Fitoterapia 75(2004) 342–354

0367-326X/04/$ - see front matter� 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.fitote.2004.03.001

HPLC-DAD-MS identification of bioactivesecondary metabolites fromFerula communis

roots

Lolita Arnoldi *, Mauro Ballero , Nicola Fuzzati ,a, b a

Andrea Maxia , Enrico Mercalli , Luca Pagnib a a

Indena S.p.A. Research and Development Laboratories, Via Don Minzoni 6, 20090 Settala (MI), Italya

Department of Botanical Science, University of Cagliari, Viale S. Ignazio 13, Cagliari, Italyb

Received 2 February 2004; accepted 11 March 2004

Abstract

A simple HPLC method was developed to distinguish between ‘poisonous’ and ‘non-poisonous’ chemotypes ofFerula communis. The method was performed on a C reverse8

phase analytical column using a binary eluent(aqueous TFA 0.01%–TFA 0.01% inacetonitrile) under gradient condition. The two chemotypes showed different fingerprints.The identification of five coumarins and eleven daucane derivatives by HPLC-diode arraydetection(HPLC-DAD) and HPLC-MS is described. A coumarin, not yet described, wasdetected.� 2004 Elsevier B.V. All rights reserved.

Keywords: Ferula communis; Daucanes; Ferutinin; Prenyl coumarins; HPLC-MS; HPLC-DAD-UV

1. Introduction

Plants from theFerula spp. have a long history of medicinal use and theirhormonal effects are well documented in both the human and the veterinary practicew1x. Biological activity is mainly attributable to ferutinin, an aromatic ester of adaucane alcohol. An Italian species ofFerula, the SardinianF. communis L. (giantfennel), has been reported to contain ferutinin, acting as a possible source of

*Corresponding author.E-mail address: lolita.arnoldi@indena.com(L. Arnoldi).

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phytoestrogens of the daucane typew2x. Unfortunately, this plant is morphologicallyindistinguishable from the poisonous chemotype ofF. communis infesting Sardinianpasturesw3x. The poisonous chemotype ofF. communis can cause a lethal haemor-rhagic disease known as ‘ferulosis’ and its toxicity was traced back to the prenylatedcoumarin ferulenol and related analoguesw4,5x. The two chemotypes are reportedto be genetically differentw6x and present a different geographic distributionw3,6x.Recently, an hystochemical analysis of fresh sample ofF. communis has been

proposed in alternative to TLC as a way to distinguish the two chemotypesw7x.However, no further analytical study to distinguish and characterise the twochemotypes has been reported yet.With the aim of providing a more general and comprehensive method to

distinguish between the two chemotypes, we have developed a HPLC method tofingerprint the two chemotypes of SardinianF. communis, identifying variouscoumarins and daucane derivatives by HPLC-diode array detection(HPLC-DAD)and HPLC-MS.

2. Experimental

2.1. Materials

Acetonitrile (MeCN) and methanol(MeOH) HPLC grade were purchased fromJ.T. Baker(Deventer, The Netherlands). Water was purified by MilliQ systemplus

from Millipore (Milford, MA, USA). Trifluoroacetic acid(TFA) was purchasedfrom Merck (Darmstadt, Germany). Formic acid was purchased from Fluka(Buchs,Switzerland). All other solvents and reagents were of analytical grade.Ferutinin and jaeskeanadiol benzoate were isolated and purified in Indena

Chemical Laboratories. Ferulenol and lapiferin were kindly provided by Prof. G.Appendino(University of Piemonte Orientale, Italy). All the authentic samples weresolubilised in MeOH.

2.2. Plants

Both ‘poisonous’ and ‘non-poisonous’ chemotypes ofF. communis roots werecollected in Sardinia(Italy), near Bonarcado(OR) and near Iglesias(CA),respectively.Voucher specimens were preserved for reference in the Department of Botany,

University of Cagliari(exiccata reference numbers: 612yb: ‘poisonous’F. communisL.; 612yc: ‘non-poisonous’F. communis L.).

2.3. Analytical procedure

2.3.1. Plant material extractionPowdered dried roots(0.5 g) were dissolved in 150 ml of MeOH, sonicated for

20 min and shaken for 2 h at 200 rev.ymin. The extracts were filtered(0.2m filter)and analysed.

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2.3.2. HPLC-DAD-UV analysisThe HPLC apparatus included a PumpySystems Controller Waters Alliance 2690;

a PDA 996 Waters Photodiode array detector and Software Millenium 32(version3.1).Separation was performed on a Zorbax XDB C8 column(250=4.6 mm ID; 5m;

100 A) set at 308C. The solvent system was: TFA 0.01% in water(solvent A) and˚TFA 0.01% in MeCN(solvent B), starting with 55% A for 5 min, then a lineargradient from 55% A to 10% A in 40 min. The flow rate was 1.0 mlymin; the PDAdetection range was 190–400 nm; the injection volume was 10ml.

2.3.3. HPLC-DAD-MSThe HPLC system included: SpectraSYSTEM P4000 pump; SpectraSYSTEM AS

3000 automatic sample injector module; SpectraSYSTEM UV6000LP diode arraydetector(DAD); equipped with a Microsoft Window NT� data system.� �

Separation conditions were the same as described in HPLC-DAD-UV analysis,using HCOOH 0.1% instead of TFA 0.01%.Mass spectral analyses were performed with a Finnigan MAT LCQ ion trap mass

spectrometer equipped both with ESI and APCI interface. ESI mass spectra wereacquired in both positive and negative-ion modes by scanning over the 100–1500mass range.For ESI-MS detection the following instrument parameters were used:

– positive mode: capillary temperature 2508C; sheath gas flow: 90 units; auxiliarygas flow: 60 units; source voltage: 4.8 kV; capillary voltage: 19 kV;

– negative mode: capillary temperature 2908C; sheath gas flow: 80 units; auxiliarygas flow: 40 units; source voltage: 4.0 kV; capillary voltage:y33 kV;

APCI positive parameters were: capillary temperature 708C; sheath gas flow: 80units; auxiliary gas flow: 50 units; source voltage: 6.0 kV; capillary voltage: 30 kV;vaporizer temperature: 3508C.For MSyMS experimentswMqHx , wM-H OqHx or wMqNax ions wereq q q

2

isolated with an isolation width of 2myz units and fragmented. Fragmentation wascarried out using an activation amplitude of 30%.

3. Results and discussion

The HPLC-DAD-UV, HPLC-ESI-MS recorded in positive and negative ion modeand the positive HPLC-APCI-MS, MS analyses of ‘poisonous’ and ‘non-poisonous’2

F. communis extracts allowed to identify eleven daucanes and five coumarins(Fig.1; Table 1).Fig. 2a shows the HPLC profile at 197 nm of the methanolic extract of the ‘non-

poisonous’ chemotype under the optimised conditions of analysis. The chromatogramshows the presence of two main peaks: ferutinin7 and the prenylated coumarinferulenol 13, both identified by comparison of the spectral and chromatographicdata with authentic samples. Further 10 minor peaks, compounds1, 2, 4–6, 8, 11,12, 15 and 16 were found. Peaks4 and 16 could be identified as lapiferin and

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Fig. 1. Daucane and prenyl coumarins derivatives of SardinianF. communis.

jaeskeanadiol benzoate, respectively, by comparison with authentic samples. Thepresence of ferutinin and other daucane derivatives in the ‘non-poisonous’ chemotypeof F. communis is described in literaturew2,6–8x. Several other daucane derivativeswere described for theFerula spp., including compounds modified at the position6 w9x.Ferutinin presents al at 256 nm, attributable to thep-hydroxy benzoatemax

chromophore. The positive ESI-MS spectrum of ferutinin(Fig. 3a) exhibits a signalat myz 341 wM-H OqHx and a fragment atmyz 203 wM-H O-(p-OH-BzOH)qq

2 2

Hx , showing a very high tendency to lose H O. On the contrary, the negative ionq2

ESI-MS spectrum shows ions atmyz 357 wMyHx and at myz 403 wM-Hqy

HCOOHx (Fig. 3b). In positive APCI-MS spectrum(Fig. 3c) ferutinin also showsy

a high tendency to lose H O, however, thewMqHx ion at myz 359 can beq2

detected. In APCI MS spectrum(Fig. 3d) of the signalwM-H OqHx the main2 q2

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342–354Table 1Main signals exhibited in the HPLC-MS and HPLC-MS spectra of compounds detected inF. communis and proposed attribution*2

Peak Rrt MW ESIqmyz ESIymyz APCIqmyz Fragment ionsmyz

1 0.50 374 357wM-H OqHxq2 419 wM-HqHCOOHx- 375 wMqHxq 357™339 wM-2H OqHxq2

wM-H O-pOHBzOHqHxq219 2 wMyHxy373 wM-H OqHxq357 2 wM-H O-pOHBzOHqHxq219 2

201 wM-2H O-pOHBzOHqHxq2

121 wpOHBenzoylxq

2 0.63 416 399wM-H OqHxq2 461 wM-HqHCOOHxy 417 wMqHxq 399™339 wM-H O-AcOHqHxq2

219 wM-AcOH-pOHBzOHqHxq wMyHxy415 wM-H OqHxq399 2 201 wM-H O-AcOH-pOHBzOHqHxq2

201 wM-H O-AcOH-pOHBzOHqHxq2 357 wM-AcOHqHxq 173; 159; 145; 131; 117: di-esterifieddaucane moiety fragments

3 0.72 382 405wMqNaxq wMyHxy381 wMqHxq383 383™365 wM-H OqHxq2

383 wMqHxq 365 wM-H OqHxq2 309, 283, 269 and 255: prenylatedwM-H OqHxq365 2 coumarin moiety fragments

4 0.80 394 wM-H OqHxq377 2 436 wMqCH CNqHxq3 377™359 wM-2H OqHxq2

277 wM-H O-AngOHqHxq2 418 wM-H OqCH CNqHxq2 3 277 wM-H O-AngOHqHxq2

217 wM-H O-AngOH-AcOHqHxq2 395 wMqHxq wM-H O-AngOH-AcOHqHxq217 2

wM-H OqHxq377 2 199 wM-2H O-AngOH-AcOHqHxq2

317 wM-H O-AcOHqHxq2

277 wM-H O-AngOHqHxq2

217 wM-H O-AngOH-AcOHqHxq2

5 0.82 386 w2MqNaxq795 409™ wM-AniOHqNaqCH CNxq298 3

450 wMqNaqCH CNxq3 274 wM-AniOHqCH CNxq3

409 wMqNaxq 257 wM-AniOHqNaxq

6 0.97 456 439wM-H OqHxq2 501 wM-HqHCOOHxy 456 wMqHxq 439™339 wM-H O-AngOHqHxq2

219 wM-AngOH-pOHBzOHqHxq wMyHxy455 wM-H OqHxq439 2 301 wM-H O-pOHBzOHqHxq2

wM-H O-AngOH-pOHBzOHqHxq201 2 357 wM-AngOHqHxq wM-H O-AngOH-pOHBzOHqHxq201 2

339 wM-H O-AngOHqHxq2 173; 159; 145; 131; 117: di-esterifieddaucane moiety fragments

7 1.00 358 wM-H OqHxq341 2 403 wM-HqHCOOHxy 359 wMqHxq 341™ wM-H O-pOHBzOHqHxq203 2

203 wM-H O-pOHBzOHxq2 wMyHxy357 wM-H OqHxq341 2 121 wpOHBenzoylxq

175; 161; 147; 133; 119: daucanemoiety fragments

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Table 1(Continued)

Peak Rrt MW ESIqmyz ESIymyz APCIqmyz Fragment ionsmyz

8 1.01 388 wM-H OqHxq371 2 433 wM-HqHCOOHxy 389 wMqHxq 371™ wM-H O-VanOHqHxq203 2

151 wVanilloylxq wMyHxy387 wM-H OqHxq371 2 151 wvanilloylxq

175; 147; 133; 119 daucanemoiety fragments

9 1.05 447wMqNaxq 425™365 wM-AcOHqHxq

424 425wMqHxq wMyHxy423 wMqHxq425 309, 283, 269 and 255: prenylated10 1.07 wM-AcOHqHxq365 365 wM-AcOHqHxq coumarin moiety fragments

11 1.15 384 wM-H OqHxq367 2 426 wM-HqHCOOHxy 385 wMqHxq

203 wM-H O-pcoumOHqHxq2 wMyHxy383 wM-H OqHxq367 2

12 1.19 402 wM-H OqHxq385 2 403 wMqHxq 385™ wM-H O-VerOHqHxq203 2

203 wM-H O-VerOHqHxq2 wM-H OqHxq385 2 165 wVeratroylxq

165 wVeratroylxq 175; 161, 147; 133; 119: daucanemoiety fragments

13 1.31 366 wMqHxq367 wMyHxy365 wMqHxq367 367™311; 297; 285, 271, 257; 243;231; prenylated coumarin moiety fragments175

14 1.35 486 509wMqNaxq wMyHxy485 wMqHxq487 487™ wM-BzOHqHxq365487 wMqHxq 365 wM-BzOHqHxq 309, 283, 269 and 255: prenylated

wM-BzOHqHxq365 coumarin moiety fragments

15 1.35 372 wM-H OqHxq355 2 373 wMqHxq 355™ wM-H O-AniOHqHxq203 2

203 wM-H O-AniOHqHxq2 wM-H OqHxq355 2 135 wAnisoylxq

175; 161, 147; 133; 119: daucanemoiety fragments

16 1.38 342 wM-H OqHxq325 2 343 wMqHxq 325™ wM-H O-BzOHqHxq203 2

203 wM-H O-BzOHqHxq2 wM-H OqHxq325 2 175; 161, 147; 133; 119: daucanemoiety fragments

The relative retention time is referred to ferutinin.pOHBzOH: p-hydroxybenzoic acid; AcOH: acetic acid; AngOH: angelic acid; AniOH: anisic acid; VanOH: vanillic acid;pCoumOH:p-coumaric acid; VerOH:

veratric acid; BzOH: benzoic acid.*Base peak is underlined.

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Fig. 2.(a) HPLC-DAD profile atls197 nm ofF. communis ‘non-poisonous’ chemotype.(b) HPLC-DAD profile atls197 nm ofF. communis ‘poisonous’chemotype.

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Fig. 3. ESI positive(a); ESI negative(b); APCI positive(c) and APCI positive MSyMS spectra(d) offerutinin (7). Proposed fragmentation pathway.

350 L. Arnoldi et al. / Fitoterapia 75 (2004) 342–354

ion at myz 203 was originated by the loss of the ester moiety from the daucaneskeleton, a process partially occurring with retention of charge as shown by thedetection of the complementary core moiety atmyz 121. Besides these two peaks,signals due to the fragmentation of the daucane skeleton are observed atmyz 119,133, 147, 161 and 175.Jaeskeanadiol benzoate16, already described as constituent of ‘non-poisonous’

chemotype of SardinianF. communis w3x, exhibits the same behaviour of ferutininin the positive ESI-MS, APCI-MS and MS spectra. In particular the presence of2

the signal atmyz 203 together with the fragments atmyz 119, 133, 147, 161 and175 are diagnostic of daucanes with the jaeskeanadiol core. Furthermore, becauseof the lack of thep-OH acidic substituent on the aromatic ester group, jaeskeanadiolbenzoate displays a maximum at 229 nm in the UV spectrum and no response inthe negative ESI-MS analysis. As occurred for ferutinin and jaeskeanadiol benzoate,the positive APCI-MS spectra of compounds8, 11, 12 and 15 show thewMqHxq

ions and thewM-H OqHx fragments. In negative ESI spectra of compounds8 andq2

11 the wMyHx ion is observed atmyz 387 and 383, while no signal is obtainedy

for compounds12 and15. This behaviour suggests the presence of an acidic groupin the structure of compounds8 and11, whereas the lack of an acidic residue couldbe inferred for compounds12 and 15. In APCI MS spectra of compounds8, 11,2

12 and 15 the occurrence of the ion atmyz 203 allows to identify the presence ofthe jaeskeanadiol polyol structure. Moreover, the signals atmyz 151, 165 andmyz135 detected in the spectra of compounds8, 12 and15, respectively, are attributableto the presence of vanillic, veratric and anisic esters. The UV spectra of compounds8, 11, 12 and15 confirmed the identification of the daucane substituents. Therefore,compounds8, 12 and 15 could be identified as the ferutinin derivatives teferin,akiferin and ferutidin, respectively. Teferin was found inF. hermonis Boiss, F.jaeschkeana Vatke, F. elaeochytris Korovin and F. rigidula Fisch. ex DC.w10x.Akiferin and ferutidin were described as constituents of SardinianF. communisw3,8x. Finally, compound11 was identified asp-coumaroyl jaeskeanadiol, a daucaneester already reported inF. rigidula w11x.Due to the presence of several fragments, the ESI and APCI positive spectra of

compounds2 and 6 are more complex than those of mono-substituted daucanes.The ESI negative spectra of both compounds showed thewMyHx signal, indicatingy

the presence of an acidic group and allowing the determination of the molecularweight. The UV spectra of2 and 6 show a singlel at 257 nm, indicating themax

presence of ap-hydroxybenzoate group. In APCI positive MS of ionswM-H Oq22

Hx , the presence of the main peak atmyz 201 together with minor peaks atmyzq

117, 131, 145, 159 and 173, 2 amu less than ions originated from mono-esterdaucane, suggests the presence of two ester groups on the daucane core moiety.Furthermore, the MS spectra of compounds2 and 6 exhibit a signal atmyz 339,2

attributable to a loss of acetic and angelic acid, respectively. Compounds2 and 6were therefore identified as acetoxyferutinin and fertidin, respectively, both describedfor F. communis L. var. brevifolia Mariz w12x, a Moroccan variety ofF. communis.

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The UV spectrum of4 presents al at 218 nm, indicating the lack of themax

aromatic chromophor. The lack of response in negative ESI-MS spectrum suggeststhe absence of an acidic group. Due to the presence of several fragments andadducts(Table 1) compound4 shows complex APCI spectra. The positive ESI-MSand APCI MS spectra shows the presence of ions atmyz 277 wM-H O-AngOHq2

2

Hx and myz 217 wM-H O-AngOH-AcOHqHx attributable to the successiveq q2

losses of angelic and acetic acid. Compound4 was identified as lapiferin, previouslydescribed inF. linkii Webb w13x, by injection of the authentic sample. Therefore,the presence of a signalmyz 359 in the APCI-MS spectrum due to the loss of two2

H O molecules was considered peculiar to the epoxy-daucane.2

The l at 257 nm in the UV spectrum of compound1 and the occurrence ofmax

the ionmyz 219, wM-H O-pOHBzOHqHx in both the positive ESI-MS and APCI-q2

MS spectra indicate the presence of ap-hydroxybenzoyl moiety. Furthermore, the2

presence of both signals atmyz 339, wM-2H OqHx and myz 201, wM-2H O-q2 2

pOHBzOHqHx suggests the presence of the epoxy group on the daucane coreq

moiety. Compound1 was therefore identified as jeaeskenin, first described inF.jaeschkeana w14x.Compound5 shows the same UV spectrum as ferutinin, suggesting the presence

of a chromophore due to an aromaticp-substituted ester. The lack of response inthe ESI negative mode indicated the lack of the hydroxy group on the chromophore.Differently from all the other daucane esters where the ionwM-H OqHx representsq

2

the main signal, the positive ESI-MS spectrum of5 exhibits wMqNax , w2Mqq

Nax and wMqNaqCH CNx adducts. No APCI positive spectrum could beq q3

recorded. Therefore the MS experiment was performed on thewMqNax ion in2 q

positive ESI ion mode. As occurred for the other daucane derivatives, the mainobserved fragments correspond to the loss of the daucane ester group. In particular,the ionswM-152qNax , wM-152qNaqCH CNx andwM-152qCH CNx indicateq q q

3 3

the presence of an anisic ester group. The daucane core moiety of compound5presents a mass of 14 amu greater than that of the unmodified daucane skeleton,attributable to a keto group in the ring. The structure of compound5 could beattributed either to siol anisate(5a) or to oxo-jaeskeanadiol anisate(5b), reportedas components of rootsw8x and seedsw15x of the ‘non-poisonous’ chemotypeF.communis, respectively.The UV spectrum of13 showsl at 202, 282 and 307 nm, attributable to themax

hydroxy-coumarin moiety. Both the positive ESI and APCI-MS spectra(Fig. 4)showedwMqHx signal atmyz 367 (Fig. 4a) while the wMyHx ion atmyz 365q y

was recorded in the negative ESI-MS spectrum(Fig. 4b). The APCI MS spectrum2

of 13 was characterised by subsequent losses of 14 amu:myz 311, 297, 285, 271,257, 243 and 231, attributable to the progressive fragmentation of an aliphatic chain.The base peak atmyz 175 was assigned to hydroxycoumarin core moiety(Fig. 4d).The coumarin13 was identified as ferulenol, a compound described as a constituentof the ‘poisonous’ chemotypew3,6–8x of F. communis, but reported to be absent inlatex w6–8x or rootsw3x of the ‘non-poisonous’ chemotype. No other peak belonging

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Fig. 4. ESI positive(a); ESI negative(b); APCI positive(c) and APCI positive MSyMS spectra(d) offerulenol(13). Proposed fragmentation pathway.

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to this class of compounds was detected in the ‘non-poisonous’ chemotype ofF.communis.The HPLC-DAD-UV fingerprint of the ‘poisonous’ chemotype ofF. communis

was totally different from that of the ‘non poisonous’ chemotype(Fig. 2b). Fivemain peaks(3, 9, 10, 13 and 14) were detected, all showing the same UV spectraof ferulenol (13), and therefore possessing a 4-hydroxy-coumarin structure. The‘poisonous’ chemotype ofF. communis is reported to contain the toxic coumarinsferulenol and itsv-hydroxy derivativesw8x. Daucane derivatives could not bedetected in the ‘poisonous’ chemotype ofF. communis.The presence ofwMqHx signals in the positive ESI-MS and APCI-MS spectraq

of compounds3, 9, 10 and14 together with thewMyHx ions in the negative ESI-y

MS spectra allow the determination of the molecular weights. Furthermore, theoccurrence of fragments atmyz 309, 283, 269, 255, 229 in all the APCI-MS2

spectra indicate the presence of a farnesyl chain. In addition the APCI MS spectrum2

of 3 shows the base ion atmyz 365 wM-H OqHx , allowing to identify3 as 129q2

or 159 hydroxyferulenol w8x. Compound9 and 10 show the same MS and MS2

spectra therefore they are isomers. In their MS spectra the base ion atmyz 3652

wM-AcOHqHx is present. Compounds9 and 10 were therefore identified as 129q

and 159-acetoxyferulenolw8x. Finally, compound14 presents an intense ion atmyz365 attributable to the loss of benzoic acid from the pseudomolecular ion atmyz487. Compound14 was tentatively identified asv-benzoyloxyferulenol, a newcompoundw16x.

4. Conclusion

A HPLC-UV method has been developed for the analysis of daucanes andcoumarins inF. communis, leading to the identification of eleven daucanes and fiveprenylated coumarins.The developed HPLC method allows a simple and rapid identification of the two

different and morphologically indistinguishable chemotypes ofF. communis growingin Sardinia. These plants present different and specific HPLC fingerprints. The ‘non-poisonous’ chemotype contains as main constituents the daucane ester ferutinin and,surprisingly, the prenyl coumarin ferulenol. The presence of toxic ferulenol in the‘non-poisonous’ chemotype ofF. communis has not been described yet. A series ofdaucane esters are also present.Five prenylated coumarins were identified in the ‘poisonous’ chemotype. Dauca-

nes were not detected in the toxic chemotype.

Acknowledgments

The authors wish to thank Prof. G. Appendino, Dipartimento di Scienze Chimiche,Alimentari, Farmaceutiche e Farmacologiche dell’Universita del Piemonte Orientale,`Novara, Italy, for his useful suggestions on the manuscript drafting.

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