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Research Article

Received: 15 November 2008,

Accepted: 11 December 2008

Published online 8 April 2009 in Wiley Interscience

(www.interscience.wiley.com) DOI 10.1002/bmc.1207

Metabolite analysis in Curcuma domestica


using various GC-MS and LC-MS separation
and detection techniques
John Wiley & Sons, Ltd.

Diran Herebian,a Jeong-Heui Choi,b A. M. Abd El-Aty,c,d Jae-Han Shimb and


Michael Spitellera*
ABSTRACT: The metabolic profile of polar (methanol) and non-polar (hexane) extracts of Curcuma domestica, a widely used
medicinal plant, was established using various different analytical techniques, including GC-FID, GC-MS, HR-GC-MS and
analytical HPLC-ESI-MS/MS by means of LTQ-Orbitrap technology. The major non-volatile curcuminoids curcumin,
demethoxycurcumin and bisdemethoxycurcumin were identified when their chromatographic and precursor ion masses
were compared with those of authentic standard compounds. In this paper we describe for the first time a GC/MS-based
method for metabolic profiling of the hydrophilic extract. We also identified 61 polar metabolites as TMS derivatives.
Copyright 2009 John Wiley & Sons, Ltd.
Keywords: Curcuma domestica; LTQ-Orbitrap; metabolite profile; (HR)-GC-MS; HPLC-ESI-MS/MS

Introduction

Biomed. Chromatogr. 2009; 23: 951965

* Correspondence to: M. Spiteller, Institute of Environmental Research,


University of Dortmund, D-44221 Dortmund, Germany. E-mail: spiteller@
infu.uni-dortmund.de
J.-H. Shim, Division of Applied Bioscience and Biotechnology, Chonnam
National University, Gwangju 500-757, Republic of Korea. E-mail: jhshim@jnu.ac.kr
a

Institute of Environmental Research, University of Dortmund, D-44221 Dortmund, Germany

Natural Products Chemistry Laboratory, Division of Applied Bioscience and


Biotechnology, Chonnam National University, 300 Yongbong-dong, Buk-gu,
Gwangju 500-757, Republic of Korea

Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong, Kwangjin-gu, Seoul
143-701, Republic of Korea

Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12211-Giza, Egypt

The first two authors contributed equally to this paper.


Abbreviations used: BDMC, bisdemethoxycurcumin; CID, collision-induced
dissociation; CURC, curcumin; DMC, demethoxycurcumin; HECD, higher
energy collisional dissociation; PFCA, perfluorocarboxylic acid; PFK,
perfluorokerosene.
Contract/grant sponsor: Korea Science and Engineering Foundation;
Contract/grant number: F03-2007-000-10020-0.

Copyright 2009 John Wiley & Sons, Ltd.

951

Turmeric (Curcuma domestica) is a widely cultivated tropical


plant of southern Asia and Central America (Yuan et al., 2005). Its
powder has long been used as a spice, coloring agent, cosmetic
and medicinal agent in Asian and Eastern cultures (Tilak et al.,
2004). It is very popular in Asian medicine for the treatment of
coryza, hepatic disorders and rheumatism (Ammon and Wahl,
1991). In Europe the rhizomes of Curcuma xanthorrhiza and C.
domestica are used in phytotherapy (Wichtl, 2002). C. domestica
contains the major diarylheptanoids, curcuminoids (curcumin,
monodemethyoxycurcumin, and bisdemethyoxycurcumin) in
the range of 2.99.1% (dry weight) (Lechtenberg et al., 2004;
Dairam et al., 2007). Curcuminoids have been shown to possess
many pharmacological properties including anti-inflammatory
and hepatoprotective activities (Srimal and Dhawan, 1973;
Hnsel, 1997; Lukita-Atmadja et al., 2002), antioxidant and cholekinetic activities (Masuda et al., 1992, 1999; Hnsel, 1997; Rasyid
et al., 2002), and anti-protease activity (Nishigaki et al., 1992; Sui
et al., 1993). In addition, they have been shown to induce apoptosis in human cancer cells (Choudhuri et al., 2002) and act as a
chemopreventive agents for major cancer targets, including the
breast, prostate, lung, stomach, duodenal and colon cancers, as
well as leukemias (Kim et al., 1998; Kelloff et al., 2000; Shao et al.,
2002; Duvoix et al., 2005), and display neuroprotective effects
(Lee et al., 2007). It has also been reported that curcumin is a
more potent free radical scavenger than vitamin E (Zhao et al.,
1989). More recently, the methanolic extract of Curcuma domestica was shown to have a better molluscicidal activity against
Bulinus camerunensis than aqueous extracts (Ndamukong et al.,
2006). However, it is still not known if the above-mentioned
compounds are the only diarylheptanoids responsible for the
activity observed in the Curcuma species (Jiang et al., 2006a).
Various methods for the analysis of curcuminoids, such as
HPLC (Tnnesen and Karlsen, 1983; Smith and Witowska, 1984;

Khurana and Ho, 1988; Cooper et al., 1994; Jayaprakasha et al.,


2002), HPLC-ESI-MS (qualitative analysis; He et al., 1998), HPLC/
GC-MS (Hiserod et al., 1996), HPLC-photodiode array detection
(Bos et al., 2007) and capillary electrophoresis (Lechtenberg
et al., 2004), have been described in previous studies. The main
objective of this study was to analyse the polar (methanol) and

D. Herebian et al.
non-polar (hexane) extracts obtained from the Curcuma species, C. domestica, using various different analytical hyphenated
detection techniques, such as GC/MS and LC-MS, which offer
clear advantages compared with the previously described methods.

Experimental
Chemicals and Reagents
Commercially available curcumin, which consists of a mixture of
three naturally occurring curcuminoids, curcumin (1) (70%), demethoxycurcumin (2) (23%) and bisdemethoxycurcumi (3) (7%), was
obtained from Sigma (St Louis, MO, USA). Methanol and acetonitrile
(HPLC grade) were purchased from Baker (Deventer, The Netherlands). n-Hexane was supplied by Merck KGaA (Darmstadt,
Germany). Formic acid and ammonium acetate (analytical grade)
were supplied by Fluka (Riedel-de Han, Seelze, Germany).
Deionized water was re-distilled in all experiments. All standards
used to confirm the identity of metabolites from GC-MS measurements as well as derivatization reagents (MSTFA and methoxyamine hydrochloride) were obtained from Sigma (St Louis,
MO, USA).
Plant Material
The fresh rhizomes of C. domestica were brought to the laboratory,
Natural Products Chemistry, Institute of Agricultural Science and
Technology, Chonnam National University, Gwangju, Republic of
Korea, on February 2005 from a local herbal market in Phnom
Pemh city, the capital of Cambodia. All rhizome samples were
chopped into small pieces and air-dried.
Extraction Procedures
Dried rhizomes of C. domestica (1.95 kg) were extracted with 80%
aqueous methanol (7 L) for 30 days. One liter of the 80% aqueous
methanol extract was taken and partitioned with hexane (1 L).
The aqueous methanol layer was freeze-dried in order to obtain
5.05 g of solid material, whereas the hexane-soluble part was
concentrated using a rotary evaporator (Bchi Rotavapor R-114,
Germany) at 40C (Bchi Waterbath B-480, Germany) resulting in
a yield of 8.99 g of solid material.
Microcapillary HPLC- LTQ-Orbitrap

952

The ESI-FT-MS/MS spectra were obtained with an LTQ-Orbitrap


mass spectrometer (Thermo Scientific, Waltham, MA, USA). The
spectrometer was operated in positive ion mode (1 spectrum/s;
mass range 50 1000) with a nominal mass resolving power
of 60,000 at m/z 400 and a scan rate of 1 Hz. Automatic gain control and three internal standardsbis(2-ethylhexyl)phthalate,
m/z = 391.284286, polydimethylcyclosiloxane-[(CH3)2SiO]6, m/z =
445.120025 and polydimethylcyclosiloxane-[(CH3)2SiO]7, m/z =
519.138816were used to provide high-accuracy mass measurements within 2 ppm deviation. In case a negative ionization
process occurred we used a PFCAs (perfluorocarboxylic acids)
solution mixture for the external calibration mode.
MS/MS experiments were performed using CID (collisioninduced dissociation) at an energy of 35 eV for the positive
ionization mode and by HCD (higher energy collisional dissociation) at an energy of 50 eV in the case of negative ionization
mode. Argon was used as a collision gas.

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The spectrometer was equipped with a Dionex HPLC system


Ultimate 3000 (Sunnyvale, CA, USA), which contained a pump,
UVvis detector ( = 430 nm), flow manager, and autosampler
(injection volume 1 L). Nitrogen was used as the sheath gas
(5 arbitrary units) and helium served as the collision gas. The
separations were performed using a Phenomenex Gemini C18
column (150 0.3 mm, 3 m; Torrance, CA, USA) with a water
(+0.1% HCOOH) (A)acetonitrile (+0.1% HCOOH) (B) gradient
(flow rate 4 L/min). Samples were analyzed using a gradient
program as follows: 100% (A) isocratic for 4 min, linear gradient
to 100% (B) over 16 min; after 100% (B) isocratic for 10 min, the
system returned to its initial condition (100% A) within 1 min,
and was then equilibrated for 9 min.
Analytical HPLC-MS/MS
HPLC analyses of the samples were performed using a Thermo
Finnigan Surveyor HPLC system (Thermo Fisher Scientific, Waltham,
MA, USA), which comprised a Surveyor MS-Pump, Surveyor
Autosampler-Plus (injection volume 5 L), and Surveyor-Plus
PDA detector (Thermo Fisher Scientific, Waltham, MA, USA). The
compounds were separated on a Synergi Fusion RP80 (150
3 mm, 4 m particle size) column from Phenomenex (Torrance,
CA, USA) and peaks were detected at 430 nm. The mobile phase
consisted of water containing 1 mM ammonium acetate and
0.1% HCOOH (A) and acetonitrile +0.1% HCOOH (B). Curcuminoids were separated using a gradient program as follows: (flow
rate of 200 L/min) 100% A isocratic for 4 min, linear gradient
to 100% B over 16 min, after 100% B isocratic for 10 min, the
system returned to its initial condition (100% A) within 1 min,
and was then equilibrated for 9 min before the next run restarted.
MS detection was performed using a TSQ Quantum Ultra AM
(Thermo Scientific, Waltham, MA, USA) equipped with an ESI ion
source operating in the negative mode. Nitrogen was employed
as both the sheath (60 arbitrary units) and auxiliary (1 arbitrary
units) gas and argon served as the collision gas with a pressure
of 1.5 mTorr. The capillary temperature for the TSQ was set to
330C, and the source voltage was set at 4.3 kV.
Quantitative determination of the three curcuminoids (1, 2
and 3) was done in the negative ESI-mode by introducing a total
flow of 200 L/min into the mass spectrometer. The standard
calibration curve for each compound was achieved from 11
standard diluted mixtures (1, 5, 10, 25, 50, 100, 200, 500, 1000,
2000 and 5000 ng/mL). Fragmentation chromatograms were
obtained by operating in the selected reaction monitoring
(SRM) mode and measuring the transitions of the two most
intense and specific fragment ions of the precursor ion (mass
window: 0.5 u). Eluted compounds of the curcuminoids (ESI
pos/neg) and their fragmentation ions were identified and confirmed in according to Jiang et al. (2006a, b).
GC-FID
A Thermo Finnigan Trace Gas Chromatograph (Thermo Fisher
Scientific Inc., Waltham, MA, USA), equipped with a fused
silica capillary column (J&W Scientific, DB-5MS, 30 m 0.25 mm
0.25 m) and FID was used for relative quantification of the
hexane extract. The carrier gas was helium (1 mL/min) and the
following temperature program was used: 60C (1 min), 60
175C at 20C/min, 175260C at 4C/min and 260C (2 min).
Injector temperature 310C; detector temperature 320C; splitless mode.

Copyright 2009 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2009; 23: 951965

Metabolite analysis in Curcuma domestica

GC-MS and High-resolution GC-MS


Analyses of the samples were performed on a Thermo Finnigan
Trace Gas Chromatograph and Trace MSPLUS detector (Thermo
Fisher Scientific Inc., Waltham, MA, USA), equipped with a fused
silica column (J&W Scientific, HP-5MS, 30 m 0.25 mm 0.25 m)
with a siltek deactivated guard column (Restek, Bellefonte, PA,
USA, 10 m 0.53 mm); carrier gas helium (1 mL/min); injection
volume 1 L; temperature programme for the non-polar phase:
60C (5 min), 60300C at 4C/min, 300C (25 min); splitless
mode; temperature program for the polar phase: 70C (1 min),
70 76C at 1C/min, 76325C at 6C/min, 325C (10 min); split,
25 mL/min. Source temperature 200C; interface temperature
250C; detector voltage 350 V; emission current 150 A; electron
energy, 70 eV; scan, m/z 40580.
The raw data files were processed with the Xcalibur (version
2.0) and AMDIS (automated mass spectral deconvolution and
identification system, version 2.65) software supplied by NIST
(National Institute of Standards and Technology, Gaithersburg,
MD, USA). The recorded spectra were first compared with a commercial available library (NIST02) and then together with the
corresponding RI values of an internally compiled library. The
identity of compounds that were not stored in the internal
library was determined using commercially available reference
compounds. The identity of substances not commercially available (non polar phase) was confirmed by comparing their fragmentation and intensities with those in the literature.
Accurate mass measurements were obtained for non-polar
compounds (hexane extraction) using DFS high-resolution
GC/MS (Thermo Fischer Scientific). We used perfluorokerosene
(PFK) as a mass calibration reagent. The temperature program
and capillary column were the same as discussed above.
Metabolite Derivatization
The methanolic extract was gently dried under a nitrogen stream
at 40C. Residues were derivitized in a two-step procedure as
follows: 50 L of methoxyamine hydrochloride (20 mg/mL in
pyridine) were added and the solution was heated to 40C for
1 h. Silylation was carried out by addition of 50 L MSTFA (Nmethyl-N-trimethylsilyl-trifluoroacetamide) at 50C. Finally 20 L
of an alkane-mixture (C10, C12, C14, C16, C18, C20, C22, C24,
C26, C28, C30, C32, C34, and C38; 0.25 mg/mL in hexane) were
added to determine of the RI values of the eluted compounds.

Results and Discussion


Analysis of Polar Metabolites by GC-MS and GC-FID

Biomed. Chromatogr. 2009; 23: 951965

Analysis of Non-polar Metabolites by GC-FID, GC-MS, and


HR-GC-MS
In general, gas chromatographymass spectrometry is still regarded
as the preferred method for measuring volatile non-polar compounds. The underivatized hexane fraction was subjected to GCMS analysis and three major peaks were detected. These major
constituents were identified as -turmerone, ar-turmerone and
-turmerone based on their mass fragmentation pattern, retention times, and retention indices. A typical chromatogram of this
extract is shown in Fig. 2.
In addition, we were able to identify eight other compounds,
which were present at minor levels. The identification of these
compounds is listed in Table 3.
GC-FID based relative quantification of these volatile compounds generated from the non-polar extract (hexane fraction)
revealed a high proportion of -turmerone (20.1%), ar-turmerone
(19.5%) and -turmerone (17.6%) (Table 3).
It has previously been shown that turmeric oil contains mainly
phyto-pharmaceuticals that are quite similar to turmerones
(Honwad and Rao, 1964). Turmerone is a kind of sesquiterpenoid
cyclic ketone (Chang et al., 2006). As reported by (He et al., 1998),
sesquiterpenoids are the major constituents of turmeric oil and
ar-turmerone is the predominant turmerone, followed by turmerone and -turmerone. The three turmerones have similar
chemical structures, physical properties and molecular weights,
even though they have different tastes (Kao et al., 2007). Since
herbal medicines constitute a rich source of bioactive chemicals
that are largely free from adverse effects, plant extracts may
have excellent pharmacological properties. For instance, it was
reported that ar-turmerone is the best local treatment for
edema, necrosis and local hemorrhage after Bothrops alternatus
envenomation (Melo et al., 2005). Moreover, ar-turmerone has
been shown to display antiplatelet activity and is a more potent
platelet inhibitor against platelet aggregation induced by collagen than aspirin (Lee, 2006). In addition ar-turmerone is
assumed to improve insulin resistance and ameliorate type 2
diabetes mellitus through the same biological mechanism as
thiazolidinedione derivatives (Kuroda et al., 2005). Furthermore,
the insect repellent and antifeeding properties of Curcuma have
been attributed to turmerones (Su et al., 1982; Lee et al., 2001)
and curcuminoids (Chowdhury et al., 2000). Therefore, we
believe that turmerone could be used as an alternative to

Copyright 2009 John Wiley & Sons, Ltd.

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953

The polar extract of C. domestica is composed of multiple classes


of chemical compounds (e.g. amino acids, organic acids, sugars,
sugar alcohols, sugar phosphates, aromatic amines). The polar
functional groups were modified in order to make these various
classes of compounds volatile and thus accessible for GC-MS
analysis. The derivatization reagent MSTFA [N-methyl-N-(trimethylsilyl)trifluoroacetamide], which is able to react with a
wide range of compound classes, was used to achieve this goal.
The carbonyl groups in sugars and their derivatives also required
chemical modification before GC-MS analysis. The methoximation reaction prevents ring formation by reducing sugars and
stabilizing the carbonyl moieties in the -position. This reaction
produces two different stereoisomers (syn and anti) that are well

resolved by gas chromatography. A GC-MS total ion chromatogram from the methanolic polar phase is shown in Fig. 1.
The chromatogram is made up of a complex pattern of major,
minor and trace peaks. In order to identify the chemical nature
of each peak their recorded spectra were compared with commercially available mass spectral libraries such as NIST or Wiley.
In addition, determination of retention indices (RI), which were
scaled with a time standard mixture of 14 hydrocarbons from an
internally compiled library, was also done for all detected compounds after their mass spectra had been deconvoluted using
the software AMDIS. The six major compounds identified were
glycerol, malic acid, citric acid, fructose, glucose, and sucrose. A
further 55 analytes of known chemical structure as TMS derivatives were identified from the minor and trace peaks (Table 1).
The chemically modified extract was also analysed by a GCFID-based method under nearly the same chromatographic
conditions used in GC-MS. The calculated relative areas of the
detected compounds are listed in Table 2.

D. Herebian et al.

Figure 1. GC-MS chromatographic profile of polar metabolites as TMS derivatives from the methanolic extract of
Curcuma domestica.

954

synthetic agrichemicals, which are used to control insects/pests,


diseases and repellents of house hold insects including mosquitoes, as aromatherapy and antiseptic agents. This would in
turn, reduce the public health hazards derived from the use of
agrichemicals.
Cineole, the monoterpene cyclic ether known as eucalyptol,
was also found in very low amounts (<1%) in the Curcuma
extracts. This component might contribute to the characteristic
fresh and camphoraceous fragrance and pungent taste of Curcuma.
As a result of this, Curcuma extracts could also be incorporated
in pharmaceutical formulations for use as an external applicant,
nasal spray, cosmetic, food flavoring and analgesic, as well as disinfectants (Miyazawa et al., 2001).
Defense responses in plants against insects are generally triggered by volatiles (Langenheim, 1994; Pare and Tumlinson,
1999; Pichersky and Gershenzon, 2002). The sesquiterpene Z-farnesene can act as a semiochemical, providing built-in protection against invading organisms (Maruyama et al., 2001).
Although a-terpinolene was found in very low concentrations
(<1%), it was previously reported that the main larvicidal (mosquito)
effect may have originated from minor constituents such as 3carene, terpinolene, -terpinene and -terpinene (Cheng et al.,
2009).
Both antimicrobial and insecticidal activities have been reported
for -himchalene as well as its oxygenated derivatives (Singh

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and Agarwal, 1988). Typical ginger oil has been characterized


as having a high content of sesquiterpene hydrocarbons, in
particular zingiberene, ar-curcumene, -bisabolene and sesquiphellandrene (Vernin and Parkanyi, 1994; Martins et al.,
2001). In the present study, the proportion of the four major
curcumenes was sesquiphellandrene (4.1%), curcumene (2.3%),
zingiberene (2.3%) and bisabolene (<1%). However, these values
were relatively lowzingiberene (4.8 2.0%), ar-curcumene
(3.2 0.7%), -sesquiphellandrene (4.4 0.9%) and -bisabolene
(1.5 0.3%)compared with ginger (Zingiber officinale Roscoe)
grown in Australia (Wohlmuth et al., 2006).
Accurate mass measurements were performed on DFS highresolution GC-MS to confirm the identity of the 11 analytes
detected. The exact measured and calculated masses, in addition to the sum formulae and mass accuracy, (expressed in
parts per million) of the identified compounds are summarized
in Table 4. In general, an accuracy of 3 ppm was achieved for all
11 detected compounds using PFK as the mass calibration
reagent.
Microcapillary HPLC-MS and -MS/MS Analysis
To confirm the identity of the precursor and product ions generated
by the linear ion trap, high-resolution and accurate mass measurements were performed using an Orbitrap mass spectrometer

Copyright 2009 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2009; 23: 951965

Metabolite analysis in Curcuma domestica

Table 1. Identified polar metabolites by GC-MS as TMS/MeOX derivatives from the methanolic extract
with their sum formulae, molecular weights and ionization form
Compounds

Biomed. Chromatogr. 2009; 23: 951965

Sum formulas

MW [g/mol]

[M]+

[M15]+

3
3
4
5/1
5/1
8
2
2
2
2
1
2
3
2
3
2
2
3
2
4
4
3
5
2
2
2
6
6
2
6
2
2
2
3
2/1
2
3
3
3
2
2
5
4/1
4
2
2
3
3
2
3
3
4
5
4
2
3
8
2
4
2
1

C12H32O3Si3
C13H30O5Si3
C18H40O7Si4
C22H55NO6Si5
C22H55NO6Si5
C36H86O11Si8
C9H22O3Si2
C9H23NO2Si2
C8H18O4Si2
C9H20O4Si2
C8H24O2Si2
C11H27NO2Si2
C9H27O4PSi3
C10H22O4Si2
C12H30O4Si3
C10H20O4Si2
C10H23NO4Si2
C13H31NO4Si3
C11H23NO3Si2
C16H42O4Si4
C16H40NO5Si4
C14H33NO4Si3
C20H52O5Si5
C15H26O3Si2
C14H24O4Si2
C15H24O3Si2
C24H62O6Si6
C24H60O7Si6
C16H26O4Si2
C24H60O6Si6
C9H24O2Si2
C8H20O3Si2
C12H29NO2Si2
C9H27NOSi3
C8H21NO3Si2
C9H23NO3Si2
C12H31NO3Si3
C11H29NO2Si3
C13H33NO3Si3
C13H22O3Si2
C14H24O3Si2
C20H52O5Si5
C19H47NO5Si4
C15H41O6PSi4
C15H27NO3Si2
C16H28O4Si2
C13H33NO2Si3
C14H32O5Si3
C13H33NO2Si3
C13H32N2O3Si3
C15H30O6Si3
C16H40O5Si4
C20H50O6Si5
C19H42O5Si4
C17H28N2O2Si2
C18H36N2O6Si3
C36H86O11Si8
C12H29NO2Si2
C21H50N2O6Si4
C7H21O4PSi2
C8H13NOSi

308
350
480
569
569
918
234
233
234
248
189
261
314
262
322
260
277
349
273
410
424
363
512
310
312
308
614
628
338
612
220
220
275
249
235
249
321
291
335
282
296
512
481
460
325
340
319
364
291
348
462
424
526
462
348
460
918
275
538
256
167

350

569
569

234
233
234
248
189
261
314
262
307
260
277
349
273
410
424
363

310
312
308

338
612

275
249
235
249

291

282
296

340
319

348
462
424

462
348

275

256
167

293
335
465
554
554

219
218
219
233
174
246
299
247
307
245
262
334
258
395
409
348
497
295
297
293

323

205
205
260

220
234
306
276
320
267
281
497

445
310
325
304
349
276
333
447
409
511
447
333
445

260
523
219
152

955

1. Glycerol
2. Malic acid
3. Citric acid
4. Fructose
5. Glucose
6. Sucrose
7. Lactic acid
8. L-Alanine
9. Oxalic acid
10. Malonic acid
11. L-Valine
12. L-Valine
13. Phosphoric acid
14. Succinic acid
15. Glyceric acid
16. Fumaric acid
17. L-Aspartic acid
18. L-Aspartic acid
19. 5-Oxo-proline
20. Erythritol
21. L-Threonic acid
22. L-Glutamic acid
23. Xylitol
24. p-Hydroxycinnamic acid
25. Vanillic acid
26. p-Coumaric acid
27. Glucitol
28. Gulonic acid
29. trans-Ferulic acid
30. myo-Inositol
31. 1,3-Propandiol
32. Acetic acid
33. L-Leucine
34. Hydroxylamine
35. Carbonic acid
36. L-Serine
37. L-Serine
38. L-Glycine
39. L-Threonine
40. p-Hydroxybenzoic acid
41. p-Hydroxyphenylacetate
42. Ribitol
43. 6-Deoxy-galactose
44. -Glycerophosphate
45. L-Tyrosine
46. Dihydroferulic acid
47. -Alanine
48. Citramalic acid
49. NAcetyl-L-serine
50. L-Asparagine
51. Aconitic acid
52. Tetronic acid
53. Ribonic acid
54. Shikimic acid
55. L-Tryptophane
56. Uridine
57. Trehalose
58. L-Isoleucine
59. N-Acetylglucoseamine
60. Monomethylphosphate (A)
61. 2-Hydroxypyridin (A)
A, Artifact.

TMS/MeOX

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D. Herebian et al.

Table 2. Identified compounds by GC-MS as TMS/MeOX derivatives from the methanolic extract
including their retention times, retention indices. Relative areas are based on GC-FID measurements
TMS/MeOX

RT

RI

Relative area
(%) (GC-FID)

Identified major compounds (GC-MS, >50% relative abundance)


1. Glycerol
3TMS
15.2
2. Malic acid
3TMS
20.0
3. Citric acid
4TMS
26.4
4. Fructose 1
5TMS 1MeOx
27.5
Fructose 2
5TMS 1MeOx
27.7
5. Glucose 1
5TMS 1MeOx
27.9
Glucose 2
5TMS 1MeOx
28.2
6. Sucrose
8TMS
38.7

1291.5
1514.3
1851.0
1918.0
1928.4
1943.5
1959.5
2716.2

7.26
5.24
3.76
7.83
6.45
11.10
2.08
9.46

Identified minor compounds (GC-MS, 550% relative abundance)


7. Lactic acid
2TMS
9.3
2TMS
10.5
8. L-Alanine
9. Oxalic acid
2TMS
11.4
10. Monomethylphosphate (A)
2TMS
12.6
11. Malonic acid
2TMS
13.3
2TMS
13.6
12. L-Valine
13. Phosphoric acid
3TMS
15.1
14. Succinic acid
5TMS
15.9
15. Glyceric acid
3TMS
16.5
16. Fumaric acid
2TMS
16.7
2TMS
18.4
17. L-Aspartic acid
18. 5-Oxo-proline
2TMS
20.5
19. Erythritol
4TMS
20.6
3TMS
20.7
20. L-Aspartic acid
4TMS
21.3
21. L-Threonic acid
22. L-Glutamic acid
3TMS
22.6
23. Xylitol
5TMS
24.8
24. p-Hydroxycinnamic acid
2TMS
24.9
25. Vanillic acid
2TMS
25.1
26. p-Coumaric acid
2TMS
28.0
27. Glucitol
6TMS
28.4
28. Gulonic acid
6TMS
29.6
29. trans-Ferulic acid
3TMS
30.4
30. myo-Inositol
6TMS
30.8

1062.1
1103.4
1135.2
1179.0
1201.4
1215.1
1289.6
1324.9
1353.6
1360.0
1441.3
1536.7
1541.7
1543.1
1574.3
1638.6
1758.4
1768.9
1774.7
1948.0
1974.6
2048.0
2104.9
2133.1

1.52
0.41
1.56
1.18
0.40
0.12
1.05
1.39
0.49
0.54
0.83
1.29
0.39
1.33
0.47
0.37
2.03
2.80
0.17
1.28
2.33
2.20
0.61
1.43

Identified trace compounds (GC-MS, <5% relative abundance)


31. 2-Hydroxypyridin (A)
1TMS
8.3
32. 1,3-Propandiol
2TMS
9.1
33. Acetic acid
2TMS
9.7
1TMS
9.9
34. L-Valine
35. Hydroxylamine
3TMS
10.7
36. Carbonic acid
2TMS 1MeOX
11.6
2TMS
14.7
37. L-Serine
38. L-Leucine
2TMS
15.0
2TMS
15.5
39. L-Isoleucine
40. L-Glycine
3TMS
15.8
41. L-Serine
3TMS
17.2
3TMS
17.8
42. L-Threonine
43. -Alanine
3TMS
18.5
44. Citramalic acid
3TMS
19.6
2TMS
20.3
45. N-acetyl-L-serine
46. Tetronic acid
4TMS
21.6
47. p-OH-benzoic acid
2TMS
22.5
48. p-OH-phenylacetate
2TMS
22.7
3TMS
23.5
49. L-Asparagine

1029.5
1054.3
1076.6
1083.5
1110.8
1141.3
1265.1
1281.5
1305.6
1317.7
1384.7
1413.0
1445.0
1495.6
1527.3
1590.0
1633.7
1648.1
1690.3

956
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Copyright 2009 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2009; 23: 951965

Metabolite analysis in Curcuma domestica

Table 2. (Continued)
TMS/MeOX
50. Ribitol
51. 6-Deoxy-galactose
52. Aconitic acid
53. -Glycerophosphate
54. Ribonic acid
55. Shikimc acid
56. L-Tyrosine
57. Dihydroferulic acid
58. N-Acetyl-glucosamine
59. L-Tryptophane
60. Uridine
61. Trehalose

5TMS
4TMS 1MeOX
3TMS
4TMS
5TMS
4TMS
2TMS
2TMS
4TMS
2TMS
3TMS
8TMS

RT

RI

Relative area
(%) (GC-FID)

24.3
24.7
24.9
25.5
25.6
26.2
27.2
27.3
30.7
32.1
35.4
39.9

1734.2
1754.2
1768.6
1797.3
1802.7
1840.0
1898.5
1908.3
2125.7
2219.5
2475.5
2818.8

TMS, trimethylsilyl. MeOx, methoxime (this procedure serves to avoid sugar ring cyclization and produces two forms of peaks, syn and anti). A, artifact.

Figure 2. GC-MS chromatographic profile of non-polar metabolites from the hexane extract of Curcuma
domestica.

Biomed. Chromatogr. 2009; 23: 951965

3 (and its metabolite, dihydro-bisdemethoxycurcumin 6), respectively (Fig. 3).


The precursor ions of curcuminoids were detected as [M + H]+
at m/z 369.13315 for 1, 339.12258 for 2 and 309.11198 for 3, or
as [MH] at m/z 367.11746 for 1, 337.10733 for 2 and 307.09808
for 3, respectively (Tables 5 and 6).
The stable chemical structures of these diarylheptanoids
exhibited a mass difference of 30 Da between CURC/DMC and
DMC/BDMC, suggesting the lack of a methoxy group. For the

Copyright 2009 John Wiley & Sons, Ltd.

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957

at a resolving power of 60,000 in internal (for positive ionization) or external (for negative ionization) calibration mode. The
high-resolution full-scan in a positive ion mode (scan range
m/z 85 380) was used to identify curcuminoids and their
metabolites. The full-scan mass spectra of the curcuminoid and
their metabolite fractions detected the presence of 21, 20 and
19 carbons for curcumin (CURC) 1 (and its metabolite, dihydrocurcumin 4), demethoxycurcumin (DMC) 2 (and its metabolite,
dihydro-demethoxycurcumin 5) and bisdemethoxycurcumin (BDMC)

D. Herebian et al.

Table 3. Identified compounds by GC-MS from the hexane


extract including their retention times, retention indices, sum
formulae and molecular weights. Relative areas are based on
GC-FID measurement
Name

RT

RI

Formula MW Relative
area (%)

Major compounds >5%


ar-Turmerone
31.3 1672 C15H20O
-Turmerone
31.4 1675 C15H22O
-Turmerone
32.3 1706 C15H22O

216
218
218

19.5
20.1
17.6

Minor compounds <5%


Cineole
a-Terpinolene
Z--Farnesene
-Himachalene
ar-Curcumene
-Zingiberene
-Bisabolene
-Sesquiphellandrene

154
136
204
204
202
204
204
204

<1
<1
<1
<1
2.3
2.3
<1
4.1

10.3
12,4
25.1
25,7
25.9
26.2
26.6
27.1

1048
1094
1086
1478
1481
1495
1508
1523

C10H18O
C10H16
C15H24
C15H24
C15H22
C15H24
C15H24
C15H24

curcuminoid homologs 4, 5 and 6 a mass shift of 2 amu was


observed, indicating that there were two additional hydrogen
atoms, which resulted from a reduction of a double bond on
the heptanoid skeleton. The same elemental compositions for
CURC, DMC and BDMC were observed in ESI-negative ionization
mode; however, none of the above-mentioned metabolites 4,
5 and 6 could be measured on account of the low sensitivity
of the negative ion mode in the Orbitrap mass spectrometer.
The same elemental compositions were observed in both the
LC positive and negative mode with a variation in the number of
hydrogen atoms (Tables 5 and 6). The values of retention times
of CURC, DMC and BDMC were 19.6, 19.4, and 19.2 min, respectively (Fig. 4).
These retention time shifts are in agreement with the corresponding molecular structures of 1, 2 and 3 on reversed-phase
HPLC using the solvent gradient profile described in the Experimental section. The fragmentation patterns of curcuminoids 1, 2
and 3 in HPLC-ESI(+)-MS/MS analysis showed some characteristic
fragment ions e.g. 285 (1), 255 (2) and 225 (3) m/z as shown in
Fig. 5. The observed decrease by 30 or 60 amu suggests the lack

of one or two methoxy groups of the curcuminoids on the aromatic rings. The MS/MS spectra were dominated by a couple of
major peaks at m/z 285, 245 and 175 for 1; 255, 245 and 175 for
2; and 225 and 147 for 3, respectively (Fig. 5). The expected loss
of water was observed in all three compounds at m/z 351 (1),
321 (2) and 291 (3). The proposed molecular structures of the
protonated major fragment ions are illustrated in Fig. 6. All other
fragments are listed in Table 7 and are in agreement with previously published results, which were based on accurate mass
FTICR-MS/MS measurements (Table 7).
The deprotonation of the curcuminoids could theoretically
occur at various positions e.g. on the keto and/or enol forms of
the heptanoid chain. In the LC-ESI-neg-MS analysis [MH] ions
were observed at m/z 367 (1), 337 (2) and 307 (3) as the phenolate, the enolate or a mixture of both forms. Additional details
regarding the deprotonation behavior of curcuminoids in the
ESI negative mode have been previously discussed (Jiang et al.,
2006a, b). The LC-ESI-neg-MS/MS spectra exhibited characteristic base peaks at m/z 134 and 158 for 1; 119 and 158 for 2; and
119, 143, and 187 for 3 (Fig. 7). The product ion spectra in the
negative mode were of high quality because of the presence of
multiple hydroxyl groups on the corresponding molecule, which
facilitates the negative ionization process in the electrospray.
Figure 7 reproduces the product ions of curcuminoids, which were
fragmented at 50 eV by HCD cell of the LTQ-Orbitrap. The HCD
spectra gave many more fragmentation peaks than the conventional CID technique. All other product ions are listed in Table 8
and the assignments of the base peaks are presented in Fig. 8.

Conclusions
Here, we report the development and application of a GC-MS
method for the identification of polar metabolites from the
methanolic extract of Curcuma domestica. To the best of our
knowledge this is the first report where this has been demonstrated. In addition we were able to identify 61 polar compounds
as TMS derivatives by means of their retention times, retention
indices and fragmentation patterns. A high-resolution GC-MS
method was also developed and utilized to confirm the identity
of the 11 non-polar compounds from the hexane extract. Both
methods can serve as analytical tools for the quality control of
various turmeric species (metabolic fingerprint) or for better
understanding in the biosynthetic pathways of turmeric and
their derivatives.

Table 4. Accurate mass measurements within 3 ppm error were obtained for non-polar compounds
(hexane-extraction) using DFS high-resolution GC/MS (Thermo Fischer Scientific)
Compounds

958

ar-Turmerone
-Turmerone
-Turmerone
Cineole
a-Terpinolene
Z--Farnesene
-Himachalene
ar-Curcumene
-Zingiberene
-Bisabolene
-Sesquiphellandrene

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RT

Formula

masstheor

massexp

ppm

31.3
31.4
32.3
10.3
12.4
25.1
25.7
25.9
26.2
26.6
27.1

C15H20O
C15H22O
C15H22O
C10H18O
C10H16
C15H24
C15H24
C15H22
C15H24
C15H24
C15H24

216.15087
218.16652
218.16652
154.13522
136.12465
204.18725
204.18725
202.17160
204.18725
204.18725
204.18725

216.15104
218.16712
218.16701
154.13532
136.12425
204.18771
204.18773
202.17212
204.18714
204.18766
204.18745

0.477
2.765
2.260
0.670
2.954
2.241
2.339
2.561
0.550
1.997
0.968

Copyright 2009 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2009; 23: 951965

Metabolite analysis in Curcuma domestica

Figure 3. Chemical structures of curcuminoids and their metabolites.

Table 5. Precursor ion masses of curcuminoids and their metabolites including elemental compositions determined by high resolution and accurate mass LTQ-Orbitrap measurements in ESI-positive ion mode
Compound, ionized form
Curcumin, [M + H]+
Demethoxycurcumin, [M + H]+
Bisdemethoxycurcumin, [M + H]+
Dihydro-curcumin, [M + H]+
Dihydro-demethoxycurcumin, [M + H]+
Dihydro-bisdemethoxycurcumin, [M + H]+

Ion

Calculated
mass

Measured
mass

ppm

Elemental
composition

1
2
3
4
5
6

369.13326
339.12270
309.11213
371.14891
341.13835
311.12778

369.13315
339.12258
309.11198
371.14890
341.13834
311.12773

0.115
0.051
0.156
0.015
0.011
0.056

C21H21O6
C20H19O5
C19H17O4
C21H23O6
C20H21O5
C19H19O4

Table 6. Precursor ion masses of curcuminoids including elemental compositions determined by high-resolution and accurate
mass LTQ-Orbitrap measurements in ESI-negative mode
Compound, ionized form
Curcumin, [MH]
Demethoxycurcumin, [MH]
Bisdemethoxycurcumin, [MH]

Ion

Calculated
mass

Measured
mass

ppm

Elemental
composition

1
2
3

367.11761
337.10705
307.09648

367.11746
337.10733
307.09808

0.155
0.280
1.594

C21H19O6
C20H17O5
C19H15O4

959

Biomed. Chromatogr. 2009; 23: 951965

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D. Herebian et al.

Figure 4. LC-MS full-scan chromatogram of curcuminoid mixture, containing curcumin 1, demethoxycurcumin 2 and
bisdemethoxycurcumin 3.

In addition, we used a high-resolution LC-MS/MS system based


on Orbitrap technology for the identification of the diarylheptanoids curcumin 1, demethoxycurcumin 2, bisdemethoxycurcumin 3, their metabolites and product ions. The corresponding
MS/MS data spectra were dominated by a couple of characteristic
major peaks and their sum formulae were used to determine the
molecular structures.
Acknowledgements
We are grateful to the Korean Science and Engineering Foundation (grant no. F03-2007-000-10020-0) for granting funds for the
study of Professor Jae-Han Shim in Germany. Moreover we are
grateful to the Ministry of Innovation, Science, Research and
Technology of the State of North Rhine-Westphalia for granting
a high-resolution mass spectrometer.

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Copyright 2009 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2009; 23: 951965

Metabolite analysis in Curcuma domestica

Figure 5. Product ion spectra of curcuminoids 1, 2 and 3 from positive ion LC-ESI-MS/MS measurements using the LTQ-Orbitrap mass spectrometer.

961

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Copyright 2009 John Wiley & Sons, Ltd.

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D. Herebian et al.

Figure 6.

Proposed molecular structures for fragment ions detected in LC-ESI(pos)-MS/MS measurements.

Table 7. Fragment ions of curcuminoids in positive ESI-mode using high resolution and
accurate mass LTQ-Orbitrap-MS/MS measurements (CID 35 eV)
Nominal mass
of fragment ions

Measured
mass

Calculated
mass

ppm

Elemental
composition

Curcumin 1
351
299
285
259
245
213
175

351.09250
299.12784
285.11246
259.09662
245.08067
213.05463
175.07536

351.12270
299.12778
285.08095
259.09648
245.08095
213.05462
175.07535

0.31
0.05
0.32
0.13
0.17
0.01
0.00

C21H19O5
C18H19O4
C17H17O4
C15H15O4
C14H13O4
C13H9O3
C11H11O2

Demthoxycurcumin 2
321
293
269
255
245
229
175
147

321.11276
293.11776
269.11673
255.10123
245.08095
229.08544
175.07512
147.04374

321.11213
293.11722
269.11723
255.10157
245.08083
229.08592
175.07535
147.04405

0.62
0.54
0.49
0.34
0.09
0.48
0.24
0.16

C20H17O4
C19H17O3
C17H17O3
C16H15O3
C14H13O4
C14H13O3
C11H11O2
C9H7O2

Bisdemethoxycurcumin 3
291
291.10178
263
263.10622
239
239.10631
225
225.09081
215
215.06998
189
189.05432
163
163.03851
147
147.04388
131
131.04894
107
107.04908

291.10157
263.10665
239.10665
225.09101
215.07027
189.05462
163.03897
147.04405
131.04914
107.04914

0.21
0.44
0.35
0.19
0.29
0.30
0.46
0.18
0.20
0.06

C19H15O3
C18H15O2
C16H15O2
C15H13O2
C13H11O3
C11H9O3
C9H7O3
C9H7O2
C9H7O1
C7H7O1

962
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Metabolite analysis in Curcuma domestica

Figure 7. Product ion spectra of curcuminoids 1, 2 and 3 from negative ion LCESI-MS/MS measurements using the LTQ-Orbitrap mass spectrometer.

963

Biomed. Chromatogr. 2009; 23: 951965

Copyright 2009 John Wiley & Sons, Ltd.

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D. Herebian et al.

Table 8. Fragment ions of curcuminoids in negative ESI-mode using high-resolution and


accurate mass LTQ-Orbitrap-MS/MS measurements (HCD 50 eV)
Nominal mass
of fragment ions

Calculated
mass

Measured
mass

ppm

Elemental
composition

Curcumin 1
201
175
173
160
158
149
134
132

201.01933
175.04007
173.06080
160.01659
158.03733
149.06080
134.03733
132.02168

201.01894
175.03957
173.06037
160.01628
158.03710
149.06136
134.03697
132.02143

1.97
2.81
2.53
1.96
1.44
2.91
2.65
1.91

C11H5O4
C10H7O3
C11H9O2
C9H4O3
C10H6O2
C9H9O2
C8H6O2
C8H4O2

Demethoxycurcumin 2
202
201
175
173
160
158
149
145
143
134
119

202.02716
201.01933
175.04007
173.06080
160.01659
158.03733
149.06080
145.02950
143.05024
134.03733
119.05024

202.02700
201.01875
175.03977
173.06025
160.01623
158.03697
149.06043
145.02906
143.04985
134.03693
119.04981

0.79
2.91
1.69
3.21
2.26
2.29
2.50
2.96
2.75
2.96
3.62

C11H6O4
C11H5O4
C10H7O3
C11H9O2
C9H4O3
C10H6O2
C9H9O2
C9H5O2
C10H7O
C8H6O2
C8H7O

Bisdemethoxycurcumin 3
187
187.04007
145
145.02950
143
143.05024
119
119.05024
117
117.03459
115
115.05532

187.03988
145.02924
143.05004
119.04997
117.03439
115.05519

1.01
1.80
1.37
2.22
1.67
1.16

C11H7O3
C9H5O2
C10H7O
C8H7O
C8H5O
C9H7

Figure 8. Proposed molecular structures for detected fragment ions from LC-ESI(neg)-MS/
MS measurements.

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Metabolite analysis in Curcuma domestica


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