Development of Microscale Separation Techniques for ...library.umac.mo/etheses/b25903743_toc.pdf · iii Abstract Microscale separations, e.g., capillary electrophoresis (CE) and capillary

Development of Microscale Separation Techniques for Quality

Control of Chinese Medicines

by

Xiao-jia Chen

Doctor of Philosophy in Biomedical Sciences

2012

Institute of Chinese Medical Sciences

University of Macau

Development of Microscale Separation Techniques for Quality

Control of Chinese Medicines

by

Xiao-jia Chen

Supervisor: Professor Yitao Wang

Doctor of Philosophy in Biomedical Sciences

2012

Institute of Chinese Medical Sciences

University of Macau

Author’s right 2012 by CHEN, Xiaojia

i

Acknowledgements

First of all, I would like to express my deep and sincere gratitude to my

supervisor Prof. Yitao Wang. His wide knowledge and deep thinking have been of

great value for me, and his understanding, encouraging and personal guidance have

provided a good basis for the present thesis. I am especially grateful to my

co-supervisor Prof. Shaoping Li, whose constant enlightenment, encouragement and

inexhaustible patience support me in completing this thesis. I would also like to

express my deeply gratitude to Dr. Frantisek Svec for his enlightening guidance and

stimulating suggestions during my study in Lawrence Berkeley National Laboratory.

I would like to express my heartfelt thanks to Prof. Ming-Yuen Lee, Prof. Ying

Zheng, Prof. Ying Bian, Dr. Qingwen Zhang, Dr. Ru Yan, Dr. Pui Man Hoi, Dr. Hao

Hu, Dr. Li-Jen Cheng and all the administrative staffs Ms. Hattie U, Ms. Chloe Lao,

Ms. Carol Lam, Mr. Leon Lai, Ms. Sandy Lao, Ms. Sio Kio Kuong, Ms. Wing Leong,

Ms. Katrina Wong, Ms. Ada Wong, Mr. Dorian Ng and Ms. Joanna Lio for their

warm support during my study and living.

Moreover, I would like to thank all the colleagues of Institute of Chinese Medical

Sciences, University of Macau. Special thanks to Peng Li, Jianbo Wan, Yuanjia Hu,

Jianli Gao, Fengqing Yang, Jia Guan, Yingbo Li, Zhengming Qian and Kun Feng for

giving me the great memory of the happiness to study and work together.

Acknowledgements

ii

My warm thanks are also expressed to all the members in Organic Facility,

Molecular Foundry, Lawrence Berkeley National Laboratory. Special thanks to Dr.

Yongqin Lu, Mr. Zhixing Lin, Dr. Fernando Maya and Dr. Natalia Blinova for the

supports and helps with my study and living in the United States.

This research was supported by grants from Macao Science and Technology

Development Fund (049/2005/A-R1 and 029/2007/A2 to Prof. Yitao Wang) and

University of Macau (UL015 to Prof. Shaoping Li).

Last but not the least, I am forever indebted to my family for their understanding,

endless patience and support during these years.

iii

Abstract

Microscale separations, e.g., capillary electrophoresis (CE) and capillary

electrochromatography (CEC), are powerful analytical techniques that provide fast

and efficient separation with low consumption of sample and reagent. Due to their

versatility and high efficiency, CE and CEC have attracted great interest of analysts

in the analysis of phytochemicals in Chinese medicines. And they have become

alternative methods to the widely used high-performance liquid chromatography. In

this study, CE and CEC analysis of flavonoids from Epimedium and licorice, as well

as nucleosides and nucleobases, were investigated. The effect of stability of internal

standard on quantitative determination and peak suppression technique for

quantification of overlapping peaks were also studied. In addition, preliminary

investigation on hypercrosslinked porous poly(styrene-co-vinylbenzyl

chloride-co-divinylbenzene) and poly(4-methylstyrene-co-vinylbenzyl chloride-co-

divinylbenzene) monolithic columns for CEC analysis of small molecules were also

performed. The major achievements of this study are summarized as follows:

(1) A capillary zone electrophoretic method for simultaneous determination of 15

flavonoids, including epimedin B, epimedin A, hexandraside F, epimedin C, icariin,

sagittatoside B, sagittatoside A, hexandraside E, 2"-O-rhamnosyl icariside II,

baohuoside VII, baohuoside I, caohuoside C, epimedoside C, baohuoside II and

kaempferol-3-O-rhamnoside, in different species of Epimedium were developed, and

the effect of stability of internal standard on quantification was also investigated.

(2) CEC methods for simultaneous determination of flavonoids in Epimedium

and licorice using packed C18 capillary column were developed, respectively. The

Abstract

iv

influence of relevant parameters such as buffer concentration, pH and proportion of

acetonitrile were investigated and optimized. Peak suppression technique was used

for the quantification of the overlapping peaks, isoliquiritin and ononin, based on the

differences between their UV absorption spectra.

(3) The utility of hypercrosslinked poly(styrene-co-vinylbenzyl chloride-co-

divinylbenzene) and poly(4-methylstyrene-co-vinylbenzyl chloride-co-

divinylbenzene) monolithic columns for CEC separation was demonstrated for the

first time. The separation of alkylbenzenes, flavonoids from Epimedium, as well as

nucleosides and nucleobases was also investigated. Although alkylbenzenes were

successfully separated, the separations of flavonoids from Epimedium and

nucleosides and nucleobases using these columns were not good. Further efforts

should be made to improve the CEC separation of phytochemicals using the

monolithic columns.

In summary, microscale separations including CE and CEC analysis of selected

phytochemicals in Chinese medicines were developed and investigated, some of

which were studied for the first time. This study is helpful for the application of CE

and CEC for quality control of Chinese medicines.

v

Table of Contents

Acknowledgements ....................................................................................................... i

Abstract...... ................................................................................................................. iii

Table of Contents ......................................................................................................... v

List of Tables ............................................................................................................. viii

List of Figures .............................................................................................................. x

List of Abbreviations ................................................................................................. xiii

Chapter 1 Introduction ................................................................................................. 1

1.1 General background ....................................................................................... 1

1.1.1 Capillary electrophoresis ..................................................................... 1

1.1.2 Capillary electrochromatography ........................................................ 6

1.2 Specific background ....................................................................................... 9

1.2.1 Sample preparation............................................................................ 10

1.2.2 Separation .......................................................................................... 14

1.2.3 Detection ........................................................................................... 24

1.3 Research goals and objectives ...................................................................... 29

1.4 Research methodology and design ............................................................... 31

1.5 Potential contributions ................................................................................. 31

1.6 Organization of the thesis ............................................................................. 33

1.7 Statement of originality ................................................................................ 34

References .......................................................................................................... 39

Chapter 2 Simultaneous quantification of 15 flavonoids in Epimedium using capillary

zone electrophoresis ................................................................................. 62

2.1 Introduction .................................................................................................. 62

Table of Contents

vi

2.2 Materials and methods ................................................................................. 64

2.2.1 Chemicals, reagents and materials .................................................... 64

2.2.2 Sample preparation ............................................................................ 67

2.2.3 CZE Analysis ..................................................................................... 68

2.2.4 Calibration curves ............................................................................. 69

2.2.5 Limits of detection and quantification .............................................. 69

2.2.6 Precision, repeatability and accuracy ................................................ 69

2.3 Results and discussion .................................................................................. 70

2.3.1 Optimization of CZE conditions ....................................................... 70

2.3.2 Selection of IS ................................................................................... 71

2.4 Conclusions .................................................................................................. 85

References .......................................................................................................... 86

Chapter 3 Capillary electrochromatographic analysis of flavonoids in Epimedium and

licorice using packed capillary columns .................................................. 89

3.1 Introduction .................................................................................................. 89

3.2 Simultaneous determination of 7 flavonoids in Epimedium ........................ 90

3.2.1 Materials and methods ...................................................................... 90

3.2.2 Results and discussion ....................................................................... 94

3.3 Simultaneous determination of 5 flavonoids in licorice ............................. 101

3.3.1 Materials and methods .................................................................... 103

3.3.2. Results and discussion .................................................................... 107

3.4 Conclusions ................................................................................................ 119

References ........................................................................................................ 119

Chapter 4 Preliminary investigation on capillary electrochromatographic separation

of phytochemicals using hypercrosslinked porous polymer monolithic

Table of Contents

vii

capillary columns ................................................................................... 123

4.1 Introduction ................................................................................................ 123

4.2 Materials and Methods ............................................................................... 127

4.2.1 Materials .......................................................................................... 127

4.2.2 Preparation of generic monolithic capillary columns ..................... 129

4.2.3 Hypercrosslinking ........................................................................... 130

4.2.4 Oxidation ......................................................................................... 130

4.2.5 Capillary electrochromatography .................................................... 131

4.3 Results and discussion ............................................................................... 131

4.3.1 Generic and hypercrosslinked monolithic columns ........................ 131

4.3.2 Oxidation of the hypercrosslinked monolithic columns ................. 136

4.3.3 Problems and perspectives .............................................................. 141

4.4 Conclusions ................................................................................................ 142

References ........................................................................................................ 143

Chapter 5 Conclusions ............................................................................................. 147

5.1 Conclusions ................................................................................................ 147

5.2 Limitations of current study ....................................................................... 149

5.3 Perspectives for future work ...................................................................... 150

References ........................................................................................................ 151

Appendix... ............................................................................................................... 153

Curriculum Vitae ...................................................................................................... 230

viii

List of Tables

Table 2.1 Summary for the tested samples of Epimedium ..................................... 66

Table 2.2 Linear regression data, LOD and LOQ of the investigated flavonoids

(using rutin as IS) ................................................................................... 72

Table 2.3 Intra- and inter-day precision of the investigated flavonoids (using rutin

as IS) ....................................................................................................... 72

Table 2.4 Repeatability of the investigated flavonoids (using rutin as IS) ............ 73

Table 2.5 Recoveries for the assay of 15 flavonoids in Epimedium (using rutin as IS)

................................................................................................................ 74

Table 2.6 Contents (mg/g) of investigated flavonoids in Epimedium using rutin as

IS ............................................................................................................ 77

Table 2.7 Stability of rutin and daidzein evaluated as icariin and their peak area

ratios ....................................................................................................... 79

Table 2.8 Linear regression data, LOD and LOQ of the investigated flavonoids

(using daidzein as IS) ............................................................................. 80

Table 2.9 Intra- and inter-day precision of the investigated flavonoids (using

daidzein as IS) ........................................................................................ 80

Table 2.10 Repeatability of the investigated flavonoids (using daidzein as IS) ...... 81

Table 2.11 Recoveries for the assay of 15 flavonoids in Epimedium (using daidzein

as IS) ....................................................................................................... 82

Table 2.12 Contents (mg/g) of the investigated flavonoids in Epimedium using

daidzein as IS ......................................................................................... 83

Table 3.1 Linear regression data, LOD and LOQ of the investigated flavonoids .. 96

Table 3.2 Intra- and inter-day precision of the investigated flavonoids ................. 97

List of Tables

ix

Table 3.3 Repeatability of the investigated flavonoids .......................................... 97

Table 3.4 Recoveries for the assay of 7 flavonoids in Epimedium ........................ 98

Table 3.5 Stability of the investigated flavonoids .................................................. 98

Table 3.6 Contents (mg/g) of investigated flavonoids in Epimedium .................... 99

Table 3.7 Summary for the tested samples of licorice ......................................... 104

Table 3.8 Linear regression data, LOD and LOQ of the investigated flavonoids 112

Table 3.9 Intra- and inter-day precision of the investigated flavonoids ............... 113

Table 3.10 Repeatability of the investigated flavonoids ........................................ 113

Table 3.11 Recoveries for the assay of five flavonoids in licorice ........................ 114

Table 3.12 Contents (mg/g) of investigated flavonoids in licorice ........................ 115

Table 3.13 Summarized methods for the analysis of flavonoids in licorice .......... 118

Table 4.1 EOF mobility of generic and hypercrosslinked monolithic columns ... 133

x

List of Figures

Figure 2.1 Chemical structures of 15 investigated flavonoids, rutin and daidzein. . 65

Figure 2.2 CZE profiles of (A) mixed standards and PLE extracts of (B) Epimedium

brevicornu; (C) E. sagittatum; (D) E. pubescens; (E) E. wushanense; (F)

E. koreanum; (G) E. acuminatum; (H) E. myrianthum; (I) E. franchetii; (J)

E. stellulatum; (K) E. zhushanense; (L) E. lishihchenii; (M) E. davidii. 75

Figure 2.3 Total contents of 15 flavonoids determined by HPLC, UHPLC and CZE.

................................................................................................................ 78

Figure 2.4 CZE profiles of (A) mixed standards and PLE extracts of (B) Epimedium

brevicornu; (C) E. sagittatum; (D) E. pubescens; (E) E. wushanense; (F)

E. koreanum; (G) E. acuminatum; (H) E. myrianthum; (I) E. franchetii; (J)

E. stellulatum; (K) E. zhushanense; (L) E. lishihchenii; (M) E. davidii; (N)

E. fargesii; (O) E. hunanense; (P) E. leptorrhizum; (Q) E. platypetalum;

and (R) E. sutchuenense. ........................................................................ 84

Figure 3.1 Effects of pH (◆), buffer concentration (■), proportion of ACN (▲),

temperature (●) and voltage (○) on (A) the resolution (Rs) between

epimedin B and C and (B) entire run-time (TR). .................................... 95

Figure 3.2 Capillary electrochromatograms of (A) mixed standards and PLE extracts

of (B) Epimedium brevicornu; (C) E. sagittatum; (D) E. pubescens from

Guangxi; (E) E. pubescens from Sichuan; (F) E. wushanense and (G) E.

koreanum. ............................................................................................. 100

Figure 3.3 Chemical structures of 5 investigated flavonoids and baicalein (IS). .. 103

List of Figures

xi

Figure 3.4 Influences of solvent, temperature, static extraction time and particle size

on pressurized liquid extraction of liquiritin ( ), isoliquiritin ( ), ononin

( ), liquiritigenin ( ) and isoliquiritigenin ( ) from licorice. ........... 108

Figure 3.5 Capillary electrochromatograms of (A) mixed standards and (B) licorice

sample GU-17 using 10 mM phosphate buffer (pH 3.0)/ACN (50:50 v/v)

as mobile phase at 20 kV of voltage and 25 oC of temperature. .......... 110

Figure 3.6 The (A) UV spectra of isoliquiritin and ononin, and capillary

electrochromatograms of mixed standards detected at 254 nm (B) without

and (C) with reference wavelength of 405 nm, as well as (D) at 360 nm.

.............................................................................................................. 111

Figure 3.7 Capillary electrochromatograms of (A) mixed standards and PLE extracts

of underground part of (B) Glycyrrhiza uralensis; (C) G. inflata; (D) G.

glabra; and (E) aerial part of G. uralensis. .......................................... 116

Figure 4.1 Scheme of the synthesis of poly(styrene-co-vinylbenzyl

chloride-co-divinylbenzene) monolith. ................................................ 126

Figure 4.2 Chemical structures of the monomers used for the preparation of

monoliths. ............................................................................................. 128

Figure 4.3 Chemical structures of the investigated nucleosides and nucleobases. 129

Figure 4.4 Electrochromatograms of thiourea and alkylbenzenes using (A)

poly(ST-VBC-DVB) and (B) poly(MST-VBC-DVB) hypercrosslinked

monolithic columns. ............................................................................. 134

Figure 4.5 Electrochromatogram of the investigated flavonoids under the optimum

conditions. ............................................................................................ 135

Figure 4.6 IR spectrogram of oxidized hypercrosslinked poly(MST-VBC-DVB)

monolithic column. .............................................................................. 136

List of Figures

xii

Figure 4.7 Electrochromatogram of thiourea and alkylbenzenes using oxidized

hypercrosslinked poly(MST-VBC-DVB) monolithic column. ............ 137

Figure 4.8 Electrochromatograms of flavonoids using oxidized hypercrosslinked

poly (MST-VBC-DVB) monolithic column. ....................................... 139

Figure 4.9 Electrochromatograms of nucleosides and nucleobases using oxidized

hypercrosslinked poly (MST-VBC-DVB) monolithic column. ........... 141

xiii

List of Abbreviations

ACN acetonitrile

AD amperometric detection

AIBN 2,2'-azobisisobutyronitrile

ANDSA 7-amino-1,3-naphthalene disulfonic acid

BMImBF4 1-butyl-3-methylimidazolium tetrafluoroborate

BMImPF6 1-butyl-3-methylimidazolium hexafluorophosphate

BGE background electrolyte

BSA bovine serum albumin

CCD central composite design

CD cyclodextrin

C4D capacitively coupled contactless conductivity detection

CE capillary electrophoresis

CEC capillary electrochromatography

CGE capillary gel electrophoresis

CIEF capillary isoelectric focusing

CITP capillary isotachophoresis

CL chemiluminescence

CMC critical micelle concentration

CTAB cetyltrimethyl ammonium bromide

CZE capillary zone electrophoresis

2-D two-dimensional

DTAC dodecyltrimethylammonium chloride

List of Abbreviations

xiv

DTAF 5-([4,6-dichlorotriazin-2-yl]amino) fluorescein

ECD electrochemical detection

ECL electrochemiluminescence

EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

eHPLC electrokinetic high performance liquid chromatography

EKC electrokinetic chromatography

EMImBF4 1-ethyl-3-methyiimidazolium tetrafluroborate

EOF electroosmotic flow

ESI electrospray ionization

FASI field-amplified sample injection

FASS field-amplified sample stacking

FI flow injection

Glc β-D-glucose

HEC hydroxyethylcellulose

HEPES N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid)

HILI hydrophilic interaction

HPC hydroxypropyl cellulose

HPLC high performance liquid chromatography

i.d. internal diameter

IL ionic liquid

IR infrared

IS internal standard

ITP isotachophoresis

LC liquid chromatography

List of Abbreviations

xv

LIF laser-induced fluorescence

LLE liquid-liquid extraction

LOD limit of detection

LOQ limit of quantification

LVSS large volume sample stacking

MEEKC microemulsion electrokinetic chromatography

MEKC micellar electrokinetic chromatography

MES 2-(N-morpholino)ethanesulfonic acid

MOPSO 3-(N-morpholino)-2-hydroxypropanesulfonic acid

MS mass spectrometry

MSS micelle to solvent stacking

NACE nonaqueous capillary electrophoresis

NBD-F 4-fluoro-7-nitro-2,1,3-benzoxadiazole

NMP 1-naphthyl-3-methyl-5-pyrazolone

OT-CEC open tubular capillary electrochromatography

pCEC pressurized capillary electrochromatography

PEG polyethylene glycol

pI isoelectric point

PLE pressurized liquid extraction

PMP 1-phenyl-3-methyl-5-pyrazolone

poly-L-SUCL polysodium N-undecenoxycarbonyl-L-leucinate

poly(MST-VBC-DVB) poly(4-methylstyrene-co-vinylbenzyl chloride-co-divinylbenzene)

poly(ST-VBC-DVB) poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene)

Rha α-L-rhamnose

List of Abbreviations

xvi

RP reversed-phase

RSD relative standard deviation

SC sodium cholate

SDC sodium deoxycholate

SDS sodium dodecyl sulfate

SEM scanning electron microscopy

SFE supercritical fluid extraction

S/N signal-to-noise ratio

SPE solid-phase extraction

TBAC tetrabutylammonium chloride

TBAP tetrabutylammonium perchlorate

TTAB tetradecyltrimethyl ammonium bromide

TLC thin layer chromatography

tris tris(hydroxymethyl)aminomethane

UHPLC ultra high-performance liquid chromatography

UV ultraviolet

vis visible

Xyl β-D-xylose