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 m 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 Parkinsons 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
-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

50C, PFV 1.0

Column: Thermo Scientif

Temperature:

30 C

Mobile Phase A:

water

Mobile Phase B:

90%a

Gradient: 020 min: 50 -9

Flow Rate:

0.5 m

Injection Volume:

10 L

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: 80C
Static time:

5min

Static cycle:

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 m 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

80C 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,
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 g/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

50C, PFV 1.0
Column: Thermo Scientific Acclaim 120 C18, 2.2m, 2.1 x 100 mm
Temperature:

30 C

Mobile Phase A:

water

Mobile Phase B:

90%acetonitrile, 10% methanol

Gradient: 020 min: 50 -90%B; hold at 90% B for 5min

Flow Rate:

0.5 mL/min

Injection Volume:

10 L
Table 1. Extraction efficiency

Data Analysis
Thermo Scientific Dionex Chromeleon Chromatography Data System software,
software 7.1
71
SR1 L.

80C
80
C, 3 cycles
80C, 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

-50

-100

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
80C 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, -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 g/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

80C
80
C, 3 cycles

90 0%
90.0%

91 3%
91.3%

90 6%
90.6%

80C, 4 cycles

96.0%

98.4%

96.5%

25.0

100

50

-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

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

2.2m, 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

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

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

10

20
30
40
Concentration (g/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 2009, 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

12.5
UV_VIS_1

9-hydroxycala
abaxanthone

WVL:320 nm

0.0

Corona Veo

CAD

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.

37.5

8-d
desoxygartanin

16.0

3-is
so-mangostin

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.; Crdenas-Rodrguez, N.; Orozco-Ibarra, M.; Prez-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 -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

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