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
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