Development of methods to improve sensitivity and portability of capillary electrophoresis for the analysis of stabilizers and drugs 1

CZE Capillary zone electrophoresis DC Direct current DPA Diphenylamine EC Ethyl centralite ECD Electrochemical detection EOF Electroosmotic flow EP European Pharmacopoeia ESI Electros

Acknowledgements First of all, I would like to express my sincere thanks to my supervisor, Professor Sam F Y Li, for his invaluable suggestions, guidance and encouragement during the course of my work

Special thanks go to all the kind staff in Chemistry Department in particular Ms Tang C N., Dr Wang T L., Mdm Lim F and Mdm Wong L K

Many thanks are due to all of my lab mates, Dr Wei H P., Dr Ng H T., Dr Fang A P., Dr Qin W D., Dr Feng H T., Mr Yu L J., Mr Law W S., Mr Roderick Borong Pernites, Miss Xu Y., Miss Lau H F., Miss Junie Tok, Mr Jiang Z J for their help and encouragement

I sincerely appreciate the National University of Singapore for providing me the financial support during my research

Last but not least, thanks very much to my parents for their endless understanding, concern and support

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TABLE OF CONTENTS

Acknowledgements……….… ….… i Table of Contents……… ii~vii Abbreviations……….viii~xi Publications……… xii~xiii Summary……… ……….xiv~xvi

Chapter 1 Introduction……… …… 1~57

1.1 Basic Theory of Capillary Electrophoresis……….… 1~6 1.2 Electroosmotic Flow (EOF)……… 6~13 1.3 Different Modes of CE……….…… 13~20 1.3.1 Capillary Zone Electrophoresis (CZE)……… 13~14 1.3.2 Micellar Electrokinetic Chromatography (MEKC)………….… 14~15 1.3.3 Capillary Gel Electrophoresis (CGE)……… 15~16 1.3.4 Capillary Isoelectric Focusing (CIEF)……….… 16~17 1.3.5 Capillary Electrochromatography (CGE)……… 17~18 1.3.6 Capillary Isotachophoresis (CITP)……… …… 18~20 1.4 Sample Introduction and Concentration……… 20~31 1.4.1 Sample Introduction………20~22 1.4.2 On-line Concentration Technique in CE……… 22~32 1.4.2.1 Sample Stacking……… 23~26

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1.4.2.2 Field Amplified Sample Injection (FASI)……… 26~28 1.4.2.3 Isotachophoresis (ITP)……… ……… 28~29 1.4.2.4 pH-related Stacking……… …… 29~31 1.4.2.5 Sweeping……….……… 31 1.5 Different Detection Methods with CE……… 31~38 1.5.1 UV Detection……… 33 1.5.2 Fluorescence Detection……… …… 33~34 1.5.3 Electrochemical Detection……… … 34~38 1.5.4 Other Detection ………….……… 38 1.6 Current Trends of CE……… …… 38~45 1.6.1 Brief History of CE……… ……… 38~40 1.6.2 General Introduction of Several Trends in CE Field……… 40 1.6.3 CE-MS……… 40~42 1.6.4 Miniaturization of CE……… 42~44 1.6.5 Methods Development of Improving Sensitivity in CE…… … 44~45 1.7 Scope of Research ………….……….…… 45~50 References……… …… 51~57

Part I Sensitivity Improvement for MEKC of Stabilizers with UV

Detection ……… 58~94 Chapter 2 Modified Field Amplification Sample Injection for MEKC of

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Neutral Compounds……… …… 59~79

2.1 Introduction……… … 59~60 2.2 Materials and Methods……… 60~63 2.3 Results and Discussion……… 63~76 2.3.1 Basic Consideration……… 63~66 2.3.2 Modified Field Amplification Sample Injection……… 66~67 2.3.3 Effect of Stability of Inclusion Complexes on Concentration

Factor……… 67~69 2.3.4 Effect of Displacer plug on Concentration Factor………69~73 2.3.5 Optimization of Injection Time………73~75 2.3.6 Evaluation of Analytical Performance of Modified Field Amplification Sample Injection……… 75~76 2.4 Conclusions………76~77 References………78~79

Chapter 3 Application of Fast Analysis of Stabilizers in Gunpowder on Portable CE with UV Detection ……….………80~94

3.1 Introduction ……….… 80~83 3.2 Materials and Methods……….… 83~85 3.3 Results and Discussion……….……… 85~92 3.3.1 Basic Consideration……….……….85~86

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3.3.2 Optimization of Buffer Concentration……….….…….86~87 3.3.3 Optimization of Sample Solvent ……… ……… …… 87~88 3.3.4 Linearity, Repeatability and Limits of Detection………….….…….88~89 3.3.5 Application and Comparison with the HPLC Results………89~92 3.4 Conclusions……….………92~93 References……… 93~94

Part II Direct Determination of non-UV Active Drugs with

Electrochemical Detection on Portable CE System………… …….95~156 Chapter 4 Direct Determination of Antibiotics Components on portable

CE System ……… …… 96~124

4.1 Introduction……….…………96~100 4.2 Materials and Methods……… 100~105 4.3 Results and Discussion of Gentamicin Experiments……….………105~113 4.3.1 Basic Consideration……….…… 105~107 4.3.2 Buffer System and pH Optimization……… ……… 107~108 4.3.3 Effect of CTAB Concentration……… 108~109 4.3.4 Buffer Concentration Optimization……… 109~113 4.4 Results and Discussion of Neomycin Experiments……… …… 113~120 4.4.1 Basic Consideration……….………….113~114 4.4.2 Separation Medium Optimization……….…………114

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4.4.3 Separation Voltage Optimization……… …….114~115 4.4.4 Linearity, Repeatability and Limits of Detection………… ……115~119 4.4.5 Application……….……119~120 4.5 Conclusion……….….120~121 References ……… …….122~124

Chapter 5 Chiral Separation of Gentamicin Components on Portable CE System ……… 125~140

5.1 Introduction………125~126 5.2 Materials and Methods……… 126~129 5.3 Results and Discussion……….… 129~137 5.3.1 Chiral Separation……….……… 129~130 5.3.2 Buffer System and pH Optimization……….130~131 5.3.3 Effect of Vancomycin Concentration………131~132 5.3.4 Linearity, Repeatability and Limits of Detection……… 132~136 5.3.5 Real Sample Analysis……….…… 136~137 5.4 Conclusion……….……… 137~139 References ……… 139~140

Chapter 6 Fast Analysis of Carnitine on Portable CE

System ……….………141~155

6.1 Introduction ……… 141~144

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6.2 Materials and Methods……… 144~146 6.3 Results and Discussion……… 146~153 6.3.1 Basic Consideration……….146~147 6.3.2 Buffer Optimization ……….147~152 6.3.2.1 Buffer System……… 147~148 6.3.2.2 Effect of pH Value……… 148~149 6.3.2.3 Linearity, Repeatability and Limits of Detection……….149~151 6.3.5 Determination of Real Sample ……….………151~153 6.4 Conclusions………153 Reference……… 154~155

Chapter 7 Concluding and Future Work ……… 156~160

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Abbreviations 2nDPA 2-Nitro Diphenylamine

4nDPA 4-Nitro Diphenylamine

β-CD-NH 2 mono(6-amino-6-deoxy)-β-cyclodextrin

AC 1-Adamantanecarboxylic acid

ACN Acetonitrile

ADAM 9-anthryldiazomethane

BGE Background electrolyte

C4D Capacitively coupled contactless conductivity detection

CCD Contactless conductivity detector

CD Cyclodextrin

CE Capillary Electrophoresis

CEC Capillary Electrochromatography

CF Concentration factor

CF-FAB Continuous-flow fast atom bombardment

CGE Capillary gel electrophoresis

CIEF Capillary isoelectric focusing

CIS Coordination ion spray

CITP Capillary isotachophoresis

CTAB Cetyltrimethylammonium bromide

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CZE Capillary zone electrophoresis

DC Direct current

DPA Diphenylamine

EC Ethyl centralite

ECD Electrochemical detection

EOF Electroosmotic flow

EP European Pharmacopoeia

ESI Electrospray ionization

FAB–MS Fast atom bombardment mass spectrometry

FASI Field amplified sample injection

FMOC 9-fluorenylmethyl chloroformate

GC Gas chromatography

GC-MS Gas chromatography–mass spectrometry

HPCE High performance capillary electrophoresis

HPLC High performance liquid chromatography

ID Internal diameter

IS Internal standard

ITP Isotachophoresis

LC Liquid chromatography

LIF Laser-induced fluorescence

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LOD Limit of Detection

LVSS Large volume sample stacking

MAA Mercaptoacetic acid

MALDI-MS Matrix-assisted laser desorption/ionization mass spectrometry

MC Methyl centralite

MEKC Micellar Electrokinetic Chromatography

mFASI Modified field amplified sample injection

MS Mass spectrometry

NaOH Sodium Hydroxide

NE1 1-naphthaleneethanol

NM1 1-naphthalenemethanol

NM2 2-naphthalenemethanol

NMR Nuclear magnetic resonance

N-nDPA N-Nitroso Diphenylamine

NSM Normal stacking mode

OD Outside diameter

OPA 1,2-phthalic dicarboxaldehyde

PAD Pulsed amperometric detection

PGD Potential gradient detection

PVA Polyvinylalcohol

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PVP Polyvinylpyrrolidone

RSD Relative standard deviation

SFC Supercritical fluid chromatography

SDS Sodium dodecyl sulfate

TLC Thin layer chromatography

USP US Pharmacopoeia

UV-Vis Ultraviolet-visible

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Publications

1 TianLin Wang; LingLing Yuan; Sam Fong Yau Li, Modified Field Amplification Sample Injection for Micellar Electrokinetic Chromatography of Neutral Compounds with Amino-Substituted Cyclodextrin as Carrier and 1-Adamantanecarboxylate as

Displacer, J Chromatogr A, 1013, 2003, 19-27

2 HuaTao Feng; LingLing Yuan; Sam Fong Yau Li, Analysis of Chinese medicine

preparations by capillary electrophoresis–mass spectrometry, J Chromatogr A,

1014, 2003, 83-91

3 Lijun Yu, LingLing Yuan, Huatao Feng, Sam Fong Yau Li, Determination of the bacterial pathogen (Edwardsiella tarda) in fish species by capillary electrophoresis

with blue light-emitting diode induced fluorescence, Electrophoresis, 25, 2004,

3139-3144

4 LingLing Yuan, HongPing Wei, Sam Fong Yau Li, Direct Determination of Gentamicin Components by Capillary Electrophoresis with Potential Gradient Detection,

Electrophoresis, 26, 2005, 196-201

5 Wai Siang Law, Pavel Kubáň, LingLing Yuan, Jian Hong Zhao, Sam Fong Yau Li, Peter

C Hauser, Determination of Tobramycin in Human Serum by Capillary

Electrophoresis with Contactless Conductivity Detection, Electrophoresis, in press

6 LingLing Yuan, HongPing Wei, HuaTao Feng, Sam Fong Yau Li, Fast Analysis of Native Neomycin Components by Capillary Electrophoresis with Potential Gradient

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Detection, Analytical and Bioanalytical Chemistry, accepted

7 LingLing Yuan, Cerenna Ng, Sam Fong Yau Li, Simultaneous Separation of Chiral and Other Components of Gentamicin on a Portable Capillary Electrophoresis System with

Potential Gradient Detectio, J Chromatogr A, submitted

8 LingLing Yuan, Sam Fong Yau Li, Fast Analysis of Carnitine on Portable Capillary

Electrophoresis System with Contactless Conductivity Detection, J Chromatogr A,

submitted

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Summary

This work mainly focused on the development of methods to improve sensitivity and portability in capillary electrophoresis systems With the high separation efficiency, high resolution and portability, CE has been proven a powerful method for the studies of many groups of compounds The improvement of sensitivity and the miniaturization of CE technique are two major trends of capillary electrophoresis which have gain more and more attention In this work, approaches to enhance sensitivity and portability of CE are investigated

A modified field amplification sample injection method was proposed and evaluated by using positively mono-charged cyclodextrin (CD) as carrier and 1-adamantanecarboxylate

as displacer for on-capillary preconcentration of neutral stabilizers in pharmaceutical products and improvement of the concentration limit of detection (LOD) in micellar electrokinetic chromatography (MEKC) In this mode a displacer plug was introduced before sample injection to reduce the length of the concentrated sample zone and increase the peak height by slowing down the forward movement of the neutral sample associated with β-CD-NH2 and the backward movement of the neutral sample partitioned in the micelles of sodium dodecyl sulfate (SDS) Stability of the inclusion complexes formed between the carrier and the solute was found to be an important factor affecting stacking efficiency in both the conventional field amplification sample injection mode and the

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modified one However, further enhancement of the stacking efficiency in the modified mode rested on the relative stability of the displacer-carrier complex to that of the solute-carrier complex

Next, a new method to test stabilizers in gunpowder was optimized, validated and applied

on the stabilizers analysis in real gunpowder samples Good sensitivity, linearity and reproducibility were obtained after optimization The results obtained by CE were compared with those of high performance liquid chromatography (HPLC), which showed the capillary electrophoresis (CE) method on our portable CE instrument can be a good alternate method for HPLC, with the advantage that it can be more readily applied in on-site analysis

A simple and fast method was developed to determine non-UV active compounds directly without derivatization on a portable capillary electrophoresis system with potential gradient detection The usefulness of the method was demonstrated by detecting the major components in aminoglycoside antibiotic mixtures Gentamicin was separated into several peaks in 15 minutes, and neomycin into three peaks even faster within 4 minutes This method showed better sensitivity (LOD 7~10 ppm) than other CE methods for determining underivatized gentamicin and neomycin with large linear range (10~1000 ppm) Because of its good repeatability (RSD <5 %) and simplicity, this new method

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could be a good alternative for the current assays given by US Pharmacopoeia (USP) and European Pharmacopoeia (EP)

A further improved method was developed to determine gentamicin’s two stereoisomer components (C2, C2a) as well as two other major components (C1 and C1a) Chiral separation was achieved by using 0.2mM CTAB, 1mM vancomycin and 1mM ammonium citrate buffer system, pH3.5 Large linear range, good repeatability and simplicity of the method could render itself a good alternate method to HPLC also The experiments were developed on the same portable capillary electrophoresis system as in the previous chapter, and potentially can be used for field analysis of chiral compounds

In addition, a fast method for the analysis of underivertized carnitine was developed on a portable capillary electrophoresis system with capacitively coupled contactless conductivity detection (C4D) Compared with potential gradient detection, the C4D method has the advantage that there is no need to custom-make a detection cell for each capillary The LOD was found to be 3ppm The linear range was between 3 to 1000 ppm, and RSD value was satisfactory, i.e < 1.5% in the same day and < 5% on different days The simplicity, low LOD, wide linearity and good repeatability of the method render itself

a good alternate method to the HPLC method recommended by USP

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