Gas chromatography of furocoumarins


Haggag, M.Y.; Hilal, S.H.

Egyptian Journal of Pharmaceutical Sciences 18(1): 71-76

1977


The GLC technique was successfully employed for the analysis of furocoumarins preseat in Ammi majus fruits, as well as, in some pharmaceutical preparations. Separation was achieved on columns packed with 3% SE-30 at 215°. Standard curves were made for xanthotoxin, imperatorin and bergapten and were used for calculating the amount of corresponding furocoumarins studied, and analysed under the same experimental conditions.

Egypt.
J.
Pharm.
Sri.,
18,
No.
1,
pp.
71-76.
(1977)
Gas
Chromatography
of
Furocoumarins
M.Y.
Haggag
and
S.H.
Hilal
Pharmacognosy
Dept.,
Faculty
of
Pharmacy,
Cairo
University,
Egypt.
THE
GLC
technique
was
successfully
employed
foe
the
ana-
lysis
of
furocoumarins
preseat
in
Ammi
majus
fruits,
as
well
as,
in
some
pharmaceutical
preparations.
Separation
was
achieved
on
columns
packed
with
3%
SE
-30
at
215
Standard
curves
were
made
for
xanthotoxin,
imperatorin
and
bergapten
and
were
used
for
calculating
the
amount
of
corresponding
furo-
coumarins
studied,
and
analysed
under
the
same
experimental
conditions.
Furocoumarins
include
a
number
of
compounds
having
important
phar-
maceutical
value.
Among
these
compounds,
Aanthotoxin
(ammoidin),
imperatorin
(ammidin)
and
bergapten
(majudin)
present
in
Arnmi
majus
occupy
a
prominent
position,
especially
xanth,otoxin,
owing
to
its
unique
antileucodermic
activity.
Owing
to
their
importance,
furocoumarins
have
been
the
subject
of
numerous
investigations('
-
5
).
Most
of
the
work
published
dealt
with
either
their
identification
and
or
their
estimation,using
different
classical
methods
(
2-5
).
Recently,
the
GLC
technique
was
used
for
the
analysis
of
different
types
of
compounds(
6
).
Few
reports,
however,
were
traced
dealing
with
the
application
of
this
technique
for
the
analysis
of
coumarins
and
coumarin-
containing
drugs(
?
-
9
).
Brown
and
Shyluk(
7
)
found
a
correlation
between
the
retention
times
and
ether
linkages
of
15
natural
phenolic
coumarins.
Warren
and
Bailey(
5
)
used
a
combined
LGC-UV
absorption
spectroscopy
and
nmr
analysis
for
characterization
of
some
plant
coumarins.
They
carried
out
the
GLC
analysis
on
copper
columns
packed
with
5%
SE
-3D
at
705°
and
the
detector
was
a
thermal
conductivity
detector.
Damjanic
art
Akacic(
9
)
used
a
preparative
gas
chromatograph
equipped
with
steal
columns
packed
with
20
%
carbowax
20
M
on
Chromosorb
W,
at
200°,
for
the
separation
of
psoralen
and
bergapten
from
Ficus
carica.
72
HAGGAG
AND
S.H.
URAL
In
thr
following
work,
the
GLC
technique
was
successfully
employed
for
the
analysis
of
furocoumarin
content
of
Ammi
majus
fruits,
as
welI
as
for
determining
the
amount
of
xanthotoxin
and
imperatorin
in
some
phar-
maceutical
preparations
containing
them.
Experimental
Material
a.
Fruit.
of
Aninii
majus
L
:
collected
in
May
1977
from
plants
cultivated
in
the
ExpeLimcntal
Station
of
Medicinal
Plants,
Pharmacognosy
Dept.,
Faculty
of
Pharmacy,
Cairo
University
at
Guita.
irults
were
air-cLied
thcn
reduced
to
a
fine
powder.
D.
Pharmlict:uticul
preparations
:
(Kindly
supplied
taro
ugh
the
.:ourtesy
of
tax
M,:mpais
ChnikaI
Co
ninny,
Cairo,
Egypt):
-
MeiadcnIn
tablets;
1:ach
tabla
-
contains
13
;rig
xanthotoxin
+
5
mg
imperatc:in
McIadenin
paint,
Each
ail
contains
7
.5
mg
xanthotoxin
+
2.5
mg
Imperatorin
-
Newn.lacienin
tableis;
each
tabIeticontains
15
mg
xantkotc
win.
c.
Authentic
reference
furocoamarins
:
Xanthotoxin,
imperatorin,
ber-
gapten,
marmesin,
and
isopimpinellin
were
used
as
reference
samples.
(The
fi
rst
3
compounds
were
kindly
supplied
by
the
Memphis
Chemical
Company,
Cairo,
Egypt,
while
the
last
2
compounds
were
separated
from
Arnmi
majus
fruits,
and
authenticated
by
the
authors).
0
.2%
solution
from
each
furocoumarin
(0
.4%
solution
of
impera-
torin)
was
prepared
in
chloroform.
Preparation
of
Arumi
majus
extract
Two
grams
of
the
powdered
Arnmi
majus
fruits
were
exhaustively
ex—
tracted
with
chloroform
in
a
SA:1kt
apparatus
(about
10
hi).
The
-
solvent
was
removed
under
vacuum
and
residue
dissolved
in
10
ml
pure
chloroform.
Pharmaceutical
preparations
Ten
tablets
from
each
of
meladenin
tablets
and
Neomeladenin
tablets
were
separately
pulverized
in
a
porcelain
mortar.
An
amount,
equivalent
to
5
tablets
from
each
preparation,
was
exhaustively
extracted
with
succes-
sive
10
ml
portions
of
chloroform
in
a
small
conical
flask.
The
combined
Egypt.
J.
Pharra.
Sci.,
18,
No.
1
(1977)
GAS
CHROMATOGRAPHY
OF
FUROCOLTMA.RINS
73
chloroforrnic
extract,
in
each
case,
was
evaporated
on
a
water
-bath
and
residue
dissolved
in
5
nil
chloroform
(1
ml
15
mg
xanthotoxin
in
neo-
meladenin,
and=
10
mg
xanthotoxin
+
5
mg
imperatorin
in
case
of
mela-
denin
tablets).
Meladenin
paint
:
3
ml
of
the
paint
were
transferred
to
a
small
separating
funnel
containing
16
ml
chloroform
+
10
ml
water
and
the
solution
was
thoroughly
shaken
then
the
chloroforraic
layer
was
transferred
to
a
clean
dry
flask.
The
aqueous
phase
was
again
shaken
with
further
3
x
10
ml
chloroform.
The
combined
chloroformic
extract
was
evaporated
as
fore
-
mentioned
and
the
residue
dissolved
in
3
ml
chloroform
(I
ml
7.5
mg
xanthotoxin
+
2
.5
mg
imperatorin).
GLC
analysis:
GLC
analysis
was
carried
out
on
a
Pye
Unicam
Gas
Chro-
matograph
series
104,
model
64,
equipped
with
dual
column
and
flame
ionization
detectors.
The
following
working
conditions
proved
most
satis-
factor
y:
Column:
coiled
glass
columns,
5
feet
long,
4
mm
internal
diameter,
packed
with
3%
SE
-30
adsorbed
on
celite
80
-
IGO
mesh;
carrier
gas,
nit-
rogen,
at
a
flow
rate
of
5G
mljmin;
hydrogen
flow
rate
50
mll
min;
air
flow
rate,
400
mlihr;
column
temperature
215°
kept
isothermal
throughout
the
analysis;
detector
to
nptrature,
240°;
attenuation,
50
1G-
2
;
chart
speed,
2
cm)
2
min.
Identification
of
furocou
narins
present
in
Am
ni
majus
extract
was
carried
out,
comparing
the
retention
times
of
their
corresponding
peaks
with
those
of
the
available
authentic
samples
cf
furccoumarins,
as
well
as,
by
adding
each
furocoumarin
separately
to
Ammi
majus
extinct
before
in-
jection
and
noticing
the
increase
in
their
corresponding
peaks.
Fig.
1
shows
the
chromatogram
obtained
for
the
available
furocoumarins,
injected
in
a
mixture
form.
The
quantitative
estimation
was
carried
out
based
on
peak
-area
measurement.
Standard
curves
for
xanthotoxin,
ammidin
and
bergapten
were
made
by
injecting
different
concentrations
(0
.5
-8
sg
xanthotoxin
and
bergapten,
2
-
20
µg
i
nperatorin)
and
calculating
the
areas
of
their
corres-
ponding
peaks.
The
mean
values
obtained
from
at
least
three
determinations
were
plotted
against
the
corresponding
concentrations
when
straight
lines
were
obtained
in
all
cases.
These
curves
were
used
for
calculating
the
amount
of
the
corresponding
furocoumarins
in
the
extract
of
Arrant
majus,
as
well
as,
in
the
Pharmaceutical
preparations
under
investigation.
Results
obtain-
ed
are
compiled
in
Tables
1
and
2.
The
percentage
of
furocou
narins
in
Egypt.
J.
Pharm.
Sci.,
18,
No.
1
(1.977k
74
M.Y.
HAGGAG
AND
S.H.
HILAL
Ammi
majus
fruits
was
also
determined
by
the
calorimetric
method
(
5
)
and
was
found
comparable
with
GLC
(Table
I).
Results
and
Discussions
Trials
for
separating
furocoumarins
present
in
Ammi
majus
fruits
by
GLC
were
carried
out
using
different
stationary
phases
and
adopting
different
►working
conditions.
Best
separation
was
achieved
on
SE
-30
at
215°
(Fig.
1).
0
ft
0
Fig.
I
Egypt,
J.
Pharm.
Sci.,
18,
No.
1
(1977)
GAS
CHROMATOGRAPHY
OF
FUROCOUMARINS
75
Under
the
working
conditions,
the
extract
of
Arnmi
rnajus
revealed
I0
peaks
(Table
I),
among
which
xanthotoxin,
bergapten,
isopixnpinellin,
marmesin
and
imperatorin
could
be
identified.
The
quantitative
determination
revealed
that
both
xanthotoxin
and
bergapten
could
be
determined
in
amounts
ranging
from
0.5
pg
-8
lig
white
imperatorin
could
be
determined
in
amounts
ranging
from
2
-2G
pg.
These
results
show
the
very
high
sensitivity
of
the
GLC
technique,
in
addition
to
being
simple
and
rapid,
compared
to
other
classical
methods.
TABLE
1.
Results
of
GLC
analysis
of
furocoumarins
present
in
Ammi
majus
fruits.
Peak
number
T
r
Authentic
of
Important
furocoumarins
1
2
0.30.
0.80
by
GLC
Calorimetric
3
1,00
xanthotoxin
1
.10
0.95
4
1.12
bergapten
0.20
0.25
5
1.26
------
6
1.45
iso
pimpinell
in
7
1.80
0
.13
8
2.28
marmesin
9
2.64
imperatorin
0.75
0
HO
10
4.56
-
Tr
=
retention
time
relative
to
that
of
xanthotoxin.
TABLE
2.
Results
of
GLC
analysis
of
furocoumarins
present
in
some
pharmaceutical
preparations.
Preparation
xanthotoxin
imperatorin
Lam.
f.am.
%
Lain.
f.am.
(mg) (mg)
(mg)
(mg)
Meladenin
tab.
Meladenin
paint
Neomeladenin
tab.
10/tab.
9.75
+97.5
5/tab.
4.91
98.0
7
.51zzli
7.20
96.0
2.5/mI
2.0
80.0.
15/tab.
14.65
+98.0
1.am.
=
labelled
amount;
f.arn.
-
found
amount;
tab.
=
tablet.
Egypt.
J.
Phi
m.
Sci.,
18,
Ho.
1
(1977)
76
HAGGAG
AND
SJH.
IILLAL
Determination
of
the
furocoumaiin
content
in
the
pharmaceutical
preparations
studied
showed
that
the
amount
of
xanthotoxin
varied
from
96
-
98%
of
the
labelled
amounts,
while
that
of
imperatorin
was
98%
of
labelled
amount
in
meladenin
tablets
and
80%
in
meladenin
paint.
This
deoi
ease
in
amount
of
imperatorin
in
meladenin
paint,
might
be
attributed
to
some
possible
decomposition.
References
I
.
Beyrich,
T.:
J.
Chromatography,
20,
173
,
(1965).
2.
Beyrich,
T.
:
Pharmazie,
22,
661,
(1967).
3.
Kramarenko,
V.P.
and
AkopyaL,
OA.,
Farfftaksevt-Zh.
Kiev,
24
(4),
52,
(1969).
4.
Balbaa,
S.1.,
Hilal,
S.H.
and
Haggag,
M.Y.,
Planta
Medica,
22
(2),
209,
(1972).
5.
filial,
S.H.
and
Haggag,
M.Y.,
T.
Pharm.
Sci.
(Egypt),
in
press.
6.
Kubeczka,
K.H.,
Arch.
Pharm.
Berl.,
11,
304,
(1971).
7.
Brown,
S.A.
and
Shyink,
J.P.,
:
Anal.
Chem.
34
(9),
1058,
(1962).
8.
Warren
Steck
and
Bailey,
B.K.
:
Can.
J.
Chem.
47
(19),
3577,
(1969).
9.
Damjanic,
A.
and
Akacic,
B.
:
Planta
Medica,
26
p),
119,
(1974).
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jtea...^A 014j1.
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%DIA
414.0
4
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-
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it
-01
y
-T.
•,
41'
r
Vic
e
j
1
2-
74.
1%
.0
•11.4Ji
J
14.1
L
,i
;4
4
..",11
Le
Sil
,
4
)A
3
c.
„11
.1
..;_411
J-00.;11
tz.
..71z_LI
1
„Is
1.
4
4,1■7
Z1
jai
1
4.
-r
1,1
-:—.J
1
Egypt.
J.
Marra,
Seim,
18,
No.
1
(1977)