LCMS/MS-based metabolites of Eurycoma longifolia (Tongkat Ali) in Malaysia (Perak and Pahang

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Journal of Chromatography B, 879 (2011) 3909– 3919

Contents lists available at SciVerse ScienceDirect

Journal of Chromatography B

j ourna l ho me page: www.elsev ier .com/ locate /chromb

LC–MS/MS­based metabolites of Eurycoma longifolia (Tongkat Ali) in Malaysia

(Perak and Pahang)

Lee Suan Chua a,∗ , Nor Amaiza Mohd Aminb , Jason Chun Hong Neo c , Ting Hun Lee a , Chew Tin Lee a ,Mohamad Roji Sarmidi a, Ramlan Abdul Aziz a

a Metabolite Profiling Laboratory, Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysiab Department of Process & Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysiac 11 Biopolis Way, #09­04 Helios, Singapore 138667, Singapore

a r t i c l e i n f o

Article history:

Received 8 September 2011

Accepted 2 November 2011

Available online 9 November 2011

Keywords:

LC–MS/MS

Eurycoma longifolia

Quassinoids

Alkaloids

Triterpenes

Biphenylneolignans

a b s t r a c t

A number of three LC–MS/MS hybrid systems (QTof, TripleTof and QTrap) has been used to profile small

metabolites (m/z 100–1000) and to detect the targeted metabolites such as quassinoids, alkaloids, triter­

pene and biphenylneolignans from the aqueous extracts of Eurycoma longifolia. The metabolite profiles

of small molecules showed four significant clusters in the principle component analysis for the aqueous

extracts of E. longifolia, which had been collected from different geographical terrains (Perak and Pahang)

and processed at different extraction temperatures (35 ◦C and 100 ◦C). A small peptide of leucine (m/z 679)

and a new hydroxyl methyl b­carboline propionic acid have been identified to differentiate E. longifolia

extracts that prepared at 35 ◦C and 100 ◦C, respectively. From the targeted metabolites identification, it

was found that 3,4«­dihydroeurycomanone (quassinoids) and eurylene (squalene­type triterpene) could

only be detected in the Pahang extract, whereas canthin­6­one­3N­oxide could only be detected in the

Perak extract. Overall, quassinoids were present in the highest concentration, particularly eurycomanone

and its derivatives compared to the other groups of metabolites. However, the concentration of canthin­

6­one and b­carboline alkaloids was significantly increased when the roots of the plant samples were

extracted at 100 ◦C.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Tongkat Ali belongs to a family of Simaroubaceae. Till today, four

species of Tongkat Ali have been reported, namely Eurycoma longi­

folia, Entomophthora apiculata, Polyathia bullata and Goniothalamus

sp. [1]. Among them, E. longifolia is the most widely used species,

particularly its roots, in South East Asian countries for centuries, as

a well known herbal folk medicine. It is a popular herb that tradi­

tionally used by the indigenous people, especially from Malaysia

and Indonesia for its aphrodisiac and vitality effects. It is believed

that the plant contains phytochemicals which could stimulate the

healing effect of the body and treat a range of common diseases.

Recently, numerous studies have reported its diverse biologi­

cal activities on antimalarial [2], antiulcer [3], antipyretic [4] and

cytotoxic to cancer cells [5]. The wide spectrum of pharmacological

activities was closely associated with various bioactive compounds

such as quassinoids, canthin­6­one alkaloids, b­carboline alkaloids,

squalene derivatives, tirucallane­type triterpenes and biphenylne­

olignans [6–11]. Quassinoids, as modified triterpenoids, are the

∗ Corresponding author. Tel.: +60 19 7214378; fax: +60 7 5569706.

E­mail address: [email protected] (L.S. Chua).

characteristic phytochemical of the Simaroubaceae family with bit­

ter taste. The presence of squalene and tirucallane­type triterpenes

might be the biological precursors of quassinoids. Canthin­6­one

and b­carboline alkaloids are naturally occurring amine compound

formed as metabolic by­products in order to repel insects and her­

bivores. However, this plant usually takes long time, from 4 to 7

years for harvesting [12]. Therefore, the type of metabolite and

its concentration in the plant extract are not only dependent on

the processing temperature, but also the geographical factor. It is

crucial to ensure the consistency of the phytochemical content,

particularly for herbal medicine efficacy study as well as for stan­

dardization.

These secondary metabolites are normally present in small

amount. Hence, the use of high sensitivity and high mass accuracy

tandem mass spectrometer is required to produce highly reliable

data. Over 150 quassinoids, mostly C18­C22 have been identified

till today [13]. Most of them were evaluated by 1H and 13C NMR

spectral analysis and supported by data from UV, IR, MS and X­

ray analysis. These methods usually require high concentration of

compounds with high purity and also extensive analysis for iden­

tification.

In the present study, the known quassinoids, alkaloids, triterp­

nes and biphenylneolignans that have been identified in previous

1570­0232/$ – see front matter ©  2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.jchromb.2011.11.002

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3910 L.S. Chua et al. / J. Chromatogr. B 879 (2011) 3909– 3919

studies were complied, screened and identified by using tandem

mass analyzers to confirm the presence of the targeted metabolites.

The results showed significant difference in the metabolite profile

of the plant species that collected from different locations (Perak

and Pahang) and processed at different temperatures of extrac­

tion (35 ◦C and 100 ◦C). This chemogeographical relationship is very

important because it would affect the bioassay activity studies.

2. Experimental

2.1. Chemicals and plant material

Formic acid and acetonitrile were bought from Fisher Scien­

tific (Pittsburg, USA). HPLC grade of methanol was purchased

from Merck (Darmstadt, Germany). 18.2 megohm­cm water was

produced from Barnstead NANOpure Diamond water purification

system (State of Illinois, USA).

The roots of E. longifolia Jack were provided by local suppliers

from Perak (Tasik Belum) and Pahang (Bentong), Malaysia. They

are dried and ground before sieved into the size of 2.4 mm. The

ground samples of E. longifolia were stored at −20 ◦C until analy­

sis. The voucher specimens (TA­TB Pr and TA­BE Ph) of the plant

samples have been deposited at the Metabolites Profiling Labo­

ratory, Institute of Bioproduct Development, Universiti Teknologi

Malaysia.

2.2. Sample preparation

The dried and ground roots of E. longifolia Jack (50 g) were

immersed in water (500 ml) at 35 ◦C overnight. The solution was

filtered and kept in a freezer at −20 ◦C. The same samples were re­

immersed with another 500 ml of water for triplicate. At day 3, the

solution (1500 ml) was combined and freeze­dried to produce 1.15

and 1.30 g of room temperature extracts of E. longifolia from Perak

and Pahang, respectively.

The 100 ◦C sample of the plant extract was prepared by boiling

the dried and ground roots of E. longifolia Jack (50 g) in water for

2 h. After cooling, the solution was filtered and sent for freeze dry.

A total amount of 1.97 and 2.06 g of plant extracts was obtained

from E. longifolia Perak and Pahang, respectively.

The aqueous extracts (0.5 mg) were dissolved in methanol

(1 ml). The solution (0.1 ml) was diluted with methanol to 10 ml

and filtered with 0.2 mm nylon membrane filter prior to LC–MS/MS

injection.

2.3. Liquid chromatography and mass spectrometer

2.3.1. Capillary LC–QTOF MS for small metabolite profiling

A capillary liquid chromatography (Dionex Corporation Ulti­

Mate 3000; Sunnyvale, CA) system was integrated with a

quadrupole and time­of­flight, QTOF mass spectrometer (AB SCIEX

QSTAR Elite; Foster City, CA) with a turbo spray ionization (TIS)

source. A C18 reversed phase Acclaim PA column (1 mm × 5 mm,

3 mm) with a flow rate of 100 ml/min was used for separation. A

binary gradient system consists of solvent A (2% acetonitrile in

water with 0.1% formic acid) and solvent B (2% water in acetoni­

trile with 0.1% formic acid). The LC gradient was: 0–30 min, 2–30%

B; 30–40 min, 30–98% B; 98% B hold for 0.1 min and 44.1–60 min,

98–8% B. The injection volume was 1 ml. All samples were filtered

with 0.2­mm nylon membrane filter prior to injection.

The QTOF mass spectrometer was used for the small metabolite

screening from m/z 100–1000. It was calibrated by using 1 pmol of

reserpine (m/z 609.2807) before used to ensure the mass accuracy.

A single Information Dependent Acquisition (IDA) method was cre­

ated to acquire both TOF MS and three dependent runs of product

ion scan with rolling collision energy. Nitrogen gas was used for

nebulizing (40 psi) and curtain gas (40 psi). Collision gas was set

at 3, the accumulation time was 1 s for TOF MS and 2 s for each

product ion scan. The voltage of ion spray was 5500 V, the declus­

tering potential was 60 V and the focusing potential was set at

200 V.

2.3.2. UFLC­triple TOF MS for high mass accuracy screening

An ultra­fast liquid chromatography (Shimadzu Corporation

UFLC XR; Kyoto, Japan) system was connected to a triple time­of­

flight mass spectrometer (AB SCIEX Triple TOF 5600; Foster City, CA)

with a duo spray ionization source. All samples were separated by a

Waters Xbridge C18 column (2.1 mm × 150 mm, 3.5 mm). A binary

solvent gradient consists of solvent A (water with 0.1% formic acid)

and solvent B (methanol with 0.1% formic acid) at a flow rate of

300 ml/min was used. The total run time was 50 min with the gra­

dient as follows: 0–3 min, 2% B; 3–30 min, 2–30% B; 30–42 min,

30–98% B; 98% B hold for 2 min; 98–2% B in 0.1 min and 2% B hold

for 5.9 min. An injection volume of 5 ml was used.

This high mass accuracy mass spectrometer was used for the

screening of targeted metabolites from the plant. A calibrant deliv­

ery system was used to ensure the accuracy of mass less than 1 ppm.

The settings of nitrogen gas for nebulizer, 50 psi; curtain gas, 25 psi;

drying solvent, 50 psi, temperature, 600 ◦C and ion spray voltage at

5500 V. An IDA method was also created to acquire both MS and

MS/MS data. The IDA criteria were as follows: peak intensity, more

than 700 cps; excluding time for former target ion, 9 s; excluding

isotope, 4.0 Da; maximum number of monitoring ions, 4; mass tol­

erance, 25 ppm and dynamic background subtraction was on. The

mass spectra were acquired at high resolution mode with a 0.2 s

ion accumulation time, declustering potential at 65 V and collision

energy at 40 V with a collision energy spread of 15 V in positive

ionization mode.

2.3.3. UPLC–QTRAP MS for targeted metabolite identification

An ultra­high performance liquid chromatography (Waters

Acquity UPLC; Milford, MA) system was coupled with a triple

quadrupole and linear­ion trap mass spectrometer (AB SCIEX 4000

QTRAP; Foster City, CA) with an electrospray ionization (ESI) source.

A C18 reserved phase Acquity column (4.6 mm × 150 mm, 1.7 mm)

protected by a guard column was used throughout this study. The

mobile phase was a binary solvent system consisting of solvent A

(water with 0.1% formic acid) and solvent B (acetonitrile). The UPLC

gradient was: 0–5 min, 10% B; 5–15 min, 10–90% B; 15–20 min,

90% B; 20–25 min, 90–10% B; 25–30 min, 10% B for final wash­

ing and equilibration of the column for the next run. Each wash

cycle consisted of 200 ml of strong wash solvent (90% acetonitrile

in water) and 600 ml of weak wash solvent (10% acetonitrile in

water). The flow rate was 0.25 ml/min and the injection volume was

5 ml.

This hybrid system was used for the identification of targeted

metabolites in high sensitivity and selectivity performance due

to the presence of small amount of the secondary metabolites.

The mass spectra were acquired using both enhanced product ion

(EPI) and multiple reactions monitoring (MRM) in positive ion­

ization mode. The EPI scan was carried out in three parallel runs

with different collision energies ranging from 10 to 50 V in order

to obtain the most information­rich fragmentation pattern. Low

energy collision­induced dissociation (CID) was used to confirm

the characteristic ion of metabolites in MRM mode. The capillary

and voltage of ion source were maintained at 400 ◦C and 4.5 kV,

respectively. All other parameters were as follows: nitrogen was

used as ion source gas for nebulization, 40 psi; for drying sol­

vent, 40 psi; curtain gas, 10 psi; collision gas, high; declustering

potential, 40 V, and collision exit energy, 10 V. The scan rate was

1000 amu/s.

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L.S. Chua et al. / J. Chromatogr. B 879 (2011) 3909– 3919 3911

Fig. 1. Score and loading plots of metabolites extracted at room temperature (RT) and 100 ◦C, and collected from Perak (TB) and Pahang (BE).

2.4. Data acquisition and processing

The high resolution mass spectrometric data was analyzed by

using PeakView software (AB SCIEX, Foster City, CA). This software

contains companion software tools such as XIC Manager, Formula

Finder and Fragment Interpretation Tool to assist in data interpreta­

tion. XIC Manager was used to screen the presence of metabolites by

importing the name and their chemical formula. The settings of XIC

Manager was; positive ion (H+), width of extracted ion at 0.01 Da,

isotopic masses at 20% tolerance, intensity threshold at 1000 cps

and minimum signal to noise at 5.

The detected metabolites were further interpreted with the

help of Formula Finder. Each possible detected compound was

confirmed by using the following criteria; the mass difference of

experimental and theoretical mass must be less than 5 ppm, the

intensity of isotopic peaks must be within 20% of its theoretical

distribution, and maximum elemental settings were carbon, 50;

hydrogen, 200; nitrogen, 10; oxygen, 20 and sulphur, 5.

In additional to MS data, the spectra from MS/MS was also ana­

lyzed by Fragment Interpretation Tool to verify the fragmentation

pattern of the detected compound. The structure of metabolites was

drawn using ChemSketch version 12.0 software under Advanced

Chemistry Development (ACD; Toronto, Canada) and imported into

PeakView software for theoretical fragmentation prediction and

matching with experimental data. The mass tolerance of the match

fragments must be less than 0.02 Da from the theoretical fragment

masses. MS Fragmenter 12.0 from ACD was also used to confirm

the fragmentation pattern of the targeted compound.

The statistical software, MarkerView 1.1 (AB SCIEX, Foster City,

CA) was used to perform sample classification by carrying out prin­

ciple component analysis (PCA).

3. Results and discussion

Mass screening was carried out for small metabolites, m/z

100–1000 using a capillary LC­QTOF mass spectrometer. QTOF mass

analyzer was chosen for the screening purpose, mainly because

of its exact mass determination capability. The mass spectra of

the aqueous extracts of E. longifolia were statistically analyzed by

an unsupervised pattern recognition method, principle component

analysis (PCA) as presented in Fig. 1.

The PCA result showed that the metabolites were significantly

different from both locations and for both temperature prepara­

tions. It showed four significant clusters in the score plot. The

first and second principle components were 45.6% and 24.0%,

respectively. The metabolites extracted at 100 ◦C were located in

the positive region of the first principle component, whereas the

metabolites extracted at room temperature were located in the

negative region of the score plot. The metabolites of E. longifolia

from Pahang extracted at 100 ◦C showed the highest positive score.

In the loading plot, it was found that m/z 679 and 271 were

metabolites could be used to differentiate E. longifolia extracts pre­

pared from different temperatures between room temperature and

100 ◦C, respectively. The electrospray ionization mass spectra of

both metabolites are presented in Fig. 2. It was found that m/z 679

is a small peptide consisting of five units of leucine (C6H13NO2, MW

131) bonded together through dehydration process with the forma­

tion of amide bonds (Fig. 2). The m/z 271 might be a hydroxyl methyl

b­carboline propionic acid, based on the match result between the

experimental data with the product ions generated by MS Frag­

menter 12.0.

To the best of our knowledge, more than eighty­five compounds

have been reported from E. longifolia till today. These compounds

are majority from the classes of quassinoids, canthin­6­one alka­

loids, b­carboline alkaloids, squalene derivatives, tirucallane­type

triterpenes and biphenylneolignans. A number of fifty­six quassi­

noids, sixteen canthin­6­one alkaloids, four b­carboline alkaloids,

six squalene­type triterpene and three biphenylneolignans were

tabulated and screened for their existence in the plant extracts

using a triple TOF mass spectrometer integrated to UFLC. Out

of these numbers, thirty­eight quassinoids, eleven canthin­6­one

alkaloids, three b­carboline alkaloids, two squalene­type triter­

pene, three biphenylneolignans and another seven metabolites that

previously reported in the other genera of the Simaroubaceous

family were also detected in E. longifolia. Among the seven metabo­

lites, six of them were quassinoids and the rest was belonged to

the class of canthin­6­one alkaloid. This was an alkaloid glycoside,

namely bruceolline G or 11­o­b­d­glucopyranosylcanthin­6­one,

which was previously detected in Brucea mollis var. Tonkinensis

reported by Okano et al. [14]. The six quassinoids were picrasi­

noside B from the barks of Picrasma javanica [15]; klaineanolide

B from the roots of Hannoa klaineana [16]; iandonoside B from

Eurycoma harmandiana [17]; 16­a­o­methylneoquassin from Quas­

sia amara [18]; samaderin B from the leaves and stems of Samadera

indica [19] and glaucarubolone from the aerial part of Castela macro­

phylla as well as the twigs and thorns of Castela polyandra [20,21].

The mass errors of all product ions of the detected metabolites were

less than 2 ppm. It demonstrated high resolving power more than

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3912 L.S. Chua et al. / J. Chromatogr. B 879 (2011) 3909– 3919

Table 1

Isotopic distribution of detected metabolites from the aqueous extracts of E. longifolia.

Quassinoids Formula Theoretical mass Isotope distribution

[M+H]+ (%) [M+2H]+ (%) [M+3H]+ (%)

Eurycolactone A C20H24O7 376.1522 377.1581 (100) 378.1605 (30.1) 379.165 (7.3)

Eurycolactone B C18H19ClO5 350.0921 351.0973 (100) 352.1017 (21.8) 353.0955 (40.4)

Eurycolactone C C18H20O6 332.126 333.1328 (100) 334.1358 (19.9) 335.1392 (4.4)

Eurycolactone D C18H22O5 318.1467 319.1532 (100) 320.1563 (29.0) 321.1577 (5.4)

Eurycolactone E C19H26O6 350.1729 351.1794 (100) 352.1824 (28.6) 353.1838 (4.5)

Eurycomalide A C19H26O6 350.1729 351.1794 (100) 352.1824 (28.6) 353.1838 (4.5)

Eurycomalide B C19H24O6 348.1573 349.1640 (100) 350.1668 (34.6) 351.1695 (8.5)

Eurycomalactone C19H24O6 348.1573 349.1599 (100) 350.1608 (35.0) 351.1675 (8.0)

6a­Hydroxyeurycomalactone C19H24O7 364.1522 365.1599 (100) 366.1624 (31.4) 367.1648 (6.1)

7a­Hydroxyeurycomalactone C19H26O6 350.1729 351.1794 (100) 352.1824 (28.6) 353.1838 (4.5)

Eurycomanone C20H24O9 408.1420 409.1489 (100) 410.1524 (40.8) 411.1532 (11.3)

13a(21)­Epoxyeurycomanone C20H24O10 424.1369 425.1440 (100) 426.1478 (35.5) 427.1533 (13.5)

12,15­Diacetyl­13a(21)­epoxy­eurycomanone C24H28O12 508.1581 509.1590 (100) 510.1599 (27.4) 511.1606 (6.0)

12­Acetyl­13,21­dihydroeurycomanone C22H28O10 452.1682 453.1690 (100) 454.1707 (25.1) 455.1711 (5.0)

15­Acetyl­13a(21)­Epoxyeurycomanone C22H26O11 466.1475 467.151 (100) 468.1528 (29.7) 469.1489 (15.7)

3,4«­Dihydroeurycomanone C19H22O11 426.1162 427.1170 (100) 428.1177 (28.8) 429.1183 (4.4)

13, 21­Dihydroeurycomanone C20H26O11 442.1475 443.1559 (100) 444.1574 (32.1) 445.1590 (8.2)

Eurycomanol C20H26O9 410.1577 411.1643 (100) 412.1673 (41.1) 413.1684 (10.9)

13b,18­Dihydroeurycomanol C20H28O9 412.1733 413.1796 (100) 414.1826 (30.3) 415.1842 (7.3)

13b, 21­Dihydroxyeurycomanol C20H28O11 444.1632 445.1693 (100) 446.1723 (28.3) 447.1735 (6.5)

Eurycomanol­2­o­b­d­glycopyranoside C26H36O14 572.2105 573.2157 (100) 574.2192 (31.5) 575.2240 (7.9)

11­Dehydroklaineanone C20H26O6 362.1729 363.1792 (100) 364.1819 (26.9) 365.1843 (5.2)

15b­Hydroxyklaineanone C20H28O7 380.1835 381.1893 (100) 382.1921 (28.1) 383.1936 (4.7)

14,15b­Dihydroxyklaineanone C20H28O8 396.1784 397.1855 (100) 398.1885 (35.5) 399.1897 (7.9)

5a,14b,15b­Trihydroxyklaineanone C20H28O9 412.1733 413.1796 (100) 414.1826 (30.3) 415.1842 (7.3)

15b­O­Acetyl­14­hydroxyklaineanone C22H30O9 438.1890 439.1881 (100) 440.1901 (25.1) 441.1922 (4.8)

6a­Acetoxy­14,15b­dihydroxyklaineanone C22H30O10 454.1839 455.1911 (100) 456.1934 (25.1) 457.1942 (5.0)

6a­Acetoxy­15b­hydroxyklaineanone C22H30O9 438.189 439.1991 (100) 440.1987 (25.1) 441.1990 (4.8)

Laurycolactone A C18H22O5 318.1467 319.1532 (100) 320.1563 (29.0) 321.1577 (5.4)

Laurycolactone B C18H20O5 316.1311 317.1376 (100) 318.1405 (24.8) 319.1424 (3.7)

Longilactone C19H26O7 366.1679 367.1746 (100) 368.1772 (30.9) 369.1801 (7.1)

Dehydroxylongilactone C19H26O6 350.1729 351.1794 (100) 352.1824 (28.6) 353.1838 (4.5)

2,3­Dehydro­4a­hydroxylongilactone C19H28O7 368.1835 369.1905(100) 370.1934 (36.0) 371.1964 (8.8)

Ailanthone C20H24O7 376.1522 377.1581 (100) 378.1605 (30.1) 379.165 (7.3)

(a/b­epoxide) Ailanthone C20H24O8 392.1471 393.1529 (100) 394.1554 (25.9) 395.1583 (5.8)

Chaparrinone (a­methyl) C20H26O7 378.1679 379.1743 (100) 380.1769 (29) 381.1821 (8.1)

3,4«­Dihydrochaparrinone C19H24O9 396.142 397.1487 (100) 398.1513 (26) 399.1538 (5.6)

Picrasinoside B C28H40O11 552.2571 553.2606 (100) 554.2611 (31.9) 555.2707 (7.1)

Klaineanolide B C27H34O8 486.2254 487.2319 (100) 488.2349 (36.4) 489.2375 (6.9)

Iandonoside B C26H38O14 574.2262 575.2311 (100) 576.2409 (29.8) 577.2505 (7.1)

16­a­o­Methylneoquassin C15H24O4 268.1675 269.1705 (100) 270.1723 (17.0) 271.1733 (2.1)

Samaderin B C19H22O7 362.1366 363.1430 (100) 364.1453 (28.4) 365.1515 (4.7)

Glaucarubolone C20H26O8 394.1628 395.1687 (100) 396.1714 (33.4) 397.1738 (7.9)

Canthin­6­one alkaloids

Canthin­6­one C14H8N2O 220.0637 221.0707 (100) 222.0735 (22.5) 223.0771 (2.3)

9­Methoxycanthin­6­one C15H10O2N2 250.0742 251.0817 (100) 252.0846 (25.8) 253.0864 (3.5)

5,9­Dimethoxycanthin­6­one C16H12N2O3 280.0848 281.0913 (100) 282.0939 (22.8) 283.0952 (3.5)

9,10­Dimethoxycanthin­6­one C16H12N2O3 280.0848 281.0913 (100) 282.0939 (22.8) 283.0952 (3.5)

11­Hydroxycanthin­6­one C14H8N2O2 236.0586 237.0658 (100) 238.0687 (29.6) 239.0703 (5.4)

1­Hydroxy­11­methoxycanthin­6­one C15H10N2O3 266.0691 267.0752 (100) 268.0778 (18.9) 269.079 (1.3)

10­Hydroxy­9­methoxycanthin­6­one C15H10N2O3 266.0691 267.0752 (100) 268.0778 (18.9) 269.079 (1.3)

11­Hydroxy­10­methoxycanthin­6­one C15H10N2O3 266.0691 267.0752 (100) 268.0778 (18.9) 269.079 (1.3)

11­o­b­d­Glucopyranosylcanthin­6­one C20H18N2O7 398.1114 399.1202 (100) 340.1221 (23.4) 341.1232 (4.0)

Canthin­6­one­3N­oxide C14H8N2O2 236.0586 237.0658 (100) 238.0687 (29.6) 239.0703 (5.4)

9­Methoxycanthin­6­one­3N­oxide C15H12N2O3 268.0848 269.0811 (100) 270.0821 (17.6) 271.0901 (2.0)

9­Methoxy­3­methylcanthin­5,6­dione C16H12N2O3 280.0848 281.0913 (100) 282.0939 (22.8) 283.0952 (3.5)

b­Carboline alkaloids

7­Hydroxy­b­carboline­1­propionic acid C14H12O3N2 256.0848 257.0915 (100) 258.0942 (21.1) 259.0958 (2.6)

b­Carboline­1­propionic acid C14H12N2O2 240.0899 241.0973 (100) 242.0998 (27.6) 243.1007 (3.3)

1­Methoxymethyl­b­carboline C13H12N2O 212.095 213.0990 (100) 214.1008 (15.3) 215.1107 (1.2)

Squalene­type triterpene

Eurylene C34H58O8 594.4132 595.4122 (100) 596.4132 (38.7) 597.4144 (8.8)

11/14­Deacetyl eurylene C32H56O7 552.4026 553.4111 (100) 554.4121 (36.4) 555.4189 (7.8)

Biphenylneolignans

2,2′­Dimethoxy­4­(3­hydroxy­1­propenyl)­4′­(1,2,3­

trihydroxypropyl) diphenyl ethers

(isomer)

C20H24O7 376.1522 377.1581 (100) 378.1605 (30.1) 379.165 (7.3)

2­Hydroxy­3,2′ ,6′­trimethoxy­4′­(2,3­epoxy­1­hydroxypropyl)­5­(3­

hydroxy­1­propenyl)­biphenyl

C21H24O7 388.1522 389.1602 (100) 390.1611 (23.8) 391.1707 (4.1)

2­Hydroxy­3,2′­dimethoxy­4′­(2,3­epoxy­1­hydroxypropyl)­5­(3­

hydroxy­1­propenyl)­biphenyl

C20H22O6 358.1416 359.1484 (100) 360.1514 (27.4) 361.1576 (6.3)

Page 5

L.S. Chua et al. / J. Chromatogr. B 879 (2011) 3909– 3919 3913

Fig. 2. Electrospray ionization mass spectra of m/z 679 (a) and 271 (b) as the metabolites to differentiate the aqueous extracts of E. longifolia extracted at 35 ◦C and 100 ◦C,

respectively.

5000 (full width at half maximum) and allowed isotopic resolu­

tion. The isotopic distribution of the molecular ions is presented in

Table 1.

The presence of the identified compounds was further con­

firmed by the high throughput and high sensitivity hyphenated

UPLC–QTRAP MS/MS. The product ions of each detected compound

were compared to the theoretical product ions generated from MS

Fragmenter 12.0 and literature values for conformation (Table 2).

Overall, the presence of quassinoids was in higher

concentration than alkaloids, triterpenes and biphenylne­

olignans (Fig. 3). Eurycomanone and its derivatives were

in the highest concentration among the detected metabo­

lites. The derivatives include 13a(21)­epoxyeurycomanone,

12,15­diacetyl­13a(21)­epoxy­eurycomanone, 12­acetyl­13,21­

dihydroeurycomanone, 15­acetyl­13a(21)­epoxyeurycomanone,

3,4«­dihydroeurycomanone and 13, 21­dihydroeurycomanone.

However, the concentration of alkaloids was significantly increased

if the plant samples were extracted at 100 ◦C. The increase was

significant for b­carboline alkaloids, particularly 7­hydroxy­

b­carboline­1­propionic acid and b­carboline­1­propionic

acid.

The results showed that 16­a­o­methylneoquassin could

only be detected in the room temperature extract in small

amount. In the geographical wise, 3,4«­dihydroeurycomanone and

eurylene could only be detected in the Pahang extract, whereas

canthin­6­one­3N­oxide could only be detected in the Perak

extract. It is also interesting to note that the concentration

of longilactone, chaparrinone, 3,4«­dihydrochaparrinone and

canthin­6­one in the Pahang extract was significantly higher than

the Perak extract at both temperatures (Fig. 3).

Eurycolactone A to E was detected in this study. As reported by

Ang et al. [6], the presence of a chlorine atom at the C3 of eurycolac­

tone B was demonstarted by an isotope peak, [M+2]+ at m/z 352. The

intensity of the isotope peak is about one third of the peak inten­

sity of the molecular ion. The detection of fragment ion at m/z 315

was also due to the loss of HCl from the molecular ion. It was found

that eurycolactone C and E have almost similar fragmentation pat­

tern because they have high similarity in their chemical structures,

except at the C1, C2, C5 and C6 (Fig. 4).

The product ions of both eurycomalide A and B have constant

difference in mass (2 Da). This was due to the presence of dou­

ble bond at the C3 and C4 in eurycomalide B. The explanation also

describes why the product ions of both laurylactone A and B hav­

ing constant mass difference. The product ions of laurycolactone A

have 2 Da more than laurycolactone B because of hydrogenation at

the C4 and C5 in laurycolactone A.

The difference in chemical structure between eurycomanone

and eurycomanol is at the C2, where the ketone group of

Page 6

3914 L.S. Chua et al. / J. Chromatogr. B 879 (2011) 3909– 3919T

ab

le

2

Qu

ass

ino

ids,

can

thin

­6­o

ne

alk

alo

ids,

b­c

arb

oli

ne

alk

alo

ids,

squ

ale

ne­t

yp

e

trit

erp

en

es

an

d

bip

hen

yln

eo

lig

nan

s

dete

cted

fro

m

the

aq

ueo

us

ex

tract

s

of

E.

lon

gif

oli

a

coll

ect

ed

fro

m

Pera

k

an

d

Pah

an

g

an

d

ex

tract

ed

at

roo

m

an

d

bo

ilin

g

tem

pera

ture

s.

Qu

ass

ino

ids

Fo

rmu

la

[M+

H]+

Pera

k

Pah

an

g

35◦C

10

0◦C

35◦C

10

0◦C

Eu

ryco

lact

on

e

A

C2

0H

24O

73

77

7.7

7

7.2

9

7.4

3

7.2

6

Eu

ryco

lact

on

e

BC

18H

19C

lO5

35

16

.25

5.2

05

.61

5.2

0

Eu

ryco

lact

on

e

C

C1

8H

20O

63

33

5.9

7

5.0

1

5.0

8

4.8

1

Eu

ryco

lact

on

e

DC

18H

22O

53

19

8.7

58

.70

8.6

68

.65

Eu

ryco

lact

on

e

EC

19H

26O

63

51

6.5

4

5.5

1

5.9

0

5.4

9

Eu

ryco

mali

de

A

C1

9H

26O

63

51

5.9

3

4.8

0

5.2

6

4.7

8

Eu

ryco

mali

de

BC

19H

24O

63

49

8.3

08

.29

8.2

8

8.2

4

Eu

ryco

mala

cto

ne

C1

9H

24O

63

49

8.9

5

8.7

0

8.6

4

8.6

3

6a

­Hy

dro

xy

eu

ryco

mala

cto

ne

C1

9H

24O

73

65

8.5

28

.24

8.3

18

.19

7a

­Hy

dro

xy

eu

ryco

mala

cto

ne

C1

9H

26O

63

51

8.4

6

8.2

7

8.3

3

8.2

2

Eu

ryco

man

on

eC

20H

24O

94

09

6.0

95

.80

5.8

85

.64

13

a(2

1)­

Ep

ox

yeu

ryco

man

on

e

C2

0H

24O

10

42

5

5.5

4

5.2

4

5.5

5

4.9

4

12

,15

­Dia

cety

l­1

3a

(21

)­ep

ox

y­e

ury

com

an

on

e

C2

4H

28O

12

50

95

.69

5.5

86

.56

.09

12

­Ace

tyl­

13

,21

­dih

yd

roeu

ryco

man

on

eC

22H

28O

10

45

3

7.7

1

7.5

9

7.6

4

7.5

7

15

­Ace

tyl­

13

a(2

1)­

ep

ox

yeu

ryco

man

on

e

C2

2H

26O

11

46

77

.60

7.6

37

.66

7.5

5

3,4

«­D

ihy

dro

eu

ryco

man

on

e

C1

9H

22O

11

42

7

no

no

7.1

2

7.1

3

13

,21

­Dih

yd

roeu

ryco

man

on

e

C2

0H

26O

11

44

3

4.8

4

4.6

9

4.6

7

4.2

0

Eu

ryco

man

ol

C2

0H

26O

94

11

6.2

05

.76

5.9

8

5.6

3

13

b,1

8­D

ihy

dro

eu

ryco

man

ol

C2

0H

28O

94

13

7.0

9

6.8

1

7.0

0

6.7

6

13

b, 2

1­D

ihy

dro

xy

eu

ryco

man

ol

C2

0H

28O

11

44

5

7.7

6

7.6

1

7.6

7

7.5

0

Eu

ryco

man

ol­

2­o

­b­d

­gly

cop

yra

no

sid

e

C2

6H

36O

14

57

3

8.3

0

8.2

5

8.3

2

8.2

4

11

­Deh

yd

rok

lain

ean

on

eC

20H

26O

63

63

4.1

9

3.9

0

4.2

5

3.8

2

15

b­H

yd

rox

yk

lain

ean

on

e

C2

0H

28O

73

81

6.8

6

6.4

9

6.7

8

6.4

9

14

,15

b­D

ihy

dro

xy

kla

inean

on

e

C2

0H

28O

83

97

5.3

5

5.1

2

5.0

2

4.8

2

5a

,14

b,1

5b

­Tri

hy

dro

xy

kla

inean

on

eC

20H

28O

94

13

6.7

46

.44

6.6

76

.33

15

b­O

­Ace

tyl­

14

­hy

dro

xy

kla

inean

on

e

C2

2H

30O

94

39

9.1

3

9.1

7

9.1

8

9.1

7

6a

­Ace

tox

y­1

4,1

5b

­dih

yd

rox

yk

lain

ean

on

e

C2

2H

30O

10

45

5

7.5

9

7.6

0

7.6

2

7.5

8

6a

­Ace

tox

y­1

5b

­hy

dro

xy

kla

inean

on

e

C2

2H

30O

94

39

3.7

7

3.5

9

3.4

8

3.1

8

Lau

ryco

lact

on

e

A

C1

8H

22O

53

19

8.9

5

8.8

9

8.8

6

8.8

4

Lau

ryco

lact

on

e

B

C1

8H

20O

53

17

8.7

5

8.8

0

8.7

7

8.7

5

Lo

ng

ilact

on

e

C1

9H

26O

73

67

7.1

2

7.1

2

7.1

6

7.1

6

Deh

yd

rox

ylo

ng

ilact

on

eC

19H

26O

63

51

8.8

18

.68

8.7

3

8.6

5

2,3

­Deh

yd

ro­4

a­h

yd

rox

ylo

ng

ilact

on

e

C1

9H

28O

73

69

5.7

2

5.1

4

5.5

8

5.0

5

Ail

an

tho

ne

C2

0H

24O

73

77

7.1

1

6.2

3

6.5

5

6.1

6

(a/b

­ep

ox

ide)

Ail

an

tho

ne

C2

0H

24O

83

93

6.7

3

6.6

1

6.6

0

6.6

3

Ch

ap

arr

ino

ne

(a­m

eth

yl)

C2

0H

26O

73

79

7.7

6

7.7

7

7.7

6

7.7

7

3,4

«­D

ihy

dro

chap

arr

ino

ne

C1

9H

24O

93

97

7.7

2

7.5

5

7.6

6

7.5

6

Pic

rasi

no

sid

e

B

C2

8H

40O

11

55

3

5.3

1

4.8

5

5.1

0

4.7

0

Kla

inean

oli

de

BC

27H

34O

84

87

8.5

6

8.8

4

8.8

9

8.5

6

Ian

do

no

sid

es

B

C2

6H

38O

14

57

5

4.9

8

4.9

1

5.2

0

4.8

0

16

­a­o

­Meth

yln

eo

qu

ass

in

C1

5H

24O

42

69

6.3

5

no

6.6

2

no

Sam

ad

eri

n

B

C1

9H

22O

73

63

8.4

4

8.3

5

8.4

0

8.3

2

Gla

uca

rub

olo

ne

C2

0H

26O

83

95

5.5

9n

o4

.61

4.3

9

Ca

nth

in­6

­on

e

alk

alo

ids

Can

thin

­6­o

ne

C1

4H

8N

2O

22

1

8.5

5

8.8

1

8.9

5

8.7

9

9­M

eth

ox

yca

nth

in­6

­on

e

C1

5H

10O

2N

22

51

1.2

7

1.2

7

1.1

9

1.2

8

5,9

­Dim

eth

ox

yca

nth

in­6

­on

e

C1

6H

12N

2O

32

81

8.4

3

8.2

6

8.2

0

8.4

0

9,1

0­D

imeth

ox

yca

nth

in­6

­on

e

C1

6H

12N

2O

32

81

8.6

2

8.4

4

8.6

7

8.6

2

11

­hy

dro

xy

can

thin

­6­o

ne

C1

4H

8N

2O

22

37

9.3

6

9.5

7

9.5

6

9.3

7

1­H

yd

rox

y­1

1­m

eth

ox

yca

nth

in­6

­on

e

C1

5H

10N

2O

32

67

1.7

3

1.5

2

1.4

4

1.7

5

10

­Hy

dro

xy

­9­m

eth

ox

yca

nth

in­6

­on

e

C1

5H

10N

2O

32

67

7.4

2

7.8

0

7.7

0

7.4

0

11

­Hy

dro

xy

­10

­meth

ox

yca

nth

in­6

­on

e

C1

5H

10N

2O

32

67

7.0

4

7.3

4

7.4

7

7.1

4

11

­o­b

­d­G

luco

py

ran

osy

lcan

thin

­6­o

ne

C2

0H

18N

2O

73

99

3.6

33

.63

3.6

6

3.6

6

Page 7

L.S. Chua et al. / J. Chromatogr. B 879 (2011) 3909– 3919 3915

Can

thin

­6­o

ne­3

N­o

xid

e

C1

4H

8N

2O

22

37

9.3

6

9.3

6

no

No

9­M

eth

ox

yca

nth

in­6

­on

e­3

N­o

xid

e

C1

5H

12N

2O

32

67

9.5

8

9.7

0

9.6

9

9.5

8

9­M

eth

ox

y­3

­meth

ylc

an

thin

­5,6

­dio

ne

C1

6H

12N

2O

32

81

10

.37

10

.61

10

.59

10

.37

b­C

arb

oli

ne

alk

alo

ids

7­h

yd

rox

y­b

­carb

oli

ne

1­p

rop

ion

ic

aci

d

C1

4H

12O

3N

22

57

8.1

4

8.0

5

8.0

8

8.0

3

b­C

arb

oli

ne­1

­pro

pio

nic

aci

d

C1

4H

12N

2O

22

41

4.3

9

7.4

7

7.3

9

4.4

0

1­M

eth

ox

ym

eth

yl­

b­c

arb

oli

ne

C1

3H

12N

2O

21

3

9.6

8

9.6

8

9.8

5

9.6

9

Sq

ua

len

e­t

yp

e

trit

erp

en

e

Eu

ryle

ne

C3

4H

58O

85

95

no

no

5.4

5.2

3

11

/14

­Deace

tyl

eu

ryle

ne

C3

2H

56O

75

53

6.1

4

5.5

2

5.8

5.3

5

Bip

he

ny

lne

oli

gn

an

s

2,2′­D

imeth

ox

y­4

­(3

­hy

dro

xy

­1­p

rop

en

yl)

­4′­(

1,2

,3­t

rih

yd

rox

yp

rop

yl)

dip

hen

yl

eth

er

C2

0H

24O

73

77

5.7

4

4.8

9

5.7

6

4.8

3

2­H

yd

rox

y­3

,2′,6′­t

rim

eth

ox

y­4′­(

2,3

­ep

ox

y­1

­hy

dro

xy

pro

py

l)­5

­(3

­hy

dro

xy

­1­p

rop

en

yl)

­bip

hen

yl

C2

1H

24O

73

89

8.1

4

8.0

6

8.0

9

8.1

0

2­H

yd

rox

y­3

,2′­d

imeth

ox

y­4′­(

2,3

­ep

ox

y­1

­hy

dro

xy

pro

py

l)­5

­(3

­hy

dro

xy

­1­p

rop

en

yl)

­bip

hen

yl

C2

0H

22O

63

59

7.9

6

7.9

9

7.9

7

7.9

6

Qu

ass

ino

ids

Fra

gm

en

t

ion

s

Ref.

Eu

ryco

lact

on

e

A

37

7/3

59

(–H

2O

)/3

31

(–C

H2O

2)/

31

1(–

C5H

6)/

29

5/2

75

/23

7/2

27

/17

1/1

37

[6]

Eu

ryco

lact

on

e

B

35

1/3

33

(–H

2O

)/3

15

(–H

Cl)

/30

5(–

CH

2O

2)/

29

7/2

79

/26

9/2

41

/23

5/2

25

/20

9/1

69

[6]

Eu

ryco

lact

on

e

C3

33

/31

5(–

H2O

)/2

97

/29

2(–

C3H

5)/

28

7(–

CH

2O

2)/

26

9/2

51

/22

3(–

C6H

6O

2)/

21

1/1

93

/13

7(–

C1

1H

16O

3)

[6]

Eu

ryco

lact

on

e

D

31

9/3

01

(–H

2O

)/2

83

/27

3(–

CH

2O

2)/

25

5/2

37

/22

7/2

09

/19

4/1

79

/13

3/1

05

[7]

Eu

ryco

lact

on

e

E

35

1/3

33

(–H

2O

)/3

15

(–2

H2O

)/3

05

(–C

H2O

2)/

29

7/2

79

(–C

15H

19O

5)/

26

9/2

35

/22

3/2

09

/18

3

[7,2

2]

Eu

ryco

mali

de

A

35

1/3

33

(–H

2O

)/3

15

(–2

H2O

)/2

97

(–3

H2O

)/2

69

(–C

O)/

25

1(–

H2O

)/2

36

(–C

H3)/

23

5(–

CH

4)/

22

7/2

20

/20

9/1

95

/18

3/1

79

/17

3/1

45

[2]

Eu

ryco

mali

de

B

34

9/3

31

(–H

2O

)/3

13

(–2

H2O

)/3

03

(–C

H2O

2)/

29

5(–

3H

2O

)/2

85

(–C

O)/

26

7(–

H2O

)/2

39

(–C

O)/

22

5/2

11

/20

9/1

69

/95

[2]

Eu

ryco

mala

cto

ne

34

9/3

31

(–H

2O

)/3

13

(–2

H2O

)/3

03

(–C

H2O

2)/

28

5/2

67

/25

7/2

39

/22

5/2

11

/13

3/1

23

[10

,23

,24

]

6a

­Hy

dro

xy

eu

ryco

mala

cto

ne

36

5/3

47

(–H

2O

)/3

29

(–2

H2O

)/3

19

(–C

H2O

2)/

30

1/2

83

/26

5/2

55

/23

7/2

07

/18

9/1

59

/12

1

[23

]

7a

­Hy

dro

xy

eu

ryco

mala

cto

ne

35

1/3

33

(–H

2O

)/3

15

(–2

H2O

)/3

05

(–C

H2O

2)/

29

7(C

4H

6)/

28

7/2

69

/24

1/2

27

/19

9(–

C9H

12O

2)/

18

7/1

71

/14

9

[10

]

Eu

ryco

man

on

e4

09

/39

1(–

H2O

)/3

73

(–2

H2O

)/3

45

(–C

O)/

33

7(–

CH

3–

CH

3O

)/3

29

/30

9/2

79

/26

9/2

51

/22

5/2

09

/19

7

[9,2

4,2

5]

13

a(2

1)­

Ep

ox

yeu

ryco

man

on

e

42

5/4

07

(–H

2O

)/3

97

(–C

O)/

38

9/3

77

(–C

H4O

2)/

36

1/3

43

/26

9/2

67

/23

9/2

23

/21

1/1

85

/14

5

[10

,24

]

12

,15

­Dia

cety

l­1

3a

(21

)­ep

ox

y­

eu

ryco

man

on

e

50

9/4

85

/47

2/4

67

(–C

2H

2O

)/4

49

(–C

2H

4O

2)/

42

1/4

03

/39

2/2

83

/17

9/1

45

[10

]

12

­Ace

tyl­

13

,21

­dih

yd

roeu

ryco

man

on

e

45

3/4

35

(–H

2O

)/4

17

(–2

H2O

)/4

09

(–C

2H

4O

2)/

39

1(–

CH

2O

3)/

34

3/3

36

/32

6/3

08

/22

6/2

09

/19

2/1

82

/14

7/1

00

[10

]

15

­Ace

tyl­

13

a(2

1)­

ep

ox

yeu

ryco

man

on

e

46

7/4

57

/44

9(–

H2O

)/4

21

(–C

H2O

2)/

40

5(–

CH

2O

3)/

36

9/3

05

/28

7/2

69

/24

1/2

27

/21

5/1

89

/13

7

[10

]

3,4

«­D

ihy

dro

eu

ryco

man

on

e

42

7/4

09

(–H

2O

)/4

06

/39

5(–

CH

4O

)/3

91

(–H

2O

)/3

86

/37

7/3

69

(–C

3H

6O

)/3

68

/35

9/3

43

/32

5/3

07

/29

5/2

51

/22

3/2

15

/20

9/1

83

/15

9/1

35

[3]

13

,21

­Dih

yd

roeu

ryco

man

on

e

44

3/4

26

(–O

H)/

42

5(–

H2O

)/4

07

(–2

H2O

)/3

95

(–C

H4O

2)/

38

9/3

71

/36

3/3

59

/34

1/3

33

/31

3/2

97

/26

9/2

39

/22

3/1

85

/14

1

[3]

Eu

ryco

man

ol

41

1/3

93

(–H

2O

)/3

75

(–2

H2O

)/3

45

(–C

H2O

)/3

39

(–C

2H

6O

)/3

29

/31

3/3

01

/28

1/2

73

/25

3/2

39

/22

5/2

09

/17

3

[8,9

,23

–2

5]

13

b,1

8­D

ihy

dro

eu

ryco

man

ol

41

3/3

95

(–H

2O

)/3

77

(–2

H2O

)/3

59

(–C

2H

6O

)/3

29

/31

1/2

85

/25

5/2

49

(–C

6H

12O

5)/

21

1/1

57

[8,2

3,2

5]

13

b, 2

1­D

ihy

dro

xy

eu

ryco

man

ol

44

5/4

29

(–C

H4)/

41

5(–

CH

2O

)/4

05

(–C

3H

4)/

39

7(–

CH

4O

2)/

34

3/2

46

/23

7/2

28

/14

9/1

17

[2]

Eu

ryco

man

ol­

2­o

­b­d

­gly

cop

yra

no

sid

e

57

3/5

72

(–H

)/5

55

(–H

2O

)/5

27

(–C

H2O

2)/

51

8/5

11

(–C

H2O

3)/

49

3/3

69

/35

1/3

41

/32

3/3

05

/29

5/2

77

/24

1/2

23

/19

5/1

46

[2,8

,23

,25

]

11

­Deh

yd

rok

lain

ean

on

e3

63

/34

5(–

H2O

)/3

17

(–C

H2O

2)/

30

9/2

67

/26

3/2

53

/24

9/2

39

/22

3/2

11

/14

9

[22

,26

]

15

b­H

yd

rox

yk

lain

ean

on

e

38

1/3

63

(–H

2O

)/3

45

/32

7/3

17

/30

1/2

99

/28

1/2

71

/25

3/2

43

/22

7/2

13

/20

1/1

87

/14

5

[22

,24

,26

]

14

,15

b­D

ihy

dro

xy

kla

inean

on

e

39

7/3

79

(–H

2O

)/3

61

(–2

H2O

)/3

51

(–C

H2O

2)/

31

5/3

05

/26

3/2

59

/18

9/1

61

[8,2

2–

24

,26

,27

]

5a

,14

b,1

5b

­Tri

hy

dro

xy

kla

inean

on

e

41

3/3

96

(–O

H)/

39

5(–

H2O

)/3

77

/35

9/3

41

/31

3/2

83

/26

5/2

49

/23

7/2

23

/19

1

[2]

15

b­O

­Ace

tyl­

14

­hy

dro

xy

kla

inean

on

e

43

9/4

21

(–H

2O

)/3

94

/37

6/3

71

/35

8/3

06

/26

7/2

49

/14

9

[10

,22

,26

]

6a

­Ace

tox

y­1

4,1

5b

­dih

yd

rox

yk

lain

ean

on

e

45

5/4

54

(–H

)/4

37

(–H

2O

)/4

36

(–H

3O

)/4

19

/40

7(–

CH

4O

2)/

39

2(–

CH

3O

3)/

37

9(–

C2H

4O

3)/

37

7(–

C2H

6O

3)/

35

5/3

38

/33

7/3

27

/22

7/2

11

(–C

11H

16O

6)/

21

0(–

C1

1H

17O

6)/

20

9(–

C1

1H

18O

6)/

18

2/1

00

[10

]

6a

­Ace

tox

y­1

5b

­hy

dro

xy

kla

inean

on

e

43

9/4

21

(–H

2O

)/3

97

(–C

2H

2O

)/3

93

(–C

H2O

2)/

38

5/3

65

/32

8/3

11

/29

9/2

83

(–C

8H

12O

3)/

26

5/2

39

/23

7/2

09

(–C

11H

18O

5)/

19

3(–

C1

1H

18O

6)

[10

]

Lau

ryco

lact

on

e

A

31

9/3

01

(–H

2O

)/2

83

(–C

2H

6O

)/2

73

(–C

H2O

2)/

25

9(–

C3H

8O

)/2

55

/22

7/2

13

/19

7/1

85

/13

3

[28

]

Lau

ryco

lact

on

e

B

31

7/3

16

(–H

)/2

99

(–H

2O

)/2

98

(–H

3O

)/2

94

/27

6(–

C3

H5

)/2

71

(–C

H2O

2)/

26

3/2

57

(C2H

4O

2)/

25

3/2

49

/23

8/2

35

/22

7/2

25

/21

0/1

97

/17

9/1

65

[6,7

,28

]

Lo

ng

ilact

on

e

36

7/3

49

(–H

2O

)/3

46

/33

7(C

H2O

)/3

31

(–2

H2O

)/3

19

/31

3/2

95

/28

5/2

67

/25

1/2

39

/22

3/2

11

/20

9/1

87

/17

1/1

59

/12

1

[7,2

2,2

6]

Deh

yd

rox

ylo

ng

ilact

on

e

35

1/3

33

(–H

2O

)/3

05

(–C

H2O

2)/

29

7(–

2H

2O

)/2

87

(–C

2H

6O

)/2

71

/26

9/2

59

/24

1/2

23

/19

9/1

71

/13

5

[10

,22

]

2,3

­Deh

yd

ro­4

a­h

yd

rox

ylo

ng

ilact

on

e

36

9/3

52

(–O

H)/

35

1(–

H2O

)/3

33

(–2

H2O

)/3

27

/30

5(–

CO

)/3

15

/29

7/2

79

/26

9(–

C5H

8O

2)/

25

1/2

35

/22

3/2

09

/19

5/1

83

/17

1/1

59

/11

9

[29

]

Ail

an

tho

ne

37

7/3

59

(–H

2O

)/3

47

(–2

CH

3)/

34

1(–

2H

2O

)/3

31

(–C

H2O

2)/

31

3(–

CH

4O

3)/

31

1/2

83

(–C

3H

10O

3)/

27

5/2

67

/25

5/2

39

/23

7/2

09

/17

1

[3,3

0]

(a/b

­ep

ox

ide)

Ail

an

tho

ne

39

3/3

75

(–H

2O

)/3

65

(–C

O)/

35

7/3

45

/33

9/3

29

/32

1/3

09

/30

1/2

95

/29

3/2

83

/28

1/2

67

/26

5/2

53

/23

7/2

09

/19

9/1

83

/17

3/1

19

[3]

Ch

ap

arr

ino

ne

(a­m

eth

yl)

37

9/3

61

(–H

2O

)/3

43

(–2

H2O

)/3

38

/33

3/3

25

/31

7/3

07

/29

7/2

79

/26

9/2

53

/25

1/2

25

/21

1/1

99

/18

7/1

75

/15

9/1

35

[3,3

0]

3,4

«­D

ihy

dro

chap

arr

ino

ne

39

7/3

79

(–H

2O

)/3

73

/36

5/3

61

/35

5/3

51

/34

3/3

33

/32

5/3

19

/31

5/2

97

/27

9/2

87

/26

9/2

51

/22

3/2

19

/19

1/1

35

[3]

Pic

rasi

no

sid

e

B

55

3/5

35

(–H

2O

)/5

06

/49

3/4

91

(–C

2H

6O

2)/

45

1(–

C4H

6O

3)/

43

3(–

C4H

8O

4)/

39

1(–

glc

)/3

73

(–C

6H

13O

6)/

37

2(–

C6H

12O

6)/

32

9/3

23

/29

5/2

65

/20

3

[15

]

Kla

inean

oli

de

B

48

7/4

69

(–H

2O

)/4

59

(–C

O)/

44

5(–

C3H

6)/

44

1(–

CH

2O

2)/

42

7(–

C3H

8O

)/4

09

/39

1/3

73

(–C

5H

6O

3)/

24

9/1

97

[16

]

Ian

do

no

sid

es

B

57

5/5

57

(–H

2O

)/5

34

(–C

3H

4)/

52

9(–

CH

2O

2)/

51

6(–

C2H

3O

2)/

49

7(–

C2H

6O

2)/

41

3(–

glc

)

[17

]

Page 8

3916 L.S. Chua et al. / J. Chromatogr. B 879 (2011) 3909– 3919

Qu

ass

ino

ids

Fra

gm

en

t

ion

sR

ef.

16

­a­o

­Meth

yln

eo

qu

ass

in

26

9/2

54

(–C

H3)/

25

1(–

H2O

)/2

41

(–C

O)/

23

7(–

CH

4O

)/2

23

(–C

2H

6O

)/2

11

(–C

3H

6O

)/1

95

(–C

3H

6O

2)/

18

0/1

77

/14

1(–

C7H

12O

2)

[18

]

Sam

ad

eri

n

B3

63

/34

5(–

H2O

)/3

27

(–2

H2O

)/3

19

(–C

O2)/

29

9/2

81

/27

1/2

67

/25

3/2

37

/22

5/2

11

/17

9[1

9]

Gla

uca

rub

olo

ne

39

5/3

77

(–H

2O

)/3

59

(–2

H2

O)/

34

9(–

CH

2O

2)/

31

1/3

01

/28

3/2

65

/26

1/2

39

/23

1/2

15

/20

3/1

87

/17

1/1

59

/14

3

[20

,21

]

Ca

nth

in­6

­on

e

alk

alo

ids

Can

thin

­6­o

ne

22

1/2

20

(–H

)/2

06

/20

3(–

H2O

)/1

95

(–C

2H

2)/

19

3(–

CO

)/1

92

(–C

HO

)/1

85

/17

9/1

75

/16

6(–

C3H

3O

)/1

61

/15

9/1

50

/14

7/1

45

(–C

6H

4)/

14

0(–

C4H

3N

O)/

10

5[3

1]

9­M

eth

ox

yca

nth

in­6

­on

e2

51

/25

0(–

H)/

23

6(–

CH

3)/

23

3(–

H2O

)/2

17

/21

6/2

15

/21

0/2

05

/19

8(–

C3H

3N

)/1

87

/18

2/1

59

/14

9(–

C7H

4N

)/1

45

(–C

7H

6O

)/1

41

/12

1

[24

,31

]

5,9

­Dim

eth

ox

yca

nth

in­6

­on

e2

81

/26

3(–

H2O

)/2

53

(–C

O)/

23

9(–

C2H

2O

)/2

38

(–C

2H

3O

)/2

35

/22

1(C

2H

4O

2)/

20

4/1

98

(–C

4H

5N

O)/

17

5(–

C9H

7N

2O

2)/

15

1/1

48

/13

3/1

20

/10

5[3

1]

9,1

0­D

imeth

ox

yca

nth

in­6

­on

e

28

1/2

80

(–H

)/2

66

(–C

H3)/

26

4(–

NH

3)/

24

5/2

37

(–C

H2N

O)/

22

1/2

04

/14

8(–

C8H

7N

O)/

11

9/1

05

[31

]

11

­hy

dro

xy

can

thin

­6­o

ne

23

7/2

36

(–H

)/2

19

(–H

2O

)/1

95

(–C

2H

2O

)/1

94

(–C

HN

O)/

14

5(–

C6H

4O

)/1

35

(–C

7H

4N

)/1

19

/10

5/9

1

[31

]

1­H

yd

rox

y­1

1­m

eth

ox

yca

nth

in­6

­on

e2

67

/25

2(–

CH

3)/

25

1(C

H4)/

24

9(–

H2O

)/2

34

(–C

H4O

)/2

21

(CH

O2)/

20

3/1

85

/17

7/1

61

(–C

7H

6O

)[3

2]

10

­Hy

dro

xy

­9­m

eth

ox

yca

nth

in­6

­on

e

26

7/2

49

(–H

2O

)/2

25

(–C

2H

2O

)/2

03

/18

5/1

57

/12

1(–

C8H

6N

2O

)/9

9(–

C1

0H

4N

2O

)

[31

]

11

­Hy

dro

xy

­10

­meth

ox

yca

nth

in­6

­on

e2

67

/24

9(–

H2O

)/2

46

/24

3/2

31

/22

4(–

CH

NO

)/2

21

/21

3(–

C3H

2O

)/2

03

/19

1/1

92

/18

5/1

75

/15

7/1

45

(–C

7H

6O

2)/

13

9/1

19

/99

(–C

10H

4N

2O

)[3

1]

11

­o­b

­d­G

luco

py

ran

osy

lcan

thin

­6­o

ne

39

9/3

81

(–H

2O

)/3

75

/36

3/3

45

/33

5/3

27

/30

9/2

99

/29

1(–

C3H

8O

4)/

28

1/2

63

/23

5(–

glc

–2

H)/

22

3/2

05

/19

1/1

59

[14

]

Can

thin

­6­o

ne­3

N­o

xid

e2

37

/23

6(–

H)/

22

2/2

20

(NH

3)/

21

9(–

OH

)/2

07

/20

1/1

96

/19

1(–

CO

)/1

79

/16

1(–

C6H

4)/

15

9/1

45

/13

5/1

21

/11

9(C

7H

4N

O)/

10

9/1

05

/93

[31

]

9­M

eth

ox

yca

nth

in­6

­on

e­3

N­o

xid

e2

67

/24

9(–

H2O

)/2

39

(–C

O)/

23

7(–

CH

2O

)/2

21

(–C

2H

6O

)/2

11

(–C

3H

4O

)/1

93

(–C

3H

5O

2)/

17

9/9

9

[31

]

9­M

eth

ox

y­3

­meth

ylc

an

thin

­5,6

­dio

ne

28

1/2

66

(–C

H3)/

26

5(–

CH

4)/

26

3(–

H2O

)/2

48

(–C

H4O

)/2

35

/22

5(–

C3H

4O

)/2

03

/19

7/1

69

/15

1

[31

]

b­C

arb

oli

ne

alk

alo

ids

7­h

yd

rox

y­b

­carb

oli

ne

1­p

rop

ion

ic

aci

d

25

7/2

39

(–H

2O

)/2

11

(–C

H2O

2)/

19

7(–

C2H

4O

2)/

19

5/1

93

/18

5/1

69

/16

7/1

65

(–C

6H

4O

)/1

57

(–C

4H

6N

O2)/

15

5/1

19

/79

[31

]

b­C

arb

oli

ne­1

­pro

pio

nic

aci

d

24

1/2

23

(–H

2O

)/1

95

(–C

H2O

2)/

18

1(–

C2H

4O

2)/

16

7(–

C3H

6O

2)/

15

4/1

40

(–C

4H

7N

O2)

[31

]

1­M

eth

ox

ym

eth

yl­

b­c

arb

oli

ne

21

3/2

12

(–H

)/2

11

(–H

2)/

19

6(–

NH

3)/

19

5/1

85

(–C

2H

4)/

17

7/1

71

(–C

3H

7)/

16

9(–

C2H

4O

)/1

57

(–C

3H

4O

)/1

55

(–C

3H

7O

)/1

41

(–C

3H

6N

O)/

13

5(–

C6H

6)/

11

9/9

1(–

C7H

8N

O)

[31

]

Sq

ua

len

e­t

yp

e

trit

erp

en

e

Eu

ryle

ne

59

5/5

77

(–H

2O

)/5

59

/55

4/5

49

/53

5(–

C3H

8O

)/5

19

(–C

3H

8O

2)/

51

7(–

C2H

6O

3)/

51

3(–

C6H

10)/

29

8/2

55

/22

3

[11

]

11

/14

­Deace

tyl

eu

ryle

ne

55

3/5

35

(–H

2O

)/5

12

/51

1(–

C2H

2O

)/4

95

(–C

3H

6O

)/4

93

(–C

2H

5O

2)/

47

7(–

C3H

8O

2)/

45

1(–

C6H

14O

)/2

97

/21

3/1

85

[11

]

Bip

he

ny

lne

oli

gn

an

s

2,2′­D

imeth

ox

y­4

­(3

­hy

dro

xy

­1­

pro

pen

yl)

­4′­(

1,2

,3­t

rih

yd

rox

yp

rop

yl)

dip

hen

yl

eth

er

37

7/3

62

(–C

H3)/

35

9(–

H2O

)/3

29

(–C

H4O

2)/

31

5(–

C2H

6O

2)/

29

5(–

C5H

6O

)/2

83

/26

1(–

C5H

8O

3)/

23

9/2

31

/21

7/2

03

/17

1/1

59

[33

]

2­H

yd

rox

y­3

,2′,6′­t

rim

eth

ox

y­4′­(

2,3

­

ep

ox

y­1

­hy

dro

xy

pro

py

l)­5

­(3

­hy

dro

xy

­

1­p

rop

en

yl)

­bip

hen

yl

38

9/3

71

(–H

2O

)/3

57

(–C

H4O

)/3

43

(–C

2H

6O

)/3

39

/31

7(–

C3H

4O

2)/

30

7(–

C5H

6O

)/2

11

/18

2/1

67

[33

]

2­H

yd

rox

y­3

,2′­d

imeth

ox

y­4′­(

2,3

­ep

ox

y­

1­h

yd

rox

yp

rop

yl)

­5­(

3­h

yd

rox

y­1

­

pro

pen

yl)

­bip

hen

yl

35

9/3

50

/34

1(–

H2O

)/3

23

/31

3(–

C2H

6O

)/3

14

(–C

2H

5O

)/2

77

(–C

5H

6O

)/2

37

/22

8/2

09

/16

6/1

48

/13

7

[33

]

Page 9

L.S. Chua et al. / J. Chromatogr. B 879 (2011) 3909– 3919 3917

Fig. 3. Bar chart of detected quassinoids (a), and alkaloids, triterpene and biphenylneolignan (b) from the aqueous extracts of E. longifolia collected from Perak at room

temperature (dark blue) and 100 ◦C (light blue), and collected from Pahang at room temperature (dark orange) and 100 ◦C (light orange). (For interpretation of the references

to color in this figure legend, the reader is referred to the web version of the article.)

eurycomanone is replaced by a hydroxyl group. Hence, euryco­

manol (C20H26O9) has two more protons than eurycomanone

(C20H24O9). In addition to the loss of water molecule, the

presence of m/z 345 was due to the loss of carbonyl (–CO)

and formaldehyde (–CH2O) from the C2 of eurycomanone

and eurycomanol, respectively. Apart from eurycomanol, two

derivatives of eurycomanol (13b,18­dihydroeurycomanol and13b,

21­dihydroxyeurycomanol) and one eurycomanol glycoside were

also detected in this study.

Besides eurycomalactone, its derivatives such as 6a­

hydroxyeurycomalactone and 7a­hydroxyeurycomalactone

were also detected. They have an additional hydroxyl group

attached to the C6 and C7 of eurycomalactone, respectively. The

different location of the hydroxyl group caused the product ions of

the derivatives having constant mass difference from the product

ions of their basic structure, eurycomalcatone. The EPI results

showed that the product ions of 6a­hydroxyeurycomalactone

would loss 2 Da, whereas 7a­hydroxyeurycomalactone would add

2 Da to the product ions of eurycomalactone.

Klaineanone is a quassinoid diterpenoid. A number of seven

reported klaineanone derivatives were detected, namely 11­

dehydroklaineanone, 15b­hydroxyklaineanone, 14,15b­ dihydro

Page 10

3918 L.S. Chua et al. / J. Chromatogr. B 879 (2011) 3909– 3919

Fig. 4. Chemical structure of eurycolactone A to E with their formula molecular weight.

xyklaineanone, 5a,14b,15b­trihydroxyklaineanone, 15b­O­acetyl

­14­hydroxyklaineanone, 6a­acetoxy­15b­hydroxyklaineanone

and 6a­acetoxy­14,15b­dihydroxyklaineanone. Among them,

6a­acetoxy­15b­hydroxyklaineanone and 6a­acetoxy­14,15b­

dihydroxyklaineanone are the lowest and highest concentration

of klaineanone derivatives in the aqueous extract of E. longifolia,

respectively.

The absence of a hydroxyl group from longilactone has produced

dehydrolongilactone which is having a characteristic ion at m/z 333

after losing a water molecule. This characteristic ion is 16 Da less

than the characteristic ion of longilactone, m/z 349 because of the

loss of an oxygen atom. However, they have the same fragment ion

at m/z 171, contributed by the breakdown of the chemical bond

between C6–C7 and C9–C10 at the ring A (6,9B).

Based on the theoretical fragments and literature values, it was

reported that the different location of the hydroxyl and methoxyl

groups at the basic structure of canthin­6­one produced different

mass spectral data. The absence of the basic fragment (m/z 221)

of canthin­6­one in the mass spectra of some of the derivatives

might be due to the low intensity of the fragment compared to

other fragments.

4. Conclusions

The approach of LC–MS/MS­based metabolites identification

showed that the aqueous extract of E. longifolia has different

profiles when extracted at different temperatures and grown

in different environments. Besides, the concentration of the

targeted metabolites such as quassinoids, alkaloids, triterpene

and biphenylneolignans was not only affected by the processing

temperature, but also the geographical factor, particularly 16­a­

o­methylneoquassin, 3,4«­dihydroeurycomanone, eurylene and

canthin­6­one­3N­oxide. The result also found that quassinoids

were significantly present in higher concentration than alkaloids.

Eurycomanone and its derivatives represent the highest amount

among the detected quassinoids. Somehow, the concentration

of alkaloids was increased when the roots of E. longifolia were

extracted at 100 ◦C.

Acknowledgements

The authors would like to thank the practical students, espe­

cially Siti Farhana binti Samsudin and Muhammad Hafiz bin Ali for

their help in data organization.

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