LC-MS and CZE of Dianthrones from

Cassia Angustifolia and

Acutifolia
H. S t u p p n e r * / S. S t u r m
Institute of Pharmacognosy, University of Innsbruck, Innrain 52, A-6020 Innsbruck, Austria

Capillary zone electrophoresis (CZE) with multiWavelength detection has been used for the separation
of the main dianthrones from Cassia angustifolia. Optimum separation has been achieved with a fused silica
capillary and 0.1 M borate-sodium hydroxide buffer
(pH 8.4). KC1 and 5 % of isopropanol has been added to
the electrolyte. Applied voltage was 25 kV and capillary
temperature constant at 20 ~ LC separation of senna
dianthrones was on reversed phase (RP 18) material
With an acetonitrile-water solvent gradient containing
0.01% trifluoracetic acid. UV detection was at 205 nm,
column temperature 44 ~ LC-electrospray ionisationMS was used for peak assignment and purity control.
New aloe-emodine dianthrone isomers could also be
identified by this technique. The established HPLC and
CZE methods are suitable for qualitative characterization and quantitative determination of sennosides
in crude senna extracts and phytopharmaceuticals.

side) as well as aloe-emodin dianthrone-diglucosides

are minor ones (Figure 1). Additionally, many other
components have been isolated from the two senna
species, e.g. rhein-anthrone-8-glucoside, rhein-8-diglucoside, aloe-emodin-8-glucoside and two naphthalene derivatives [1]. Senna pods and leaves are
present in numerous laxative preparations and are sold
all over the world. Despite the pharmaceutical importance of this plant only a few papers dealing with
analytical aspects of senna dianthrones have been published. Most of the pharmacopoeias determine the total
senna leaf and pod glycosides in terms of sennoside B.
The procedure involves extraction of the glycosides,
their hydrolysis and oxidation to rhein and aloe-emodin
and finally their spectrophotometric determination
(modified Borntr~iger reaction) [2]. However, the
qualitative results obtained are poorly reproducible
(standard deviations > 10 %). More precise are thinlayer chromatography (TLC) [3, 4], HPLC [5-8] and gas
chromatography (GC) [9] methods, which allow separation and quantitative determination of single constituents. In this paper, CZE and HPLC separations as
well as quantitative determination of sennosides will be
presented. The influence of different parameters on the
separation of dianthrones is discussed and results obtained with both techniques are compared. Additionally,
the potential utility of an HPLC-electrospray combination for the analysis of senna dianthrones will be shown
[10].

Introduction

Experimental

Two species of Cassia (Caesalpiniaceae) are used as

herbal medicines: Cassia senna L. (Alexandrian senna)
and Cassia angustifolia Vahl (Tinnevelly senna). The
plant parts used medicinally are the dry leaves and the
mature pods [1]. Both, leaves and pods contain mainly
dianthrone glycosides (sennosides) as active components: sennoside A, A1 and B (mesomeric forms of
rhein-dianthrone-8,8'diglucoside) are major constituents (> 80 %), sennoside C, D and D1 (mesomeric
forms of rhein-aloe-emodin dianthrone-8,8'-digluco-

Materials

Key Words
Column liquid chromatography-mass spectrometry
Capillary zone electrophoresis
Dianthrones and sennosides
Cassia acutifolia and Cassia angustifolia

Summary

Original
0009-5893/96/06 697-07 $ 03.00/0

Methanol, acetonitrile, trifluoracetic acid, boric acid,

KCI, NaOH, 1-propanol and 2-propanol were from
Merck (Darmstadt, Germany). The references (sennosides A, A1, B, C, D and D1) were from Dr. Grimminger, Fa. Madaus AG, (K61n, Germany). Plant
material of C. acutifolia (pods) and C. angustifolia
(leaves and pods) was from Kottas, Austria (batch no.
KL-3304, KL-6104, KL-2924). Voucher specimens are

Chromatographia Vol.42,No. ll/12,June 1996

9 1996 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

697

deposited at the Institute of Pharmacognosy, University

of Innsbruck (Austria). Pharmaceutical preparations A
and B: Colonorm (A) (Mundipharma (Austria)) and
Bekunis (B) (Roha Arzneimittel (Germany)) were purchased in a Pharmacy.

Sample Preparation (HPLC)

Sennosides A, A1, B, C, D and D1 (1.11 mg each compound) were dissolved in 50.00 mL aqueous methanol
(50 %). Powdered pods and leaves (4.00 g) of C.
acutifolia and. C. angustifolia were extracted three times
with 30 mL 50 % aqueous methanol (stirring at room
temperature for 15 rain). The suspensions were

6-D-GIc-O

B-D-GIc-O
8-D-GIc-O

OH

OH

B-D-GIr

OH

B-D-GIc-O O
~
~

OH

H/" " H " ~

B-D-GIc-O

A, A1, B

For CZE analyses samples prepared for HPLC were

diluted 1:4 with water.

Calibration (HPLC)

Calibration (CZE)

COOH
COOH

Calibration curves were obtained from standard solutions containing the sennosides A and C in concentrations between 1.1-600.0 gg mL -1 (solvent: 15 % aqueous methanol) (Table I).

Analytical Methods

C, C1, D, D1

CZE: Experiments were performed with an HP 30

COOH
cI'LzOH

H/ ~

Sample preparation (CZE)

Calibration curves were obtained from standard solutions containing'the sennosides A and C in concentrations between 5.6-1110.0 lag mL -1 (solvent: 50 % aqueous methanol) (Table I).

OH

~
H/

centrifuged and decanted. The combined extracts were

diluted to 100.0 mL with 50 % aqueous methanol. Four
Colonorm tablets and two Bekunis dragees were
ground up and extracted in the same way as plant
material. Before analysis each solution was filtered
through a 0.45 pm filter (Sartorius, G6ttingen, Germany).

AED1,2,3
-CH2OH

CH2OH

OH

Figure 1
Structures of sennosides A, A1, B, C, C1, D, D1 and aloe emodin
dianthrone-diglucosides(AED1, AED2 and AED3).

Capillary Electrophoresis System (Hewlett Packard)

equipped with a diode array detector, an automatic injector, a temperature controlled column cartridge with a
fused silica capillary (66 cm 50 ~tm I.D.), an autosampler and a printer. Detection was at 205 nm. All experiments were carried out at 20 ~ and at 25 kV. Injections were made using the pressure mode (50 mbar) for
3 s each. Electrolyte solution (0.1 M): 50 mL solution A
(6.18 g boric acid dissolved in 1000 mL 0.1 M KCI),
8.6 mL solution B (0.1 M NaOH) and 5 mL isopropanol
were diluted with water to 100.0 mL (pH 8.4). For those
experiments, where the effect of electrolyte pH o~a
resolution was studied pH values between 7.0-9.2 were
used. Buffer solutions were filtered through 0.45 pm filters (Sartorius). Between runs, the capillary was washed
with 0.1 M NaOH (3 rain) and water (3 min), followed
by equilibration with running buffer (3 min).
HPLC: Experiments were performed with an HP 1090
Series II Liquid Chromatograph equipped with diode
array detector (HP 1040), automatic injector,
autosampler, column oven and printer. Detectiorl

TableI. Regressionequationsand correlationcoefficientsofsennosidesA and C.

Sennoside
A
C

698

Regression
equation
for HPLC
y = 69.5756 + 1.6153x
Y = 153,1341 + 1.6311x

Regression
equation
for CZE
Y = 0.16663x- 0.01604
y = 0,23799x- 0.00988

Chromatographia Vol.42, No. ll/12,June 1996

r2 for
HPLC

r2 for
CZE

0.9961
0.9973

0.9990
0.9992

Original

wavelength was 205 rim. Column: Zorbax SB-C-18, 1504.6, particle size 5 ~tm (Rockland Technology, USA).
Senna dianthrones were separated with a water (A)acetonitrile (B) solvent gradient containing 0 . 0 1 % triflouracetic acid: gradient: 90 % A (1 min), in 1 min to
85 % A, 85 % A (8 rain), in 10 min to 82 % A, in 3 min
to 80 % A, in 2 min to 79 % A, in 6 min to 65 % A and
in 4 rain to 10 % A. The injection was 5/.tL. All experiments were at 44 ~ For those experiments, where the
impact of column temperature on resolution was
studied column temperatures of 25-60 ~ were used.
Between runs, the column was washed with 90 %
acetonitrile-water containing 0.01% trifluoracetic acid,
followed by equilibration (10 min).
Mass spectrometry: Finnigan MAT SSQ 7000 with Digital DEC 3000 data station; electrospray interface 3.1 V,
7.3 mA, negative mode, temperature of heated capillary:
200 ~ sheath gas: 80 psi; CID offset: 0-50 V; the LC
flow (1 mL min-1) split 1:5.

15-

10

I]A1

5-

C,D,D1

X1

L
' 17.8

rain

Figure 2
Electropherogram of aqueous-methanolicextract of pods (B) of
C. angustifolia. Sennoside A, A1, B, C, D and D, rhein (R) and
unknown compounds (X1, X2). Running electrolyte:0.1 M boratesodium hydroxidebuffer (pH 8.4) containing0.1 M KCI and 5 %
isopropanol; column: fused silica 66 cm x 50 gm I.D., injection:
pressure mode, 3 s at 50 mbar; voltage: 25 kV; detection: UV
205 rim; temperature: 20 ~

Results
CZE
An electropherogram of an aqueous methanolic extract
of pods of C. angustifolia is shown in Figure 2. Baseline
separation of the three major sennosides A, A1 and B
(migration times 10.4, 10.5 and 10.7, respectively) could
be achieved using a fused silica capillary with a 0.1 M
borate-sodium hydroxide buffer (pH 8.4) containing
5 % isopropanol. The applied voltage was 25 kV and the
capillary thermostat 20 ~ UV detection was at 205 rim.
Sennosides C, D and D1, the minor sennosides, coeluted
as a single peak at 8.0 min and could not be separated
from each other. The aglycone rhein has a migration
time of 13.6 min. Assignment of the peaks was established by comparison of migration times with those of
authentic references. Separation of the sennosides A, A1
and B was mainly influenced by two parameters: the pH
of the electrolyte solution and the proportion of organic
solvent in the buffer system.
To ensure an adequate degree of dissociation of the sennosides (weak acids), a basic boric acid-NaOH buffer
was chosen. Sennosides are negatively charged and consequently migrate based on the strong electroosmotic
flow to the cathode [11]. To keep the ionic strength constant over a large pH range KCI was added to the buffer
system. The resolutions (Rs) of adjacent substance pairs
at 8 different pH values of the borate-sodium hydroxide
buffer are shown in Figure 3. The compounds X1 and X2
could only be separated from sennosides B and A,
respectively, within a pH range 8.2-8.6. The best resolutions of the sennoside pairs B-A1 and A1-A were obtained using a pH of 8.4 which was therefore selected as
the buffer pH.
The influence of different organic solvents as buffer additives on the separation of the sennoside pair A1-B is
shown in Figure 4. Without addition of organic solvents
Original

6~ 1

,0/

J / /

" ""

4.0

Rs
3.0

2.0

I,~

0,0

7.0

7.4

7-R

8,2

8.4

8.6

8.8

g.2

pH
Figure 3
Effect of pH (7.0-9.2) on resolution (Rs) of pairs A-A1, B-A1,
B-X1 and A-X2. Conditionsas Figure 2.

to the buffer the peaks B and A1 overlap (Rs 0.6). When

adding 5 or 10 % methanol, ethanol, 1-propanol, 2propanol or acetonitrile resolution of the substance pair
increases. The best resolution of A1 and B could be
achieved using electrolytes containing 10 % isopropanol (Rs 1.8). Addition of high concentrations of organic solvents to the electrolyte can decrease
reproducibility due to evaporation effects. Therefore a
lower concentration of 5 % isopropanol in the buffer
was preferred which resulted in sufficient resolution
(> 1.2) of the A1-B pair.

Chromatographia Vol.42, No. 11/12,June 1996

699

2.0-

Nomt

1.8-

40O

1.61.4~

1,2-

300

R s ~.o0.8-

20O

0.~
0.40.2-

1110

0.0-

I III
2OO

Figure 4
Effect of organic solvents on resolution (Rs) of sennoside pair
A1-B. Other conditions as Figure 2.

i j i l l l l
250

l l | l [
3OO

ii

ill

350

ii11
400

i
nm

Figure 5
UV spectra of sennosides A, A1, and C recorded on-line (HPLC)
by DAD detector.

~U
400-

t00

A1C

Figure 6
LC chromatogram of aqueous methanolic extract of leaves of C angustifolia. Column: Zorbax SB-C-18,
150--4.6, 5 ~m; solvent gradient water (A) acetonitrile (B) containing 0.01% triflouracetic acid: 90 % A
(1 min),in 1 min to 85 % A, 85 % A (8 min),in 10 rain to 82 % A, in 3 min to 80 % A, in 2 min to 79 %
A, in 6 rain to 65 % A and in 4 rain to 10 % A.; flow rate: 1 mL min-l; injection volume: 5 ~L; detection:
205 nm; column temperature; 44 ~

HPLC
The H P L C chromatogram of a methanolic extract of the
leaves of C angustifolia is shown in Figure 6. The following peaks could be assigned by means of the retention
time and the U V spectrum (Figure 5) compared with
authentic references: sennosides A, A1, B, C, D and D1.
Baseline separation of these compounds as well as some
other dianthrones could be achieved in less than 25 min
using a reversed phase system (RP 18) and a solvent
gradient of water-acetonitrile containing 0 . 0 1 % trifluoracetic acid - the acid of choice for LC-MS analyses
- which gave better separations than phosphoric acid.
The optimum concentration of trifluoracetic acid

700

( 0 . 0 1 % ) was established empirically. Concentrations of

0.005, 0.025 and 0.05 % resulted in p o o r e r resolution of
the sennosides A and C (Rs < 1). Detection was at
205 nm, column temperature 44 ~ Peak purity checked
by the D A D proved to be always higher than 99.8 %.
The column temperature had a strong influence on the
separation of the crucial substance pairs X-D1, D1-B
and B-D as shown in Figure 7 (X is an unknown compound). Best results could be obtained with column
temperature 44 ~ A b o v e this, resolution of the peak
pair X-D1 decreased, below 40 ~ separation of the
pairs X-D1 and B-D was no longer acceptable.

Chromatographia Vol.42,No. 11/12,June 1996

Original

HPLC-MS

* X-DI
+DI-B
--a-- B-D

3.5

3.0

The primary interest in the LC-MS technique was to ensure the purity of LC peaks and to identify additional
dianthrones, LC was connected to the MS via an
electrospray interface (ESI). Data acquisition was performed in the negative ionisation mode which gave by
far better mass spectra for the sennosides than the positive m o d e . The ion c h r o m a t o g r a m within the range m / z
300-1000 is shown in Figure 8. Mass spectra of some of
the dianthrone peaks are shown in Figure 9. Without
collision induced dissociation (CID) mainly adducts
with trifluoracetic acid [M-H+TFA]- are produced
whereas the molecular ion [M-HI- and fragments e.g.

2.5
2.0

Rs
1.5
1.0

0.5
0,0 ~ ' - ~ /
25

35

45

55

Figure 7
Effect of column temperature on resolution (Rs) of pair X (unknown compound) sennoside D1 and pairs D1-B and B-D.
Temperature: 20-60 ~ Other conditions as Figure 6.

max

30-50
[M/2-H]-,

"C

[M-H-Glc]-,

[M-H-CO2]-

or

[M-H-GIc-

CO2]- are only generated when CID values are set to

kV.
Through the use of the single ion monitoring (SIM)
technique almost all dianthrone peaks could be as-

SM 7

300>1000

I00

E+05

3.614

80
60
40
20
0

SH

ta/z:833,947

100

E+04

5,046

80
60
40
20
....

Jl

j~..t,.

_ A

m/z:f147,961

i00

E+05

2.332

80
60

40
20
0

A~

SM 7

m/z:861,975

I00

4. 189

80
60
40
20
0

'

4:10

8:20

12:30

16:40

'

--'

20:50

'

25:00

Figure 8
ESI analysis of aqueous methanolic extract of leaves of C angustifolia. LC parameters see
Figure 6; ESI parameters: 3.1 V, 73 mA, negative mode, temperature of heated capillary:
200 ~ sheath gas: 80 psi; CID offset: 0 V; flow rate: 200 ktL rain-1.

Original

Chromatographia Vol. 42,No. 11/12,June 1996

701

liO.'P

A (CID=0V)

lO0

A (CID=30V)

II0

I0

u,

OO

'~

4D

20

20.

~lJl ,'/

I~
t,

. . . . . . . . +bo " "

I00

..,

' /
.

/
.

am

| 7D7.1

. . . . . . .

,Ira

.......
14;0.1 1 1 ~ too

C
14' '~

II0

i, ,

"d~ . . . . . . . .

l.I..,,
.......

.....

idoo

*ha . . . . . . .

~?+0

AED2

10

60

40

40

III.I

20 22p.!

20

ILlt

311+.|

4"1+1.S

1++,1.3
15~4.~s!+.2
4"7,.+J,r
+...J.,h
..J,

.......

l~

........

u + . 0 7 p ...?
........

.
a+6

~+2~:2
"'+

471.S

+1~'.I

s;+.I122.'~

I.

++e.= *~ +
i~0

Figure 9

LC-MS of sennosides A (CID offset 0 V and 30 V, respectively), C and AED2. Parameters see Figure 8.

signed. As expected three peaks were obtained in the

ion trace for m/z 861/975 ([M-H]-/[M-H+TFA]-) assignable to the sennosides A, A1 and B (Figure 8). In comparison, the ion trace for m/z 847/961 showed four peaks.
Three of these could be assigned to the sennosides C, D
and D1. The fourth with a retention time of 17:03 min is
obviously sennoside C1, an isomer which, as far as we
know, has not been identified yet. Three peaks in the ion
chromatogram of m/z 833/947 could be identified as
aloe-emodin dianthrone isomers ( A E D 1-3). According
to the literature only one of these has been isolated so
far [12]. As shown in Figure 8, sennoside B and one of
the aloe-emodin dianthrones are coeluting components
which, through selected ion profiles, can be separated
and, if necessary, quantified. In contrast, in the UV
chromatogram a single homogeneous peak with a D A D
matching factor of 1000 appeared.
Quantitative

Analysis

Quantitative information on C. a n g u s t i f o l i a
and
plant material as well as two pharmaceutical
preparations were obtained by using the external standard method. C Z E and LC calibration were performed
with sennosides A and C. The calibration curve obtained
for C Z E was linear in the range 1.1-200/ag m L -1 the
one for H P L C in the range of 5.55-1110 pg m L -1 (Table
I). Detection limits were approximately 0.6 pg m L -1
( C Z E ) and 2.0 ~tg m L -I (HPLC). For quantitation of
rhein dianthrones the calibration curve of sennoside A,
acutifolia

702

for quantitation of rhein-aloe-emodin dianthrones as

well as aloe-emodin dianthrones the one for sennoside
C was applied.
Table II shows the results of quantitation of plant
material as well as phytopharmaceuticals obtained with
C Z E and HPLC. The relative standard deviations for
the quantitative analyses (six experiments) were 0.56.0 % for all compounds studied. In Figure 10 the total
sennoside content of senna plant material is compared
with that obtained with the classical m e t h o d of the
E u r o p e a n pharmacopoeia [2].

Discussion
The H P L C method developed provides baseline separation of almost all sennosides and can be applied for
quantitation of each of the individual anthronoids in
senna plant material as well as pharmaceutical preparations. The data obtained are in agreement with the
literature and comparable with data obtained by CZE.
However, C Z E separates only the major sennosides A,
A1 and B, but leaves the minor ones unresolved. Nevertheless, the total sennoside content determined by C Z E
and H P L C is comparable. In contrast the m e t h o d of the
pharmacopoeia results in values which, depending on
the investigated plant material, can be significantly
lower (Figure 10). This is in agreement with the literature [4]. From the point of view of the sennoside separation H P L C is the more selective technique and there-

Chromatographia Vol. 42, No. 11/12, June 1996

Original

Table il. Ouanlitalivc determination of sennosides.

sennosides

C. angustifolia pods

C. angustifolia leaves

C. acutifolia pods

pharm, preparation A

pharm, preparation B

g 100 g-I

g 100 g-1

g 100 g-I

mg tablet -1

mg tablet -1

HPLC
A
AI
B
C
C1
D
DI
AED2
AED3
)2 C, C1, D, D1
E sennosides

0.65 (2.4)
0.26 (0.3)
1.03 (2.5)*
0.06 (6.0)
0.04 (7.8)
0.08 (2.3)
0.06 (2.0)
2.18

CZE

HPLC

CZE

0.64 (3.5) 1.05(0.8) 0.92 (4.7)

0.26 (3.9) 0,31 (1.1) 0.26 (3.2)
1.06 (3.9) 1.47(0.7)* 1.26 (4.4)
0.37 (0.8)
0.20 (0.7)
0.36 (0.5)
0.33 (0.7)
0.08 (2.0)
0.08 (4.6)
0.23 (2.5)
1.03 (3.8)
2.19
4.25
3.47

HPLC

CZE

HPLC

CZE

HPLC

CZE

1.38 (0.5)
0.39 (0.8)
1.56 (0.6)*
0.16 (1.2)
0.07 (1.2)
0.13 (0.7)
0.12 (0.3)

1.17 (2.0)
0.35 (2.3)
1.57 (2.2)

5.6 (4.0)
1.5 (3.6)
6.2 (3.7)*
0.3 (5.0)
0.3 (2.6)
0.5 (3.2)
0.4 (3.5)

5,2 (2.8)
1.3 (4.5)
6.0 (3.8)

8.0 (1.2)
3,1 (1.6)
13.3 (1.0)*
0.3 (6.7)
0.5 (4.3)
1.0 (2.0)
0.7 (2.6)

6.3 (4.7)
2.4 (3.8)
11.0 (4,7)

0.06 (3.1)

3.87

0.2 (5.0)
0.41 (3.5)
3.50

15.0

1,5 (3.4)
14.0

26.9

2.0 (4.8)
21.7

Data: means of six replicates; relative standard deviations in parentheses.

*sennoside B overlapping with AED 1.

Acknowledgements

4-

3.5

We gratefully a c k n o w l e d g e Dr. W o l f g a n g G r i m m i n g e r
( M a d a u s AG, K61n, G e r m a n y ) for kindly supplying sennosides A, A1, B, C, D a n d D1.

3
2.5

2
1.5

I1
0.5
0

References
C. ang.
(leaves)

C. eng.
(pods)

C. acut.
(pods)

Figure 10
Total sennoside content of C. angustifolia and acutifolia determined
by CZE, HPLC and aecording to European pharmacopoeia
(EUPH).

fore p r e f e r r e d w h e n e v e r y o u n e e d to quantify m i n o r
dianthrones. O n the o t h e r h a n d C Z E analyses are significantly faster. Both, H P L C and C Z E p r o v i d e acceptable analysis times, but d u e to cleaning and equilibration p r o c e d u r e s H P L C analyses take 20 min longer. A d ditionally, C Z E analyses are m u c h cheaper. Therefore, if
attention is f o c u s e d on the m a j o r sennosides and on the
exact total sennoside d e t e r m i n a t i o n C Z E could be a
quite valuable analytical technique.
E l e c t r o s p r a y mass s p e c t r o m e t r y in c o m b i n a t i o n with
H P L C p r o v e d to be an useful tool for solving varied
types of analytical p r o b l e m s (e.g. p e a k purity), confirmed structure identity and p r o v i d e d mass spectral inf o r m a t i o n f r o m previously u n a p p r o a c h a b l e sennosides.

[1] R. H i*nsel, K. Keller, H. Rimpler, G. Schneider, Hagers Handbuch der pharmazeutischen Praxis, 5. Edition, Vol. 4, Springer
Verlag, Berlin, 1992, p. 713.
[2] Fructus
Sennae
angustifoliae,
Gehaltsbestimmung,
Europ~iisches
Arzneibuch, Vol. III, Verlag der
Osterreichischen Staatsdrnckerei, Amtliche Osterreichische
Ausgabe 1990.
[3] H. Miething, W. Boventer, R. Hiinsel, Pharm. Ztg. 131, 747
(1986).
[4] H. Miething, W. Boventer, R. Hansel, Dtsch. Apoth. Ztg. 126,
2158 (1986).
[5] K. Shimura, T. Yamada, K. Kenzo, Y. Mori, T. Ishizu, Mie-ken
Eisei Kenkyusho Nemnpo 34, 87 (1989).
[6] S. Kitanaka, A. Matsuura, M. Takido, H. Shirai, K. Kagei,
Shoyakugaku Zasshi 39, 106 (I985).
[7] V. K. Srivastava, M. L. Maheshwari, S. Mandal, Indian J.
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Received: Mar 18,1996

Accepted: May 8, 1996

Original

Chromatographia Vol. 42, No. 11/12, June 1996

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