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Journal of Chromatography A, 1206 (2008) 131–139

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

haracterization of polyprenylated xanthones in Garcinia xipshuanbannaensissing liquid chromatography coupled with electrospray ionization quadrupoleime-of-flight tandem mass spectrometry

an Zhou, Quan-Bin Han, Jing-Zheng Song, Chun-Feng Qiao, Hong-Xi Xu ∗

ong Kong Jockey Club Institute of Chinese Medicine, Shatin, New Territories, Hong Kong, China

r t i c l e i n f o

rticle history:eceived 20 March 2008eceived in revised form 29 July 2008ccepted 4 August 2008vailable online 15 August 2008

eywords:olyprenylated xanthonesarcinia xipshuanbannaensisPLC–QTOF-MS/MS/MS

a b s t r a c t

A reliable and sensitive on-line high-performance liquid chromatography (HPLC) coupled with electro-spray quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS/MS) method has beenoptimized and established for the analysis of polyprenylated xanthones in the plant Garcinia xipshuan-bannaensis. Collision induced MS/MS techniques were used to fragment the precursor molecular ionsand MS/MS/MS techniques based on cone voltage fragmentation were used to further break down theresulting product ions sequentially. It was found that Retro–Diels–Alder rearrangement occurred fromthe xanthone skeleton in the MS/MS/MS process and produced characteristic fragment ions, which areuseful for differentiating some positional isomers containing the prenyl unit on the A ring or B ring. Com-plementary fragmentation information, for instance the successive loss of prenyl residues, is also valuablefor the identification of this class of xanthones. Under optimized HPLC–MS/MS/MS method, a total of 15prenylated xanthones could be separated within 10 min. This method also provided information about

the molecular formula of a precursor molecule and its fragments, which could be used for dereplicationof known or likely new prenylated xanthones in Garcinia plants before the purification and structural

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elucidation process.

. Introduction

Garcinia L. (Guttiferae family) is a large genus of polygamousrees or shrubs, distributed in tropical Asia, Africa, and Polyne-ia. It consists of 450 species, of which 21 species spread acrosshina [1]. Among them, gamboge (Garcinia hanburyi Hook. f.) isne of the most commonly used species, which is well known toossess anti-tumor activities [2–8]. The most notable lead com-ound derived from gamboge is gambogic acid (GA), which haseen tested as a wide spectrum anti-tumor drug in clinical trialhase II in China [9–12]. To identify new gambogic acid derivativesnd to search for substitute plant resource of import G. hanburyi inhina, a series of researches on the cytotoxicity [12–14], stability15], and quality control [16] of Garcinia plants were conducted inur laboratory.

Polyprenylated xanthones were found to be the majornticancer components of Garcinia plants [17,18]. In particu-ar, G. xipshuanbannaensis, a rare and endemic species in theishuanbanna district of Yunnan Province [19,20], contains preny-

∗ Corresponding author.E-mail address: xuhongxi@hkjcicm.org (H.-X. Xu).

bacimbus

021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2008.08.042

© 2008 Elsevier B.V. All rights reserved.

ated xanthones as its main bio-active components [21]. Thetructure–activity relationship indicated that xanthones withnsaturated prenyl group have stronger anticancer activity [21].herefore, systematic bio-assay guided isolation of different Gaciniapecies including G. xipshuanbannaensis was conducted. However,big problem encountered in our research was the repeated dis-

overy of known compounds in isolation and structural elucidation.ereplication therefore may circumvent the time-consuming and

edious discovery process on known structures [22–25]. For thaturpose, fragmentation mechanisms and empirical formula cal-ulations should be used in addition to other available analyticalethods to identify these xanthones.High-performance liquid chromatography (HPLC) with small

articles and high pressures is an incremental change, has proveno be an effective separation tool to fractionate complex extractsecause of its high selection and sensitivity [26,27]. While othernalytical methods each have their own advantages, they alsoontain major shortcomings that compromise the separation and

dentification of compounds. MS, especially ESI-IT-MSn, provides

ore structural information for structure identification [28–31],ut only gives nominal mass and so may not be effective inntargeted metabolite profiling. Quadrupole time-of-flight masspectrometry (Q-TOF), on the other hand, allows the generation

132 Y. Zhou et al. / J. Chromatogr. A 1206 (2008) 131–139

Table 1Element constituents of major ions observed in on-line UPLC–MS/MS spectra of Garcinia xipshuanbannaensis

Peaks Rt. Compounds Formula Calculated Observed Error (ppm)

1 1.06 Bannaxanthone H C28H33O7 481.2226 481.2229 0.6C21H25O5 357.1702 357.1706 1.1C23H25O5 341.1025 341.1034 2.6C18H17O5 285.0399 285.0413 4.9C14H9O5 257.0450 257.0445 −1.9C17H15O5 229.0501 229.0513 5.2

2 1.41 1,3,5,6-Tetrahydroxy-2-(3-methylbut-2-enyl)xanthone C18H17O6 329.1025 329.1032 2.1C14H9O6 273.0399 273.0402 1.1C12H7O5 231.0293 231.0287 −2.2C7H5O4 153.0188 153.0180 −5.2

3 1.65 Bannaxanthone F C28H33O9 513.2125 513.2131 1.2C28H29O7 477.1913 477.1916 0.6C24H21O7 421.1287 421.1307 4.7C21H25O8 405.1549 405.1541 −2.0C21H23O7 387.1444 387.1438 −1.5

4 1.81 Garcinone C C23H27O7 415.1698 415.1710 2.9C19H17O6 341.1025 341.1024 −0.3C15H9O6 285.0399 285.0407 2.8C14H9O5 257.0450 257.0461 4.3C13H9O4 229.0501 229.0492 −3.9C8H5O4 165.0188 165.0195 4.2

5 2.10 1,3,6,7-Tetrahydroxy-8-(3-methylbut-2-enyl)xanthone C18H17O6 329.1025 329.1026 0.2C29H29O8 273.0399 273.0395 −1.1C28H29O7 245.0450 245.0455 2.1C28H27O6 217.0501 217.0504 1.4

6 2.40 Bannaxanthone G C28H31O8 495.2019 495.2023 0.8C28H29O7 477.1913 477.1921 1.7C28H27O6 459.1808 459.1826 3.9C24H25O8 441.1549 441.1550 1.0C24H21O6 405.1338 405.1345 1.5C21H17O5 351.1232 351.1252 5.7

7 2.89 Bannaxanthone B C23H25O7 413.1600 413.1602 0.5C19H15O6 339.0869 339.0874 1.5C15H9O6 285.0399 285.0385 −4.9C14H9O5 257.0450 257.0451 0.4C12H7O5 231.0293 231.0281 −5.2

8 3.20 �-Mangostin C23H25O6 397.1651 397.1641 −2.5C19H17O6 341.1025 341.1024 −0.3C15H9O6 285.0399 285.0392 −2.5C14H9O5 257.0450 257.0442 −3.1C8H5O4 165.0188 165.0180 −4.8

9 5.17 Garcinone E C28H33O6 465.2277 465.2277 0C24H25O6 409.1651 409.1654 0.7C20H17O6 353.1025 353.1027 0.6C16H9O6 297.0399 297.0406 2.4C13H7O5 243.0293 243.0297 1.6

10 7.26 Bananxanthone E C28H31O7 479.2070 479.2067 −0.6C24H21O6 405.1338 405.1324 −3.5C24H19O5 387.1232 387.1224 −2.1C12H11O4 219.0657 219.0665 3.6

11 7.80 Allanxanthone C C28H33O6 465.2277 465.2270 −1.5C21H25O4 341.1753 341.1758 1.5C17H17O4 285.1127 285.1124 −1.1C16H17O3 257.1178 257.1181 1.2C13H9O4 229.0501 229.0507 2.6

12 9.14 Bannaxanthone D C28H31O6 463.2121 463.2119 −0.4C24H23O6 407.1495 407.1513 4.4C20H15O6 351.0869 351.0886 4.8C20H13O5 333.0763 333.0760 −0.9C19H15O5 323.0919 323.0908 −3.4

13 2.74 1,3,5,6-Tetrahydroxy-7-(3-methylbut-2-enyl)xanthone C18H17O6 329.1025 329.1027 0.6C29H29O8 273.0399 273.0389 −3.7C28H29O7 245.0450 245.0453 1.2C28H27O6 217.0501 217.0503 0.9

Y. Zhou et al. / J. Chromatogr. A 1206 (2008) 131–139 133

Table 1 (Continued )

Peaks Rt. Compounds Formula Calculated Observed Error (ppm)

14 6.61 Xanthone V1a C23H25O6 397.1651 397.1645 −1.5C19H17O6 341.1025 341.1029 1.2C15H9O6 285.0399 285.0404 1.8C12H9O5 233.0450 233.0441 −3.9C7H7O4 155.0344 155.0340 −2.6

15 7.13 Nigrolinexanthone V C24H25O6 409.1651 409.1654 0.7C20H17O6 353.1025 353.1034 2.5

19H15O18H12O16H11O9H7O4

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f mass information with greater accuracy and precision, and soas been used to determine the molecular formula at low ppm.recursor ions could be fragmented in the ES source with nitrogenollision gas at high declustering potential. ESI-QTOF-MS/MS/MSas been recently introduced for compound identification, since

t provides abundant structural information and exact mass [32].owever, it may be difficult to detect trace compounds in theomplex crude extracts without HPLC separation, due to the sup-ressing effect. Many MS parameters including RF lens value,ampling cone voltage, nitrogen gas flow rate, collision energy,nd cell entrance may also influence the in-source fragmentation.n-line LC–QTOF-MS/MS/MS method remains difficult to handle.lthough controlling HPLC and Q-qTOF separately can also achieveigh quality mass spectra [33], the operation is inconvenient andannot be used for the automatic analysis of large amounts of sam-les.

Compared to the above-mentioned methods, the on-linePLC–QTOF-MS/MS/MS method is more effective. In this study,e have optimized and established a reliable HPLC–QTOF-S/MS/MS method for the simultaneous analysis of prenylated

anthones in the crude extracts of G. xipshuanbannaensis. Com-lex extracts could be separated rapidly within 10 min by HPLC,hich minimizes the suppressing effect for analytes of low

oncentration during ESI process. Adjusting the cone voltagearameter could fragment precursor ions. Then, each targeted

bmis

Fig. 1. Cone voltage fragmentation for compound 2. (A) Cone volta

5 323.0919 323.0924 1.55 308.0685 308.0693 2.66 299.0556 299.0575 6.4

179.0344 179.0345 0.6

roduct ions was selected to pass through the first quadrupoleQ1) for the second fragmentation, which was carried out in

collision cell (Q2) to generate MS/MS/MS fragment ions, ofhich m/z values were analyzed by QTOF. As a result, the

haracteristic fragmentation of prenylated xanthones was sum-arized. Based on the information, some trace xanthones were

dentified.

. Experimental

.1. Chemicals

Acetonitrile (HPLC–MS grade) was purchased from Fishier Sci-ntific (Loughborough, UK) and formic acid (spectroscopy grade)as purchased from Sigma–Aldrich (MQ, USA). Water was purified

in-house’ using a MilliQ SP Regent Water system (Millipore, MA,SA), Leucine-enkephalin was obtained from Sigma–Aldrich.

.2. Sample preparation

The twigs of G. xipshuanbannaensis were collected in Xishuang-anna Prefecture, Yunnan Province, China in 2006. The plantaterial was identified by Prof. Wang Hong, Xishuangnanna Trop-

cal Botanical Garden, Chinese Academy of Sciences. A voucherpecimen (CMED-0471) was deposited in the Hong Kong Jockey

ge = 40 V, (B) cone voltage = 60 V and (C) cone voltage = 80 V

134 Y. Zhou et al. / J. Chromatogr. A 1206 (2008) 131–139

xtrac

CtucrAib

(tb

Fig. 2. Structures assigned in the e

lub Institute of Chinese Medicine. The fine powder (0.2 g) ofhe dry plant material was extracted with methanol (3× 5 ml)sing supersonic washer for 10 min. The extraction solutions were

ombined, and diluted to 25 ml with methanol in a volumet-ic flask, and then filtered through a 0.22 �m PTFE syringe filter.n aliquot of each filtrate (2 �l) was injected into the UPLC

nstrument for analysis. Twelve reference compounds, namelyannaxanthone H (1) [21], 1,3,5,6-tetrahydroxy-2-prenylxanthone

btltc

ts of Garcinia xipshuanbannaensis.

2) [34], bannaxanthone F (3) [21], garcinone C (4) [35], 1,3,6,7-etrahydroxy-8-prenylxanthone (5) [36], bannaxanthone G (6) [21],anaxanthone B (7) [21], �-mangostin [37] (8), garcinone E (9) [37],

annaxanthone E (10) [21], allanxanthone C [38] (11), banaxan-hone D (12) [21] were isolated from G. xipshuanbannaensis in thisaboratory and were identified based on IR, UV, and NMR spec-roscopy analyses. They were dissolved in acetonitrile to give aoncentration of 0.1–2 �g/ml.

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Y. Zhou et al. / J. Chroma

.3. Liquid chromatography

HPLC was performed using a Waters ACQUITY UPLCTM systemWaters Corp., MA, USA), equipped with a binary solvent deliveryystem, autosampler, and a PDA detector. The chromatography waserformed on a Waters ACQUITY BEH C18 column (50 mm × 2.1 mm,.7 �m, Waters Corp., Ireland). The mobile phase consisted of (A).1% formic acid in water and (B) acetonitrile containing 0.1% formiccid. The HPLC eluting conditions were optimized as follows: iso-ratic at 50% B (0–0.5 min), linear gradient from 50 to 70% B0.5–3 min), linear gradient from 70 to 80% B (3–6 min), linear gra-ient from 80 to 95% B (6–9 min), isocratic at 95% B (9–10 min), and

inear gradient from 95 to 50% B (10–11 min). The flow rate was.3 ml/min. The column and autosampler were maintained at 35nd 10 ◦C, respectively. Each wash cycle consisted of 200 �l of strongolvent (90% acetonitrile) and 600 �l of weak solvent (50% acetoni-rile). The injection volume of the standards and sample is 2 �l.AD detector was set to scan from 200 to 400 nm, and 310 nm wassed as detection wavelength for the polyprenylated xanthonesnalysis.

.4. Mass spectrometry

Mass spectrometry was performed using a Waters Q-TOF Pre-ier (Micromass MS Technologies, Manchester, UK) operating in

ositive ion mode. The nebulization gas was set to 600 l/h atemperature of 300 ◦C, the cone gas set to 50 l/h, and the sourceemperature set to 80 ◦C. The capillary voltage and cone voltageere set to 2700 and 45 V, respectively. The Q-TOF premier acqui-

ition rate was set to 0.2 s, with a 0.02 s inter-scan delay. Argonas employed as the collision gas at a pressure of 5.3 × 10−5 Torr.

he instrument was operated with the first resolving quadrupole

n a wide pass mode with the collision cell operating at twolternative energies: 5 and 30 eV. The data was collected intowo separated data channels, with the instrument spending 0.1 sn data acquisition for each channel and a 0.02 s inter-channelelay.

wmwci

Fig. 3. Fragmentation pathways of [M+

1206 (2008) 131–139 135

The MS/MS experiments were carried out by setting the Q-OF premier quardrupole to allow ions of interest to pass prior toragmentation in the collision cell with argon collision gas at appro-riate collision energies to produce abundant product ions beforeetection in the TOF analyzer. The molecular masses of the pre-ursor ion and of the product ions are accurately determined witheference compound Leucine-enkephalin in the LockSpray modem/z 556.2771) at a concentration of 50 pg/�l and an infusion flowate of 10 �l/min. A dwell time of 0.1 s was employed with an inter-cquisition delay of 0.02 s.

For MS/MS/MS experiment, RF lens value, cone gas, samplingone voltage, nitrogen gas flow rate, collision energy, and cellntrance were adjusted for each sample to optimize signal andbtain maximal structural information from the ion of interest. Byesting different sampling cone voltage, the best cone voltage frag-

ent mass spectra was obtained at 60 V from the precursor [M+H]+.hus, precursor ions were first fragmented through cone voltageragmentation, and then the targeted product ion was selected toass through Q1 and fragmented in Q2 to generate MS/MS/MSragment ions. Observed masses were corrected for accurate mea-urement by using the reference compound Leucine-enkephalin inhe LockSpray.

. Results and discussion

.1. Optimization of HPLC conditions and cone voltageragmentation of standards

In order to obtain the optimal elution conditions for the separa-ion and determination of the constituents, various linear gradientsf aqueous solution and acetonitrile at a flow rate of 0.3 ml/min

ere investigated. By the optimal gradient elution, all of the 12ain peaks could be well separated within 10 min. 0.1% formic acidas added to both aqueous solution and acetonitrile to improve

hromatographic behavior, reduce the peak tailing and facilitateonization. The mixtures of acetonitrile and water solution con-

H]+ (A) for bannaxanthone H (1).

136 Y. Zhou et al. / J. Chromatogr. A 1206 (2008) 131–139

F (2) an(

t0o

pcvwmD

tc[cF

ig. 4. MS analysis of positional isomers of 1,3,5,6-tetrahydroxy-2-prenylxanthone11).

aining different concentrations of formic acid at a flow rate of.3 ml/min at 35 C were screened in the method development. Theptimized chromatographic conditions are described in Section 2.3.

Among the various MS parameters including RF lens value, sam-ling cone voltage, nitrogen gas flow rate, collision energy, and

ell entrance, which can influence the in-source dissociation, coneoltage is a key factor. When ions were produced by ESP, the ionshich were accelerated by the cone voltage collide with nitrogenolecules, and the cone voltage fragmentation may be observed.esired product ions generated through cone voltage fragmenta-

3

f

d 1,3,6,7-tetrahydroxy-8-prenylxanthone (5), garcinone E (9) and allanxanthone C

ion were mass selected in Q1 and fragmented in Q2 to obtainorresponding MS/MS/MS spectra. Fortunately, fragmentation ofM+H]+ ions occurred readily by cone voltage fragmentation. Theone voltage fragmentation by analyzing compound 2 is shown inig. 1.

.2. Fragmentation behavior of the prenylated xanthones

Authentic samples of 12 polyprenylated xanthones isolatedrom G. xipshuanbannaensis were studied by means of QTOF-MS

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Y. Zhou et al. / J. Chroma

n positive mode. Their structures are depicted in Fig. 2 and theirandem mass data are shown in Table 1. The fragmentation path-ays of the polyprenylated xanthones were found to follow these

eneral trends: all compounds showed abundant [M+H]+ ions, andome [M+Na]+ ions. All compounds yielded successive loss of prenylesidues C4H8 (56 Da) in the MS/MS spectra, which would be help-ul to identify the numbers of prenyl units. Some product ions withelatively high abundance observed in the MS spectrum were frag-ents of the [M+H]+ ions produced through in-source dissociation.otably, despite the community of MS fragmentation pattern of

he polyprenylated xanthones, compounds with trivial structuralifference may also result in different MS behavior.

The tandem mass spectra of bannaxanthone H (1) provide aepresentative example of above fragmentation pathways. As aompound characterizing oxygenated six member rings at C-7–C-8f ring B, which showed typical fragmentation patterns. In MS/MSpectrum, the fragment ion at m/z 341 appeared to be characteristicor the [M+H]+ at m/z 481, which was formed through McLaffertyearrangement to lose the prenyl group (69 Da) and further disso-iated through the Retro–Diels–Alder rearrangement at the doubleond C-7–C-8 to lose C4H7O (71 Da). In MS/MS/MS spectrum, the

on at m/z 341 was further fragmented to produce the fragment ionst m/z 285, 257, 229 by losing a prenyl residue C4H8, C4H8 + CO,6H8O2, respectively. The proposed fragmentation mechanism ishown in Fig. 3.

In the MS/MS/MS experiment, the Retro–Diels–Alder rearrange-ent at the double bond C-8a–C-10a or C-4a–C-9a was commonly

bserved, which was helpful to differentiate some positional iso-ers. 1,3,5,6-tetrahydroxy-2-(3-methylbut-2-enyl)xanthone (2),

nd 1,3,6,7-tetrahydroxy-8-(3-methylbut-2-enyl)xanthone (5) pre-ented the same protonated ion at m/z 329 corresponding to theolecular formulae C18H17O6. The prenyl unit is at C-2 of ring A

or compound 2, while the prenyl unit is at C-8 of ring B for com-ound 5. For compound 2, in the MS/MS spectrum of the [M+H]+

t m/z 329, the most abundant fragment peak (m/z 273) was dueo the loss of a prenyl unit C4H8 (56 Da). This fragment furtherissociated through the Retro–Diels–Alder rearrangement at theouble bond C-8a–C-10a to produce main fragment ion at m/z31 (Fig. 4A). For compound 5, the main fragment ion at m/z 273as also detected in the MS/MS spectrum of the precursor ion

M+H]+ at m/z 329, and this fragment was further dissociated toroduce the fragment ions at m/z 245 and 217, by the loss of CO and4H8 (56 Da), respectively. The ion at m/z 217 was formed throughetro–Diels–Alder rearrangement at the double bond C-8a–C-10aFig. 4B). Thus, MS/MS/MS tandem mass spectra could be helpful

o differentiate the isomers with the prenyl unit at ring A or ring Bn the xanthone skeleton.

Isomers garcinone E (9) and allanxanthone C (11) eluted in UPLCt 5.1 and 7.8 min, presented the same protonated ion [M+H]+ at m/z65 corresponding to the molecular formulae C28H33O6. Since the

wmmrM

Fig. 5. UPLC–QTOF-MS chromatograms of the crude extract of G. xipshuanban

1206 (2008) 131–139 137

renyl units are located at different positions, their fragmentationatterns are also different. For garcinone E (9), the major product

ons of [M+H]+ at m/z 465 were at m/z 409, 353, and 297 by elim-nation of a prenyl residue C4H8, two prenyl residues 2C4H8, andhree prenyl residues 3C4H8, respectively. A fragment ion at m/z43 from the precursor ion at m/z 353 was observed abundantlyn the MS/MS/MS spectrum, indicating that the Retro–Diels–Alderearrangement at C-4a–C-9a double bond occurs more readily afterhe prenyl units have dissociated (Fig. 4C and D). As for allanxan-hone C (11), the product ion at m/z 341 of [M+H]+ at m/z 465 wasormed by eliminating a prenyl group C5H9 and then undergoingetro–Diels–Alder rearrangement at the C-4a–C-9a double bondy loss of C7H8O2. Further dissociation of the ion at m/z 341 inS/MS/MS spectrum produced product ions at m/z 285, 257, 229

y the loss of C4H8, C4H8 + CO, 2C4H8, respectively (Fig. 4E and F).hus, the application of detailed MS/MS/MS analysis allowed theifferentiation of the two isomers.

.3. Chemical profiles of Garcinia xishuanbannaensis

UPLC–DAD–QTOF-MS/MS/MS was employed to analyze therenylated xanthones in the extract of G. xishuanbannaensis.his class of xanthones had similar UV maximal absorptionavelengths of around 250–330 nm. The chromatograms of G.

ishuanbannaensis included main peaks 1–12, namely bannaxan-hone H (1), 1,3,5,6-tetrahydroxy-2-(3-methylbut-2-enyl)xanthone2), bannaxanthone F (3), garcinone C (4), 1,3,6,7-tetrahydroxy-8-3-methylbut-2-enyl)xanthone (5), bannaxanthone G (6), banaxan-hone B (7), �-mangostin (8), garcinone (9), bannaxanthone E (10),llanxanthone C (11), and banaxanthone D (12) (Fig. 5).

The peak 13 showed an accurate mass of [M+H]+ ion at m/z29.1027 gave the molecular formula C18H17O6. Fragment ionsbserved for 13 were closely similar to compound 5, indicatinghat they have the same skeleton. The MS/MS spectra containedn abundant fragment ion at m/z 273, formed by the loss of C4H856 Da). The MS/MS/MS spectrum of the precursor ion at m/z 273isplayed fragment ions at m/z 245, 217, 189, and 171. The diag-ostic ion at m/z 217 formed by Retro–Diels–Alder rearrangementt the double bond C-8–C-8a indicated that the prenyl unit is oning B. According to this finding as well as other supporting evi-ence from literature, compound 13 could be tentatively assigneds 1,3,5,6-tetrahydroxy-7-(3-methylbut-2-enyl)xanthone [21]. Par-ial fragmentation pathways of 13 are illustrated in Fig. 6A and.

The trace peak 14 eluted in the chromatography at Rt. 6.61 min,

hich gave a [M+H]+ ion at m/z 397.1645 corresponded to theolecular formula C24H25O6. Its MS/MS spectra yielded ions at/z 341 and 285, which were generated by the loss of a prenyl

esidue C4H8 (56 Da) and two prenyl residues 2C4H8 (112 Da). TheS/MS/MS spectra of the ion at m/z 285 produced the ion at m/z

naensis. (A) UPLC chromatogram and (B) total ion chromatogram (TIC).

1 togr. A

2bi[a

syCmi

rtocf

L

38 Y. Zhou et al. / J. Chroma

33, formed by Retro–Diels–Alder rearrangement at the doubleond C-4a–C-9a, which validated that the two prenyl units existed

n ring A. Thus, this peak was tentatively identified as xanthone V1a39]. Partial fragmentation pathways of 14 are illustrated in Fig. 6Cnd D.

Peak 15 (Rt. 7.13) gave a [M+H]+ ion at m/z 409.1658 corre-ponded to the molecular formula C24H25O6. Its MS/MS spectra

ielded a dominant ion at m/z 353 by elimination of a prenyl residue4H8 (56 Da). This ion was further broken down to produce frag-ent ions at m/z 323, 308, 299, and 179. The characteristic fragment

ons at m/z 299 and 179 were formed from Retro–Diels–Alder rear-

coei

Fig. 6. MS analysis of 1,3,5,6-tetrahydroxy-7-(3-methylbut-2-enyl)xa

1206 (2008) 131–139

angement at the double bonds C-4a–C-9a, and C-6–C-7, respec-ively. The formation of the ion at m/z 299 indicated the prenylatedxygenated six member ring existed at C-6–C-7. Therefore, peak 15ould be tentatively identified as nigrolinexanthone V [40]. Partialragmentation pathways of 15 are illustrated in Fig. 6E and F.

It should be noteworthy that these LC–MS/MS andC–MS/MS/MS techniques complement each other, and their

ombination provides an effective method for the dereplicationf known compounds in Garcinia species. Incidentally, differ-nt Garcinia species contain different kinds of compounds, fornstance, G. hanburyi mainly contains cadged xanthones, and

nthone (13), xanthone V1a (14), and nigrolinexanthone V (15).

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Y. Zhou et al. / J. Chroma

arcinia lancilimba, Garcinia yunnanensis, and Garcinia oblongifoliaainly contain polyprenylated benzoylphloroglucinols. Thus, this

ewly HPLC–QTOF-MS/MS/MS method was applied to study ninearcinia species. As a result, we constructed a database containing

he uniform HPLC and MS information of the polyprenylatedanthones, benzophenones and cadged xanthones obtained fromarcinia plants in our laboratory.

However, it should be pointed out that although there has beenajor progress made in the application of ESI-QTOF-MS/MS/MS to

he analysis of natural products compounds, the connection of theubstituents in some isomers and some stereochemistry still cannote solved. For example, compounds 5 and 13 presented very similarS spectra, since the prenyl unit is located in A ring, thus tandemS cannot dedifferentiate the two compounds. Moreover, using

his combined technology, only the product ions with peaks of suffi-iently large intensity could be selected for subsequent MS/MS/MSxperiment.

. Conclusion

A number of conclusions may be drawn from this investiga-ion in which on-line UPLC–ESI-QTOF-MS and detailed MS/MS,

S/MS/MS analyses were used to study the polyprenylatedanthone for the first time. A total of 12 xanthones from G. xipshuan-annaensis were separated within 10 min, and were unequivocallydentified via comparisons with authentic standards. Fragmen-ation pathways of the polyprenylated xanthone were proposedo rationalize the observed behavior. Specific fragment ions gen-rated under the present MS/MS/MS conditions allowed thestablishment of indicators for structural features, such as theetro–Diels–Alder rearrangement in the xanthones skeleton, whichould be useful to differentiate positional isomers with prenyl unit

n ring A or ring B.To solve the practical difficulty in repeated discovery of known

ompounds in the G. xipshuanbannaensis, the UPLC–MS/MS/MSethod was optimized and established for selective and reliable

lucidation of known xanthones based on the retention time, accu-ate mass analyses, and tandem mass spectra. The three traceanthones observed in G. xipshuanbannaensis could be tentativelydentified by interpretation of their [M+H]+ and fragment ionshrough related standards.

cknowledgements

This research was supported by the Hong Kong Jockey Club Char-ties Trust Fund. The authors are grateful to Ms. Swee Lee Yap of

aters Corp. for discussion of building MS method.

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