JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 2003; 38: 607–617Published online 25 April 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.470

Direct-temperature mass spectrometric detectionof volatile terpenoids and natural terpenoid polymersin fresh and artificially aged resins

Dominique Scalarone,1 Jerre van der Horst,2 Jaap J. Boon2 and Oscar Chiantore1∗

1 Department of Chemistry IPM, University of Turin, Via P. Giuria 7, 10125 Turin, Italy2 FOM Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands

Received 28 October 2002; Accepted 10 February 2003

Electron impact (EI) ionization and ammonia chemical ionization (NH3/CI) direct-temperature massspectrometry (DTMS) was used to characterize five natural terpenoid resins: dammar, mastic, colophony,Manila copal and sandarac. Compositional differences were highlighted by the identification of lowmolecular mass compounds, ranging from di- to triterpenoids, and polymeric components, based onpolycadinene and polycommunic acid. Photo-ageing processes occurring under accelerated indoor andoutdoor exposure conditions were also investigated. NH3/CI and tetramethylammonium hydroxide EIwere applied to increase the sensitivity towards highly oxidized molecules. Oxidation and cross-linkingreactions were found to affect mostly triterpenoid resins and diterpenoid abietane and pimarane molecules.Oxidation proceeds through a radical mechanism, generally starting from conjugated double bonds.Oxygen atoms are incorporated in the terpenoid structures in the form of alcohols, ketones and carboxylicacids. Oxidized cadinene oligomers released by pyrolytic degradation of the polycadinene fraction ofdammar were detected even in unaged samples. Evidence is given indicating the occurrence of cleavagesin the cross-linked polycommunic structure of aged sandarac and Manila copal. Bond scissions produceoligomeric fragments based on the communic acid structure and sufficiently volatile to be desorbed at lowtemperature in DTMS measurements. Copyright 2003 John Wiley & Sons, Ltd.

KEYWORDS: direct temperature mass spectrometry; terpenoids; natural polymers; resins; artificial ageing

INTRODUCTION

The identification of the materials in works of art is offundamental importance for any conservation task. Oneof the most important challenges of analytical chemistryapplied to the characterization of painting materials is therecognition of organic binding media and varnishes. Naturalsubstances used by Old Masters, e.g. siccative oils, animalglue, casein, egg, waxes and resins, are complex and unstablemixtures. During ageing they undergo structural changesdue to photoxidation and/or interaction with metal oxidesand salts present in the paint and ground layers as pigmentsor driers.

Gas chromatography/mass spectrometry (GC/MS) hasbeen successfully applied to identify the chemical structuresof many lower molecular mass terpenoids in paint varnishes.The composition and ageing mechanisms of triterpenoid andditerpenoid resins have been investigated and a detailedliterature on this subject is already accessible.1 – 8 In contrast,there is less literature on cross-linked resins, such as copals9

ŁCorrespondence to: Oscar Chiantore, Department of ChemistryIPM, University of Turin, Via P. Giuria 7, 10125 Turin, Italy.E-mail: oscar.chiantore@unito.itContract/grant sponsor: Foundation for Fundamental Research onMatter (FOM).

and sandarac,10 especially with respect to the polymericcomponents.

This paper is intended to highlight the potential ofdirect temperature-resolved mass spectrometry (DTMS)for the characterization of terpenoid resins utilized byrestorers now, and by artists in the past, to produce paintvarnishes. Although traditional recipes for the preparationof varnishes include several treatments to enhance clarityand transparency and to improve handling properties, theresults reported here refer to the unprocessed raw materialsavailable commercially.

During DTMS measurements the sample is graduallyheated inside the ion source. Low molecular mass com-pounds, such as di- and triterpenoids, are volatilized at lowtemperature, while polymeric materials undergo pyrolyticdegradation at higher temperature. All the volatile prod-ucts so formed are ionized. Moreover, as pyrolysis isaccomplished on-line under vacuum, thermal dissociationproducts, once formed, evaporate quickly, while the proba-bility of molecular collisions is very low and the occurrenceof secondary reactions is therefore minimal.

As separation in DTMS does not occur at the com-pound level, such as in GC/MS, the main problem asso-ciated with this technique is the interpretation of the MSsummation spectra of multi-component mixtures produced

Copyright 2003 John Wiley & Sons, Ltd.

608 D. Scalarone et al.

by volatilization and thermal degradation. In the presentwork the identification of the compounds constituting thesecomplex mixtures was achieved by comparison with ref-erence mass spectra and with data previously obtained byGC/MS2,11 and thermally assisted hydrolysis and methyla-tion (THM)/GC/MS.12

From a molecular point of view, terpenoid resins areextremely disperse systems that consist of apolar terpenes,polar terpenoids and high molecular mass compounds. Thelast type can result from cross-linking reactions of the originalcomponents or they can be already present in the nativematerial as polymeric fractions. As the degree of oxidationand cross-linking varies according to the ageing conditions,we tested two different types of photooxidative ageing.Fluorescent tubes and UV filters were used to simulateindoor exposure conditions, and a xenon lamp was used forexternal conditions. The latter case is unmatched in any realpaint sample as it is assumed that paintings are kept indoors,where sunlight should not reach them directly. However,xenon lamps can simulate extreme situations in which all thedegradation reactions (i.e. oxidation, cross-linking, cleavage)occur to an extent that would never be reached by naturalageing in museums.

By investigating the structural changes caused by selectedmodes of artificial ageing, and comparing them withreference data on naturally aged resins and varnishes, itwas possible to evaluate how exposure parameters, suchas wavelength range and light intensity, affect the ageingprocesses on a molecular level.

The main objective of this study was the detailedcharacterization of highly oxidized and high molecular masscompounds in complex mixtures of terpenoid molecules,such as natural resins. We investigated five different rawresins (dammar, mastic, colophony, Manila copal andsandarac) using DTMS under electron impact (EI) ionizationand chemical ionization (CI).

EXPERIMENTAL

MaterialsDammar, mastic, colophony, Manila copal and sandaracwere purchased from Phase (Florence, Italy) and usedwithout further purification. The samples for ageing wereapplied as thin films (70–80 µm) supported on glassslides.

Ageing procedureA Suntest CPS high-speed exposure unit (Heraeus, Ger-many), equipped with a xenon lamp and a UV filter thatabsorbs wavelengths lower than 295 nm, was used to simu-late outdoor solar exposure. Irradiance was set at 765 W m�2,and the maximum temperature on the samples was kept at45 °C by forced air circulation. A set of fluorescent tubesplaced behind a UV filter to cut off radiation below 400 nmwere used to reproduce indoor light exposure. The environ-mental conditions within the box were recorded as about 5 °Cabove room temperature and ¾10% below ambient relativehumidity (RH). The average light intensity was measured as22 000 lux, which is ¾34.5 W m�2 assuming a monochromatic

source at 555.02 nm (note that photometric units are normal-ized radiometric units weighted with the spectral response ofthe eye;13 at the maximum value of the luminosity function,555.02 nm, the normalizing factor is 683 lm W�1).

Direct temperature-resolved mass spectrometryThe samples were homogenized in a mini-mortar and madeinto a suspension with a few drops of ethanol. Volumes of2–3 µl of the suspension were applied to the Pt–Rh (9 : 1)filament of a direct insertion probe. Samples on the probewere evaporated to dryness and then introduced in the ionsource. The probe filament was temperature programmed ata rate of either 1 A min�1 in EI or 0.5 A min�1 in CI, to anend temperature of about 800 °C. The compounds releasedfrom the filament were continuously ionized at an ionizationenergy of 16 eV. Ions were generated either by EI or ammoniaCI (NH3/CI) in an ionization chamber kept at 180 °C, andaccelerated to 8kV.

The mass spectrometer, a JEOL SX-102-102A four-sectordouble-focusing mass spectrometer (BEBE), was scannedfrom m/z 20 to 1000 for EI and from m/z 60 to 1000 for CI.

RESULTS AND DISCUSSION

The natural resins investigated in this study are mainlycomposed of cyclic di- and triterpenoids whose structuresare shown in Scheme 1. Further compositional details oneach resin are listed in Tables 1–4, together with the m/zvalues of the most characteristic fragment ions obtained byEI-DTMS and NH3/CI-DTMS.

DTMS total ion currents (TIC), such as shown in Fig. 1,give information on the volatile and high molecular masscomponents of the samples. Further information on the mainageing phenomena, in terms of oxidation, polymerization orscission reactions, can be drawn by comparing the shapes,relative intensities and the position of the evaporation (1)and the pyrolysis event (2).

Summation mass spectra in Figs 2–4 provide a completemass spectrometric overview of the samples and, bycomparison with reference mass spectra, a number of markercompounds suitable for the identification of natural resinscan be identified.

Total ion currents of unaged and light-aged naturalterpenoid resins are shown in Fig. 1. The most relevantconsequences of light ageing for dammar, mastic andcolophony are oxidation and cross-linking, albeit in differentproportions depending on the kind of resin and theperformed type of ageing. The shifting of the volatilizationpeak (1 in Fig. 1) to higher scan numbers, and so towardshigher temperature, is evidence of the formation of polarcompounds, in this case oxidized tri- and diterpenoids. Theoccurrence of cross-linking reactions is demonstrated by theincreasing height of the pyrolysis peak (2 in Fig. 1).

The total ion currents of sandarac and Manila copalhave a completely different shape. As these natural resinsare composed of a considerable amount of polymericmaterial, even when unaged, their TIC curves show a highintensity pyrolysis event at scan numbers 30 to 50. Themost important effect of light ageing is the production

Copyright 2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 607–617

DTMS of natural volatile and polymeric terpenoids 609

Diterpenoids

COOH

1

18

20

19

5 14

18

17

4 6

10

COOH

COOH

COOH

R

COOH

COOH

1

18

20

19

17

8

11

R=COOH agathic acid

R=CH2OH agatholic acid

R=CH2OAc acetoxyagatholic acid

trans-communic aciddehydroabietic acid

abietic acidsandaracopimaric acidpimaric acid

Triterpenoids

O

1

12

1819

9

6

2928

3

2221

27

26

17

O

OH OH

OH3CO

dammaradienone hydroxydammarenone dammarenolic acid

R1=H R2=CH3 R3=CHO oleanonic aldehyde

R1=CH3 R2=H R3=CHO ursonic aldehyde

R1=H R2=CH3 R3=COOH oleanonic acid

R1=CH3 R2=H R3=COOH ursonic acid

R1=H R2=CH3 R3=COOH moronic acid*

(*) double bond located at C18

R2

R3

O

R1

O

COOH

CH3OCO

COOH

3-O-acetyl-3-epi(iso)masticadienolic acid(iso)masticadienonic acid

Scheme 1. Structures of some diterpenoids and triterpenoids.

of lower molecular mass compounds (Fig. 1(d) and 1(e)).This is surprising when compared with the ageing effectsobserved for dammar, mastic and colophony and it meansthat cleavage of chemical bonds, evidenced by the volatilemolecules desorbed by DTMS, plays an important role inthe photochemical degradation mechanism of sandarac andcopals. In the aged samples the pyrolysis peak remainsintense, but slightly wider or translated towards lower scannumbers.

DTMS of fresh terpenoid resinsMany of the compounds detected in the DTMS summationspectra of dammar (Table 1) and mastic (Table 2) wereidentified by comparison with the GC/MS results obtainedon the same samples. Triterpenoids are detected betweenm/z 400 and 500 as molecular ions or as radical cations,[M � H2O]Cž, if hydroxyl groups are present. Fragment ionsare detected at lower m/z values: fragments at m/z 109derive from the aliphatic side-chain by scission in position

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610 D. Scalarone et al.

Table 1. List of the most characteristic m/z values in the DT summation mass spectra of unaged and artificially aged dammar(assignments are based on GC/MS results and literature data2,3)

Artificially aged

No. Compound Mr

CharacteristicEI fragmentions (m/z)

CharacteristicNH3/CIfragment

ions (m/z) Unaged

Indoorexposure

(600 h)

Outdoorexposure

(100 h)

1/2 Nor-˛-amyrone/nor-ˇ-amyrone 410 410, 204 428p p

3 Dammaradienone 424 424, 205, 109 442p p

4 Dammaradienol 426 426, 109p p p

5 Hydroxydammarenone 442 424, 355, 315, 109 442p p

6 Dammarenolic acid 458 440, 109 476, 458p p p

7/8 Oleanonic/ursonic acid 454 454, 409, 248, 203, 189 472p p

9/10 Oleanonic/ursonic aldehyde 438 438, 409, 232, 203 456p p

11 20,24-Epoxy-25-hydroxy-3,4-seco-4(28)- 474 143 143p p

dammaren-3-oic acid12 20,24-Epoxy-25-hydroxy-dammaren-3-one 458 399, 143 143

ptra

13/14 3,4-Seco-28-nor-olean-12-en-3-oic acid/ 428 428, 204 490p

3,4-Seco-28-nor-urs-12-en-3-oic acid15/16 3,4-Seco-28-nor-olean-12-en-3,28-dioic acid/ 472 472, 248, 203 446

p3,4-Seco-28-nor-urs-12-en-3,28-dioic acid

a tr means in traces.

Table 2. List of the most characteristic m/z values in the DT summation mass spectra of unaged and artificially aged mastic(assignments are based on GC/MS results and literature data2–4)

Artificially aged

No. Compound Mr

Characteristic EIfragment ions

(m/z)

CharacteristicNH3/CIfragment

ions (m/z) Unaged

Indoorexposure

(600 h)

Outdoorexposure

(100 h)

17 Unidentified oleanadiene 408 408 409p p

tra

2 Nor-ˇ-amyrone 410 410, 204 428p p

5 Hydroxydammarenone 442 424, 355, 315, 109 442p p

7/18 Oleanonic/moronic acid 454 454, 409, 248, 203, 189 472p p p

9 Oleanonic aldehyde 438 438, 409, 232, 203 456p p

19 Oxidized oleanane-type molecule 468 468 486p p

20 (Iso)masticadienonic acid 454 454, 439 472p p

tra

21 3-O-Acetyl-3-epi(iso)masticadienonic acid 498 498 516 tr12 20,24-Epoxy-25-hydroxydammaren-3-one 458 399, 143 143 tr

ptra

13/22 3,4-Seco-28-nor-olean-12-en-3-oic acid/ 428 428, 204 446p

3,4-Sseco-28-nor-olean-18-en-3-oic acid15/23 3,4-Seco-28-nor-olean-12-en-3,28-dioic acid/ 472 472, 248, 203 490

p3,4-Seco-28-nor-olean-18-en-3,28-dioic acid

24 28-nor-olean-18-en-3-one 410 410, 191, 163 428p p p

a tr means in traces.

C-17 of dammarane-type molecules (3, 4, 5, 6), peaks at m/z248, 232, 203 and 189 indicate the presence of moronic acid(18) and oleanonic/ursonic acids (7, 8) and aldehydes (9, 10),and those at m/z 315 and 355 are typical fragment ions ofhydroxydammarenone (5) (Fig. 2).

Both dammar and mastic consist of dammarane andoleanane compounds, although in different proportions, butcan be distinguished by comparing the relative intensities ofmass peaks attributed to dammarane (m/z 424, 426, 440, 339,355, 315, 143, 109) and oleanane (m/z 454, 438, 409, 248, 232,

189) molecules, respectively. Although this method workswell for fresh resins, it fails with aged or mixed resins.

The marker compounds that can be used to discriminatefresh dammar from mastic are moronic acid (18), (iso)mastica-dienonic acid (20) and 3-O-acetyl-3-epi(iso)masticadienolicacid (21). Moronic and (iso)masticadienonic acids havethe same molecular mass of oleanonic and ursonic acids,which are also present in dammar, and hence they can-not be distinguished by DTMS. Under EI, 3-O-acetyl-3-epi(iso)masticadienolic acid gives a low molecular ion peak at

Copyright 2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 607–617

DTMS of natural volatile and polymeric terpenoids 611

Table 3. List of the most characteristic m/z values in the DT summation mass spectra of unaged and artificially aged colophony(assignments are based on GC/MS results and literature data5,7)

Artificially aged

No. Compound Mr

CharacteristicEI fragmentions (m/z)

CharacteristicNH3/CI fragment

ions (m/z) Unaged

Indoorexposure

(600 h)

Outdoorexposure

(100 h)

25/26/27/28/29/30 Pimaric/isopimaric/sandaracopimaric/ 302 302, 287 303, 320p p p

Palustric/abietic/neoabietic acid31 Dehydro-DHA 298 298 316

ptra

32 Dehydroabietic acid (DHA) 300 300, 285 318 trp p

33/34 7-Hydroxy-DHA/15-hydroxy-DHA 316 316 334p

tra

35 Unidentified dihydroxy-DHA 332 332 350p

tra

36 7-Oxo-15-hydroxy-DHA 330 330 348p

tra

37 7-Oxo-DHA 314 314 332p

tra

a tr means in traces.

Table 4. List of the most characteristic m/z values in the DT summation mass spectra of unaged and artificially aged sandarac andManila copal (assignments are based on GC/MS results)

Artificially aged

No. Compound Mr

CharacteristicEI fragmentions (m/z)

CharacteristicNH3/CI fragment

ions (m/z) Unaged

Indoorexposure

(600 h)

Outdoorexposure

(100 h)

38 1,4a,6-Trimethyl-1,2,3,4,4a,7,8,8a- 222 161, 177, 222 240p p p

octahydronaphthalencarboxylic acid39 1,4a,6-Trimethyl-5-methylene-1,2,3,4,4a,5,8,8a- 234 173, 188, 234 252

p p poctahydronaphthalencarboxylic acid

40 1,4a,5,6-Tetramethyl-1,2,3,4,4a,7,8,8a- 236 161, 177, 236 254p p p

octahydronaphthalencarboxylic acid41 cis/trans-Communic acid 302 302, 287, 221, 175 320

p p p27 Sandaracopimaric acid 302 302 320

p p p42 Agathalic acid 318 318, 289 336

p p p43 Monomethyl agathate 348 348, 333, 316 366

p p p44 Acetoxyagatholic acid 362 362, 302 380

p p p45 Agathic acid 334 333, 316, 289 352

p p p46 Agatholic acid 320 289 338

p p p47 19-Nor-labda-8(20),13-dien-15-oic acid 290 290, 275 308 tr

p p

m/z 498, which can be hardly identified in EI-DT mass spec-tra, and an intense fragment ion peak at m/z 438, assignedto the radical cation [M � HCOOCH3]Cž resulting from theloss of the acetoxy group. Unfortunately the molecular ionof oleanonic and ursonic aldehydes (M D 438 Da) also givesa peak at the same m/z value.

In conclusion, in EI-DTMS the only marker for mastic wasfound to be the peak at m/z 163 that has been attributed to acharacteristic fragment ion of 28-norolean-18-en-3-one (24),a pyrolytic degradation product of moronic acid (Table 2).

EI-DT mass spectra of diterpenoid resins showed char-acteristic peaks at about m/z 300. Fresh colophony is almostentirely composed of free abietane and pimarane acids(Table 3), while the volatile fraction of fresh Manila copaland sandarac consists mostly of labdane-type diterpenoids(Table 4).

Peaks at m/z 362, 318 and 348 are representative of themolecular ions of acetoxyagatholic acid (44), agathalic acid(42) and agathic acid monomethyl ester (43), respectively

(Fig. 3(a)). Aghatic acid and its monomethyl ester alsogive fragment ions of the type [M � CH3]C at m/z 333and [M � CH3OH]Cž at m/z 316. The intense peak at m/z302 can be assigned to an ionized pyrolysis fragment ofpolycommunic acid, but also to free communic acid (41) andto the EI fragment [M � HOCOCH3]Cž of agatholic acid (46).

Identification of high molecular mass componentsin terpenoid resinsTerpenoid resins can also contain polymeric material indifferent proportions depending on their botanical andgeographical origin. The poly-ˇ-myrcene14 fraction of mastic,for instance, can be detected only after separation from theraw resin, as its concentration is very low. Other resins,such as sandarac and copals, are almost entirely composedof polymeric material as demonstrated by their total ioncurrents in DTMS analysis (Fig. 1).

Polymeric material was also observed in dammar. Theisolation of a polysesquiterpene with a cadinene (C15H26)

Copyright 2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 607–617

612 D. Scalarone et al.

2

1

scan no.

100

80

60

40

20

00 20 40 60 80 100 120

(a)

rel.

int.

(%)

21

scan no.

100

80

60

40

20

00 20 40 60 80 100 120

(d)

rel.

int.

(%)

21

scan no.

100

80

60

40

20

00 20 40 60 80 100 120

(e)

rel.

int.

(%)

2

1

scan no.

100

80

60

40

20

00 20 40 60 80 100 120

(c)

rel.

int.

(%)

2

1

scan no.

100

80

60

40

20

00 20 40 60 80 100 120

(b)

rel.

int.

(%)

Figure 1. EI-DTMS total ion currents of (a) dammar, (b) mastic, (c) colophony, (d) Manila copal and (e) sandarac. Solid curves,unaged; dotted curves, aged 600 h under fluorescent tubes (� > 400 nm); dashed curves, aged 100 h under a xenon lamp(� > 295 nm).

carbon skeleton from both fossil and recent dammarresins has been demonstrated and the identification of itsthermal degradation products by pyrolysis/GC/MS hasalready been discussed.15 – 17 Pyrolytic depolymerization ofpolycadinene proceeds through the cleavage of bonds inthe allylic position, resulting in the formation of C15, C30

and C45 fragments. Maturation of polycadinene involves

isomerization, cross-linking, cyclization and aromatizationreactions, but until now no oxidation products have everbeen observed.

In this work, DTMS applied to the analysis of freshand light-aged samples of dammar allowed the detectionof cadinene oligomers with different degrees of oxidationdepending on the type of ageing.

Copyright 2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 607–617

DTMS of natural volatile and polymeric terpenoids 613

834 143

880864

816

850

644628

612

408 373

442

426

109248

204

0

20

40

60

80

100

0 100 200 300 400 500 600 700 800 900

m/z

rel.

int.

(%)

(c) x20

816832

644

628

612

355359315

109

143

204

248

454

440

426408

424

0

20

40

60

80

100

0 100 200 300 400 500 600 700 800 900

m/z

rel.

int.

(%)

(b)x20

0

20

40

60

80

100

0 100 200 300 400 500 600 700 800 900

m/z

rel.

int.

(%)

(a)cadinene

644

204 Da204 Da

204 Da

832

628

816

612355

315

232 248

189

109

204

203

408

454

x20426

440

424

Figure 2. EI-DT mass spectra of dammar: (a) unaged; (b) aged600 h under fluorescent tubes (� > 400 nm); (c) aged 100 hunder a xenon lamp (� > 295 nm).

Figure 2(a) shows the complete EI-DT mass spectrum offresh dammar. Peaks at m/z 204, 408, 612 and 816 representthe molecular ions of cadinene monomers, dimers, trimersand tetramers, respectively, and the sequences of peaks atm/z 612, 628 and 642 and at m/z 816 and 832 indicate thatpartial oxidation has already occurred, even in the unagedresin. Formation of new oxygenated functional groupsimplies a molecular mass increase of 14 or 16, depending onthe type of reaction. According to the well known mechanismof radical autoxidation, the reaction of atmospheric oxygenwith a methylene radical leads to hydroperoxides and, aftertheir homolytic cleavage, to alkoxy radicals. These can thenreact to give alcohols, with a total mass gain of 16, or ketones,with a mass gain of 14.

During ageing, the number of oxygen atoms incorporatedin the terpenoid structure increases and the greater structural

variety leads to mass increments ranging from 12 to 18(Fig. 2(b) and (c)).

NH3/CI allowed the detection of highly polar moleculesin the form of ammonia adducts (Fig. 4). Fragments at m/z646, 662, 680 and 696 were attributed to [3M C nO C NH4]C

ions, with n ranging from 1 to 4. In the aged samples therelative intensity of these peaks increased, but no compoundswith higher oxygen content were detected. This suggeststhat also under extreme ageing conditions no more than twooxygen atoms can be added to each cadinene monomericunit.

In a similar way, peaks between m/z 800 and 900should represent oxidized cadinene tetramers, but in fact thesituation looks more complicated. The molecular masses ofsome of the most intense peaks (m/z 854, 868, 886 and 916) donot correspond to the expected values for [4M C nO C NH4]C

ions, which are detected at m/z 850, 866, 882, 898, 914and 930. As triterpenoid dimers,18 if present, should beobserved in the same mass range where cadinene tetramersare expected, there is a high probability that, especially inlight-aged dammar, both types of ions are revealed, giving amixture of signals that are difficult to interpret.

Figure 3(b) shows the EI-DT summation mass spectrumof the polymeric fraction of sandarac, identified as poly-communic acid. The comparison with the mass spectra ofthe other diterpenoid molecules identified in the sample byGC/MS suggests that marker peaks for communic acid arethose at m/z 302, 256, 221 and 175. Apart from the firstpeak, which represents the molecular ion, the other peaksrepresent fragment ions resulting from the loss of formic acid([M � 46]Cž), from the cleavage of the aliphatic side chainon C-9–C-11 ([M � 81]C) and from the cleavage of methylgroups ([M � 15]C). Anyway, as pyrolysis is applied to anal-yse the polymeric material, the resulting DT mass spectrum isa combination of fragment ions of depolymerized communicacid and smaller thermal degradation products. Elsewhereit has already been reported19 that three characteristic pyrol-ysis products of polycommunic acid are compounds 38, 39and 40 (Table 4).

The polymeric nature of the sample is definitely high-lighted by methylation with TMAH (Fig. 3(d)). The loss ofthe methyl group on C-10 ([M � 15]C) and of the methylester group ([M � 59]C) from methyl communate (M D 316)leads to ions at m/z 301 and 257. Peaks at m/z 316, 632and 948, with mass increments of D 316, represent methylcommunate monomer, dimer and trimer, respectively, andthe satellite peaks at m/z 604, 618 and 646 and at m/z 920,934 and 962 ( D 14) indicate that fragments with molecularmasses different from those expected for methyl communatedimers and trimers are also detected. An increment of 14 inthe molecular mass of abietane diterpenoid molecules hasalready been observed and explained by the oxidation of amethylene to a carbonyl group.3 Likewise the same decreasein mass values can be attributed to the loss of an oxygen atomby reduction of ketones. This explanation does not fit wellin the case of polycommunic acid. Theoretically, oxidationof communic acid could occur by reaction with atmosphericoxygen, but no ketone derivative has been observed experi-mentally, either under normal or UV irradiation. On the other

Copyright 2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 607–617

614 D. Scalarone et al.

302 100(a)

80re

l. in

t. (%

) 135 60 289

121 175189221

235 109

40 274

316 20 348

362

00 100 200 300 400

m/z

302 221

175

161 188

135 119

234109

100(b)

80

rel.

int.

(%)

60

40

20

00 10050 200150 250 300 350

m/z

0

20

40

60

80

100

50 150 250 350 450 550 650 750 850 950

rel.

int.

(%)

(d)

m/z

x50

316 Da

316

316 Da

175 235 632

x100 1121 61

288303

948 189 362

618 646

392

37634

7

0

20

40

60

80

100

50 150 250 350 600 700 800 900

rel.

int.

(%)

(c)

m/z

302 Da

3366 80320

338

302 Da 308

x50252

240254

622

924

Figure 3. (a) EI-DT mass spectrum of unaged sandarac; (b) EI-DT mass spectrum of the cross-linked fraction (40 < scan No. < 60);(c) NH3/CI-DT mass spectrum of unaged sandarac; (d) TMAH-EI-DT mass spectrum of unaged sandarac. Similar spectra wereobtained from the cross-linked fraction of Manila copal.

hand diterpenoids with conjugated double bonds in the side-chain are known to polymerize to a cross-linked structurewhose degree of cross-linking increases with age.20,21 Sucha polymer may degrade by pyrolysis into monomer, dimer,trimer, etc., but it may also undergo scission of nearbyC—C bonds to give fragments with a different number ofmethylene groups (M D 14).

Identification of oxidized and degradedcomponents in aged terpenic resinsVan der Doelen and co-workers have already applied DTMSto the characterization of triterpenoid varnishes2 and to the

study of their ageing mechanisms under different exposureconditions.8

The composition of mastic is similar to that of dammarexcept for the presence of (iso)masticadienonic (20) acidand 3-O-acetyl-3-epi(iso)masticadienolic acid (21). Theirstructure is characterized by a side-chain, as for dammaranemolecules, but with a carboxylic acid group (Scheme 1).However, in contrast to dammarane molecules such asdammarenone and hydroxydammarenone, no oxidationproducts of (iso)masticadienonic acid and 3-O-acetyl-3-epi(iso)masticadienolic acid have been identified in agedmastic. Their disappearance during ageing, monitored by

Copyright 2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 607–617

DTMS of natural volatile and polymeric terpenoids 615

0

20

40

60

80

100

50 150 250 350 450 550 650 750 850 950

m/z

rel.

int.

(%)

205

428

442456

460

476

409 613

817

646

662868

886898

(a)

204 Da

204 Da 204 Da

x30

Figure 4. NH3/CI-DT mass spectra of unaged dammar.

GC/MS, could be due to cross-linking and to formation ofhigher molecular mass compounds that cannot be eluted byGC. The DT mass spectra are in good agreement with thishypothesis as ageing leads to a decrease of peaks attributedto (iso)masticadienonic acid and a simultaneous increase inthe relative intensity of peaks at m/z ³ 900, where dimersare expected.

The EI-DT mass spectra of dammar and mastic resinsartificially aged, and also of aged varnishes from paintings,exhibit two marker peaks at m/z 143 and 399 that havebeen identified as fragment ions of oxidized dammaranecompounds with a hydroxyisopropylmethyltetrahydrofuranside-chain (11, 12). Except for these two peaks, the DT massspectra of dammar and mastic, aged under fluorescent tubesto reproduce inside exposure conditions, do not show anyrelevant difference in composition. On the other hand, thesame resins aged under a xenon lamp (� > 295 nm) givea very different fingerprint. In the triterpenoid mass range,peaks at m/z 424, 440 and 454 have a lower intensity orhave totally disappeared, indicating the degradation of theoriginal triterpenoid compounds. All of these compoundshave a keto group at the C-3 position of the A-ring that,under UV irradiation, is susceptible to ˛-scission accordingto the Norrish I reaction. The A-ring oxidation results in acarboxylic acid group at C-2 and an isopropyl group at C-5.Actually, several Norrish products, 13, 14, 15 and 16, havebeen identified by GC/MS in UV irradiated dammar layers(Fig. 2(c)). In the NH3/CI-DT mass spectra (Table 1), all theNorrish products have been detected as ammonia adductions, [M C NH4]C, at m/z 446 and 490 and in the EI/DT massspectra as molecular ions, [M]Cž, at m/z 428 and 472. Becauseof fragmentation the relative intensities of the latter peaksare low.

The DT mass spectra of mastic layers irradiated with axenon lamp also show the same peaks but, in contrast todammar, they are not attributed to ursane molecules, as theyare not identified in GC/MS measurements, but to oleanemolecules, including the 3,4-seco-olean-18-en-3,28-dioic acid(M D 472, 23), the Norrish product of moronic acid (Table 2).

The composition of naturally and artificially agedcolophony has been determined by thermally assistedhydrolysis and methylation with tetramethylammonium

hydroxide (TMAH).12 As suggested by the TIC curves inFig. 1(c), the main degradation process of colophony is oxi-dation. The precise mechanisms involved are still unclearbut the reaction path proposed by Pastorova et al.5 is in goodagreement with the analytical results reported in the litera-ture. According to this ageing mechanism, oxidation startson the conjugated double bonds of abietane diterpenoids toform dehydroabietic acid (DHA) and continues by furtherincorporation of oxygen.

The DT mass spectra of aged colophony are much morecomplicated than those of the fresh resin but they can beinterpreted fairly easily by comparison with the knownspectra of diterpenoid acids.

The molecular ions of DHA (32) and dehydro-DHA(31) are detected at m/z 300 and 298, respectively, whilepeaks at m/z 314 and 316 and at m/z 330 and 332 resultfrom oxidized DHA molecules with one or two additionaloxygen atoms in the form of keto or hydroxyl groups (33,34, 35, 36, 37) (Table 3). During pyrolysis, fragmentation ofditerpenoid acids also takes place, producing diterpenoidpyrolysis fragments (m/z 254, 256) by loss of carboxylicacid groups. Further fragmentation during EI leads to thecleavage of a methyl group (m/z 239, 241).

Under outside exposure conditions oxidation remainsthe most important ageing phenomenon but cross-linkingoccurs to a greater extent, as demonstrated by the increasingheight of the pyrolysis peak in the total ion current (Fig. 1(c)).The broadening of the volatilization peak suggests that alsocleavage reactions, probably activated by UV light, are likelyto occur when an extremely powerful light source is appliedto accelerate the ageing.

The chemical mechanisms involved in the ageing ofsandarac and copals have never been deeply investigated,but they may be similar to the maturation process ofpolylabdanoids in fossil resins, which has been studiedin some detail by FTIR,22,23 13C NMR21,24 and pyrolysisGC/MS.25,26 The authors of these papers drew the conclusionthat the complex multicyclic chemical structure of fossilresins is the result of cross-linking, isomerization, inter- andintramolecular cyclization reactions and defunctionalizationby loss of acidic groups.

Unpublished results demonstrate that in Manila copaland sandarac the degree of cross-linking increases withage, but it is counterbalanced by the decrease in themolecular mass of the polymeric material. The occurrenceof cleavages is also supported by the relative amount ofvolatile molecules desorbed by DTMS (Fig. 1(d) and 1(e)).The only low molecular mass ageing product identified inboth resins by GC/MS and DTMS is 19-norlabda-8(20),13-dien-15-oic acid (47), whose main fragment ions are detectedat m/z 290 ([M]Cž) and 275 ([M � CH3]C) under EI, and atm/z 308 (M C NH4]C) under NH3/CI (Fig. 3). However, 19-norlabda-8(20),13-dien-15-oic acid on its own cannot justifythe increase in the volatilization peak of the total ion currentsof aged samples. Cleavage of the cross-linked polycommunicstructure in fragments still undetectable by GC/MS, but shortenough to be desorbed at low temperature in DTMS shouldbe also possible, and the SIM (single ion mass) curves in Fig. 5support this hypothesis. Under EI both free and polymeric

Copyright 2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 607–617

616 D. Scalarone et al.

0 20 40 60 6040200

rel.

int.

m/z 275

m/z 290

m/z 175

m/z 221

scan no.scan no.

m/z 275

m/z 290

m/z 175

m/z 221

(a) (b)

x8.2

x11

x1.0

x1.6

x1.8

x3.3

x1.3

x1.0

Figure 5. Single ion mass profiles at m/z 175, 221, 275 and290 during the EI-DTMS analysis of Manila copal: (a) unaged;(b) aged 600 h under fluorescent tubes.

communic acid are recognized by two characteristic fragmentions represented by peaks at m/z 175 and 221. Their currention profiles have been used to verify that volatile communicacid oligomers are produced by ageing through cleavagereactions, as demonstrated by the increasing height of thesignal at scan numbers between 25 and 35.

CONCLUSIONS

The possibility of applying DTMS to a wide varietyof samples, ranging from volatile low molecular masscompounds to polymers, makes it a leading techniquein the analysis of terpenoid resins that, as many othernatural products, are a complex mixture of molecules withdifferent chemical structure, size and polarity. Because of itssensitivity, the absence of chemical workup and the shortanalysis time, DTMS has been successfully applied in theidentification of natural resins in microscopic samples ofpaint varnishes.5,7,27,28

It is well known that the ageing mechanisms and theperformance of painting materials based on natural resinsdepend largely on the exposure conditions.

EI-DTMS total ion curves were used to monitor thechanges in the degree of oxidation and cross-linking offive selected resins aged under indoor and outdoor solarexposure.

Compositional information at a molecular level wasobtained from the EI-DT summation mass spectra. The m/zvalues of the most significant fragment ions allowed therecognition of the main terpenoids present in the investigatedresins.

NH3/CI-DTMS was applied to obtain complementarystructural information and to increase the sensitivity towardsoxidized molecules, as they exhibit a higher ammonia affinitythan apolar compounds.

The identification of di- and triterpenoids was carriedout by comparison with literature data, with knownfragmentation mechanisms and with the GC/MS datacollected on the same samples. As far as high molecular

mass compounds (either polymeric, cross-linked or highlyoxidized) are concerned, assignments of peaks were onlypartially justified by the results obtained with other analyticaltechniques (SEC, THM/GC/MS). Most of these peakswere attributed on the basis of the m/z values expectedfor terpenoids with a higher degree of oxidation andcross-linking than the original components. Only MS/MSmeasurements, not performed in this phase of the work,could allow the precise identification of the chemicalstructures involved.

AcknowledgementsThis work was performed in cooperation with the MOLART Studiesresearch programme embedded in the FOM approved program49, supported by FOM (Foundation for Fundamental Research onMatter), which is a subsidiary of the Dutch Organization for ScientificResearch (NWO).

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