Fast Analysis of Selected Xanthones in Mangosteen Pericarp Using

Fast Analysis of Selected Xanthones in Mangosteen
Pericarp Using Accelerated Solvent Extraction and
Ultra High Performance Liquid Chromatography
Qi Zhang, Bruce Bailey, Marc Plante and Ian Acworth
Thermo Fisher Scientific, Chelmsford, MA, USA
Overview
Extraction:
Purpose: Development of a fast and robust sample preparation method using
accelerated solvent extraction (ASE) and UHPLC separation for quantitative
d t
determination
i ti off selected
l t d xanthones
th
iin mangosteen
t
pericarp.
i
Methods: This poster note describes an accelerated solvent extraction method for
extraction of xanthones from mangosteen pericarp and a gradient UHPLC-UV method
for quantitative determination
Results: The ASE method presented in this poster takes only 30min with >96%
recovery and excellent reproducibility. The ASE and UHPLC-UV method was used to
quantitatively determine five selected xanthones in a commercial pericarp powder
sample. An alternative method using charged aerosol detection is also discussed.
Weigh 0.5g of each
diatomaceous earth
cell (equipped with tw
Extract the loaded ce
separate 25mL volum
with 0.2 µm filter an
Liquid Chromatography:
Thermo Scientific™ Dionex
•DPG-3600RS pump
•WPS-3000TRS
WPS 3000TRS autosam
a tosam
Introduction
Many tropical plants are important sources of biologically active compounds that have
potential therapeutic effects. Mangosteen (Garcinia mangostana L) is a tropical fruit
that is indigenous to Southeast Asia, where it has been historically used to treat
abdominal pain, diarrhea, dysentery, inflammation, wound infection, suppuration, and
chronic ulcer.1 Recently mangosteen has been proposed as a homeopathic therapy in
the treatment of Parkinson’s disease.2 Such therapeutic
p
benefits have been mostly
y
attributed to a unique family of compounds referred to as xanthones that are most
abundant in the pericarp of the fruit.3
The method presented here for the analysis of xanthones in mangosteen pericarp
combines accelerated solvent extraction and UHPLC separation. Conventional
extraction methods for mangosteen pericarp
pericarp, such as soxlet
soxlet, are time consuming,
consuming labor
intensive and do not always deliver the desired reproducibility. ASE is an automated
extraction technique that rapidly performs solvent extractions using high temperature
and pressure. The ASE system has the advantages of short extraction time, low
solvent consumption, high extraction efficiency and excellent reproducibility. The ASE
extraction
e
t act o desc
described
bed in tthis
s poste
poster requires
equ es o
only
y 30 mins
sa
and
d de
delivers
e s >96%
96% recovery
eco e y
with excellent reproducibility.
Reverse phase HPLC with UV detection is widely applied for the analysis of
xanthones.4,5 The UHPLC method for xanthone presented in this poster employs a
Dionex Acclaim RSLC C18 column and is completed in 25mins. Comparative data
using charged aerosol detection s also discussed
discussed. Five major xanthones
xanthones, including
α-mangostin, 3-isomangostin, gartanin, 9-hydroxycalabaxanthone, and
8-desoxygartanin, were quantitatively determined in mangosteen pericarp. The
structures of the selected xanthones are shown in Figure 1.
•3000 TCC-3000RS col
•DAD-3000RS diode ar
•Thermo Scientific™ Dion
50°C, PFV 1.0
Column: Thermo Scientif
Temperature:
30 ºC
Mobile Phase A:
water
Mobile Phase B:
90%a
Gradient: 0–20 min: 50 -9
Flow Rate:
0.5 m
Injection Volume:
10 µL
Data Analysis
Thermo Scientific™ Dionex
SR1 L.
Figure 1. Structure of se
Methods
Sample
Sa
p e Preparation:
epa at o
Equipment and Materials:
•Thermo Scientific™ Dionex™ ASE™ 350 Accelerated Solvent Extractor system
•10mL stainless extraction cells
•Cellulose filters
•Clear collection vials, 60mL,
•Dionex ASE Prep Diatomaceous Earth
Accelerated Solvent Extraction Conditions:
Solvent:
95% ethanol
Temperature: 80°C
Static time:
5min
Static cycle:
4
Flush:
60%
Purge :
90s
2 Fast Analysis of Selected Xanthones in Mangosteen Pericarp Using Accelerated Solvent Extraction and Ultra High Performance Liquid Chromatography
vent extraction method for
a gradient UHPLC-UV method
s only 30min with >96%
PLC-UV method was used to
mmercial pericarp powder
tection is also discussed.
y active compounds that have
gostana L) is a tropical fruit
istorically used to treat
d infection, suppuration, and
as a homeopathic therapy in
benefits have been mostly
y
xanthones that are most
s in mangosteen pericarp
paration. Conventional
xlet
xlet, are time consuming,
consuming labor
ibility. ASE is an automated
ions using high temperature
hort extraction time, low
llent reproducibility. The ASE
a
and
d de
delivers
e s >96%
96% recovery
eco e y
d for the analysis of
d in this poster employs a
5mins. Comparative data
major xanthones
xanthones, including
xanthone, and
gosteen pericarp. The
e 1.
ated Solvent Extractor system
Weigh 0.5g of each mangosteen pericarp powder sample and mix with
diatomaceous earth . Transfer the mixture into a separate 10mL stainless steel
cell (equipped with two cellulose filters on the bottom) to nearly fill the cell
cell.
Extract the loaded cells with the above conditions and transfer the extracts into
separate 25mL volumetric flasks to volume with 95% ethanol. Filter the extract
with 0.2 µm filter and dilute 50 fold with 50% acetonitrile before analysis.
Accelerated Solvent Extractio
95% ethanol was used as the e
optimized in terms of temperatu
optimized procedure requires on
Each sample was extracted for
80°C with four extraction cycles
samples
l with
ith excellent
ll t reproduc
d
of HPLC chromatograms of first
conditions. There was essential
Equal or higher extraction efficie
°C without loss of xanthones. B
the extract cools.
cools Also precipitat
acetonitrile for direct analysis on
Liquid Chromatography:
Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC system including:
•DPG-3600RS pump
•WPS-3000TRS
WPS 3000TRS autosampler
a tosampler
•3000 TCC-3000RS column thermostat
HPLC –UV Method
•DAD-3000RS diode array detector, 320 nm
The extracted sample was direc
A gradient UHPLC-UV
UHPLC UV method w
selected xanthones including, α
hydroxycalabaxanthone, and 8column and the analysis time is
standard of five selected xanth
sample.
p Figure
g
5 shows the cali
range of 1 to 50 µg/mL. Table 2
xanthones as extraction yield of
mangosteen is the major compo
UV response. The amount of g
xanthones and was not quantita
•Thermo Scientific™ Dionex™ Corona™ Veo™ charged aerosol detector, evaporation
50°C, PFV 1.0
Column: Thermo Scientific™ Acclaim™ 120 C18, 2.2µm, 2.1 x 100 mm
Temperature:
30 ºC
Mobile Phase A:
water
Mobile Phase B:
90%acetonitrile, 10% methanol
Gradient: 0–20 min: 50 -90%B; hold at 90% B for 5min
Flow Rate:
0.5 mL/min
Injection Volume:
10 µL
Table 1. Extraction efficiency
Data Analysis
Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System software,
software 7.1
71
SR1 L.
80°C
80
C, 3 cycles
80°C, 4 cycles
Figure 2. Comparison of chro
pericarp powder sample.
sample
Figure 1. Structure of selected xanthones.
250
1 - 20130617 #37
2 - 20130617 #38
mAU
200
150
3-isso-mangostin
paration method using
ration for quantitative
carp.
Results
Extraction:
100
50
0
-50
-100
2
1
0.0
.
2.0
4.0
6.0
8.0
Table 2. Summary of quan
g
pericarp
p
p pow
p
mangosteen
3-iso-mang
extraction yield* (%db)
0.32 ± 0.0
Relative UV area ((%))
1.76 ± 0.0
*Dry weight basis of original sam
Values are the mean ± std devia
Thermo Scientific Poster Note • PN70991_PITTCON_2014_E_02/14S 3
Results
Accelerated Solvent Extraction Method
mAU
600
Each sample was extracted for a second time to evaluate the extraction efficiency. At
80°C with four extraction cycles, extraction efficiency was above 96% for all three
samples
l with
ith excellent
ll t reproducibility,
d ibilit as shown
h
iin T
Table
bl 1
1. Fi
Figure 2 shows
h
comparison
i
of HPLC chromatograms of first extraction and second extraction with optimized
conditions. There was essentially no xanthones recovered in the second extraction.
Equal or higher extraction efficiency can be achieved at higher temperature of 100-120
°C without loss of xanthones. But the extract may contain material that precipitates when
the extract cools.
cools Also precipitates can form when diluting filtered extract in 50%
acetonitrile for direct analysis on HPLC.
C system including:
20130617 #18
700
95% ethanol was used as the extraction solvent in this experiment
experiment. ASE conditions were
optimized in terms of temperature, static time, flush volume and number of cycles. The
optimized procedure requires only about 30min and about 20-25 mL solvent.
3-iso-ma
angostin
powder sample and mix with
to a separate 10mL stainless steel
e bottom) to nearly fill the cell
cell.
ditions and transfer the extracts into
with 95% ethanol. Filter the extract
% acetonitrile before analysis.
Figure 3. UV chromatog
standards.
500
400
300
200
100
HPLC –UV Method
The extracted sample was directly analyzed by HPLC after simple filtration and dilution.
A gradient UHPLC-UV
UHPLC UV method was developed for quantitative determination of the five
selected xanthones including, α-mangostin, 3-isomangostin, gartanin, 9hydroxycalabaxanthone, and 8-desoxygartanin. This method uses a Dionex C18
column and the analysis time is 25min. Figures 3 and 4 show chromatograms for the
standard of five selected xanthones and a 50-fold diluted mangosteen pericarp extract
sample.
p Figure
g
5 shows the calibration cures for the standards in the concentration
range of 1 to 50 µg/mL. Table 2 summarizes quantitave results for the selected
xanthones as extraction yield of the original sample dry weight and total UV area. Alphamangosteen is the major component of the extract, and accounts for over 60% of total
UV response. The amount of gartanin was not significant compared to the other four
xanthones and was not quantitatively determined with this method.
ol
5min
Sample 1
Sample 2
Sample 3
80°C
80
C, 3 cycles
90 0%
90.0%
91 3%
91.3%
90 6%
90.6%
80°C, 4 cycles
96.0%
98.4%
96.5%
25.0
100
50
0
-50
-100
20130617 #38
15.0
5.0
-3.0
0.0
2.0
45
40
First extraction
35
1st extract
Second extraction
.
1
2nd extract
min
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
22.0
25.0
Table 2. Summary of quantitative results for four selected xanthones in
g
pericarp
p
p powder.
p
mangosteen
3-iso-mangostin 8-desoxygartanin
6.0
Figure 5. Calibratio
2
0.0
4.0
UV_VIS_1
UV_VIS_1
WVL:320 nm
9-h
hydroxycalabaxantthone
3-isso-mangostin
150
angostin
alpha-ma
200
6.0
mAU
10.0
ASE 80oC 4-cycle, 0.5mg sample2, 2nd extract, 1:50
ASE 80oC 4-cycle, 0.5mg sample3, 1st extract, 1:50
3-iso-mangostin im
mpurity
8-desoxygartanin
250
4.0
20.0
Figure 2. Comparison of chromatogram of first and ASE extract of mangosteen
pericarp powder sample.
sample
1 - 20130617 #37
2 - 20130617 #38
mAU
2.0
Figure 4. UV chromatog
pericarp powder sample
Table 1. Extraction efficiency of ASE method for mangosteen pericarp powder.
atography Data System software,
software 7.1
71
0.0
3-iso-mangostin
2.2µm, 2.1 x 100 mm
-50
alphamangostin
9-hydroxycalabaxanthone
extraction yield* (%db)
0.32 ± 0.01
0.31 ± 0.01
7.33 ± 0.20
0.57 ± 0.02
Relative UV area ((%))
1.76 ± 0.01
1.27 ± 0.01
64.05 ± 0.11
2.35 ±0.02
*Dry weight basis of original sample of pericarp powder.
Values are the mean ± std deviation (n=3)
4 Fast Analysis of Selected Xanthones in Mangosteen Pericarp Using Accelerated Solvent Extraction and Ultra High Performance Liquid Chromatography
UV area (mA
AU*min)
arged aerosol detector, evaporation
30
25
20
15
10
5
0
0
10
Figure 3. UV chromatogram showing separation of five selected xanthone
standards.
20130617 #18
700
50ug/mL
300
200
100
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
22.0
25.0
ASE 80oC 4-cycle, 0.5mg sample3, 1st extract, 1:50
15.0
10.0
25.0
12.5
WVL:320 nm
1
2.0
min
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
4.0
6.0
Conclusion
16.0
18.0
20.0
22.0
Figure 5. Calibration curves for UV response of five selected xanthones.

A 30 min ASE meth
powder was develo
reproducibility.

A 25 min gradient
extracted samples
determined.
25.0
40
First extraction
2nd extract
min
20.0
22.0
25.0
UV area (mA
AU*min)
Second extraction
18.0
2. Jung, H.A.; Su, B.
xanthones from the
Food Chem 2006:
35
1st extract
30
3-isomangostin
25
8-desoxygartanin
20
gartanin
alpha-mangostin
15
9-hydroxycalabaxanthone
10
alphamangostin
9-hydroxycalabaxanthone
7.33 ± 0.20
0.57 ± 0.02
64.05 ± 0.11
2.35 ±0.02
5
0
R f
References
1. Pedraza-Chaverri,
J.M. Medicinal prop
Chemical Toxicolog
45
r four selected xanthones in
r.
0.0
UV_VIS_1
UV_VIS_1
WVL:320 nm
9-h
hydroxycalabaxantthone
e2, 2nd extract, 1:50
le3, 1st extract, 1:50
nin
2
5.0
-3.0
16.0
0.0
-20.0
and ASE extract of mangosteen
14.0
37.5
UV_VIS_1
9-hydroxycala
abaxanthone
3-iso-mangostin
20.0
alpha
a-mangostin
mAU
3-iso-mangostin impurity
25.0
20130617 #38
8--desoxygartanin
90 6%
90.6%
96.5%
1 - 20130617 #62 [normalized]
2 - 20130617 #62
pA
min
Figure 4. UV chromatogram showing separation of the extract of mangosteen
pericarp powder sample and detection of four xanthones.
Sample 3
1 3%
1.3%
60.0
3-is
so-mangostin
-50
or mangosteen pericarp powder.
8.4%
FIGURE 6. Comparison
mangosteen pericarp e
50.0
PLC after simple filtration and dilution.
quantitative determination of the five
mangostin, gartanin, 9his method uses a Dionex C18
and 4 show chromatograms for the
diluted mangosteen pericarp extract
he standards in the concentration
titave results for the selected
e dry weight and total UV area. Alphact, and accounts for over 60% of total
gnificant compared to the other four
with this method.
mple 2
9-hydroxycalabaxa
9
anthone
400
3-iso-m
mangostin impurity
8-desoxygartanin
3-iso-ma
angostin
500
alpha-mangostin
a
WVL:320 nm
600
valuate the extraction efficiency. At
ncy was above 96% for all three
T
Table
bl 1
1. Fi
Figure 2 shows
h
comparison
i
cond extraction with optimized
covered in the second extraction.
ved at higher temperature of 100-120
contain material that precipitates when
diluting filtered extract in 50%
The use of the Corona Ve
of xanthones in mangost
Corona Veo detector chro
identical results except fo
mangosteen and other pe
advantage over UV, char
volatile analytes with sim
to measure levels of unkn
a suitable chromophore.
UV_VIS_1
mAU
garta
anin
this experiment
experiment. ASE conditions were
sh volume and number of cycles. The
nd about 20-25 mL solvent.
HPLC-Charged Aerosol
0
10
20
30
40
Concentration (µg/mL)
50
60
3. Kosem, N.; Youn-H
activities of methan
33, 83-292
4. Walker, E.B. HPLC
Sci. 2007, 30, 1229
5. Pothitirat,, W. and G
determination of α2009, 42, 7-12.
6. Zarena, A.S. and U
(Garcinia mangost
phosphomolybden
All trademarks are the prope
This information is not intend
infringe the intellectual prope
Thermo Scientific Poster Note • PN70991_PITTCON_2014_E_02/14S 5
n of five selected xanthone
HPLC-Charged Aerosol Detection
The use of the Corona Veo, a charged aerosol detector, was evaluated for detection
of xanthones in mangosteen pericarp. Figure 5 shows the comparison of UV and
Corona Veo detector chromatograms for this sample
sample. The two detectors give almost
identical results except for the difference in relative response between alphamangosteen and other peaks. Although in these experiments there is no appreciable
advantage over UV, charged aerosol detection with its ability to measure all nonvolatile analytes with similar response independent of chemical structure, can be used
to measure levels of unknown analytes in mangosteen pericarp
pericarp, even those that lack
a suitable chromophore.
UV_VIS_1
9
9-hydroxycalabaxa
anthone
WVL:320 nm
FIGURE 6. Comparison of UV and Corona Veo detection for xanthones in the same
mangosteen pericarp extract sample.
UV-CAD ASE sample3 50%
UV-CAD ASE sample3 50%
50.0
min
18.0
20.0
22.0
25.0
25.0
n of the extract of mangosteen
anthones.
1st extract, 1:50
0
12.5
UV_VIS_1
9-hydroxycala
abaxanthone
WVL:320 nm
0.0
Corona Veo
CAD
2
UV UV
1
-20.0
min
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
22.0
25.0
Conclusion
min
16.0
18.0
20.0
22.0
e of five selected xanthones.
0
37.5
8-d
desoxygartanin
16.0
3-is
so-mangostin
0
UV_VIS_1
CAD_1
9-hydroxycalabaxanthone
9
1 - 20130617 #62 [normalized]
2 - 20130617 #62
pA
angostin
alpha-ma
60.0
25.0

A 30 min ASE method for the extraction pf xanthones from mangosteen pericarp
powder was developed . Extraction efficiency was above 96% with excellent
reproducibility.

A 25 min gradient UHPLC-UV method was developed for the analysis of
extracted samples and the yield of five selected xanthones was quantitatively
determined.
R f
References
1. Pedraza-Chaverri, J.; Cárdenas-Rodríguez, N.; Orozco-Ibarra, M.; Pérez-Rojas,
J.M. Medicinal properties of mangosteen (Garcinia mangostana). Food and
Chemical Toxicology. 2008, 46, 3227-3239.
2. Jung, H.A.; Su, B.N.; Keller, W.J.; Mehta, R.G.; and Kinghorn, A.D. Antioxidant
xanthones from the pericarp of Garcinia mangostana (Mangosteen). J. Agric
Food Chem 2006: 54, 2077-2082.
3-isomangostin
8-desoxygartanin
gartanin
alpha-mangostin
9-hydroxycalabaxanthone
60
3. Kosem, N.; Youn-Hee, H.; and Moongkarndi, P. Antioxidant and cytoprotective
activities of methanolic extract from Garcinia mangostana hulls.
hulls Sci Asia.
Asia . 2007,
2007
33, 83-292
4. Walker, E.B. HPLC analysis of selected xanthones in mangosteen fruit. J. Sep
Sci. 2007, 30, 1229-34.
p , W. HPLC quantitative
q
analysis
y for the
5. Pothitirat,, W. and Gritsanapan,
determination of α-mangostin in mangosteen fruit rind extract. Thai J. Agric Sci.
2009, 42, 7-12.
6. Zarena, A.S. and Udaya, S.K. Screening of xanthones from mangosteen
(Garcinia mangostana L.) peels and their effect on cytochrome c reductase and
phosphomolybdenum activity.
activity J.
J Nat
Nat. Prod
Prod. 2009,
2009 2,
2 23
23-30.
30
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might
infringe the intellectual property rights of others.
PO70991_E 03/14S
6 Fast Analysis of Selected Xanthones in Mangosteen Pericarp Using Accelerated Solvent Extraction and Ultra High Performance Liquid Chromatography
www.thermoscientific.com
©2014 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization.
All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as
an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products
in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to
change. Not all products are available in all countries. Please consult your local sales representative for details.
Africa +43 1 333 50 34 0
Australia +61 3 9757 4300
Austria +43 810 282 206
Belgium +32 53 73 42 41
Brazil +55 11 3731 5140
Canada +1 800 530 8447
China 800 810 5118 (free call domestic)
400 650 5118
Denmark +45 70 23 62 60
Europe-Other +43 1 333 50 34 0
Finland +358 9 3291 0200
France +33 1 60 92 48 00
Germany +49 6103 408 1014
India +91 22 6742 9494
Italy +39 02 950 591
Japan +81 6 6885 1213
Korea +82 2 3420 8600
Latin America +1 561 688 8700
Middle East +43 1 333 50 34 0
Netherlands +31 76 579 55 55
New Zealand +64 9 980 6700
Norway +46 8 556 468 00
Thermo Fisher Scientific, Sunnyvale, CA
USA is ISO 9001:2008 Certified.
Russia/CIS +43 1 333 50 34 0
Singapore +65 6289 1190
Sweden +46 8 556 468 00
Switzerland +41 61 716 77 00
Taiwan +886 2 8751 6655
UK/Ireland +44 1442 233555
USA +1 800 532 4752
PN70991_E 02/14S