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CHEM. RES. CHINESE U.

Available online at www.sciencedirect.com

2007, 23 ( 5 ) , 6 2 5 - 6 2 7 Article ID 1005-9040(2007) -05-625413

I

ScienceDirect ~

Investigation of Aconitine-type Alkaloids from Processed Tuber of Aconitum carmiechaeli by HPLC-ESI-MS/MS" * YUE HaoIy2,PI Zi-feng' , ZHAO Yu-feng"' , SONG Feng-mi' , LIU Zhi-qiang' and LIU Shu-ying' * * 1. Changchun Center of Mass Spectrometry , Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , Changchun 130022, P. R. China ; 2. Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China Received Nov. 29, 2006

Keywords Aconitine-type alkaloids ; HPLC-ESI-MS/MS" ; Aconitum carmiechaeli

Introduction Aconitine-type alkaloids isolated from the roots of

Aconitum carmiechaeli show a potential toxicity and a broad spectrum of b i o a ~ t i v i t y " ~ ' .On the basis of the C,-substituent of C,,-diterpenoid skeleton, aconitinetype alkaloids can be divided into diester-diterpenoid alkaloids ( DDAs ) , monoester-diterpenoid alkaloids ( MDAs) , and lipo-alkaloids( Fig. 1 ) . By usual chemical separation methods, isolation of some unknown active or toxic compounds might not be performed because the isolation procedure is time-consuming , expensive to perform, and involves limited content. High-performance liquid chromatography( HPLC ) coupled with electrospray ionization mass spectrometry ( ESI-MS ) has been widely employed to detect and identify the compounds in natural products, because of their high sensitivity, short analysis time, and low levels of sample

In this study, a method HPLC-ESIMS/MS" was developed for identifying the aconitinetype alkaloids from the extracts of A. carmiechaeli.

Experimental 1 Instrument HPLC system consisted of a pump ( Waters 2690, Milford, MA, USA) and a photodiode-array 969 detector ( Millenniium3' workstation ) . The chromatographic conditions were as follows: an Agilent RP18 column ( 150 mm x 2.6 mm x 5 p m ) , acetonitrile ( A ) , and water ( 0. 2 % triethylamine and 0. 5% acetic acid )

( B ) . The gradient elution of mobile was 0-30 min 20% -50% A , 3 0 6 0 min 50% -15% A. The flow rate was 0. 6 mWmin , and the column temperature was at 23 "c. LCQ ion trap mass spectrometer (Finnigan MAT, San Jose, CA, USA) was equipped with an electrospray source in the positive-ion mode. The electrospray voltage was 5.0 kV. The capillary temperature was 260 T . Nitrogen gas was used as sheath gas and auxiliary gas. The collision gas used was helium gas, and the collision energy ( % ) for the MS" analyses ranged from 25% to 4 2 % .

2

I

OCH3 OCH? Hypaconitine, [ M conitine, [ M

+

+

+ HI , r n h : +

616, R, = CH,, R, = H ; deoxya-

630, R , = C, H,, R, = H ; mesaconi-

+ H] , m/z: 632, R, = CH, , R, = OH; aconitine, [ M + , m / z : 646, R, = C, H, , R, = OH. When C, is occupied by

tine, [ M H]

+ H ] , m/z:

+

OH group, monoester-diterpenoid aconitines ( MDAs ) are formed, when occupied by a fatty acyl, lip0 alkaloids are formed corresponding to diester-diterpenoid alkaloids.

Fig. 1 Structures of some known aconitine-type alkaloids in Aconitum Carmiechaeli

Extraction of Alkaloids The roots of A. carmiechaeli purchased from Si-

chuan province was powdered and extracted with 10% aqueous ammonia and ether. The extract was diluted with methanol and filtered through a syringe filter( 0.45 p m ) before analysis. All standards were prepared in methanol at a concentration of 0. 1 mg/mL.

Results and Discussion On the basis of total ion chromatograms it is difficult to distinctly distinguish every compound. However, in this study extracted ion chromatograms ( EIC )

* Supported by the National Natural Science Foundation of China( Nos. 30472134 and 30672600) and the National Basic Research Program of China( No. 2006CB5047060). * * To whom correspondence should be addressed. E-mail: mslab@ ciac. jl. cn

CHEM. RES. CHINESE U.

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was used to detect and identify a total of 70 compounds from the extracts of A. carmiechaeli. The compound eluted at 23.57 rnin was easily identified as mesaconitine by comparing the retention time, [ M + H ] - , and the characteristic fragment ions with authentic mesaconitine standards. The compound observed at 26.53 min and [ M + H I + at m/z 618, yielded some fragments of [ M -H,O + H I + , [ M -CH30H + H I + ,

Vol. 23

benzoylmesaconine , which is a major component in Aconitum herb.

"1--

[ M - HZO - CH3OH + H I + , [ M - 2CH30H + H I + , [ M -H,O -2CH,OH + H ] + , and [ M -3CH,OH + H ] + in MS/MS spectrum ( Fig. 2 ) . According to the fragmentation pathway of a~onitine'~' , the C, position was occupied by a methoxyl group in this compound, whereas it was occupied by an acetyl group in aconitine. So the compound was identified as 8-methoxyl-14benzoylaconine , which has been found in A. pseudostapfiianum[']. Furthermore, the high-resolution MS data showed the proposed formula C,,H,NO,, and mass error - 1.01 to approve the above-mentioned conclusion. Similarly, six diester-diterpenoid alkaloids ( DDAs ) were confirmed and two of them have not been reported in A. canniechaeli to the best of our knowledge. 100 h

$.

80

u

60

H 5

-2 CI

.2

%

40

s

2

c

oc

20

I

]J

,

1

N N

W 0

0 460

500

540

180 mlz

620

660

700

Fig. 2 MS2 spectrum of 8-methoxyl-14-benzoylaconine

Monoester-diterpenoid alkaloids ( MDAs ) can be produced by hydrolyzation of DDAs( Fig. 1) . So MDAs were easily identified based on comparing the MS" data with those of the corresponding DDAs. Seven MDAs were identified and elucidated by comparing the ESIMS/MS data of related DDAs with those of MDAs. Compound eluted at 14.39 min exhibited [ M + H 1 + ion at m / z 590 in ESI-MS, and the compound's daughter ions were at m/z 572 [ M - H,O + H I + , 558 [ M CH3OH + H I + ,540 [ M - HZO - CH3OH t H I + , 526 [ M -2CH30H + H I + , 522 [ M - 2H,O - CH3OH + H I ' , 508 [ M H, 0 - 2CH3OH + H I ' , 494 [ M -2H,O - C H 3 0 H -CO + H I + , 490 [ M -2H,O 2CH,OH + H] , and 476 [ M -H,O - 3CH30H + H ] + . Comparing the MS/MS spectrum of the mesaconitine with that of the compound eluted at 14. 39 min( Fig. 3) , the compound could be identified as 14-

-

+

m/z

100 h

5

80

u

60

-2 g

40

tii

20

8

5

.-

c2

m. N W m

0

600 m lz

640

680

Fig. 3 MS2 spectra of 14-benzoylmesaconine( A ) and mesaconitine( B )

Lipo-alkaloids are the other important type of alkaloids and their structures are characterized by C, position occupied by a fatty acid acyl grouptg1. In general, the MS3 spectra of the predominant [ M - lipo] + lipoalkaloids is the same as that of the [ M - AcOH] + of relative DDAs. On the basis of the characterization fragmentation behavior of known lipo-alkaloids in ESIMS/MS, more than 50 lipo-alkaloids in Aconitum were found and characterized in this lab~ratory""~~.In this study, highly selective and sensitive HPLC-ESI-MS/ MS technique was applied to identify more unknown lipo-alkaloids. For example, there were three compounds at m/z 850 observed at retention time of 45.70, 57.46, and 63.35 rnin. The MS/MS spectra of the three compounds showed predominant product ions at m/z 556, 572, and 570, corresponding to the neutral losses of nonadecadienoic acid ( ndd , 294 ) , linolenic acid( linolen, 278) , and linoleic acid( lino, 280) , respectively. In the MS3 spectrum of the compound at 47. 10 min is the same as that of HA. It can be concluded that the compound observed at 45.70 min was 8ndd-benzoylhypaconine Similarly, 8-linolenbenzoylmesaconine and 8-1ino-benzoyldeoxyaconinecorrespond to the compounds observed at 57.46 and 63.35 min, respectively. Compound at 68.38 min corresponds to the ion at m/z 784, exhibited the characteristic predominant daughter ion [ M - AcOH + H] + at m / z 556( in Fig. 4 ) . So, the compound was confirmed as

.

YUE Hao et al.

No. 5

8-ttda-benzoylhypaconine, based on the neutral loss of tertradecanoic acid and characterization fragments in

MS/MS spectrum. Accordingly, 21 unknown lipo-alkaloids were detected and characterized in this study. In addition, seven known lipo-alkaloids were first obtained in A. carmiechaeli[81. inni I""

I

c

d

3 mlz

Fig. 4 MS' spectrum of 8-ttda-benzoylhypaconine

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