Electrospray tandem mass spectrometric analysis of duboscic acid, exploring the structural features of a new class of triterpenoids, dubosane

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International
Journal
of
Mass
Spectrometry
310 (2012) 77–
80
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ScienceDirect
International
Journal
of
Mass
Spectrometry
j
our
na
l
ho
me
page:
www.elsevier.com/locate/ijms
Short
communication
Electrospray
tandem
mass
spectrometric
analysis
of
duboscic
acid,
exploring
the
structural
features
of
a
new
class
of
triterpenoids,
dubosane
Syed
Ghulam
Musharrafa,∗,
Madiha
Gohera,
Pascal
Wafob,
Ramsay
S.T.
Kamdemb
aH.E.J.
Research
Institute
of
Chemistry,
International
Center
for
Chemical
and
Biological
Sciences,
University
of
Karachi,
Karachi
75270,
Pakistan
bDepartment
of
Organic
Chemistry,
Higher
Teachers
Training
College,
University
of
Yaounde
I,
PO
Box
47,
Yaoundé,
Cameroon
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
5
October
2011
Received
in
revised
form
11
November
2011
Accepted
11
November
2011
Available online 22 November 2011
Dedicated
to
Prof.
Dr.
Muhammad
Iqbal
Choudhary
H.I.
S.I.,
T.I.
(at
the
occasion
of
his
52nd
birthday).
Keywords:
Duboscic
acid
Triterpenoids
Tandem
mass
spectrometry
Duboscia
macrocarpa
ESI-QqTOF
a
b
s
t
r
a
c
t
Duboscic
acid
belongs
to
a
new
class
of
triterpenoids
dubosane,
isolated
from
Duboscia
macrocarpa.
Gas-
phase
fragmentation
of
duboscic
acid
was
studied
using
positive
ion
electrospray
ionization
quadropole
time-of-flight
mass
spectrometry
(ESI-QqTOF-MS/MS)
hybrid
instrument.
ESI-QqTOF-MS
(positive
ion
mode)
showed
the
presence
of
the
protonated
molecule
[M+H]+which
under
low-energy
collision-
induced
dissociation
tandem
mass
spectrometric
(CID-MS/MS)
analysis
showed
the
characteristic
losses
of
methoxy,
hydroxyl
and
carboxylic
groups.
Our
results
demonstrated
the
characteristic
fragments
of
this
new
class
of
triterpenoids
which
are
formed
due
to
the
cleavage
of
seven
membered
unsaturated
ring
C.
The
fragmentation
pathways
of
characteristic
fragments
were
proposed
with
the
aid
of
HRMS
and
computational
studies.
The
knowledge
of
the
fragmentation
pattern
and
key
fragment
ions
of
duboscic
acid,
i.e.
gas
phase
fragmentation
behavior
of
unique
dubosane
structure,
will
be
useful
for
further
explo-
ration
of
the
related
species
of
the
same
genera
for
the
characterization
of
novel
members
of
this
class
of
compounds.
© 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Triterpenoids
are
ubiquitous
non-steroidal
secondary
metabo-
lites
that
are
found
in
terrestrial
and
marine
flora
and
fauna,
occurring
in
the
free
form
as
well
as
in
the
forms
of
ether,
ester
and
glycoside
[1].
Triterpenoids
exhibit
important
biological
proper-
ties
such
as
anti-tumor,
anti-viral,
antibacterial,
anti-inflammatory,
immune-regulatory
[2],
anti-HIV
protease
[3],
antiandrogenic
[4],
antioxidant
[5],
anticomplement
[6],
antimicrobial
[7]
and
angiotensin
converting
enzyme-inhibitory
activities
[8].
We
have
recently
isolated
duboscic
acid,
a
terpenoid
with
unique
carbon
backbone
from
Duboscia
macrocarpa
Bocq.
(Tiliaceae).
Different
parts
of
this
plant
are
traditionally
used
for
the
treatment
of
tuber-
culosis,
cough,
tooth
and
abdominal
problems.
It
is
also
used
as
ver-
mifuge
for
children
[9].
It
is
the
first
member
of
a
new
class
of
triter-
penoids,
for
which
the
name
“dubosane”
is
proposed.
Duboscic
acid
has
a
potent
␣-glucosidase
inhibition,
and
its
structure
was
unam-
biguously
deduced
by
a
single-crystal
X-ray
diffraction
study
[10].
∗Corresponding
author.
Tel.:
+92
21
34824924-5/4819010;
fax:
+92
21
34819018-9.
E-mail
address:
musharraf1977@yahoo.com
(S.G.
Musharraf).
To
our
knowledge,
only
one
report
on
the
phytochemical
inves-
tigation
of
Duboscia
macrocarpa
has
been
published
by
utilizing
classical
phytochemical
method
[10].
This
trivial
phytochemical
method
consumes
large
amounts
of
plant
extracts
which
obtained
from
bulk
raw
material
(in
tons).
However,
to
preserve
the
endemic
and
non
endemic
plant
species
and
their
sustainability,
the
quan-
tity
of
plant
material
has
to
be
limited
to
the
analytical
level.
Therefore,
a
sensitive
analytical
and
high-throughput
strategy
like
LC–MS/MS
for
the
characterization
of
compounds
in
complex
mix-
tures
is
needed
to
be
developed.
Electrospray
ionization
mass
spectrometry
(ESI-MS)
with
collision
induced
dissociation
(CID)
has
been
developed
as
a
powerful
technique
for
the
identification
and
characterization
of
molecules
in
a
mixture
[11,12].
However,
to
structurally
characterize
a
compound
from
its
MS/MS
data,
a
previous
knowledge
of
the
fragmentation
pathways
of
homolo-
gous
compounds
exhibiting
a
conserved
structural
core
is
required
[13],
while
ESI-MS/MS
characterization
of
duboscic
acid
has
not
been
studied
yet.
Therefore,
to
obtain
sufficient
information
on
the
structure
elucidation
of
this
new
class
of
compound,
the
detailed
fragmentation
patterns
of
duboscic
acid
was
studied
using
ESI-
quadropole
time-of-flight
mass
spectrometry
in
positive
ion
mode.
Characteristic
fragments
of
duboscic
acid
will
be
helpful
for
the
identification
of
its
congeners
in
other
plant
species.
1387-3806/$
–
see
front
matter ©
2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijms.2011.11.007

78 S.G.
Musharraf
et
al.
/
International
Journal
of
Mass
Spectrometry
310 (2012) 77–
80
Table
1
Calculated
energies
for
protonated
duboscic
acid
at
basis
set
6-31G*:
.
Neutral
E
(Hartree)
Cation
E
(Hartree)
Energy
difference
(Hartree)
from
(A)
−1736.79713 A
−1737.18048
0.00
B
−1737.16750
0.01298
C
−1737.15700
0.02348
2.
Experimental
2.1.
Chemicals
and
reagents
Chemicals
and
solvents
were
of
analytical
and
HPLC
grades,
respectively
and
were
purchased
from
Aldrich–Sigma
(USA).
Deionized
water
(Milli-Q)
was
used
throughout
the
study.
The
iso-
lation
procedure
and
spectroscopic
data
of
the
duboscic
acid
has
already
been
reported
[10].
2.2.
ESI-QqTOF-MS
analysis:
The
compound
was
dissolved
in
methanol
(0.2
␮g
␮L−1)
and
working
dilution
was
prepared
in
95:5
acetonitrile–water
con-
taining
0.1%
formic
acid
and
analyzed
by
electrospray
ionization
(ESI)
and
collision-induced
dissociation
(CID),
positive
ion
mode,
on
Qq-TOF-MS/MS
instrument
(QSTAR
XL
mass
spectrometer
Applied
Biosystem/MDS
Sciex,
Darmstadt,
Germany)
at
room
temperature.
High-purity
nitrogen
gas
was
used
as
the
curtain
gas
and
colli-
sion
gas
delivered
from
Peak
Scientific
nitrogen
generator.
The
ESI
interface
conditions
were
as
follows:
ion
spray
capillary
voltage
of
5500
V,
curtain
gas
flow
rate
20
L
min−1,
nebulizer
gas
flow
rate
30
L
min−1,
DP1
60
V,
DP2
15
V,
and
focusing
potential
of
265
V.
The
collision
energy
was
swept
from
05
to
35
eV
for
MS/MS
anal-
ysis.
Calibration
was
performed
using
internal
calibration
process.
Sample
was
introduced
into
the
mass
spectrometer
using
a
Har-
vard
syringe
pump
(Holliston,
MA)
at
a
flow
rate
of
5
␮L
min−1.
MS2
experiment
was
conducted
by
selecting
the
product
ion.
Compu-
tational
studies
were
performed
using
DFT
at
the
B3LYP
level
with
6-31G*
basis
set
in
Spartan
08
v
1.2.0
(Wavefunction,
CA,
USA).
The-
oretical
fragmentation
of
protonated
duboscic
acid
was
evaluated
by
using
ACD/MS
Fragmenter
software
(ACD
Labs).
3.
Results
and
discussion
ESI-QqTOF-MS
analysis
of
duboscic
acid
showed
[M+H]+at
m/z
533.3489
corresponding
to
the
protonated
molecular
formula
C31H49 O7(calcd
533.3472).
MS/MS
analysis
of
[M+H]+ion
showing
interesting
fragmentation
pattern
and
the
product
ion
abundance
were
found
to
be
significantly
influenced
by
the
variation
of
col-
lision
energy.
Therefore,
MS/MS
spectra
of
duboscic
acid
were
screened
against
laboratory
collision
energies
ranging
from
5
to
35
eV.
It
was
observed
that
the
fragment
ions
that
are
formed
due
to
the
substituent
losses
are
best
appeared
at
collision
energy
20
eV
(Fig.
1A)
while
the
collision
energy
35
eV
is
the
optimum
energy
for
the
fragment
ions
formed
due
to
the
cleavage
of
seven
membered
ring
C
(Fig.
1B).
The
HR-ESI-MS
data
of
characteristic
fragments
is
summarized
in
Supplementary
material
Table
1.
Major
characteristic
peaks
were
observed
by
the
sequen-
tial
removals
of
substituent
groups
from
the
[M+H]+.
A
characteristic
removal
of
MeOH
that
is
[M+H-32]+was
observed
as
a
base
peak
(m/z
501)
at
collision
energy
15
eV.
Other
characteristic
peaks
were
observed
due
to
the
fur-
ther
losses
of
multiple
hydroxyl
and
carboxylic
groups
at
m/z
483
[M+H−MeOH−H2O]+,
465
[M+H−MeOH−2H2O]+,
455
[M+H−MeOH−HCOOH]+,
437
[M+H−MeOH−H2O−HCOOH]+,
419
[M+H−MeOH−2H2O−HCOOH]+,
391
[M+H−MeOH−H2O−
2HCOOH]+and
373
[M+H−MeOH−2H2O−2HCOOH]+
(Supplementary
material
Fig.
1A).
The
seven
membered
unsatu-
rated
ring
C
has
a
great
influence
on
the
fragmentation
pattern
of
this
new
class
of
compound.
The
characteristic
fragments
at
m/z
277,
263,
237
and
223
were
observed
due
to
the
cleavage
of
this
ring
C.
While
the
other
fragments
that
appeared
at
m/z
259,
245,
219,
217,
213,
191,
177,
173,
159
and
199
were
formed
due
to
the
Fig.
1.
(A)
Relative
abundances
of
fragment
ions
which
are
formed
due
to
the
sub-
stituent
losses
vs.
collisional
energies
of
duboscic
acid,
(B)
relative
abundances
of
fragment
ions
formed
due
to
the
cleavage
of
seven
membered
ring
C
vs.
collisional
energies
of
duboscic
acid.

S.G.
Musharraf
et
al.
/
International
Journal
of
Mass
Spectrometry
310 (2012) 77–
80 79
Scheme
1.
Proposed
fragmentation
pathway
for
the
fragments
formed
due
the
loss
of
substituents.
further
losses
of
hydroxyl
and
carboxylic
groups
from
the
above
fragments
(Supplementary
material
Fig.
1B).
In
silico
studies
were
performed
to
investigate
the
most
probable
protonation
site
in
duboscic
acid.
Minimum
energy
conformation
of
the
neutral
molecule
was
first
optimized
and
every
possible
protonation
site
was
then
individually
analyzed.
There
are
three
possible
protonation
sites
in
duboscic
acid,
the
methoxy
oxygen
at
C-12
(A),
the
hydroxyl
oxygen
at
C19
(B)
and
the
hydroxyl
oxygen
at
C3
(C).
The
energy
optimization
was
done
by
DFT
at
basis
set
6-31G*
level.
It
was
found
that
the
protonation
at
methoxy
oxygen
(A)
showed
minimum
energy
and
therefore
it
was
the
most
favourable
site
for
protonation
among
the
three
possi-
ble
sites,
while
the
hydroxyl
oxygen
at
C19
(B)
was
the
second
most
favourable
site
of
protonation.
Form
A
showed
energy
of
−1737.18048
Hartree
while
form
B
showed
energy
of
−1737.16750
Hartree.
Both
forms
have
an
energy
difference
of
approximately
0.01298
Hartree
(8.14
kcal
mol−1)
(Table
1).
Therefore,
the
major
fragmentation
pathway
of
duboscic
acid
was
most
likely
to
be
ini-
tiated
from
A.
As
depicted
by
energy
calculations
the
most
feasible
site
for
pro-
tonation
is
at
methoxy
oxygen
in
ring
C
therefore
loss
of
methanol
was
observed
from
[M+H]+to
form
fragment
A1
at
m/z
501
followed
by
the
loss
of
water
molecule
either
from
C-3
or
C-19
to
give
the
product
ion
at
m/z
483
(fragment
A2)
but
the
loss
from
C19
seems
to
be
more
feasible
because
it
forms
conjugate
system.
This
frag-
ment
at
m/z
483
could
also
be
simultaneously
formed
by
another
pathway
in
which
protonation
occurs
at
hydroxyl
at
C-19.
The
protonated
form
A
could
also
leads
to
the
formation
of
fragment
A9
by
losing
water
molecule
at
m/z
515.
The
ion
abundance
of
frag-
ment
A9
is
low
while
the
fragment
A1
appeared
as
a
base
peak
at
collision
energy
15
eV.
Fragment
A7
at
m/z
419
could
be
formed
by
both
pathways.
In
one
of
which
fragment
A7
is
formed
by
the
sequential
loss
of
water
molecule
fragment
from
A2
giving
product
ion
at
m/z
465
(fragment
A3)
followed
by
the
loss
of
formic
acid.
While
in
other
possible
pathway
there
is
a
loss
of
formic
acid
to
gives
the
fragment
A4
at
m/z
455
from
fragment
A1
followed
by
the
removals
of
two
water
molecules
to
gives
the
fragment
A7.
After
the
formation
of
product
ion
at
m/z
419
further
loss
of
formic
acid
gives
fragment
A8
at
m/z
373.
Fragment
A8
could
also
be
formed
by
the
water
removal
from
A6
at
m/z
391
which
formed
from
fragment
A5
by
the
removal
of
formic
acid
(Scheme
1).
The
ion
abundances
of
fragments
A6
and
A8
are
low
at
20
eV
and
these
fragments
are
more
prominent
on
increasing
collision
energy.
Fragments
which
are
formed
due
to
the
cleavage
of
seven
mem-
bered
ring
C
are
more
prominent
on
increasing
collision
energy.
Only
the
fragment
A11
at
m/z
237
also
appeared
on
low
collision
energy.
MS/MS
analysis
of
fragment
A1
at
m/z
501
showed
the
cleavage
of
ring
C
which
leads
to
the
formation
of
fragment
A10
(m/z
263),
A11
(m/z
237),
A18
(m/z
223)
and
A21
(m/z
277).
Frag-
ments
A10
and
A11
showed
further
losses
of
formic
acid
and
water
molecules
to
afforded
fragments
A12
(m/z
217),
A13
(m/z
199),
A14
(m/z
245)
and
A15
(m/z
191),
A16
(m/z
173),
A17
(m/z
219),
respec-
tively.
In
a
similar
manner
fragments
A18
and
A21
showed
further
losses
of
formic
acid
and
water
molecules
to
afforded
fragments
Scheme
2.
Proposed
fragmentation
pathway
for
the
fragments
formed
by
the
cleavage
of
ring
C.

80 S.G.
Musharraf
et
al.
/
International
Journal
of
Mass
Spectrometry
310 (2012) 77–
80
A19
(m/z
177),
A20
(m/z
159)
and
A22
(m/z
259),
A23
(m/z
213),
respectively
(Scheme
2).
4.
Conclusion
In
conclusion,
fragmentation
pattern
of
new
class
of
compound,
duboscic
acid
has
been
studied
using
ESI-QqTOF-MS/MS.
It
has
been
observed
that
many
characteristic
neutral
losses
and
formation
of
key
fragment
ions
can
provide
important
structural
informa-
tion
of
the
basic
skeleton
having
seven
membered
ring
and
its
substituents.
The
knowledge
of
fragmentation
pattern
of
this
new
class
will
be
helpful
for
the
rapid
identification
and
characteriza-
tion
of
dubosane
type
triterpenoids
through
liquid
chromatography
coupled
with
mass
spectrometry
in
complex
mixtures
such
as
plant
extracts
or
herbal
formulations
by
utilizing
their
analytical
amount.
Acknowledgements
The
authors
are
grateful
to
Mr.
Arslan
Ali,
(H.E.J.
Research
Insti-
tute
of
Chemistry,
International
Center
for
Chemical
and
Biological
Sciences,
University
of
Karachi)
for
useful
discussions.
Appendix
A.
Supplementary
data
Supplementary
data
associated
with
this
article
can
be
found,
in
the
online
version,
at
doi:10.1016/j.ijms.2011.11.007.
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