PHARMACEUTICAL ANALYSIS BY CAPILLARY ELECTROPHORESIS Capillary Isoelectric Focusing (CIEF) and Capillary

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  • PHARMACEUTICAL ANALYSIS BY

    CAPILLARY ELECTROPHORESIS

    by Catherine Meehan, BSc,

    submitted for the degree of Master in Science,

    Institute of Technology Sligo, Ballinode,

    Sligo,

    supervised by Dr. Fiona Me Ardle

    Submitted to the Higher Education And Training Awards Council, 2003

  • ACKNOWLEDGMENTS

    I would like to thank Dr. Fiona Me Ardle for her guidance and help throughout this research project.

    I would like to express my sincere appreciation to all research colleagues, technical staff and teaching

    staff at the Institute of Technology Sligo.

    Thankyou to my parents and family for their support and encouragement during my academic years.

    For my husband Brian and son Conal, thankyou for your continuous, unconditional love.

  • ABSTRACT

    This project focused on developing and validating analytical methods for a range of pharmaceutical

    compounds using Capillary Electrophoresis.

    Analytical methods for the determination o f tromethamine, Oxytetracycline and polypyrrolidone

    contained in pharmaceutical products were developed. Approaches to method development included

    consideration of appropriate mode of capillary electrophoresis, detection technique, suitability of

    electrolyte, effect of electrolyte pH, effect of voltage, temperature and electrolyte ionic strength variation.

    Validation of the methods developed for tromethamine and Oxytetracycline included conducting

    investigations into linearity, linear range and limit of detection. Repeatability, precision and accuracy

    were also examined as well as the specifity and recovery of each method developed.

  • CONTENTS

    Page No. CHAPTER 1 INTRODUCTION 1.

    1.1 Introduction to Capillary Electrophoresis 1. 1.2 Principles of Separation by Capillary Electrophoresis 2. 1.3 Origin of Capillary Electrophoresis 3. 1.4 Modes of Capillary Electrophoresis 3. 1.5 Comparison ofCE with other Techniques 4. 1. 6 Analytes of Interest in this Study 5. 1.7 Applications of Capillary Electrophoresis 6 .

    CHAPTER 2 THEORY 11.

    2.1 Capillary Zone Electrophoresis 12.

    2.1.1 Order of Elution 12. 2.1.2 Fundamental Features Required for Separation 12. 2.1.3 Capillaries 13.

    2.1.3.1 Fused Silica Capillaries 13. 2.1.3.2 Capillary Dimensions I 13.

    2.1.4 Injection Techniques 14. 2.1.4.1 Pressurised and Vacuum Injection 15.

    2.1.5 Detection 15. 2.1.5.1 Direct UV Detection 15. 2.1.5.2 Indirect UV Detection 16.

    2.1.6 Electroosmotic Flow and Electrophoretic Mobility 17. 2.1.6.1 Electroosmotic Flow 17. 2.1.6.2 Reversal of EOF 18. 2.1.6.3 Measurement o f EOF 18. 2.1.6.4 Electroosmotic Mobility 19. 2.1.6.5 Electrophoretic Mobility 19. 2.1.6 . 6 Migration Time 20.

    2.1.7 Background Electrolyte 21. 2.1;7.1 Analyte Ionisation 21. 2.1.7.2 Electrolyte pH 22.

    2.1.8 Joule Heat 23. 2.1.8.1 Effects of Joule Heat 23.

    2.1.9 Ionic Strength 24. 2.1.10 Temperature 24. 2.1.11 Organic Solvent Addition 25. 2.1.12 Organic Modifiers 25. 2.1.13 Voltage 25.

  • 2.1.14 Efficiency, Selectivity and Resolution 26.

    2.2 Micellar Electrokinetic Capillary Chromatography 28. 2.2.1 Principles of Separation in MECC 28.

    2.2.2 Migration of Analytes 29. 2.2.3 Electroosmotic Flow in MECC 30.

    2.2.3.1 Reversal of EOF 31. 2.2.4 Resolution in MECC 31. 2.2.5 Efficiency in MECC 32. 2.2.6 Selectivity in MECC 32.

    2.2.6.1 Capacity Factor 32. 2.2.7 Electroosmotic Velocity 33. 2.2.8 Surfactants Used in MECC 33. 2.2.9 Organic Modifiers 34.

    2.3 Comparing CZE and MECC 34.

    2.4 Ion Chromatography 35. 2.4.1 Principle Components of an IC System 35. 2.4.2 Ion Exchange Columns 36. 2.4.3 Mechanism of Cation Analysis 36. 2.4.4 Order Elution 37.

    CHAPTER 3 BENZOIC ACID 38.

    3.1 Introduction 39. 3.2 Literature Review 39. 3.3 Method Application 41.

    3.3.1 Materials and Operating Conditions 41. 3.3.2 Results 42.

    CHAPTER 4 INORGANIC CATIONS 44.

    4.1 Introduction 45. 4.2 Literature Review 45. 4.3 CE Method Application 47.

    4.3.1 Materials and Operating Conditions 47. 4.3.2 Results 48.

    4.4 IC Method Application 48. 4.4.1 Materials and Operating Conditions 48. 4.4.2 Results 49.

    CHAPTER 5 TROMETHAMINE 51.

    5.1 Introduction 52.

  • 5.2 Literature Review 53. 5.3 Method Development 56.

    5.3.1 Choosing an Electrolyte 56. 5.3.1.1 Copper Sulphate 57. 5.3.1.2 Imidazole 5 8 . 5.3.1.3 DDP 60.

    5.3.2 Electrolyte pH Variation 62. 5.3.3 Voltage Variation 64. 5.3.4 Temperature Variation 65. 5.3.5 Ionic Strength Variation 65.

    5.4 Method Validation 6 6 . 5.4.1 Linearity, Linear Range and Limit o f Detection 6 6 . 5.4.2 Repeatability 67. 5.4.3 Precision and Accuracy 67. 5.4.4 Specificity 6 8 . 5.4.5 Recovery 6 8 .

    CHAPTER 6 OXYTETRACY CLINE 70.

    6.1 Introduction 71. 6.2 Literature Review 72. 6.3 Method Development 76.

    6.3.1 Solubility 76. 6.3.2 UV Measurements 76. 6.3.3 Choosing a Suitable Electrolyte 77. 6.3.4 Optimising UV Absorbance 79. 6.3.5 Changing Sample Solvent 79. 6.3.6 Varying Injection Volume 80. 6.3.7 Electrolyte pH Variation 80. 6.3.8 Ionic Strength Variation 81. 6.3.9 Electrophoretic and Electroosmotic Mobility 81. 6.3.10 Electrolyte Composition 82.

    6.3.10.1 Addition of Methanol 82. 6.3.10.2 Addition of SDS 83.

    6.3.11 V oltage V ariation 84. 6.4 Method Validation 85.

    6.4.1 Linearity, Linear Range and Limit of Detection 85. 6.4.2 Repeatability 85. 6.4.3 Precision and Accuracy 8 6 . 6.4.4 Recovery 8 6 . 6.4.5 Specificity 8 8 .

    6.4.5.1 Separation of OTC and PVP 8 8 .

  • CHAPTER 7 POVIDONE 90.

    7.1 Introduction 91. 7.2 Literature Review 91. 7.3 Method Development 93.

    7.3.1 Confirming PVP Response 93. 7.3.2 Addition of Organic Solvent 94. 7.3.3 Electrolyte pH Variation 94. 7.3.4 Capillary Wall Modification 95. 7.3.5 Investigating Linearity 96.

    7.3.5.1 Limit of Detection 96. 7.3.6 Effects of Product Ingredients 97. 7.3.7 Electroosmotic Mobility 97.

    CHAPTER 8 DISCUSSION 98.

    8.1 Benzoic Acid Analysis 99. 8.2 Inorganic Cation Analysis 99. 8.3 Tromethamine Analysis 99.

    8.3.1 Selection of Detection System 99. 8.3.2 Choosing a Suitable Electrolyte 100. 8.3.3 Identification of Unknown Peaks 101. 8.3.4 DDP Electrolyte 101. 8.3.5 Electrolyte Composition 102. 8.3.6 Effect of Electrolyte PH 102. 8.3.7 Effect of Voltage Variation 103. 8.3.8 Effect of Temperature Variation 103. 8.3.9 Effect of Ionic Strength Variation 104. 8.3.10 Linearity, Linear Range, Limit o f Detection 104. 8.3.11 Repeatability 104. 8.3.12 Precision and Accuracy 105. 8.3.13 Specificity 105. 8.3.14 Recovery 105.

    8.4 Oxytetracycline Analysis 105. 8.4.1 Choice of CE Technique 105. 8.4.2 Selection of Detection System 106. 8.4.3 Choice of Suitable Electrolyte 106. 8.4.4 Electroosmotic Flow 106. 8.4.5 Peak Efficiency 107. 8.4.6 Electrolyte Composition 107.

    8.4.6.1 Effect of Methanol Addition 108. 8.4.6.2 Effect of SDS Addition 108.

    8.4.7 Effect of Electrolyte pH 108.

  • 8.4.8 Effect of Voltage Variation 108. 8.4.9 Effect o f Temperature Variation 109. 8 .4.10 Effect of Ionic Strength Variation 109. 8.4.11 Linearity, Linear Range and Limit o f Detection 110. 8.4.12 Repeatability 110. 8.4.13 Precision and Accuracy 110. 8.4.14 Recovery 110. 8.4.15 Specificity 111.

    8.5 Povidone Analysis 111. 8.5.1 Choice of CE Technique 111. 8.5.2 Selection of Detection System 112. 8.5.3 Choice of Suitable Electrolyte 112. 8.5.4 Electroosmotic Flow 113. 8.5.5 Organic Solvent Addition 113. 8.5.6 Electrolyte pH Variation 113. 8.5.7 Capillary Wall Modification 113. 8.5.8 Effects of Product Ingredients 114.

    CHAPTER 9 CONCLUSION 115.

    9.1 Summary of Results 116. 9.2 Benefits o f the Study 117. 9.3 Future Investigations 117.

    REFERENCES

    APPENDICES

  • CHAPTER 1

    INTRODUCTION

    1

  • 1.1 Introduction to Capillary Electrophoresis

    Capillary Electrophoresis (CE), is an analytical technique, which permits rapid and efficient separation of

    charged components. Separations are based on the differences in electrophoretic mobilities o f chared

    species that take place inside small capillary tubes.

    CA PILL A R Y

    I UFFLR ANODE

    SOURCE VIAL SAMPLE VIAL

    IN T E G R A T O R O R ..

    C O M P U T E R

    B JFFER 11 C A T H O D E

    DESTINATION VIAL

    High Voltage Power Supply

    Diagram 1.1 Schematic Diagram of CE Instrumentation

    1 . 2 Principles of Separation by CE

    To perform a CE separation, the capillary is filled with an electrolyte solution and sample is introduced at:

    the source vial. The capillary ends are dipped into source and destination vials containing the electrolytic:

    and a potential is applied across the capillary. The bulk electrolyte travels by electroosmotic flow (EOF)'

    and ionic species in the sample migrate with an electrophoretic mobility (EPM) determined by their

    massxharge ratio. The analyte ions eventually pass the cell window and a response from the UV detector

    is generated and passed onto an integrator. Signals generated follow Beer-Lambert Law.

    A = s c l (Eqn. LI)!

    A = Absorbance (nm), e = Molar absorptivity coefficient, c = Concentration (mg/L) and I = path cell length (cm)

    2

  • Normally the system is configured with the source vial anodic and the destination vial cathodic. As a

    potential is applied across the two electrodes, ions in the buffer travel through the capil

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