Electrospray ionization tandem mass spectrometric analysis of ent-6,7-seco-kaurane diterpenoids from...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2009; 23: 138–146

) DOI: 10.1002/rcm.3864
Published online in Wiley InterScience (www.interscience.wiley.com
Electrospray ionization tandem mass spectrometric

analysis of ent-6,7-seco-kaurane diterpenoids from the

Isodon species

Yan Zhou1,3, Bin-Li Yang2, Jing Yang4, Sheng-Xiong Huang2, Han-Dong Sun2,

Hong-Xi Xu3* and Li-Sheng Ding1**1Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, Sichuan Province, P.R. China2State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences,

Kunming 650204, Yunnan Province, P.R. China3Hong Kong Jockey Club Institute of Chinese Medicine, Shatin, Hong Kong, P.R. China4School of Chemistry and Chemical Technology, Shanghai Jiao Tong University, Shanghai 200240, P.R. China

Received 17 June 2008; Revised 5 November 2008; Accepted 6 November 2008

*CorrespoChinese ME-mail: x**CorrespoChinese Avince, P.RE-mail: lsContract/of the P30572254Contract/Fund.

Electrospray ionization tandemmass spectrometry (ESI-MSn) using an ion trap instrument and accurate

mass measurement on a quadrupole time-of-flight (Q-TOF) mass spectrometer has aided the structural

characterization and differentiation of the enmein and spiro-lactone types of ent-6,7-seco-kaurane

diterpenoids from Isodon species. The mass spectral fragmentation data from both techniques was

compared to obtain the mass spectrometric fragmentation pathways of the ent-6,7-seco-kaurane diter-

penoids with high confidence. The analysis revealed that losses of CH2O and CO2 are the predominant

process for the enmein type of ent-kauranes in negative ion mode, and the loss of CO2 is typical for the

spiro-lactone type in positive ion mode. In addition, compounds of the spiro-lactone type with a

conserved core structure but different substituent groups, such as acetyl, hydroxyl, and aldehyde

moiety, resulted in diagnostic product ions by means of successive losses of AcOH, H2O, and CO,

respectively. The fragmentation knowledge will facilitate the analysis and identification of the ent-6,7-

seco-kauranes in future plant research. Copyright # 2008 John Wiley & Sons, Ltd.

ent-6,7-seco-Kaurane diterpenoids are a class of natural

products isolated from the genus Isodon. So far, more than

100 ent-6,7-seco-kaurane diterpenoids have been isolated

from this genus.1 Regarded as the products of the oxidative

cleavage of the C6–C7 bond of the 7,20-epoxy-ent-kauranes,

these diterpenoids can be divided into two types, namely

enmein type (1,7-lactone type) and spiro-lactone type (7,20-

lactone type).1,2 Among them, enmein was reported to have

antibacterial and antitumor activity,3,4 and its active site was

related to the cyclopentanone conjugated with an exo-

methylene unit.5 From then on, many ent-kauranes with

a-methylenecyclopentanone moiety were found to possess

antibacterial and antitumor activity.6–8 Other ent-6,7-seco-

kaurane diterpenoids, such as sculponeatins A–C, exhibited

antitumor activities both in vitro and in vivo.9

In continuation of our research on the mass spectrometric

characterization and the fragmentation routes of natural

ndence to: H.-X. Xu, Hong Kong Jockey Club Institute ofedicine, Shatin, Hong Kong, P.R. China.

[email protected] to: L. S. Ding, Chengdu Institute of Biology,cademy of Sciences, Chengdu 610041, Sichuan Pro-

. [email protected] sponsor: National Natural Sciences Foundation

eople’s Republic of China; contract/grant number:.grant sponsor: Hong Kong Jockey Club Charities Trust

product compounds,10–13 we present for the first time the

ion trap electrospray ionization tandem mass spectrometric

(IT-ESI-MSn) study of ent-6,7-seco-kaurane diterpenoids.

We also report the low-energy collision-induced dis-

sociation MS/MS on a quadrupole time-of-flight (Q-TOF)

accurate mass spectrometer. The ion trap mass analyzer has

allowed us to obtain spectra up to MS5 that are rich in

information, but the mass data are only nominal mass. The

Q-TOF mass spectrometer, on the other hand, can provide

accurate mass of the molecular ions in MS and the product

ions in MS/MS. The two sets of data were combined

and used to construct fragmentation pathways of these

compounds.

EXPERIMENTAL

MaterialsEpindosin (1),14 epinodosinol (2),15 rabdosin A (3),14 isoja-

ponins A (4),14 sculponeatin A (5),16 sculponeatin B (6),16

macrocalyxoformin A (7),16 isodocarpin (8),17 rabdosin B

(9),14 isodonoiol (10),16 inflexusin (11),18 trichorabdal H

(12),19 isodonal (13), acetylexidonin (14),14 isojaponins B

(15),14 and trichorabdal B (16)19 were obtained as previously

reported, and the purity of these authentic compounds was

over 95% by high-performance liquid chromatography

(HPLC) detection.

Copyright # 2008 John Wiley & Sons, Ltd.

ent-6,7-seco-Kaurane diterpenoids from the Isodon species 139

Methanol (HPLC grade) was obtained from Aldrich.

Deionized water (Milli-Q) was used throughout the study.

Mass spectrometryMass and tandem mass spectra were obtained on a LCQDECA

ion trap mass spectrometer (ThermoFinnigan, San Jose, CA,

USA) equipped with an ESI source, and operated in positive/

negative ion mode. The mass spectrometric conditions were

as follows: nebulizer sheath gas, N2 (20 units); capillary

temperature 2508C; spray voltage 4.5 kV; capillary voltage

fixed at �15.0 V; lens voltage, 18 V in (�) ESI, –16 V in (þ) ESI,

collision energies of 25–45%; the precursor ion isolation

window was set at 5 Da to maximize signal/noise in the

fragment ion spectra; full scan (m/z 50–1000) using 500 ms

collection time; all samples was introduced from the syringe

pump into the ESI source. All data acquired were preceded

by Finnigan XcaliburTM core data system Rev. 1.2 (Thermo-

Quest Corporation, San Jose, CA, USA).

High-resolution ESI-MS/MS experiments were performed

on a Waters Q-TOF Premier mass spectrometer equipped

with an ESI source (Micromass MS Technologies, Manche-

Figure 1. ent-6,7-seco-Kaurane diterpenoids from


ster, UK) in positive and negative ion mode, respectively.

The nebulization gas was set to 600 L/h at 3008C, the cone gas

set to 50 L/h, and the source temperature set to 808C. The

capillary voltage and cone voltage were set to 2700 V and

45 V, respectively. The Q-TOF Premier acquisition rate was

set to 0.2 s, with a 0.01 s inter-scan delay. Argon was

employed as the collision gas at a pressure of 5.3� 10�5 Torr.

The MS/MS experiments were carried out by setting the Q-

TOF Premier quadrupole to allow ions of interest to pass

prior to fragmentation in the collision cell with collision

energies varying between 20 and 35 eV. Analytes were

acquired using the LockSprayTM to ensure accuracy and

reproducibility. Leucine-enkephalin was used as the lock

mass (m/z 556.2771) in positive mode, and (m/z 554.2615) in

negative mode, at a concentration of 50 pg/mL with an

infusion flow rate of 10mL/min. Data were centroided

during acquisition, and dynamic range enhancement

(DRETM) was applied throughout the MS experiments to

ensure accurate mass measurement over a wider dynamic

range. All data acquired were preceded by Masslynx 4.1

(Waters).

the genus Isodon investigated in this study.


DOI: 10.1002/rcm

Table

1.Molecularionsandmain

productionsobservedbyESI-MSnanalysis

ofent-6,7-secoditerpenoids

No

.[M

-H]�

m/z

(%)

[Mþ

Na]

þm

/z

(%)

MS

2m

/z

(%)

MS

3m

/z

(%)

MS

4m

/z

(%)

Co

mp

ou

nd

s

136

1(10

0)33

1(10

0)31

3(9)

,287

(100

),26

9(20

),20

7(68

)26

9(10

0),

259(

18),

243(

48),

225(

10)

epin

do

sin

(1)

317(

27)

287(

23)

236

3(10

0)34

5(10

0),3

33(3

6),

327(

100)

,315

(34)

,30

1(63

),28

3(45

)29

9(30

),28

3(10

0),

253(

35)

epin

do

sin

ol

(2)

337

5(10

0)34

5(10

0)30

1(10

0),2

71(3

3),

269(

25),

239(

20)

287(

19),

273(

28),

271(

100)

,253

(21)

rab

do

sin

A(3

)33

1(80

)4

377(

100)

359(

100)

,34

1(10

0),3

29(3

0),

327(

25),

315(

50),

297(

15)

313(

20),

309(

34),

297(

100)

iso

jap

on

ins

A(4

)34

7(28

)5

359(

100)

329(

20)

285(

33),

259(

100)

,24

1(10

0),2

31(4

0),

215(

36)

scu

lpo

nea

tin

A(5

)31

5(10

0),

303(

35),

636

1(10

0)33

1(26

),31

7(41

),30

5(10

0)28

7(31

),24

3(10

0),

225

scu

lpo

nea

tin

B(6

)7

361(

100)

331(

30),

317(

35),

305(

100)

287(

26),

243(

100)

225

mac

roca

lyx

ofo

rmin

A(7

)8

345(

100)

315(

45),

301(

100)

297(

15),

287(

32),

285(

20),

271(

100)

,24

1is

od

oca

rpin

(8)

947

1(10

0)41

1(10

0)39

3(8)

,351

(100

),33

3(14

),31

5(4)

333(

75),

307(

25),

295(

5)ra

bd

osi

nB

(9)

10

429(

100)

369(

100)

351(

50),

339(

100)

,32

5(30

)32

1(10

0),2

95(2

0)is

od

on

oio

l(10)

11

557(

100)

497(

100)

437(

100)

377(

100)

,333

(20)

infl

exu

sin

(11)

12

413(

100)

353(

100)

335(

100)

,323

(20)

,30

9(39

)27

9tr

ich

ora

bd

alH

(12)

335(

40)

13

427(

100)

409(

35)

349(

100)

321(

40),

305(

100)

iso

do

nal

(13)

367(

100)

14

513(

100)

453(

100)

409(

100)

,393

(19)

,33

3(5)

349(

100)

,289

(20)

acet

yle

xid

on

in(14)

15

431(

100)

371(

100)

353(

20),

341(

33),

327(

100)

309(

20),

291(

100)

iso

jap

on

ins

B(15)

16

427(

100)

367(

100)

349(

100)

321(

25),

305(

100)

tric

ho

rab

dal

B(16)

140 Y. Zhou et al.

Copyright # 2008 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2009; 23: 138–146

DOI: 10.1002/rcm


Computational methodAll calculations were carried out using the Gaussian03

program package.20 The B3LYP hybrid density functional

and 6-31G(d) basis set were used.21,22 For all optimized struc-

tures, vibrational spectra were calculated to ensure that no

imaginary frequencies for energy minimum were obtained.

RESULTS AND DISCUSSION

The authentic samples of sixteen ent-6,7-seco-kaurane diter-

penoids isolated from Isodon species were studied by means

of ESI-MSn in both positive and negative mode. Their

structures are depicted in Fig. 1 and the accurate mass

measurements and elemental compositions of molecular ions

and main product ions are shown in Table 2.

In the ESI-MS experiments, all standards were ionized as

deprotonated molecules [M–H]� in negative mode, and as

sodium adducted molecules [MþNa]þ in positive mode,

respectively. Main dissociation routes of the enmein type and

spiro-lactone type were different. The fragmentation infor-

Figure 2. Tandem mass spectra of epindosin (1): (a) MS2 spectru

spectrum ofm/z 331 from the precursorm/z 361; (c) MS4 spectrum o

m/z 317 from the precursor m/z 361.


mation is thus useful for differentiating the two types of ent-

6,7-seco-kaurane diterpenoids.

Fragmentation of enmein typeThe enmein type features five characteristic rings, with ring B

being a 1,7-lactone and ring E being a C6–C20 hemiacetal. This

class of compounds shows very similar fragmentation

mechanism in negative mode. The characteristic fragment

ion corresponded to [M–H–30]� in the tandem mass spectra,

which was interpreted as proceeding via cleavage of the C10–

C20 and C6–O bonds to eliminate the neutral fragment CH2O

in ring E.

Epindosin (1) is a representative of these enmein type ent-

6,7-seco-kauranes. By using the deprotonated molecular ion

[M–H]� at m/z 361 as the precursor ion, under 35% collision

energy, in negative ESI-MS2 of a standard sample of 1

(as shown in Fig. 2), a predominant ion [M–H–CH2O]� at

m/z 331 and a competing minor ion [M–H–CO2]� at m/z 317

were detected. The diagnostic fragment ion at m/z 331 was

m of the deprotonated molecular [M�H]� at m/z 361; (b) MS3

fm/z 287 from the precursorm/z 331; and (d)MS3 spectrum of


DOI: 10.1002/rcm

142 Y. Zhou et al.

interpreted as originating via cleavage of the C10–C20

and C6–O bonds to eliminate the neutral fragment CH2O

in the E-ring. The MS3 spectrum of the precursor ion at

m/z 331 displayed an abundant fragment ion atm/z 287 which

was formed by the loss of CO2; the MS3 spectrum of

the precursor ion at m/z 317 showed a major fragment ion at

m/z 287 by the loss of CH2O. The MS4 spectrum of the

precursor ion at m/z 287 produced fragment ions at m/z 269

and 243 by losses of H2O and CO2, respectively. The

fragmentation mechanism is proposed in Scheme 1(a).

It should be pointed out that the fission of the C10–C20 and

C6-O bonds to eliminate the neutral fragment CH2O might

correspond to three possible pathways. The first one is

through connecting the C6–O–C11 bond to form a six-

membered ring; the second pathway is via hydrogen at C5

transferring to C6 to form the C5–C10 double bond; the third

Scheme 1. Main fragmentation pathways of (a


one is via cleavage of the C8–C9 bond to form a 8,9-seco ent-

kaurane. By our calculation (Gaussian03 program), based on

the product stability, product 1c has the advantage of

139.1 kJ/mol over product 1a and 118.5 kJ/mol over product

1b. So, to lose a molecule of formaldehyde from the reactant,

pathway 3 is more reasonable (Fig. 3).

The elemental compositions of molecular ions and main

product ions of 1–8 are given in Table 1. All these compounds

revealed similar common fragmentation pathways to

compound 1, but the mass difference was due to a different

structural pattern. Therefore, it could be used to differentiate

some isomers of the enmein type. Sculponeatin B (6) presented

the same deprotonated ion at m/z 361 corresponding to the

molecular formula C20H25O6, which was characterized by a

five-membered ring formed through connecting the C6–C19

bond. In the MS2 spectrum of the [M�H]� at m/z 361, a

) epindosin (1) and (b) sculponeatin B (6).


DOI: 10.1002/rcm

Figure 3. The relative product energies (DE) of the three

possible pathways of fission of the C10–C20 and C6–O bonds

to eliminate the neutral fragment CH2O of epindosin (1).

Table 2. Accurate mass measurements and elemental composition

analysis of for ent-6,7-seco diterpenoids

Compounds Measured mass CE (eV)

epindosin (1) 361.1653 30

331.1552 30

317.1745 30

287.1649 30

269.1550 30

epindosinol (2) 363.1801 30

345.1722 30

333.1695 30

327.1591 30

315.1601 30

301.1793 30

rabdosin A (3) 375.1803 25

345.1709 25

331.1898 25

301.1812 25

271.1703 25

isojaponins A (4) 377.1960 35

359.1866 35

347.1865 35

329.1750 35

315.1966 35



diagnostic fragment ion at m/z 305 was observed besides the

ions at m/z 331 and 317, which was formed by loss of 2CO.

This ion may further dissociate to produce the fragment ion

at m/z 243 by loss of CO2 and H2O in the MS3 spectrum. Its

fragmentation patterns are shown in Scheme 1(b).

Fragmentation pattern of spiro-lactone typeESI mass spectra of the spiro-lactone type recorded in

positive and negative mode allowed elucidation of the

molecular weight, however, for secondary ion mass spectra

(MS2), poor signal-to-noise (S/N) ratio was obtained in

negative mode. During further studies, MSn (n¼ 2–5) spectra

of all spiro-lactone types were recorded only in positive

mode. As compared to the fragmentation pathways of the

enmein type, the spiro-lactone type exhibited typical mass

fragmentation patterns. Therefore, it was possible to

differentiate the two types of ent-6,7-seco-kaurane diterpe-

noids by ESI-MSn analysis. The spiro-lactone type were

ionized as sodium-adducted molecules [MþNa]þ, and the

elemental compositions of molecular ions and main product

ions of compounds 8–15 are summarized in Table 2.

The tandem mass spectra of rabdosin B (9) (Fig. 4) provide

a representative example of these pathways. Its MS spectrum

showed a [MþNa]þ ion at m/z 471. The MS2 spectrum

(Fig. 4(a)) showed a dominant peak at m/z 411 [MþNa-

AcOH]þ, which was further dissociated to produce the

product ions at m/z 351 and 333 by loss of AcOH

and H2OþAcOH, respectively, in MS3 spectra (Figs. 4(b)

and 4(d)). Notably, the MS4 spectrum (Fig. 4(c)) displayed a

fragment ion at m/z 307 from the precursor m/z 351, and the

ion at m/z 289 from the precursor m/z 333 was due to a

different substitution pattern (for fragmentation pathway,

see Scheme 2). The main product ions of sodium-adducted

molecules of this spiro-lactone type revealed common

s of molecular ions and main product ions by HR-ESI-MS/MS

Calulated mass Elemental composition Error (ppm)

361.1651 C20H25O�6 0.8

331.1545 C19H23O�5 2.1

317.1753 C19H25O�4 �2.5

287.1647 C18H23O�3 0.7

269.1542 C18H21O�2 3.0

363.1808 C20H27O�6 �1.9

345.1702 C20H25O�5 5.8

333.1702 C19H25O�5 �2.1

327.1596 C20H23O�4 �1.5

315.1596 C19H23O�4 1.6

301.1804 C19H25O�3 �3.7

375.1808 C21H27O�6 �1.3

345.1702 C20H25O�5 2.0

331.1909 C20H27O�4 �3.3

301.1804 C19H25O�3 2.6

271.1698 C18H23O�2 1.8

377.1964 C21H29O�6 �1.1

359.1858 C21H27O�5 2.2

347.1858 C20H27O�5 2.0

329.1753 C20H25O�4 �0.9

315.1960 C20H27O�3 1.9

(Continues)


DOI: 10.1002/rcm

Table 2. (Continued)

Compounds Measured mass CE (eV) Calulated mass Elemental composition Error (ppm)

303.1893 35 303.1960 C19H27O�3 �2.3

285.1847 35 285.1855 C19H25O�2 �2.8

sculponeatin A (5) 359.1490 35 359.1495 C20H23O�6 �1.4

329.1385 35 329.1389 C19H21O�5 �1.2

315.1603 35 315.1596 C19H23O�4 2.2

303.1609 35 303.1596 C18H23O�4 4.3

259.1707 35 259.1698 C17H23O�2 3.5

sculponeatin B (6) 361.1657 30 361.1651 C20H25O�6 1.7

331.1556 30 331.1545 C19H23O�5 3.3

317.1745 30 317.1753 C19H25O�4 �2.5

305.1756 30 305.1753 C18H25O�4 1.0

243.1739 30 243.1749 C17H23O� �4.1macrocalyxoformin A (7) 361.1655 30 361.1651 C20H25O�

6 1.1

331.1550 30 331.1545 C19H23O�5 �1.5

317.1742 30 317.1753 C19H25O�4 �3.5

305.1767 30 305.1753 C18H25O�4 4.6

243.1754 30 243.1749 C17H23O� 2.1isodocarpin (8) 345.1698 30 345.1702 C20H25O�

5 �1.2

315.1603 30 315.1596 C19H23O�4 2.2

302.1819 30 301.1804 C19H25O�3 5.0

271.1690 30 271.1698 C18H23O�2 �3.0

241.1600 30 241.1592 C17H21O� �3.3rabdosin B (9) 471.1991 25 471.1995 C24H32O8Naþ �0.8

411.1787 25 411.1784 C22H28O6Naþ 0.7351.1580 25 351.1572 C20H24O4Naþ 2.3333.1472 25 333.1467 C20H22O3Naþ 1.5307.1663 25 307.1674 C19H24O2Naþ �3.6295.1668 25 295.1674 C18H24O2Naþ �2.0289.1573 25 289.1568 C19H22ONaþ 1.7

isodonoiol (10) 429.1890 25 429.1889 C22H30O7Naþ 0.2369.1685 25 369.1678 C20H26O5Naþ 1.9351.1559 25 351.1572 C20H24O4Naþ �3.7339.1588 25 339.1572 C19H24O4Naþ 4.7325.1766 25 325.1780 C19H26O3Naþ �4.3321.1455 25 321.1467 C19H22O3Naþ �3.7297.1838 25 297.1830 C18H26O2Naþ 2.7

inflexusin (11) 557.2358 20 557.2363 C28H38O10Naþ �0.9497.2139 20 497.2151 C26H34O8Naþ �2.4437.1946 20 437.1940 C24H30O6Naþ 1.4363.1750 20 377.1729 C22H26O4Naþ 5.6

trichorabdal H (12) 413.1945 25 413.1940 C22H30O6Naþ 1.2353.1741 25 353.1729 C20H26O4Naþ 3.4335.1615 25 335.1623 C20H24O3Naþ �2.4323.1613 25 323.1623 C19H24O3Naþ �3.1309.1824 25 309.1830 C19H26O2Naþ �1.9291.1719 25 291.1725 C19H24ONaþ �2.1279.1733 25 279.1725 C18H24ONaþ 2.9

isodonal (13) 427.1731 28 427.1733 C22H28O7Naþ �0.5409.1634 28 409.1627 C22H26O6Naþ 1.7367.1513 28 367.1521 C20H24O5Naþ �2.2339.1580 28 339.1572 C19H24O4Naþ 2.4323.1611 28 323.1623 C19H24O3Naþ �3.7305.1529 28 305.1517 C19H22O2Naþ 3.9295.1682 28 295.1674 C18H24O2Naþ 2.7

acetylexidonin (14) 513.2097 25 513.2101 C26H34O9Naþ �0.8453.1898 25 453.1889 C24H30O7Naþ 2.0409.1978 25 409.1991 C23H30O5Naþ �3.2393.1662 25 393.1678 C22H26O5Naþ �4.1349.1788 25 349.1780 C21H26O3Naþ 2.3333.1461 25 333.1467 C20H22O3Naþ �1.8289.1574 25 289.1568 C19H22ONaþ 2.1

isojaponins B (15) 431.2042 28 431.2046 C22H32O7Naþ �0.9371.1828 28 371.1834 C20H28O5Naþ �1.6353.1737 28 353.1729 C20H26O4Naþ 2.3341.1741 28 341.1729 C19H26O4Naþ 3.5327.1944 28 327.1936 C19H28O3Naþ 2.4

(Continues)

Copyright # 2008 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2009; 23: 138–146

DOI: 10.1002/rcm

144 Y. Zhou et al.

Table 2. (Continued)

Compounds Measured mass CE (eV) Calulated mass Elemental composition Error (ppm)

309.1822 28 309.1830 C19H26O2Naþ �2.6291.1735 28 291.1725 C19H24ONaþ 3.4

trichorabdal B (16) 427.1730 25 427.1733 C22H28O7Naþ �0.7367.1532 25 367.1521 C20H24O5Naþ 3.0349.1407 25 349.1416 C20H22O4Naþ �2.6321.1473 25 321.1467 C19H22O3Naþ 1.9305.1504 25 305.1517 C19H22O2Naþ �4.2


fragmentation pathways similar to compound 9, and the

mass difference was due to a different substitution pattern.

Therefore, it could be used to identify different substituents

of this class of compounds.

Since most of the spiro-lactone types of the ent-6,7-seco-

kaurane diterpenoids contain acetyl units, the successive loss

of AcOH (60 Da) was detected in MSn (n¼ 2–5) spectra.

Therefore, the number of the acetyl groups could be

elucidated by detailed tandem mass analysis. Shikokianal

acetate (10), trichorabdal H (12), and trichorabdal B (16)

Figure 4. Tandem mass spectra of rabdosin B (9): (a) MS2 spect

(b) MS3 spectrum of m/z 411 from the precursor m/z 471; (c) MS4 s

spectrum of m/z 333 from the precursor m/z 411.


contain one acetyl unit, and [MþNa�AcOH]þ was detected

as the base peak in their MS2 spectrum from the precursor

[MþNa]þ. Rabdosin B (9) and isodonal (13) contain two

acetyl units, and [MþNa�2AcOH]þ was detected abun-

dantly in their MS3 spectra from the precursor

[MþNa�AcOH]þ. Acetylexidonin (14) is comprised of three

acetyl units. The product ion at m/z 453 ([MþNa�AcOH]þ)

formed by the loss of AcOH (60 Da) was detected in the MS2

spectrum from the precursor m/z 513 [MþNa]þ; the product

ion at m/z 393 ([MþNa�2AcOH]þ) formed by the further loss

rum of the sodium-adducted molecular [MþNa]þ at m/z 471;

pectrum of m/z 351 from the precursor m/z 411; and (d) MS3


DOI: 10.1002/rcm

Scheme 2. Main fragmentation pathway of rabdosin B (9).

146 Y. Zhou et al.

of AcOH from the precursor m/z 453 in MS3 spectra;

moreover, the product ion at m/z 333 ([MþNa�3AcOH]þ)

was formed by the further loss of AcOH from the precursor

m/z 393 in MS4 spectra, successively. However, if the

acetyl unit is located at C15, it would be difficult to

dissociate the AcOH from the skeleton. For example,

inflexusin (11) contains four acetyl units with one at

C15; [MþNa�4AcOH]þ was not detected in the MS4 or

MS5 spectra.

Shikokianal acetate (10), trichorabdal H (12), and iso-

japonins B (15), which have a hydroxy group at C5, showed a

characteristic fragment ion formed by elimination of a

neutral CH2O (30 Da) in the MS3 spectrum.

Trichorabdal B (16), characterized by an aldehyde group

at C6, showed a characteristic fragment ion formed by

elimination of a CO (28 Da) unit in the MS3 spectrum.

CONCLUSIONS

Electrospray ionization tandem mass spectrometry (ESI-

MSn) was utilized to study the ent-6,7-seco-kaurane diterpe-

noids from Isodon species for the first time. ESI-MSn data and

accurate mass measurements were combined to create the

fragmentation pathways of the two types of ent-6,7-seco-

kauranes. Main dissociation routes of the enmein type and

the spiro-lactone type have been determined and rational-

ized. The diagnostic fragment ions of the enmein type and

the spiro-lactone type were derived from elimination

of CH2O, CO2, and CO2, respectively. The fragmentation

information is very useful for differentiating substituents of

the spiro-lactone type, which are derived from the cleaved

side-chain moiety. The combination of ion trap and accurate

mass measurement gives the proposed fragmentation path-

ways to a higher degree of confidence. We speculate that the

information on mass fragmentations of the ent-6,7-seco-

kaurane will be useful for elucidating the structures of

metabolites and analogs in crude extracts of Isodon species.

AcknowledgementsThe project was supported by the National Natural Sciences

Foundation of the People’s Republic of China (No. 30572254)

and the Hong Kong Jockey Club Charities Trust Fund.


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DOI: 10.1002/rcm

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