vii
TABLE OF CONTENTS
CHAPTER
1
2
TITLE
PAGE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENTS
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
xi
LIST OF FIGURES
xii
LIST OF ABBREVIATIONS
xvii
LIST OF SYMBOLS
xviii
LIST OF APPENDICES
xix
INTRODUCTION
1.1
Background of Study
1
1.2
Steroid Analysis
4
1.3
Problem Statement
5
1.4
Objectives of Study
7
1.5
Scope of Study
8
1.6
Significance of Study
8
LITERATURE REVIEW
2.1
Steroids
11
2.1.1
Type of Steroids
12
2.1.1.1
Anabolic Steroids
12
2.1.1.2
Androgenic Steroids
13
viii
2.1.1.3
Corticosteroids
14
2.2
Analysis of Steroids
15
2.3
Electrophoresis
17
2.4
Capillary electrophoresis
18
2.4.1
Instrumentation
19
2.4.2
Electroosmotic Flow
21
2.5
2.6
Modes of CE
22
2.5.1
Capillary Zone Electrophoresis
22
2.5.2
Electrokinetic Chromatography
23
2.5.3
Capillary Gel Electrophoresis
24
2.5.4
Capillary Isoelectric Focusing
24
2.5.5
Capillary Isotachophoresis
24
2.5.6
Micellar Electrokinetic Chromatography
25
2.5.6.1
27
Application of MEKC
Improving Detection
28
2.6.1
28
Off-Line Preconcentration in
Capillary Electrophoresis
2.6.2
On-Line Preconcentration in
29
Capillary Electrophoresis
2.7
AFMC
2.7.1
31
Micellar Electrokinetic Dilution and
32
Micelle Collapse
2.7.2
Optimizing Parameters on AFMC
35
2.7.2.1
35
Effect of Sample Injection
Length (Linj)
2.7.2.2
Effect of Conductivity Ratio
35
(BGS/S)
2.7.2.3
Effect of Surfactant
36
Concentration in the S
2.7.2.4
Effect of Organic Solvent
36
Content in the BGS
2.7.3
Critical Micelle Concentration
37
2.7.3.1
Conductometry
38
2.7.3.2
Spectrophotometry
39
ix
2.7.3.3
CE Method in CMC
461
Determination
3
RESEARCH METHODOLOGY
3.1
Introduction
43
3.2
Chemical and Reagents
44
3.3
Conductivity and pH Measurement
45
3.4
Instrumentation
45
3.5
Conditioning the Capillary
46
3.6
Methods for MEKC Separation of Selected Steroids
46
3.7
AFMC-MEKC Separation of Selected Steroids
46
3.8
Determination of CMC
47
3.9
Extraction Procedure
47
3.9.1
Pharmaceutical Tablets
48
3.9.2
Urine Samples
48
Validation Procedure
49
3.10
4
RESULTS AND DISCUSSIONS
4.1
Separation of Steroids by MEKC
50
4.1.1
Optimization Parameters
51
4.1.1.1
Wavelength Detection
52
4.1.1.2
Type of Buffer
53
4.1.1.3
Capillary Length
54
4.1.2
Analytical Performance of the Optimized
55
MEKC Method
4.2
CMC Determination
56
4.3
Separation of Selected Steroids by AFMC-MEKC
57
4.3.1
Optimization Parameters
58
4.3.1.1
Conductivity Ratio
59
4.3.1.2
SDS Concentration
62
Analytical Performance of the Optimized
64
4.3.2
AFMC-MEKC Method
4.4
Separation of Selected Steroids in Urine Matrix
by AFMC-MEKC
65
x
4.5
5
Application of AFMC-MEKC to Samples
68
4.5.1
Pharmaceutical Tablet
68
4.5.2
Urine Sample
70
CONCLUSIONS AND RECOMMENDATIONS
5.1
Conclusions
76
5.2
Recommendations
77
REFERENCES
79
Appendices A-B
87
xi
LIST OF TABLES
TABLE NO.
TITLE
PAGE
4.1
Linearity, repeatability, LODs in optimized
56
MEKC method.
4.2
SEF of AFMC-MEKC at conductivity ratio
61
(BGS/S) of (a) 0.66, (b) 0.51 and (c) 0.34
4.3
SEF
of
AFMC-MEKC
at
SDS
64
concentration of (a) 7 mM, (b) 8 mM and
(c) 10 mM in sample solution.
4.4
Linearity, repeatability, LODs and SEF in
65
optimized AFMC-MEKC method.
4.5
Linearity, repeatability, LODs (S/N = 3)
68
and SEF of the optimized AFMC-MEKC
in urine matrix.
4.6
Percent recovery and repeatability of
prednisolone
and
betamethasone
70
in
pharmaceutical tablet by AFMC-MEKC.
4.7
SPE-AFMC-MEKC application to urine
71
samples spiked with selected steroids.
4.8
Percent recovery and repeatability of all
analytes in urine sample by SPE-AFMCMEKC.
75
xii
LIST OF FIGURES
FIGURE NO.
TITLE
2.1
Steroid Nucleus
2.2
Chemical
PAGE
11
Structures
of
Anabolic
13
Steroids ; (a) testosterone, (b) 4androstene-3,17-dione and (c) 17-αmethyltestosterone
2.3
Chemical Structures of Corticosteroids ;
14
(a) prednisolone , (b) prednisone and (c)
betamethasone
2.4
Typical CE instrumentation
20
2.5
Flow of EOF in (a) Plug Flow, (b)
21
Parabolic Flow
2.6
Schematic Illustration of Principle of
26
MEKC
2.7
Evolution of Zones in Analyte Focusing
31
By Micelle Collapse (AFMC)
2.8
The effective separation length in
33
normal mode (NM)-MEKC
2.9
The effective separation length in
33
AFMC NM-MEKC
2.10
Variation of specific conductivity vs.
surfactant
concentration
for
conductometric determination of the
CMC
38
xiii
2.11
Variation of absorption wavelength vs.
surfactant
concentration
for
spectroscopic measurements
of the
40
CMC
2.12
Variation of electrophoretic mobility of
naphthalene
with
42
surfactant
concentration, for the determination of
CMC of SDS
4.1
Single run of 100 mg/L (1) prednisone,
51
(2) prednisolone, (3) betamethasone, (4)
4-androstene-3,17-dione,
testosterone
and
(5)
(6)
17-α-
methyltestosterone. Conditions: buffer
25 mM sodium borate (pH 9.0),
micellar solution: 20 mM SDS and 10
% v/v methanol, capillary 64.5 cm (56.5
cm to detector), +25 kV, temperature
25ºC,
detection
at
240
nm
and
hydrodynamic injection at 40 mbar for
1 second.
4.2
Optimization of Detection Wavelength
52
4.3
MEKC separation of a mixture of (1)
53
100 mg/L prednisone, (2) 100 mg/L
prednisolone, (3) 100 mg/L
betamethasone, (4) 25 mg/L 4androstene-3,17-dione, (5) 100 mg/L
testosterone and (6) 150 mg/L 17α5methyltestosterone. Conditions:
buffer 25 mM sodium borate (pH 9.0),
micellar solution: 20 mM SDS and 10
% v/v methanol, capillary 64.5 cm (56.5
cm to detector), +25 kV, temperature
25ºC, detection at 240 nm and pressure
injection at 40 mbar for 1 second.
xiv
4.4
Separation of selected steroid using 25
54
mM sodium acetate (pH 9.0), micellar
solution: 20 mM SDS and 10 % v/v
methanol. The other conditions are the
same as in Figure 4.3.
4.5
Separation of selected steroid using 25
55
mM sodium acetate, micellar solution:
20 mM SDS and 10 % v/v methanol
with (a) long-end capillary (Ld = 56 cm,
Lt = 64.5 cm), and (b) short-end
capillary (Ld = 40 cm, Lt = 48.5 cm).
Other conditions are the same as in
Figure 4.3.
4.6
Variation
of
surfactant
conductivity
versus
concentration
for
57
conductometric determination of the
CMC
4.7
AFMC-MEKC separation of a mixture
58
of (1) 100 mg/L prednisone, (2) 100
mg/L prednisolone, (3) 100 mg/L
betamethasone,
(4)
25
mg/L
4-
androstene-3,17-dione, (5) 100 mg/L
testosterone and (6) 150 mg/L 17-αmethyltestosterone. Conditions: BGS:
25 mM sodium acetate (pH 9.0),
micellar solution: 20 mM SDS and 10
% v/v methanol, sample matrix 250 mM
acetate and 7 mM SDS, capillary 48.5
cm (40.0 cm to detector), +25 kV,
temperature 25ºC, detection at 240 nm
and pressure injection at 40 mbar for 1
second.
4.8
Effect of conductivity ratio (BGS/S) on
AFMC-MEKC separation. Conditions:
60
xv
BGS = 25 mM sodium acetate (pH 9.0
with 20 mM SDS and 10 % v/v
methanol, S = 100 mg/L of prednisone
(1), 100 mg/L prednisolone (2), 100
mg/L betamethasone (3), 25 mg/L 4androstene-3,17-dione (4), 100 mg/L
testosterone (5) and 150 mg/L 17-αmethyltestosterone (6) in 7 mM SDS
with 200 mM (a), 225 mM (b), 250 mM
(c) sodium acetate, pH 9. The other
conditions were the same as in Figure
4.7.
4.9
Effect of SDS concentration in sample
63
solution by AFMC-MEKC. Conditions:
BGS = 25 mM sodium acetate (pH 9.0
with 20 mM SDS and 10 % v/v
methanol, S = 100 mg/L of prednisone
(1), 100 mg/L prednisolone (2), 100
mg/L betamethasone (3), 25 mg/L 4androstene-3,17-dione (4), 100 mg/L
testosterone (5) and 150 mg/L 17-αmethyltestosterone (6) in 250 mM
sodium acetate, pH 9 with 7 mM (a), 8
mM (b), 10 mM (c) SDS. Other
conditions were the same as in Figure
4.8.
4.10
AFMC-MEKC separation of a mixture
of (1) 100 mg/L prednisone, (2) 100
mg/L prednisolone, (3) 100 mg/L
betamethasone,
(4)
25
mg/L
4-
androstene-3,17-dione, (5) 150 mg/L
testosterone and (6) 100 mg/L 17-αmethyltestosterone in
urine matrix.
Other conditions were the same as
66
xvi
Figure 4.9a.
4.11
AFMC-MEKC
separation of blank
67
urine. Other conditions were the same
as Figure 4.9a.
4.12
Electropherogram of determination of
69
prednisolone in pharmaceutical tablet.
Other conditions were the same as in
Figure 4.9a.
4.13
Electropherogram of determination of
70
betamethasone in pharmaceutical tablet.
The conditions are the same as in Figure
4.9a.
4.14
Electropherogram of determination of
71
prednisone in spiked urine sample.
Other conditions are the same as in
Figure 4.10.
4.15
Electropherogram of determination of
72
prednisolone in spiked urine sample.
Other conditions are the same as in
Figure 4.10.
4.16
Electropherogram of determination of
73
betamethasone (a) and 4-androstene3,17-dione (b) in spiked urine sample.
Other conditions are the same as in
Figure 3.11.
4.17
Electropherogram of determination of
testosterone
(a)
and
17-α-
methyltestosterone (b) in spiked urine
sample. Other conditions are the same
as in Figure 4.10.
74
xvii
LIST OF ABBREVIATIONS
AFMC
-
Analyte Focusing by Micelle Collapse
BGS
-
Background Solution
CE
-
Capillary Electrophoresis
CGE
-
Capillary Gel Electrophoresis
CIEF
-
Capillary Isoelectric Focusing
CITP
-
Capillary Electrochromatography
CMC
-
Critical Micelle Concentration
CZE
-
Capillary Zone Electrophoresis
EOF
-
Electroosmotic Flow
HPLC
-
High-Performance Liquid Chromatography
LIF
-
Laser Induced Flourescence
LOD
-
Limit of Detection
MEECK
-
Micro-Emulsion Electrokinetic Chromatography
MEKC
-
Micellar Electrokinetic Chromatography
NaOH
-
Sodium Hydroxide
S
-
Neutral Analytes
SC
-
Sodium Cholate
SDS
-
Sodium Dodecyl Sulphate
UV
-
Ultraviolet
xviii
LIST OF SYMBOLS
cm
-
Centimeter
ºC
-
Degree Celcius
μm
-
Micrometer
µA
-
Micro Ampere
µL
-
Micro Litre
nL
-
Nano Litre
i.d
-
Inner Diameter
l
-
Effective Capillary Length
M
-
Molarity
N
-
Efficiency
pI
-
Isoelectric Point
ppm
-
Part Per Million
T
-
Temperature
tm
-
Migration Time
V
-
Volt
ϭ
-
Standard deviation
xix
LIST OF APPENDICES
APPENDIX
TITLE
PAGE
Calibration curve of separation of
87
NO.
A1
prednisone,
prednisolone,
betamethasone
and
MEKC
concentration
with
testosterone
by
range
between 12.5 mg/L and 120 mg/L
A2
Calibration curve of separation of 4-
88
androstene-3,17-dione by MEKC with
concentration range between 3.125
mg/L and 30 mg/L
A3
Calibration curve of separation of 17-α-
88
methyltestosterone by MEKC with
concentration range between 18.75
mg/L and 180 mg/L
A4
Calibration curve of separation of
prednisone,
betamethasone
AFMC-MEKC
89
prednisolone,
and
with
testosterone
by
concentration
range between 12.5 mg/L and 120 mg/L
A5
Calibration curve of separation of 4androstene-3,17-dione
MEKC
with
by
AFMC-
concentration
between 3.125 mg/L and 30 mg/L
range
89
xxi
A6
Calibration curve of separation 17-α-
90
methyltestosterone by AFMC-MEKC
with concentration range between 18.75
mg/L and 180 mg/L
A7
Calibration curve of separation of
prednisone,
90
prednisolone,
betamethasone and testosterone in urine
matrix
by
AFMC-MEKC
with
concentration range between 12.5 mg/L
and 120 mg/L
A8
Calibration curve of separation of 4-
91
androstene-3,17-dione in urine matrix
by AFMC-MEKC with concentration
range between 3.125 mg/L and 30 mg/L
A9
Calibration
curve
of
17-α-
91
methyltestosterone in urine matrix by
AFMC-MEKC
with
concentration
range between 18.75 mg/L and 180
mg/L
B1
List of Publication from This Study
92