LC-MS and CZE of Dianthrones from Cassia Angustifolia and Acutifolia

H. S tuppner* / S. S turm

Institute of Pharmacognosy, University of Innsbruck, Innrain 52, A-6020 Innsbruck, Austria

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

Summary Capillary zone electrophoresis (CZE) with multi- Wavelength detection has been used for the separation of the main dianthrones from Cassia angustifolia. Op- timum 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 ionisation- MS 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 charac- terization 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-di- glucoside, aloe-emodin-8-glucoside and two naph- thalene derivatives [1]. Senna pods and leaves are present in numerous laxative preparations and are sold all over the world. Despite the pharmaceutical impor- tance of this plant only a few papers dealing with analytical aspects of senna dianthrones have been pub- lished. 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 thin- layer chromatography (TLC) [3, 4], HPLC [5-8] and gas chromatography (GC) [9] methods, which allow separa- tion and quantitative determination of single con- stituents. 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 ob- tained with both techniques are compared. Additionally, the potential utility of an HPLC-electrospray combina- tion 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 com- ponents: sennoside A, A1 and B (mesomeric forms of rhein-dianthrone-8,8'diglucoside) are major con- stituents (> 80 %), sennoside C, D and D1 (mesomeric forms of rhein-aloe-emodin dianthrone-8,8'-digluco-

Materials

Methanol, acetonitrile, trifluoracetic acid, boric acid, KCI, NaOH, 1-propanol and 2-propanol were from Merck (Darmstadt, Germany). The references (sen- nosides A, A1, B, C, D and D1) were from Dr. Grim- minger, 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

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0009-5893/96/06 697-07 $ 03.00/0 �9 1996 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

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 pur- chased in a Pharmacy.

Sample Preparation (HPLC)

Sennosides A, A1, B, C, D and D1 (1.11 mg each com- pound) 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

H /

B-D-GIc-O

o OH ~ A, A1, B

COOH ~ COOH

O OH

8-D-GIc-O O OH ~ ~ C, C1, D, D1

COOH H / ~ cI'LzOH

B-D-GIr 0 OH B-D-GIc-O O OH

~ ~ AED1,2,3

H/" " H "~ -CH2OH ~ CH2OH

B-D-GIc-O O OH

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

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, Ger- many).

Sample preparation (CZE)

For CZE analyses samples prepared for HPLC were diluted 1:4 with water.

Calibration (HPLC)

Calibration curves were obtained from standard solu- tions containing'the sennosides A and C in concentra- tions between 5.6-1110.0 lag mL -1 (solvent: 50 % aque- ous methanol) (Table I).

Calibration (CZE)

Calibration curves were obtained from standard solu- tions containing the sennosides A and C in concentra- tions between 1.1-600.0 gg mL -1 (solvent: 15 % aque- ous methanol) (Table I).

Analytical Methods

CZE: Experiments were performed with an HP 30 Capillary Electrophoresis System (Hewlett Packard) equipped with a diode array detector, an automatic in- jector, a temperature controlled column cartridge with a fused silica capillary (66 cm • 50 ~tm I.D.), an auto- sampler and a printer. Detection was at 205 nm. All ex- periments were carried out at 20 ~ and at 25 kV. Injec- tions 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 fil- ters (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

Table I. Regression equations and correlation coefficients of sennosides A and C.

Sennoside Regression equation Regression equation r 2 for r 2 for for HPLC for CZE HPLC CZE

A y = 69.5756 + 1.6153x Y = 0.16663x - 0.01604 0.9961 0.9990 C Y = 153,1341 + 1.6311x y = 0,23799x- 0.00988 0.9973 0.9992

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

wavelength was 205 rim. Column: Zorbax SB-C-18, 150- 4.6, particle size 5 ~tm (Rockland Technology, USA). Senna dianthrones were separated with a water (A)- acetonitrile (B) solvent gradient containing 0 .01% tri- flouracetic 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 experi- ments 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 Digi- tal 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.

Results

15- B

10 I]A1

5-

C,D,D1 X1 L �9 ' 1 7 . 8 r a i n

Figure 2 Electropherogram of aqueous-methanolic extract 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 borate- sodium hydroxide buffer (pH 8.4) containing 0.1 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 ~

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 estab- lished 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 sen- nosides (weak acids), a basic boric acid-NaOH buffer was chosen. Sennosides are negatively charged and con- sequently migrate based on the strong electroosmotic flow to the cathode [11]. To keep the ionic strength con- stant 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 resolu- tions of the sennoside pairs B-A1 and A1-A were ob- tained using a pH of 8.4 which was therefore selected as the buffer pH.

The influence of different organic solvents as buffer ad- ditives on the separation of the sennoside pair A1-B is shown in Figure 4. Without addition of organic solvents

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. Conditions as Figure 2.

to the buffer the peaks B and A1 overlap (Rs 0.6). When adding 5 or 10 % methanol, ethanol, 1-propanol, 2- propanol or acetonitrile resolution of the substance pair increases. The best resolution of A1 and B could be achieved using electrolytes containing 10 % iso- propanol (Rs 1.8). Addition of high concentrations of or- ganic 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.

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2.0-

1.8-

1.6-

1.4~

1,2-

Rs ~.o-

0.8-

0.~ 0.4-

0.2-

0.0-

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

Nomt

40O

300

20O

1110

I I I I i j i l l l l l l | l [ i i i l l i i 1 1 i 2OO 250 3OO 350 400 nm

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

~ U

400-

t00 D 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 ~

H P L C

The HP LC chromatogram of a methanolic extract of the leaves of C angustifolia is shown in Figure 6. The follow- ing peaks could be assigned by means of the retention time and the UV 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 % tri- fluoracetic acid - the acid of choice for LC-MS analyses - which gave bet ter separations than phosphoric acid. The optimum concentrat ion of trifluoracetic acid

(0 .01%) was established empirically. Concentrat ions of 0.005, 0.025 and 0.05 % resulted in poore r resolution of the sennosides A and C (Rs < 1). Detect ion was at 205 nm, column temperature 44 ~ Peak purity checked by the DAD 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 com- pound). Best results could be obtained with column temperature 44 ~ Above this, resolution of the peak pair X-D1 decreased, below 40 ~ separation of the pairs X-D1 and B-D was no longer acceptable.

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

3.5 * X-DI + D I - B

3.0 --a-- B-D

2.5

2.0 Rs

1.5

1.0

0.5

0,0 ~ ' - ~ / J

25 35 45 55

"C

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

H P L C - M S

The primary interest in the LC-MS technique was to en- sure the purity of LC peaks and to identify additional dianthrones, LC was connected to the MS via an electrospray interface (ESI). Data acquisition was per- formed in the negative ionisation mode which gave by far better mass spectra for the sennosides than the posi- tive mode. The ion chromatogram 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. [M/2-H]-, [M-H-Glc]- , [ M - H - C O 2 ] - or [M-H-GIc- CO2]- are only generated when CID values are set to 30-50 kV. Through the use of the single ion monitoring (SIM) technique almost all dianthrone peaks could be as-

max 300>1000 SM 7

I00

80

60

40

20

0 t a / z : 8 3 3 , 9 4 7 S H 7

_ E + 0 5

3.614

100

80

60

40

20

0 m/z:f147,961

. . . . J l . . . . . . j ~ . . t , . _ A

7

_ E + 0 4

5,046

i00

80

60

40

20

0 m/z:861,975

i _

SM 7

A~

_ E+05

2.332

I00

80

60

40

20

0 4:10 8:20 12:30 16:40

' ' --' I ' I 20 :50 25 :00

_ E§

4. 189

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

I00

II0

60

C

A (CID=0V) liO.'P

~lJl ,'/

I~ , ' / / k | 7D7.1 t , . . . . J � 9 - . . , . . . . . . . i

. . . . . . . . +bo " " am ,Ira

I0

OO

4D

] 20.

A (CID=30V) ~

. . . . . . . i, , l . I . . , , I . . . . . . . . . . . . "d~ . . . . . . . . r . . . . . . . *ha . . . . . . . idoo

40

20

14' '~

14;0.1 1

ILlt 311+.| 4"1+1 .S 1++,1.3 15~4.~s!+.2 u + . 0 7 p . ? 4"7,.+J,r +...J.,h . . J , .. .

. . . . . . . l ~ . . . . . . . . ~ . . . . . . . . a +6

AED2

I I I . I

" '+ I.

1~ too

10

40

20

lO0

II0

u,

'~ 20

22p.! 471.S

~+2~:2 +1~'.I I s;+.I122.'~ ++e.= *~ +

~ ? + 0

i~0

F i g u r e 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]-) assig- nable to the sennosides A, A1 and B (Figure 8). In com- parison, 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 DAD matching factor of 1000 appeared.

Q u a n t i t a t i v e A n a l y s i s

Quantitative information on C. a n g u s t i f o l i a and a c u t i f o l i a plant material as well as two pharmaceutical preparations were obtained by using the external stand- ard method. C ZE and LC calibration were performed with sennosides A and C. The calibration curve obtained for CZE was linear in the range 1.1-200/ag mL -1 the one for HP LC in the range of 5.55-1110 pg mL -1 (Table I). Detect ion limits were approximately 0.6 pg mL -1 (CZE) and 2.0 ~tg mL -I (HPLC). For quantitation of rhein dianthrones the calibration curve of sennoside A,

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 quantitat ion of plant material as well as phytopharmaceuticals obtained with CZE and HPLC. The relative standard deviations for the quantitative analyses (six experiments) were 0.5- 6.0 % for all compounds studied. In Figure 10 the total sennoside content of senna plant material is compared with that obtained with the classical method of the European pharmacopoeia [2].

Discussion The H P LC method developed provides baseline separa- tion of almost all sennosides and can be applied for quantitation of each of the individual anthronoids in senna plant material as well as pharmaceutical prepara- tions. The data obtained are in agreement with the literature and comparable with data obtained by CZE. However, CZE separates only the major sennosides A, A1 and B, but leaves the minor ones unresolved. Never- theless, the total sennoside content determined by CZE and H P LC is comparable. In contrast the method 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 litera- ture [4]. From the point of view of the sennoside separa- tion HPLC is the more selective technique and there-

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

Table il. Ouanlitalivc determination of sennosides.

sennosides C. angustifolia pods C. angustifolia leaves

g 100 g-I g 100 g-1

HPLC CZE HPLC CZE

C. acutifolia pods pharm, preparation A pharm, preparation B

g 100 g-I mg tablet -1 mg tablet -1

HPLC CZE HPLC CZE HPLC CZE

A 0.65 (2.4) 0.64 (3.5) 1.05 (0.8) 0.92 (4.7) AI 0.26 (0.3) 0.26 (3.9) 0,31 (1.1) 0.26 (3.2) B 1.03 (2.5)* 1.06 (3.9) 1.47 (0.7)* 1.26 (4.4) C 0.06 (6.0) 0.37 (0.8) C1 0.04 (7.8) 0.20 (0.7) D 0.08 (2.3) 0.36 (0.5) DI 0.06 (2.0) 0.33 (0.7) AED2 - 0.08 (2.0) AED3 - 0.08 (4.6) )2 C, C1, D, D1 0.23 (2.5) 1.03 (3.8) E sennosides 2.18 2.19 4.25 3.47

1.38 (0.5) 1.17 (2.0) 5.6 (4.0) 5,2 (2.8) 8.0 (1.2) 6.3 (4.7) 0.39 (0.8) 0.35 (2.3) 1.5 (3.6) 1.3 (4.5) 3,1 (1.6) 2.4 (3.8) 1.56 (0.6)* 1.57 (2.2) 6.2 (3.7)* 6.0 (3.8) 13.3 (1.0)* 11.0 (4,7) 0.16 (1.2) 0.3 (5.0) 0.3 (6.7) 0.07 (1.2) 0.3 (2.6) 0.5 (4.3) 0.13 (0.7) 0.5 (3.2) 1.0 (2.0) 0.12 (0.3) 0.4 (3.5) 0.7 (2.6)

0.06 (3.1) 0.2 (5.0) - 0.41 (3.5) 1,5 (3.4) 2.0 (4.8)

3.87 3.50 15.0 14.0 26.9 21.7

Data: means of six replicates; relative standard deviations in parentheses. *sen noside B overlapping with AED 1.

4-

3.5

3

2.5

% 2

1.5

I1

0.5

0

Figure 10

C. ang. C. eng. C. acut. (leaves) (pods) (pods)

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

fore prefer red w h e n e v e r you need to quant i fy minor dianthrones. O n the o the r hand C Z E analyses are sig- nificantly faster. Both, H P L C and C Z E provide accept- able analysis times, but due to c leaning and equil ibra- tion p rocedures H P L C analyses take 20 min longer. Ad- ditionally, C Z E analyses are much cheaper. Therefore , if a t tent ion is focused on the m a j o r sennosides and on the exact total sennoside de te rmina t ion C Z E could be a quite valuable analytical technique.

E lec t rospray mass spec t romet ry in combina t ion with H P L C p roved to be an useful tool for solving varied types of analytical p rob lems (e.g. peak purity), con- f i rmed s t ructure ident i ty and p rov ided mass spectral in- fo rmat ion f rom previously unapproachab le sennosides.

Acknowledgements

We grateful ly acknowledge Dr. Wol fgang G r i m m i n g e r (Madaus AG, K61n, G e r m a n y ) for kindly supplying sen- nosides A, A1, B, C, D and D1.

References

[1] R. H i*nsel, K. Keller, H. Rimpler, G. Schneider, Hagers Hand- buch 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. Pharm. Sci. 45, 230 (1983).

[8] W. Grimminger, K. Witthohn, Pharmacology 47, 98, 1993. [9] D.C. Lewis, T. Shibamoto, J. HRCC 8, 280 (1985).

[10] S. E g Li, Capillary Electrophoresis, Elsevier, London, New York, Tokyo, 1992; p. 14, 289,

[11] Finnigan MAT: TSQ/SSQ, Atmospheric Pressure Ionization Operators Manual, Rev. A, San Jose, California.

[12] J. Lemli, Fitoterapia, LVII, 33 (1986).

Received: Mar 18,1996 Accepted: May 8, 1996

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