Table of Contents
Analysis of Normal and Modified Urinary Nucleosides:
Researching Rapid Detection Methods for Cancer.......................................................................................
1
INTRODUCTION.................................................................................................................................
1
EXPERIMENTAL CONDITIONS......................................................................................................
1
RESULTS AND DISCUSSION............................................................................................................
2
CONCLUSIONS...............................................................................................................................
3
ACKNOWLEDGEMENTS..................................................................................................................
3
REFERENCES ......................................................................................................................................
3
ProteomeLaTM—From Tissues to Targets......................................................................................................
5
Analysis of Basic Components in Crude Extracts of Natural Products for Pharmaceuticals
and Nutriceuticals by CE-MS/MS..................................................................................................................
6
NATURAL PRODUCTS FOR PHARMACEUTICALS
AND NUTRICEUTICALS....................................................................................................................
6
ANALYTICAL PROCEDURE.............................................................................................................
6
METHOD OF HERB EXTRACTION ...............................................................................................
8
SAMPLES ANALYZED ......................................................................................................................
8
RESULTS AND DISCUSSION ...........................................................................................................
8
MDQ Tip: Cleaning the Interface Block and Ejectors .................................................................................
10
REQUIRED SUPPLIES .......................................................................................................................
10
PREPARATION.....................................................................................................................................
10
CLEANING...........................................................................................................................................
10
REINSTALLATION..............................................................................................................................
10
Winner of the 2003 “Putting CE to Work” Award....................................................................................
11
CE-MS IN THE PHARMACEUTICAL, BIOTECHNOLOGICAL,
AND BIOMEDICAL ENVIRONMENT FIFTH BELGIAN
CE-USERS GROUP MEETING...................................................................................................................
12
e 7, Issue 2 • September 20
03
Volum
The worldwide newsletter
for capillar y electrophoresis
Analysis of Normal and Modified Urinary Nucleosides:
Researching Rapid Detection Methods for Cancer
GUO-WANG XU, YU-FANG ZHENG,
PU-DUN ZHANG, JIAN-HUI XIONG,
SHEN LV
NATIONAL CHROMATOGRAPHIC
R&A CENTER, DALIAN INSTITUTE
OF CHEMICAL PHYSICS,
CHINESE ACADEMY OF SCIENCES,
DALIAN, P. R. CHINA
DICP402@MAIL.DLPTT.LN.CN
EXPERIMENTAL CONDITIONS
EQUIPMENT
AND
REAGENTS
All experiments were performed
with a P/ACE™ MDQ Capillary
Electrophoresis system from
Beckman Coulter. This system is
equipped with a UV detector/diode
array detector and a 50 µm i.d. ×
50 cm bare fused-silica capillary
with an effective detection length
INTRODUCTION
odified nucleosides, derived
predominantly from tRNA,
have been detected in
abnormal amounts in the urine of
cancer patients[1-3]. People have been
interested in examining their biomedical significance as potential tumor
markers. Reversed phase high performance liquid chromatography[4-7] and
immunoassays[8-9] are the main analytical methods for these nucleosides.
Capillary electrophoresis (CE) has
gradually been applied in clinical
research due to its high efficiency,
high speed, and small sample size
requirements. In this paper, we
report a capillary electrophoresis
(CE) method for the separation of
thirteen normal and modified nucleosides and apply it to the analysis of
urine from 25 healthy adults and
31 subjects with cancer. A principal
component analysis (PCA) technique
was used to classify healthy adults
from those with cancer.
M
Fourteen nucleoside standards
including the internal standard
3-deazauridine (3-Dzu) were obtained
from Sigma (St. Louis, Mo, USA).
Sodium dodecyl sulfate (SDS) was
obtained from HuaMei biological
engineering company of China.
Affi-Gel* 601 was from Bio-Rad
(Richmond, CA, USA). Ammonium
acetate, methanol, ammonia, sodium
dihydrogenphosphate monohydrate
(NaH2PO4·H2O) and sodium
tetraborate (NaB4O7·10 H2O) were
analytical pure reagents purchased
from China. Urine specimens were
collected from 25 healthy donors
from the authors’ institute (age range,
25-63 years) and from 31 cancer
patients in the first and second affiliated hospital of Dalian Medical
University.
Nucleosides were isolated from
urine with the use of a phenylboronate gel-affinity column before
introduction into the CE system for
analysis[5-7].
CE CONDITIONS
of 40 cm. A 100 × 800 µm aperture
was used for detection. Data
acquisition was achieved with P/ACE
MDQ software version 2.3. A φ 210
pH meter from Beckman Coulter was
employed for the preparation of the
buffer.
The buffer contained 300 mM
SDS, 25 mM NaB4O7 and 50 mM
NaH2PO4. The pH value was adjusted
to 6.9 with 1 N HCl.
The capillary chamber was
thermostatted at 29°C using a
recirculating liquid coolant. The sample was introduced under 0.5 psi for
15 seconds at the anode by positive
pressure. Electrophoresis was carried
out at 7 kV (positive at the inlet end).
Volume 7, Issue 2 • September 2003
STANDARD CURVE
The concentrations of the nucleosides in urine were calculated based
on the calibration curves. The linear
correlation between nucleoside concentrations and peak areas, their corresponding correlation coefficients,
and the detection limits (expressed
as the signal-to-noise ratio of three)
are listed in Table 1.
QUANTITATION
NUCLEOSIDES
Figure 1. Capillary electropherogram of standard nucleosides. Capillary: 52 cm × 50 µm i.d.; Vacuum-injection: 0.5 psi × 15
seconds; buffer: 300 mmol/L SDS-25 mmol/L borate-50 mmol/L phosphate (pH 6.9); temperature: 29°; running voltage: 7.0
kV; UV detection: 254 nm; Peak identification—1: Pseu; 2: U; 3-C; 4: mU 5 I ; 6: m1I; 7: ac4C; 8: G; 9: m1G ; 10: A; 11: 3Dzu; 12: X; 13: m2G; 14: m6A.
Nucleosides were measured by UV
detection at 254 nm with an acquisition data rate of 4 Hz. To ascertain
the best separation and reproducibility, the capillary was regenerated by
flushing with 0.1 M sodium hydroxide for 1 minute, followed by a
1-minute water rinse and another
2-minute buffer rinse after each run.
The levels of the urinary nucleosides were calculated by the calibration curves; then these were transformed into nmol/µmol creatinine.
For the determination of urinary creatinine levels, urine was thawed at
room temperature, diluted eight-fold,
and then introduced into the
capillary. The separation was carried
out with 30 mM phosphate buffer
(pH 6.0) at 20 kV with diode array
detection at 245 nm.
RESULTS AND DISCUSSION
OPTIMIZATION
CONDITIONS
OF
CE
In order to establish the method
for determination of urinary nucleosides, the separation conditions were
optimized by selecting the parameters, including buffer and its
concentration, pH, running voltage,
and the wavelength of detection[12,15].
Thirteen normal and modified nucleosides were separated using a 50-cm
bare fused-silica column (50 µm i.d.,
40 cm effective length) with a
300 mM SDS-25 mM NaB4O7–50 mM
NaH2PO4 buffer (pH 6.9), 7 kV
running voltage, as shown in
Figure 1.
OF
URINARY
The precision of the method
was determined by analyzing a
standard nucleoside sample six
times (Table 2). Using the optimized
method, spontaneous urine samples
from 25 healthy controls and 31 cancer patients were analyzed. Figure 2
shows the typical electropherogram
of urinary nucleosides from a cancer
patient using the conditions optimized. The levels of the thirteen
urinary nucleosides from normal and
cancer subjects and the results of the
t test are listed in Table 3. By selecting p values <0.05 as statistically
significant, it can be seen that the
levels of eleven nucleosides were significantly elevated in the cancer
Table 1. The Linear Correlation Between Relative
Nucleoside Concentrations and Relative Peak Areas
as Well as Their Limits of Detection (LOD)a
No Compound
The linear correlation
R
LOD (µmol/L)
1
Pseu
y = 1.0901x-0.1662
0.9901
64.8
2
U
y = 1.4133x+0.0466
0.9673
12.5
3
C
y = 1.4239x-0.001
0.9978
10.0
4
mU
y = 1.272x+0.0003
0.9985
6.0
5
I
y = 2.0159x-0.008
0.9906
12.0
6
m1I
y = 3.6999x-0.1574
0.9901
8.0
7
ac4C
y = 3.0578x-0.0302
0.9766
7.8
8
G
y = 2.3277x+0.0277
0.9871
5.4
9
m1G
y = 2.8663x+0.0042
0.9832
6.8
10
A
y = 3.0763x+0.0087
0.9970
7.0
11
X
y = 1.5911x+0.0151
0.9786
14.6
12
m2G
y = 4.6331x-0.0238
0.9837
8.4
13
m6A
y = 4.7735x-0.0412
0.9920
15.2
a
y: peak area relative to that of the internal standard, x: individual nucleoside
concentration relative to that of the internal standard.
2
Table 2. Reproducibility of Migration Times and Peak
Areas for a Standard Solution of 13 Nucleosidesa
Compound
Mean time (min)
RSD%
Mean area
RSD%
Pseu
U
C
mU
I
m1I
ac4C
G
m1G
A
X
m2G
m6A
17.86
19.51
20.61
21.08
22.06
22.89
23.90
24.44
25.23
27.22
29.53
34.58
39.54
0.4
0.5
0.6
0.6
0.6
0.7
0.9
0.8
0.8
0.9
0.6
0.8
1.0
442350
14489
15772
14077
35042
37494
28741
15637
32416
27885
20946
35991
30972
2.4
2.0
3.6
4.6
3.0
3.8
3.4
4.1
4.0
3.6
3.5
4.2
4.0
The results are mean values from six repeated analyses of the standard sample.
a
patients (except for m6A which had
a concentration that was too low).
Figure 3 shows this situation clearly.
For an individual urine sample,
even in the same kind of cancer, the
increase in each nucleoside concentration is different. In this study, we
used the PCA technique[5-7,13] to classi-
fy cancer and non-cancer. This
approach gave us a single value
representing the summation of all
thirteen nucleosides determined for
one person. By using thirteen nucleosides as data vectors, 84% of cancer
patients were distinguishable from
healthy controls (Figure 4).
Figure 2. Capillary electropherogram of nucleosides in extracted urine. Peak identification—1: Pseu; 3: U; 4: C; 5: mU; 8: I;
10: m1I; 11: ac4c; 12: G; 13: m1G; 16: A; 18: 3-Dzu; 19: X; 23: m2G; 25: m6A.
3
CONCLUSION
Our research, although preliminary, shows the potential for combining urinary nucleoside analysis by
MECC with principle component
analysis to differentiate healthy
subjects from those with cancer.
The potential of this approach as a
diagnostic will be explored further in
our laboratory. Further information
can be found from references 5-7
and 10-18.
ACKNOWLEDGEMENTS
The authors would like to thank
the National Natural Science Foundation of China (No.29775024), the
Knowledge Innovation Program of
the Chinese Academy of Sciences,
and the Foundation of Dalian City for
financial support. We especially
thank the students and faculty volunteers from the Dalian Institute of
Chemical Physics for the normal
urine samples.
REFERENCES
1. Itoh, K., Konno, T., Sasaki, T.,
Ishiwata, S., Ishida, N., Misugaki, M.
Clin. Chim. Acta., 206, 181-189
(1992).
2. Speer, J., Gehrke, C. W., Kwo, K. C.,
et al. Cancer, 44, 2120-2123
(1979).
3. Fischbein, A., Sharma, O. K.,
Selikoff, I. J., Borek, E. Cancer
Res., 43, 2971-2974 (1983).
4. Nakano, K., Nakao, T., Schram, K. H.,
Hammargren, W. M., McClure, T. D.,
Katz, M., Petersen, E. Clin. Chim.
Acta, 218, 169-183 (1993).
5. Xu, G., Stefano, C. D., Liebich, H. M.,
Zhang, Y., Lu, P., J. Chromatogr. B,
732, 307-313 (1999).
6. Xu, G., Enderle, H., Liebich, H.,
Lu, P. Chromatographia, 52, 152158 (2000).
7. Xu, G.W., Lu, X., Zhang, Y. K., Lu,
P. C., Stefano, C. D., Lehmann, R.,
Liebich, H. M. Chinese J.
Chromatogr., 17, 97-101 (1999).
Volume 7, Issue 2 • September 2003
Figure 3. Mean excretion of normal and modified nucleosides in urine from normal subjects and cancer patients.
Figure 4. Principal component analysis (PCA) based on thirteen nucleosides from healthy controls (+) and subjects with cancer (x).
Table 3. Average Nucleoside Levels Excreted in Urine by
Both Normal and Cancer Subjects
Pseu
U
C
mU
I
m1I
ac4C
G
m1G
A
X
m2G
m6A
Normal Subjects
mean ± sd
(nmol/µmol creatinine)
17.55 ± 4.65
0.32 ± 0.15
0.29 ± 0.20
0.31 ± 0.19
0.34 ± 0.20
0.10 ± 0.27
0.49 ± 0.14
0.11 ± 0.07
0.81 ± 0.17
0.42 ± 0.13
0.83 ± 0.42
0.41 ± 0.16
0.05 ± 0.08
Subjects with Cancer
mean ± sd
(nmol/µmol creatinine)
43.02 ± 20.79
0.74 ± 0.37
0.69 ± 0.53
0.39 ± 0.36
0.64 ± 0.38
2.49 ± 1.35
1.31 ± 0.69
0.22 ± 0.21
1.87 ± 1.04
1.01 ± 0.59
1.33 ± 0.67
1.36 ± 0.84
0.14 ± 0.26
4
P
?0.001
?0.001
?0.001
?0.05
?0.001
?0.001
?0.001
?0.05
?0.001
?0.001
?0.05
?0.001
?0.10
8. Masnda, M., Nishihira, T., Itah, K.,
et al. Cancer, 72, 3571-3578
(1993).
9. Reynaud, C., Bruno, C., Boullanger,
P., et al., Cancer Lett., 61, 255-262
(1992).
10. Xu, G., Liebich, H., Lehmann, R.
Methods of Molecular Biology,
Volume 162—Capillary
Electrophoresis of Nucleic Acids,
Volume 1: Introduction to the
Capillary Electrophoresis of
Nucleic Acids. Keith Mitchelson
and Jing Cheng, Eds. Totowa, New
Jersey: Humana Press, 2000, 459.
11. Zhao , R., Xu, G., Yue, B., Liebich,
H. M., Zhang, Y. J. Chromatogr. A.,
828, 489 (1998).
12. Liebich, H. M., Xu, G., Di Stefano, C.,
Lehmann, R. Chromatographia,
V45, 396-401 (1997).
13. Xu, G., Schmid, H. R., Liebich, H. M.,
Zhang, Y., Lu, P. Biomed.
Chromatogr. 14, 1 (2000).
14. Liebich, H. M., Xu, G., Di Stefano,
C., Lehmann R. J. Chromatogr. A.,
793, 341 (1988).
15. Liebich, H. M., Lehmann, R., Xu, G.,
Wahl, H. G., Haring, H.-U. J.
Chromatogr. B., 745, 189 (2000).
16. Xu, G., Liebich, H. Am. Clin. Lab.,
20, 22 (2001).
17. Xu, G., Xin, L. , Zhueng , Y., Liu, D.,
Zhang, Yun, Zhang, Yukui, Lu, P.,
Liebich , H. M. Pittcon 2001,
New Orleans, LA, USA. March 4-8,
No.1219.
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decades. We are the only
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testing continuum from
research through
diagnosis. Building upon
key Beckman Coulter technologies
including automation, capillary
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Volume 7, Issue 2 • September 2003
Analysis of Basic Components in Crude Extracts
of Natural Products for Pharmaceuticals and
Nutriceuticals by CE-MS/MS
analytical processes are the major
causes of the quality-related
problems found in the herbal
medicine market today.
atural products of botanical
origin (herbal products) have
played a major role in health
care in human history. They provide
a major source for many drugs with
well-defined structures, and they continue to be one of the major
resources for the modern
pharmaceutical industry.
N
Nutriceuticals are food or food
ingredients considered to provide
medical or health benefits, including
the prevention of diseases. Examples
have included:
• Quinine from Cinchona bark
(1820)
• Salicylic acid from Willow bark
(1860)
• Ephedrine from Ma Huang (1920)
• Taxols from Pacific Yew tree
(1971)
• Ginseng as tonic (101 B.C.)
However, the use of herb-based
natural products for pharmaceuticals
and nutriceuticals poses many analytical challenges since the sample
matrix is typically very complex, and
the active components are usually
not well defined and may be present
in trace amounts.
1) Analysis was performed on a CE
system (P/ACE™ MDQ, Beckman
Coulter, Inc.) with a 75 µm ×
80 cm fused-silica capillary. The
outlet of the capillary is integrated
into the ESI spray needle that is
OH
OH
R
OH
H
N
H
N
S
CH3
S
R
CH3
CH3
S
CH3
1R, 2S: l-N-methyl-ephedrine (-)
Naturally occurring species, < 5%
1S, 2S: d-pseudoephedrin (+)
Decongestant, naturally occurring
species, 10 to 25%
1R, 2S: l-ephedrine (-)
Bronchodilator, naturally occurring
species, 75 to 85%
CH3
N
CH3
S
CH3
Figure 1. Ma Huang structures.
50 µm × 40 cm; 0.5 psi/5 sec injection; 5% HS-b-CD in 25 mM phosphate, pH 2.5, 15 kV, 130 µA
PDA: detector from 190 nm to 450 nm (200 nm as monitored)
0.20
0.012
0.18
0.14
0.12
0.010
1: l-ephedrine (-)
2: d-pseudoephedrine (+)
3: methyl-l-ephedrine (-)
4: ?
0.16
PTS
0.008
0.006
0.10
0.08
0.004
0.06
0.04
0.002
1
0.02
2
3
0.00
0
The lack of a well-defined active
ingredient as well as simple and
reliable sample preparation and
ANALYTICAL PROCEDURE
AU
NATURAL PRODUCTS FOR
PHARMACEUTICALS AND
NUTRICEUTICALS
In this paper, we propose the use
of CE-MS/MS methodologies to
manage the characteristics and analysis of natural product extracts.
Traditionally, most herbal
medicines are “brewed” as aqueous
extracts. These hydrophilic species
are ideally suited for capillary
electrophoresis analysis. Yet, ironically, most scientific publications use an
extraction procedure that favors the
isolation of neutral and hydrophobic
species where the active component
is most likely not to be found. Once
again, this is because, in most herbal
A200 nm
FU-TAI A. CHEN
preparation, the “active” is extracted
in water and the remaining elements
are usually discarded.
1
2
3
4
5
6
0.000
4
7
8
9
Minutes
Figure 2. Chiral Separation of Ephedrine in Crude Extract of Ma Huang.
6
10
11
12
13
14
15
CE: 75 µm × 80 cm; 0.5 psi/10 sec injection; 50 mM NH 4OAc in water/methanol (75/25), pH 4.0, 30 kV, 43 µA
LCQ: ESI/Sheath gas: 10, spray voltage: 4.5 kV
RT: 2.00 - 33.50
6.19
100
95
NL: 6.10E6
Base Peak m/z=
100.0-1000.0 F: +
c NSI Full ms [
100.00-2000.00]
MS m2x
8 major components
90
85
332.9
100
m2x#303 RT: 6.13 AV: 1 NL: 2.85E6 F:
+ c NSI Full ms [ 100.00-2000.00]
626.7
234.9
628.7
822.4
50
0
62
269.1
m2x#347 RT: 7.37 AV: 1 NL: 1.78E6 F:
+ c NSI Full ms [ 100.00-2000.00]
515.0
596.9
1
678.9
924.7
80
0
30
75
Relative Abundance
Relative Abundance
60
55
2
50
5'
7.22
45
0
54
733.9
1095.2
m2x#442 RT: 9.92 AV: 1 NL: 1.54E6 F:
+ c NSI Full ms [ 100.00-2000.00]
637.3
801.6
m2x#630 RT: 15.60 AV: 1 SB: 73
12.80-15.13 NL: 8.20E5 F: + c NSI Full
ms [ 100.00-2000.00]
565.3
529.2
595.3
929.0
0
26
35
9.84
30
4
494.3
360.2
971.4
615.4
0
17
3
7
15
511.5
25
m2x#977-1022 RT: 25.52-26.66 AV: 16
SB: 83 27.96-30.79 NL: 2.78E6 F: + c
NSI Full ms [ 100.00-2000.00]
647.4
861.3
435.7
0
15
20
Time (min)
925.3
823.2
50
0
10
m2x#944 RT: 24.67 AV: 1 SB: 15
23.53-24.32 NL: 4.72E5 F: + c NSI Full
ms [ 100.00-2000.00]
453.6
8
5
705.4
451.5
348.8
0
97
10
1023.6
903.3
10
22.79
5
m2x#876 RT: 22.79 AV: 1 SB: 53
19.97-21.89 NL: 7.31E5 F: + c NSI Full
ms [ 100.00-2000.00]
1007.4
10
15.68
1119.0
985.2
6
25
20
1054.9
257.3
l-ephedrine
40
510.0
166.1
180.2
0
29
=82
m2x#395 RT: 8.70 AV: 1 SB: 27
7.55-8.97 NL: 8.49E5 F: + c NSI Full ms
[ 100.00-2000.00]
275.0
26.58
1088.5
261.0
70
65
=98
920.3
30
1186.9
500
1000
m/z
Figure 3. CE-MS of Crude Extract of Ma Huang Capsule.
CE: 75 µm × 80 cm; 0.5 psi/10 sec injection; 50 mM NH4OAc in water/methanol (75/25), pH 4.0, 30 kV, 43 µA
LCQ: ESI/Sheath gas: 10, spray voltage: 4.5 kV
100
SM: 3B
6.19
SIM
NL: 3.33E6
Base Peak m/z= 332.5-333.5
F: + c NSI Full ms [
100.00-2000.00] MS m2x
50
0
100
7.22
15.68
8.70
10.86
Relative Abundance
0
100
9.84
50
15.60
0
100
22.70
24.35
7.51
9.70
15.45
23.86
0
100
26.20
50
m2x#397 RT: 8.75 AV: 1 NL:
7.85E4 F: + c d Full ms2
260.96@35.00 [ 60.00-535.00]
8
20
0
6
m2x#441 RT: 9.90 AV: 1 NL:
2.89E6 F: + c d Full ms2
166.08@35.00 [ 35.00-180.00]
148.1
239.0
m2x#632 RT: 15.66 AV: 1 NL:
4.18E5 F: + c d Full ms2
257.25@35.00 [ 60.00-270.00]
985.6
0.02
645.7
741.6
466.6
0.00
5
0
0.12
949.6
m2x#937 RT: 24.48 AV: 1 NL:
3.46E5 F: + c d Full ms2
903.19@35.00 [ 235.00-915.00]
727.2
453.1
m2x#1000 RT: 26.10 AV: 1 NL:
8.07E3 F: + c d Full ms2
823.29@35.00 [ 215.00-835.00]
647.0
824.0
0.05
0
0.00
5
10
15
20
Time (min)
25
m2x#866-896 RT: 22.52-23.32 AV: 9
NL: 2.11E3 F: + c d Full ms2
985.23@35.00 [ 260.00-1000.00]
2
NL: 4.04E6
Base Peak m/z= 822.7-823.7
F: + c NSI Full ms [
100.00-2000.00] MS m2x
26.27
82
260.6
242.1
NL: 4.48E5
Base Peak m/z= 902.8-903.8
F: + c NSI Full ms [
100.00-2000.00] MS m2x
24.67
50
0.0
44
24.90
15.60
0
100
m2x#355 RT: 7.57 AV: 1 NL:
6.81E5 F: + c d Full ms2
269.03@35.00 [ 60.00-550.00]
0
0.03
NL: 6.56E5
Base Peak m/z= 984.7-985.7
F: + c NSI Full ms [
100.00-2000.00] MS m2x
50
186.8
0.5
NL: 9.27E5
Base Peak m/z= 256.8-257.8
F: + c NSI Full ms [
100.00-2000.00] MS m2x
50
3.55
0
1.2
m2x#305 RT: 6.18 AV: 1 NL:
6.50E6 F: + c d Full ms2
332.88@35.00 [ 80.00-345.00]
98
5
NL: 1.67E6
Base Peak m/z= 165.5-166.5
F: + c NSI Full ms [
100.00-2000.00] MS m2x
l-ephedrine
0
100
0
10
NL: 9.05E5
Base Peak m/z= 260.5-261.5
F: + c NSI Full ms [
100.00-2000.00] MS m2x
50
234.7
50
NL: 2.35E6
Base Peak m/z= 268.5-269.5
F: + c NSI Full ms [
100.00-2000.00] MS m2x
50
0
100
100
Relative Abundance
RT: 1.00 - 32.00
30
500
m/z
Figure 4. CE-MS of the Eight Major Components in Ma Huang Capsule.
7
1000
Volume 7, Issue 2 • September 2003
CE: 75 µm × 80 cm; 0.5 psi/10 sec injection; 50 mM NH4OAc in water/methanol (75/25), pH 4.0, 30 kV, 29 µA
LCQ: ESI/Sheath gas: 10, spray voltage: 4.5 kV
RT: 2.00
2.00 -- 31.00
31.00
RT:
NL: 3.25E7
10.80
100
626.6
332.8
100
5.82min
528.7
95
50
234.9
334.8
90
0
20
85
gs2#316 RT: 5.82 AV: 1 NL:
7.01E6 F: + c NSI Full ms [
100.00 -2000.00]
724.4
822.4
1018.2
175.2
9.41min
80
gs2#451 RT: 9.41 AV: 1 NL:
1.43E6 F: + c NSI Full ms [
100.00 -2000.00]
10
75
349.3
545.3
Relative Abundance
70
65
0
33
60
20
55
10
50
0
26
697.4
871.7
337.5
10.42min
359.4
194.2
695.3
499.5
10.80min
45
40
10
25
521.4
0
96
30
Absent in N. American
ginseng
5.82
20
805.3
11.74min
50
0
15
666.4
827.3
360.0
66
13.59min
10.42
10
40
9.41
5
20
0
gs2#553 RT: 11.74 AV: 1
NL: 6.69E6 F: + c NSI Full ms
[ 100.00 -2000.00]
806.3
499.7
13.53
gs2#499 RT: 10.62 AV: 1
SB: 50 14.11 -15.66 NL:
1.85E6 F: + c NSI Full ms [
100.00 -2000.00]
337.5
194.2
35
gs2#493 RT: 10.48 AV: 1
NL: 2.34E6 F: + c NSI Full ms
[ 100.00 -2000.00]
gs2#628 RT: 13.59 AV: 1
NL: 4.61E6 F: + c NSI Full ms
[ 100.00 -2000.00]
701.9
325.0
365.2
706.9
1043.7
0
5
10
15
20
25
30
500
1000
m/z
Time (min)
Figure 5. CE-MS of Crude Extract of Asian Ginseng.
coupled to an MS/MS system
(LCQ* Advantage, Thermofinnigan, Inc.).
2) The system is controlled by
Xcalibur* software (Thermofinnigan, Inc.) that integrates the
CE and MS/MS systems with a single-point software control.
3) Buffer for CE analysis: 50 mM
NH4OAc in 75:25 water:methanol,
pH 4.0. Sheath liquid for ESI:
5 mL/min of 1% HOAc in 80:20
methanol:H2O. Applied potential
in CE : 25.5 kV/39-43 mA. ESI
potential: 4.5 kV.
METHOD OF HERB
EXTRACTION
100 mg of each pulverized sample
was dispersed in 1 mL water in a
capped 2-mL vial and heated at
100ºC/1 hr. The aqueous extract was
filtered and introduced directly to
the CE system for analysis. (Circa
101 B.C., “The herbal classic of the
divine plowman” or “Shen Nong Ben
Cao Chien” written about 101 B.C.)
SAMPLES ANALYZED
MA HUANG
An antiasthmatic herb that contains
the active ingredient ephedrine at
about 1.0 to 1.5% of dry weight.
GINSENG
Recognized as the most popular
medicinal herb used in traditional oriental medicine. The tonic essences
are extracted in hot water.
Two major species: Panax
ginseng (Asian version) and Panax
quiquefolius (North American
version) were used to compare the
distribution of compounds in water
extracts.
RESULTS AND DISCUSSION
Aqueous extract of herbal
product provides water-soluble compounds. Among them, the basic components in the crude extract are the
most abundant species that can be
readily analyzed in an open-tube capillary electrophoresis system.
8
The active ingredient in Ma
Huang extract is characterized as
ephedrine and its isomers by CE-UV
using chiral analysis, while the
presence of ephedrine is identified
by CE-MS/MS analysis.
Major basic components in
Ginseng from two species were characterized and some of the basic components were identified. Ginsengosides are neutral species in ginseng
extracts and were not detected by
the present CE-MS/MS procedure.
The present system combines CE
and MS/MS systems with a singlepoint software control that provides
an easy-to-use, robust solution to
applications that require high-resolution separation, confirmatory characterization, and quality control.
CE: 75 µm × 80 cm; 0.5 psi/10 sec injection; 50 mM NH4OAc in water/methanol (75/25), pH 4.0, 30 kV, 39 µA
LCQ: ESI/Sheath gas: 10, spray voltage: 4.5 kV
RT: 2.00 - 34.00
5.82
100
Base Peak m/z=
332.5 -333.5 F: + c NSI
Full ms [
100.00 -2000.00] MS gs2
50
9.41
Relative Abundance
0
100
8.04
10.75
10.42
NL: 2.94E6
Base Peak m/z=
336.9 -337.9 F: + c NSI
Full ms [
100.00 -2000.00] MS gs2
50
10.92
0
100
10.80
NL: 3.25E7
Base Peak m/z=
498.9 -499.9 F: + c NSI
Full ms [
100.00 -2000.00] MS gs2
50
0
100
11.74
NL: 6.69E6
Base Peak m/z=
804.5 -805.5 F: + c NSI
Full ms [
100.00 -2000.00] MS gs2
50
13.40
0
100
0
3
NL: 1.43E6
Base Peak m/z=
174.5 -175.5 F: + c NSI
Full ms [
100.00 -2000.00] MS gs2
50
5.82
50
13.53
NL: 5.42E6
Base Peak m/z=
359.5 -360.5 F: + c NSI
Full ms [
100.00 -2000.00] MS gs2
50
gs2#315 RT: 5.80 AV: 1 NL:
6.64E6 F: + c d Full ms2
332.92@35.00 [ 80.00 -345.00]
82
136.8
158.0
gs2#450 RT: 9.39 AV: 1 SB: 4
8.78 -9.86 NL: 2.10E5 F: + c d
Full ms2 175.28@35.00 [
35.00 -190.00]
1
Relative Abundance
0
100
234.7
85
NL: 6.74E6
0
6
4
319.0
257.1
175.1
gs2#492 RT: 10.47 AV: 1 SB: 4
10.15 -11.04 NL: 4.57E5 F: + c d
Full ms2 337.46@35.00 [
80.00 -350.00]
2
320.1
0
80
50
481.1
- glucose
239.1
gs2#513 -534 RT: 10.91 -11.33
AV: 7 NL: 6.26E6 F: + c d Full
ms2 499.49@35.00 [
125.00 -510.00]
419.1
337.4
483.2
0
48
787.2
gs2#555 RT: 11.79 AV: 1 NL:
3.75E6 F: + c d Full ms2
805.34@35.00 [ 210.00 -820.00]
20
769.1
256.0
0
100
463.1
400.2
625.1
324.8
gs2#633 RT: 13.70 AV: 1 NL:
7.86E6 F: + c d Full ms2
360.27@35.00 [ 85.00 -375.00]
50
162.8
0
342.0
0
5
10
15
20
25
30
200
400
Time (min)
600
800
m/z
Figure 6. CE-MS/ of Crude Extract of Asian Ginseng.
OH
OH O
OH O
HO
HO
O
OH
OH
HO
O
HO
CH
H
Arginyl-frucosylglucose
: 498
HO
NH
OH
O
C
H2
C
H2
C
H2
C
H
N
C
NH
OH
NH
N H2
CH
Arginyl-frucosyl : 336
O
C
OH
Figure 7. Structures of Ginseng.
9
H2
C
H2
C
H2
C
H
N
C
NH
N H2
Volume 7, Issue 2 • September 2003
MDQ Tip: Cleaning the Interface Block and Ejectors
REQUIRED SUPPLIES
Before you begin, you will need:
• 2 mL vial with cap
• Tissue towelettes (such as
Kimwipes*)
• Mirror
• Pen light
• Cotton swabs
• Distilled and deionized water
Interface
Block
PREPARATION
1. Lift the cartridge cover door.
2. Loosen the two thumbscrews and
lift the insertion bar.
3. Remove the capillary cartridge
from the interface block.
4. Remove the ejector covers and
ejectors for cleaning as shown in
Figure 1.
CLEANING
1. Using cotton swabs, clean
interface block, electrodes and
ejector surfaces with water
followed by methanol, then allow
to dry.
2. Refer to Figure 2. Wet a towelette
with DI water. Place the towelette
over the top of the capped 2 mL
vial. Raise the vial over the
electrode and up to the interface
block. Rotate the vial so the wet
towelette can clean the grooves
on the underside of the interface
block (A). Remove the vial and
inspect the interface block using
a mirror and pen light (B). Repeat
this process until the interface
block is clean.
Ejector/
Electrode
Locations
Ejector Covers
and Spring
Retainers
Figure 1. Interface Block and Ejectors
REINSTALLATION
1. Reinstall the ejector in front of
the electrode.
2. Reinstall the spring retainer and
ejector cover.
3. Reinstall the capillary cartridge in
the interface block.
4. Lower the insertion bar and tighten the two thumbscrews.
5. Close the cartridge cover door.
B
A
Figure 2. Cleaning Interface Block
10
Winner of the 2003 “Putting CE to Work” Award
ur congratulations go to
Laura Bindila from the
Institute for Medical Physics
and Biophysics, University, of
Münster, Germany, for her winning
electropherogram titled “Characteri-
O
zation of peptides by capillary zone
electrophoresis and electrospray
ionization quadrupole time-of-flight
tandem mass spectrometry.”
The electropherogram was voted
upon by those scientists attending
11
the “CE in the Biotechnology and
Pharmaceutical Industries 5th
Symposium on the Practical Applications for the Analysis of Proteins,
Nucleotides, and Small Molecules,”
August 23–25, San Francisco, CA.
Volume 7, Issue 2 • September 2003
CE-MS
PHARMACEUTICAL, BIOTECHNOLOGICAL, AND
BIOMEDICAL ENVIRONMENT
BELGIAN CE-USERS GROUP MEETING
IN THE
FIFTH
JANSSEN PHARMACEUTICA N.V., BEERSE – BELGIUM
OCTOBER 16, 2003
he Belgian CE-Users group
within the Royal Flemish
Chemical Society (KVCV) is
organizing the "Fifth Belgian CEUsers group meeting; CE-MS in the
pharmaceutical and bio-technological
and -medical environment " on October 16th, 2003, at Janssen
Pharmaceutica n.v., Beerse - Belgium.
T
The meeting is a joint initiative of
industrial partners together with
academia, the Royal Flemish
Chemical Society, and the equipment
vendors, highlighting the practical
impact.It is aimed to discuss the
status and the usefulness of CE-MS
through presentations of interesting
applications in the pharmaceutical,
biotechnological, and biomedical
environment (special focus on
proteomics).
The presentations will be of high
scientific level and provided by
renowned world-class experts in the
field. The use of CE-MS will always be
the key topic of the day in the
presentations, however other related
techniques may be discussed for
comparison and demonstration of
suitability.
Please register through our
website at:
http:/www.kvcv.be/analytische.htm#
CEMS
or contact:
Ilias Jimidar, Ph. D.
Johnson & Johnson Pharmaceutical
Research and Development
Beerse, Belgium
Tel: 32(0)14-603387
email: ijimidar@prdbe.jnj.com
* Affi-Gel is a registered trademark of Bio-Rad Laboratories. LCQ and Xcalibur are trademarks of ThermoFinnigan Corporation.
Kimwipe is a registered trademark of Kimberly-Clark. All other trademarks are the property of their respective owners.
Developing innovative solutions in genetic
analysis, drug discovery, and instrument systems.
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