1.2 Extraction from plasma...............................................................................................................34
 
2.1.2.1 Liquid-liquid extraction (LLE)..............................................................................................35
 
2.1.2.1.1 Degradation of sildenafil and trazodone during LLE with diethyl ether.....................38
 
2.1.2.1.2 Conclusion..............................................................................................................................42
 
2.1.2.2 Solid phase extraction (SPE)..................................................................................................43
 
2.1.2.2.1 Instruments and materials....................................................................................................43
 
2.1.2.2.2 Chromatography....................................................................................................................43
 
2.1.2.2.3 Optimisation of the SPE process........................................................................................43
 
2.1.3 Pre-validation...............................................................................................................................50
 
2.1.3.1 Stability of stock solution in glass and plastic containers.................................................52
 
2.1.4 HPLC-UV assay method validation.........................................................................................54
 
2.1.4.1 Blank plasma screening...........................................................................................................54
 
2.1.4.2 Planning of calibration (STD) and quality control (QC) standards.................................54
 
2.1.4.3 Preparation of calibration and quality control samples......................................................56
 
2.1.4.3.1 Calibration standards (STD)...............................................................................................57
 
2.1.4.3.2 Quality control standards (QC)...........................................................................................58
 
2.1.4.4 Preparation of internal standard and system performance verification samples...........59
 
2.1.4.5 Mobile phase preparation........................................................................................................60
 
2.1.4.6 Compiling the validation batches..........................................................................................60
 
2.1.4.6.1 Intra-day validation batch....................................................................................................61
 
2.1.4.6.2 Inter day validation batches.................................................................................................61
 
2.1.4.1.7 Extraction of analyte from plasma.....................................................................................62
 
2.1.5 Results and discussion................................................................................................................63
 
2.1.5.1 Linearity.....................................................................................................................................63
 
2.1.5.2 Accuracy and precision...........................................................................................................64
 
2.1.5.3 Analyte stability........................................................................................................................66
 
2.1.5.3.1 On-instrument stability.........................................................................................................66
 
2.1.5.3.2 Freeze and thaw stability:....................................................................................................67
 
2.1.5.3.3 Bench-top stability test:.......................................................................................................68
 
2.1.5.4 Analyte recovery:.....................................................................................................................69
 
2.1.6 Conclusion....................................................................................................................................70
 
2.2 LC–MS/MS ASSAY METHOD FOR SILDENAFIL..............................................................................72
 
2.2.1 Method development...................................................................................................................72
 
2.2.1.1 Instrumentation.........................................................................................................................72
 
2.2.1.2 Mobile phase optimisation......................................................................................................72
 
2.2.1.2.1 Optimisation of an alternative mobile phase....................................................................77
 
2.2.1.3 Optimisation of the LC-MS/MS system...............................................................................78
 
2.2.1.4 Extraction from plasma...........................................................................................................81
 
2.2.1.4.1 Protein precipitation.............................................................................................................82
 
2.2.1.4.2 Reproducibility of the precipitation method....................................................................85
 
2.2.1.4.3 Effect of injection volume...................................................................................................86
 
2.2.1.5 Matrix effect..............................................................................................................................86
 
2.2.1.6 Pre-validation............................................................................................................................88
 
2.2.2 LC-MS/MS Method validation.................................................................................................90
 
2.2.2.1 Blank plasma screening...........................................................................................................90
 
2.2.2.2 Planning of calibration and quality control standards........................................................90
 
2.2.2.3 Preparation of calibration standards and quality control standards.................................92
 
2.2.2.3.1 Calibration standards (STD)...............................................................................................92
 
2.2.2.3.2 Preparation of quality control standards...........................................................................93
 
2.2.2.4 Preparation of System performance verification samples and mobile phase.................95
 
2.2.2.5 Compiling the validation batch..............................................................................................95
 
2.2.2.5.1 Intra-day validation batch....................................................................................................96
 
2.2.2.5.2 Inter day validation batches.................................................................................................96
 
2.2.2.6 Extraction of analyte from plasma........................................................................................96
 
2.2.3 Results and discussion................................................................................................................97
 
2.2.3.1 Linearity.....................................................................................................................................97
 
2.2.3.2 Accuracy and precision...........................................................................................................98
 
2.2.3.3 Analyte stability......................................................................................................................100
 
2.2.3.3.1 Long term matrix stability test:.........................................................................................100
 

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