MS

Anal Bioanal Chem DOI 10.1007/s00216-016-9772-9

RESEARCH PAPER

Analysis of diterpenic compounds by GC-MS/MS: contribution to the identification of main conifer resins Clara Azemard 1 & Matthieu Menager 1 & Catherine Vieillescazes 1

Received: 4 May 2016 / Revised: 13 June 2016 / Accepted: 5 July 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract The three principal types of molecules composing diterpenic resins are the abietanes, pimaranes and labdanes. The study of their fragmentation was performed by gas chromatography coupled to an ion trap mass spectrometer, on standards and resins used in paint varnishes: colophony and sandarac. We found that the general fragmentation pattern was mostly governed by the location of the double bonds on the different cycles and the presence of functional groups, and not by the nature of the C13 group in the case of abietanes and pimaranes. As for the labdanes, the loss of their alkyl chain is very specific. This study develops an analytical strategy using tandem mass spectrometry (MS/MS) experiments to validate the proposed mechanisms of fragmentation and to find the ions of interest for the identification of diterpenic molecules. Keywords MS/MS . Diterpene . Fragmentation . Abientane . Pimarane

Introduction Terpenic plant resins are defined as lipid-soluble mixture of terpenoid secondary compounds [1]. Depending on the Clara Azemard and Matthieu Menager contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-9772-9) contains supplementary material, which is available to authorized users. * Matthieu Menager [email protected] 1

Equipe Ingénierie de la Restauration des Patrimoines Naturel et Culturel–IMBE UMR 7263 CNRS–IRD 237, Université d’Avignon et des Pays de Vaucluse, 84000 Avignon, France

biochemical pathway, two families of non-volatile compounds can be synthesised: diterpenes with 20 atoms of carbon or triterpenes with 30 carbons [1]. Conifer resins are diterpenic ones with differences in composition specific to the family or the genus of the original tree. They are used since Bronze Age [1] in a large variety of fields: therapies and sacred rituals [2–4], naval stores [5–8], food transports [9], mummification [10–12], artwork materials [13–20] and various other ones. Thus, in archaeometry, it is now crucial to be able to identify terpenes present in artistic and archaeological remains in order to determine the plant used. Advances in diterpenes identification directly interest many other scientific fields as geochemistry [1, 21, 22], environmental [23, 24] and therapeutical [25–27] chemistry. Main conifer resins used over time come from the families of the following: (i) Cupressaceae (sandarac), (ii) Araucariaceae (especially Manila Copal), (iii) Pinaceae (various turpentines and one distillation product—colophony). There are only a few notable differences between the compositions of the different types of turpentine and colophony, all coming from the Pinaceae family [28]. Their compositions include abietanes like abietic, palustric and dehydroabietic acids, and pimaranes like pimaric, sandaracopimaric and isopimaric acids [17, 20, 29]. Only a few studies of sandarac (Tetraclinis articulata) have been done. This resin is constituted of an important proportion of polycommunic acid coming from its communic acid present in the Bfresh^ resin [30]. The three major compounds of sandarac are sandaracopimaric, agathic and communic acids [30–32]. Other acids as callistric, sandaracinic and agatholic have also been found with labdanes and phenols (totarol, ferruginol and manool) [17, 30, 31, 33, 34]. Manila copal was much less used than the two other resins and its composition is very close to sandarac resin, with mainly sandaracopimaric, agathic and communic acids. All structures are given in Fig. 1. In all

C. Azemard et al.

Fig. 1 Structures of the diterpenes studied in this paper

resins, the biomarkers undergo numerous degradation reactions and oxidation [14, 28, 30, 31, 34–37]. In this way, many oxidation products of abietanes and pimaranes were identified in previous studies [28, 30, 36, 38]. But many of

them remain unidentified due to the lack of understanding in the fragmentation reactions. These complex mixtures of terpenes are mainly analysed using gas chromatography coupled with mass spectrometry

Analysis of diterpenic compounds by GC-MS/MS

(GC-MS). In order to identify such molecules, the fragmentation of terpenes by mass spectrometry was studied in the second part of the twentieth century, notably by Budzikiewick et al. [39] in 1963 and by vegetal biology researchers [40]. Pyrolysis-GC-MS (Py-GC-MS) is often used in order to analyse the polymeric fraction of the resin [30, 37, 41, 42]. Other techniques as thermally assisted hydrolysis and methylationGC-MS (THM-GC/MS) [15, 28, 30], direct temperatureresolved mass spectrometry (DTMS) [43, 44] or graphiteassisted laser desorption/ionisation (GALDI) and matrixassisted laser desorption ionisation-time of fly-mass spectrometry (MALDI-TOF/MS) were also used for resin analysis [30, 45–47]. However, if the study of the fragmentation of triterpenes has been extensively developed, only few publications look into the diterpenes fragmentation [14, 48–50] and none use MS/MS. Enzell et al. propose general fragmentation mechanisms for all types of terpenes. In their work, the importance of the position of the double bonds is often pointed out and some rearrangements well developed [49]. The proposed fragmentations of labdanes were observed and developed for Jatobá resin’s labdanes [48]. The fragmentation of the usual pimaranes and abietanes is proposed in different works on diterpenic resins [14, 50, 51], yet no global fragmentation ways are given for those two families. Few studies involve nuclear magnetic resonance (NMR) in order to elucidate chemical structure [46]. However, such technique is time consuming and often not possible as it requires large amount of pure compound. The advantages of MS/MS are obviously the great sensibility of the techniques and the possibility of coupling with a chromatography. In this study, for the first time, MS/MS was used in order to improve the identification of abietanes, pimaranes and labdanes (structures in Fig. 1). The mass spectra were taken from commercial standards, fresh and photochemically aged resins thanks to a gas chromatography coupled to an ion trap mass spectrometer. We sorted the molecules according to specific molecular units leading to common fragmentation reactions. On the basis of these data, we have done interpretations of the fragmentation of some unknown by-products. Fragmentation mechanisms are proposed for the two types of molecular skeletons: tricyclic (abietanes and pimaranes) and bicyclic diterpenes (labdanes). Furthermore, we propose an easy and efficient way to distinguish abietanes and pimaranes for both commercial standards and identified photoproducts.

Aesar). We also studied natural resins, with a special attention given to the botanical origin of the sandarac (T. articulata— EMIGA, S.A.) and the colophony (Pinus—Kremer Pigmente GmbH & Co KG). Molecules photoproducts were identified using commercial standards, literature, mass spectrum database and with the help of fragmentations. All chemical markers origins and identification are given in Table 1. Samples preparation The commercial resins were also dissolved in ethanol and spread on a microscope slide then aged under artificial sunlight (SUNTEST CPS +, Atlas) during at least 300 h under the following conditions: 35 °C, 765 W/m2. All samples (aged and commercial ones) were trimethylsilylated or methylated before injections. For the trimethylsilylation, the solutions were evaporated to dryness and mixed with 200 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) +1% trimethylchlorosilane (TMCS). Such solutions were let 30 min at 70 °C. Afterwards, the trimethylsilylated extracts were dried under a stream of nitrogen, dissolved in 1 mL of hexane, and filtered on 0.22 μm filters (Sartorius Stedim Biotech, Goettingen, Germany). The methylation procedure was similar to the trimethylsilylation one (30 min, 70 °C) with trimethylsulfonium hydroxide (TMSH) as reactant. GC conditions The analyses were performed on a Thermo Fisher Focus GC coupled to an ion trap ITQ 700 mass spectrometer on chemical standards or resins after the derivation step. We used Excalibur software to treat our data. The analyses were carried out under a helium flux of 1.2 mL/min. Injections (1 μL, splitless) were done with a 30-m capillary column, 0.25 mm internal diameter, wallcoated with TR 5 MS (Thermo, 5 % phenyl, 95 % dimethylpolysiloxane), film thickness 0.25 μm. The GC temperature programme was held to 50 °C (2 min), ramped at 7 °C min − 1 until 250 °C, and finally increased at 2.5 °C min−1 until 330 °C. The injector and the transfer line temperatures were set, respectively, at 250 and 300 °C. Mass conditions

Material and methods Standards We used standard molecules of pimaric, isopimaric, sandaracopimaric, palustric, dehydroabietic and 7-oxodehydroabietic acids (Helix Biotech) and abietic acid (Alfa

Electronic impact ionisation, with source temperature set at 250 °C, was used for every MS and MS/MS analyses. Each spectrum was measured at least three times. All analyses were made with two different sets of electron energy/lens tension parameters: (i) 70 eV/10 V the usual ones, also used for the MS/MS experiments, (ii) 17 eV/50 V in order to work on some heavy mass weight ions and, sometimes, to have a

C. Azemard et al. Table 1 Origin and identification of the molecules presented in the paper

Compound

Origin

Identification

Pimaric acid

Helix Biotech

Commercial standard

Isopimaric acid DHA

Sigma Aldrich Helix Biotech

Commercial standard Commercial standard

7-oxo-DHA

Helix Biotech.

Commercial standard

Sandaracopimaric acid

Helix Biotech

Commercial standard

Abietic acid

Helix Biotech

Commercial standard

Neoabietic acid Palustric acid

Colophony Colophony

(Mathe, 2003) (Mathe, 2003)

Dehydro-DHA

Colophony

(Clark, 2006)

7α and β-hydroxy-DHA 12-Hydroxy-DHA

Colophony Colophony

(Franich and Holland, 1985) Interpretation of mass spectrum

15-Hydroxy-DHA 7,15-DihydroxyDHA

Colophony Aged colophony

(van den Berg, 2012) (van den Berg, 2012)

7-oxo-DHA 15-Hydroxy-7-oxo-DHA

Helix Biotech Aged colophony

Commercial standard (van den Berg et al., 2000; Pastorova et al., 1997)

18 nor-pimaranes and abietanes Agathic acid 3-Hydroxy-eperuic acid

Aged colophony Sandarac Sandarac

Interpretation of mass spectrum Molart report 10 (van den Berg, 2012) Molart report 10 (van den Berg, 2012)

Pinifolic acid

Sandarac

Molart report 10 (van den Berg, 2012)

different vision of some fragmentations. For both parameters the emission current was of 250 μA. For MS/MS experiments, various precursor ions were chosen, they will always be mentioned in the text of the publication. MS/MS analyses were achieved in SCAN mode, with three microscans and an activation time of 30 ms. After preliminary experiments, we chose to let the collision energy on automatic mode with a medium excitation energy to have a better homogeneity of all MS/MS spectra.

Fragmentation of tricyclic diterpenes: abietanes and pimaranes All mass spectra were obtained after trimethylsilylation of the resins or commercial standards, with confirmation of fragmentation reactions and structures by methylation of samples. With such sample preparations, the loss of carboxyl functions (-COOTMS or -COOCH3) present respectively a loss of -118 or -60 as the fragment capture a hydrogen in beta or gamma position (possibility of forming a double bond) and hydroxyls (-OTMS or -OCH3) a loss of -90 or -32 on the mass spectrum. One double bond on the cycle B First, one abietane and one pimarane with a double bond on the cycle B were analysed: abietic and isopimaric acids. Both present a fragmentation dominated by the cations (a1) m/z = 256 and (b1) m/z = 241 as presented on Fig. 2 and in

the Electronic Supplementary Material (ESM) in appendix A. According to MS/MS experiments on these fragments (Figs. 2 and 5), a two-step fragmentation occurs: (i) the loss of the -COOTMS function, (ii) a retro-Diels Alder reaction (RDA) leading to the opening of cycle A as shown on the mechanism proposed in Fig. 3. The ion (b1) m/z = 241 get fragmented into ions (c1) m/z = 213, (d1) m/z = 199, (e1) m/z = 185 and related ones m/z = 171, 157, 143, 129, 128. They are most probably stabilised by the resonance with the double bond at C7 which explains their high proportion. The cations (a1) to (e1) only differ in mentioned diterpenes (abietic, isopimaric, sandaracopimaric, pimaric, neoabietic acids) by the localisation of their double bonds. For isopimaric acid, the ions m/z = 359 (M•+−15) and (b1) (ESM, appendix A) are more important than for abietic acid. Indeed, it seems that the loss of the methyl group can also happen on the C13 leading to a decrease of the formation of the fragment (a) m/z = 256 and resulting ones. Those affirmations have been corroborated by the study of the spectra after methylation, with a molecular peak at m/z = 316 and same ions (a1)–(e1) (data not shown). Moreover, the spectra made with an electron energy 17 eV (ESM, appendix A) confirmed the high predominance of such mechanisms and its importance in the formation of almost all the other fragments. Absence of double bond on the cycle B In this part, sandaracopimaric, pimaric and neoabietic acids were analysed. Figure 4a shows the results obtained for the

Analysis of diterpenic compounds by GC-MS/MS Fig. 2 a Mass spectrum of abietic acid; b MS/MS spectrum of fragment (b1) from abietic acid at m/z = 241

a

b

pimaric acid, data from other compounds are given in appendix B in the ESM. Thus, the absence of double bond on the cycle B allows a different fragmentation to occur, leading to important fragments at m/z = 121 (g), 120 (g′), 105 (h) and 91 (i). A possible way leading to fragment (g) implies the formation of the intermediate (f) at m/z = 239 (181 after methylation) by a Diels Alder reaction, as presented in Fig. 4c. It is noticeable that this fragmentation is only possible when there is no double bond on cycle B and one double bond in C8 on the cycle C. Such mechanism was validated by MS/MS experiments on fragments m/z = 239 (Fig. 4b). Furthermore, the ion fragment (g) at m/z = 121 leads to the (h) and (i) fragments. Differences of one unit of the m/z between different experiments were observed in ions (f′) m/z = 238 and (g′) m/z = 120. They are most probably due to the conservation of the radical on the two fragments, the MS/MS spectrum of (f′) (ESM, appendix B) only differs by one unit of the (f) one. Finally, major ion fragment at m/z = 121 and 120 are specific to the double bond on C8=C14 and, thus, give precious structural information.

As mentioned in Fig. 4a, the spectrum shows that the previous fragmentation (Fig. 3) also occurs, however, in a lower extend. Moreover, a second mechanism seems to involve the rearrangement of the cation (a1′) m/z = 257, with a similar structure to (a1) without the radical. MS/MS experiments on (a1′) (ESM, appendix B) show that its degradation undergoes the same type of fragmentation, without one double bond, leading to a radical cation (b1′) m/z = 242 and cations (j) m/z = 227, (c1′) m/z = 215, (d1′) m/z = 201, (e1′) m/z = 187 and other related ones (m/z = 173, 161, 147, 133). This pathway is only effective for the two pimaranes. Two or three double bonds on the same cycle Palustric, dehydroabietic (DHA) and dehydro-dehydroabietic (DH-DHA) acids possess respectively two and three double bonds on the C cycle. Their spectra show intense peaks (ESM, appendix C) corresponding to fragments: (b1) m/z = 241 for palustric acid, (b2) m/z = 239 for DHA, (b3)

C. Azemard et al.

R2

R2

R2 R2

2

COOR1

1

- CH3

-HCOOTMS

(a)

R2

R2

1 RDA

or 2

- C3H6

RDA - C2 H 4

(d)

(b)

- C2H 4 (e)

(c)

Fig. 3 Proposed fragmentation mechanism for abietanes and pimaranes with m/z values for R1 = TMS, 1abietic, isopimaric, sandaracopimaric, pimaric, neoabietic acids; 2dehydroabietic acid; 3dehydro-dehydroabietic acid

m/z = 237 for DH-DHA. The formation of these fragments most probably involves the mechanism previously described in Fig. 3. Indeed, MS/MS spectra of ions (a1)–(a3) (data not shown) show fragmentation patterns similar to fragment (b1) (Fig. 2b) with 2 units of difference indicating similar structure with one (or two) double bond added. The importance of fragment (b) for those molecules is directly related to the stability of such structures with the aromatic cycle C (DHA and DH-DHA)

or the presence of three conjugated double bonds (for the palustric acid). Differentiation between pimaranes and abietanes (MS/MS) When dealing with unidentified compounds, it is important to be able to make the distinction between pimarane and abietane

a

b

c R2

(g) m/z=121 (f) R1 =TMS, m/z=239 RDA -HCOOR1 COOR1

-C

2H 6

COOR1

(i) m/z=91

-CH 4

(h) m/z=105

Fig. 4 a Mass spectra of pimaric acid with electron energy; b MS/MS spectra of (f) m/z = 239; c proposed mechanism for the formation of (g) m/z = 121

Analysis of diterpenic compounds by GC-MS/MS

bond should show the same fragments with similar intensities respectively minus 2 or plus 2 (ESM, appendix D).

Degradation products of abietanes and pimaranes Abietanes and pimaranes suffer degradation, particularly under the action of light. Irradiation under artificial sunlight and natural degradation lead to the formation of hydroxylated compounds, ketones and lower molecular weight molecules, as nor-abietanes and nor-pimaranes. We observed this hydroxylated derivative after photodegradation of colophony resin and verify the similarity with DHA by looking at the MS/ MS spectra of the relevant fragments. Hydroxylated derivatives of DHA Fig. 5 Comparison of the MS/MS spectra of the fragment (a1) at m/z = 256 for pimaranes and abietanes

skeleton. Sometimes, on mass spectra, it can be done thanks to the -28 loss, coming from the vinyl function loss in the pimaranes. Unfortunately, in most pimarane molecules, this fragment is absent or very weak. In order to characterise those two chemical families, we used the MS/MS spectra of the fragment (a) at m/z = 256. Indeed, it can give precious information, as these ions are present in most of these terpenes. As presented on Fig. 5, the MS/MS spectrum of fragment (a1) of isopimaric acid presents a cation (j) m/z = 227 which is absent for the abietic acid. The same observations and the same MS/MS spectra were found for all pimarane and abietane molecules. Structures with one more or less double

a

According to the literature, the hydroxyl groups are more probably placed on C7, C12 or C15 of DHA [21, 23, 28, 36, 52]. Their fragmentations differ according to the position of the hydroxyl group, placed on a saturated or aromatic cycle, or on the isopropyl group. For all molecules, we have found a loss of the hydroxylated group(s) followed by the mechanisms proposed in Fig. 3. All fragment differing only by the presence or position of a hydroxyl group will be named as (XOHz), with X for the name of the fragment and z for the position of the hydroxyl group. Hydroxyl group on a cycle For the compounds hydroxylated on a cycle (C7 and C12), all mass spectra share similar fragments (l) at m/z = 299. As

R3

b

R3

- CH(CH3)2

R1 =TMS, m/z=460

R3

- CH4

- HCOOTMS

R2 COOR1

R3

R2

R2

R2

(m), m/z=283

COOR1

(k), m/z=417

(l), m/z=299

RDA - C3H4 R3

R3

R2 R2

(n), m/z=243

Fig. 6 a MS/MS spectrum of fragment (l) m/z = 299. b Proposed mechanism for the formation of fragment (l) m/z = 299 and (n) m/z = 243

C. Azemard et al. Fig. 7 Mass spectrum of 7α and β-hydroxy-DHA

7 -hydroxy-DHA

OTMS COOTMS

7 -hydroxy-DHA

OTMS COOTMS

described on Fig. 6b, one fragmentation pathway mechanism begins with the loss of the isopropyl group followed by the loss of the derived carboxyl group. All cations (l) exhibit the same MS/MS fingerprint, given in Fig. 6a, with a strong fragment (n) at m/z = 243, most probably coming from a retroDiels-Alder reaction (Fig. 6b). It is worth noticing, the slight occurrence of the ion at m/z = 209 (-HOTMS) showing the presence of the hydroxyl group on the fragment (l). MS/MS mass spectrum of fragment (n) (data not shown) shows notably fragments at m/z = 169 (-HOTMS) due to the conservation of the hydroxyl group. The fragmentations of 7-α- and β-hydroxy-DHA are characterised by fragments at 417, 299, 253, 237 and 191. The two mass spectra are obviously similar with variations in the fragments proportions (cf Fig. 7). Their fragmentation nicely fit the observations of Franich & Holland with TMSi ether of methyl α and β 7-hydroxy-dehydroabietate [53]. The α and β were designed according to the proportions of the ions (p) and (o) [53]. Indeed, as described in Fig. 8, we can expect a similar retrodiels-Alder reaction, leading to the radical ion (o) at m/z = 234, followed by deisopropylation, which would lead to the formation of cation (p) at m/z = 191. Such fragmentation brings an unequivocal identification of a hydroxyl in position 7. A common fragmentation reaction implies the loss of the OTMS group and the formation of a cation similar to DHDHA. After this step, on the basis of MS/MS results, three other paths of fragmentation can be proposed: (i) the formation of the ions (a3) m/z = 252 and (b3) m/z = 237 as described

in Fig. 3; (ii) another process leading to a radical cation (a3′) m/z = 253, similar to (a3) without the radical, which undergoes the formation of cations at m/z = 211, 197, 183, 169, 155 (ESM, appendix E); (iii) the mechanism proposed in Fig. 6b leading to (l)OH7. DHA can also possibly be hydroxylated on the aromatic cycle C. As previously described for phenols and various other compounds [54, 55], aromatic cycles substituted with a TMS ether are very stable and do not lose their OTMS group during fragmentation. Thus, the mass spectra of 12-hydroxyDHA (Fig. 9) exhibit fragments from three different pathways: (i) the main mechanism is similar to Fig. 3 and leads to ion (a4)OH12 m/z = 343, a huge (b4)OH12 m/z = 327 and related ions, with a similar structure to all ions (a) and (b) with a derived hydroxyl group, (ii) the formation of cation (l)OH12 as other hydroxylated DHA (Fig. 6), (iii) a specific fragmentation, given on Fig. 9, notably involving two retro-Diels-

-CH(CH3)2

RDA

OTMS COOTMS

R1 =TMS, m/z=460

OTMS

(o), m/z=234

OTMS

(p), m/z=191

Fig. 8 Proposed mechanism of the formation of fragment at m/z = 234 and m/z = 191

Analysis of diterpenic compounds by GC-MS/MS Fig. 9 Mass spectrum of 12hydroxy-DHA and proposed fragmentation path

OTMS

COOTMS

OTMS

OTMS

RDA

RDA - C2H3

(r) m/z=247

(q) m/z=274

-C2H2 OTMS OTMS

-CH 4

(t) m/z=205

Alder reactions, leading to fragments (q) m/z = 274, (r) m/z = 247, (s) m/z = 221 and (t) m/z = 205. As for DHA, DHDHA and others (Fig. 3), the stability of the structure of ion (b4)OH12 leads to a very important peak at m/z = 327 (with silylation) and 269 (with methylation).

Hydroxyl group on the isopropyl group For the 15-hydroxy-DHA, the hydroxyl group is on the isopropyl function. Such molecules appear to give very stable M •+ −15 fragments [36] leading to an intense peak, at m/z = 445 for 15-hydroxy-DHA (ESM, appendix F). In this way, the same behaviour was observed for methyl and TMS derivates. For minor fragments of TMS compounds, the fragmentation reactions are mainly focused on the loss of final hydroxylated isopropyl chain or derived carboxylic acid, as noticed on the mass spectrum in appendix F in the ESM. It is worth noticing the formation of ions (a3) and (b3) after a deshydroxylation step. Finally, the absence of fragment (l) is

a7

,15-dihydroxy-DHA OTMS

OTMS COOTMS

b

7 ,15-dihydroxy-DHA OTMS

OTMS COOTMS

Fig. 10 Mass spectra of a 7β,15-hydroxy-DHA; b 7α,15-hydroxy-DHA

(s) m/z=221

in complete accordance with the proposed fragmentation mechanism (Fig. 6). Di-hydroxy-DHA Natural degradation of colophony implies the formation of two 7,15-di-hydroxy-DHA. Firstly, as 15-OTMS-DHA derivatives, they exhibit an intense peak at m/z = M•+−15 = 533 as presented on Fig. 10. Different fragments at m/z = 253, 237, 211, 191 present the same MS/MS spectra that respective ions (b3), (a3′), (c3′) and (p). Indeed, such molecules present a radical cation (u) at M•+−90 most probably followed by a fragmentation similar to the mechanisms depicted in Figs. 3 and 6 and the formation of cations (b5) Δ 1 5 , O H 7 α , (b5)Δ15,OH7β, leading, after deisopropylation to fragments (k)OH7α and (k)OH7β. Fragment (u) can also lead to radical cation (o′) and cation (p) as illustrated in Fig. 8. It is worth noticing the presence of fragments (b3), (a3′), (c3′) and related fragments. One possible explanation for their formation is the loss of the second OTMS and the COOTMS groups.

C. Azemard et al. Fig. 11 Proposed mechanism of fragmentation for the formation of fragment (b4)OH7

-CO2 O

OTMS

O O

Ketonic derivative All fragments differing only by the presence or position of a ketone group will be named as (XOz), with X for the name of the fragment and z for the position of the ketone group. Some of the diterpenes found in resins possess a ketonic function, mostly on the C7 position. 7-oxo-DHA involves similar mechanisms to those presented in Fig. 3, leading to (a6)O7 m/z = 268 and (b6)O7 m/z = 253, with the conservation of the ketone group along the fragmentation, as presented in appendix G in the ESM. Cations at m/z = 285, 327 and 345 could be explained by a transfer of the TMS group from the carboxylic function to the ketone group as presented on Fig. 11. Similar reaction was observed on hydroxylcarboxylic and -bicarboxyllic acids trimethylsilyl derivatives [56]. Such transformation does not take place in experiments at 17 eV (data not shown). 15-Hydroxy-7-oxo-DHA presents similar reactivity to other 15-hydroxy-DHA derivatives (ESM, appendix G). Nevertheless, after the loss of the OH in position 15, it most probably leads to mechanisms similar to Fig. 11, leading to ions at m/z = 341, 325, 299 and 285. It is worth noticing that the MS/MS spectrum of the fragment at m/z = 299 is the same as Fig. 6a, which implies a structure (i)OH7 and the formation of ion (n) m/z = 243. Nor-abietanes and nor-pimaranes During their degradation, diterpenes can also lose their carboxyl groups, in order to form the associated 18-norpimaranes or abietanes. As the loss of this function is often a key step of the fragmentation of abietanes and pimaranes, the mass spectra of the corresponding nor-compounds highly decrease in specificity. Their main fragmentation mechanisms are most probably similar to the mechanism described in Fig. 3, obviously starting at fragment (a). Thus, it was not possible to find pertinent identification criterion for these compounds. Nevertheless, to identify the different nor-compounds, we looked at the molecular weight, which indicates

Si

(b4)OH7 m/z=342

the number of double bonds: 2, 3 or 4. We also manage to differentiate pimaranes from abietanes based on the MS/MS spectra of ions 258/256 (depending on the number of double bonds). In fact, as described in BDifferentiation between pimaranes and abietanes (MS/MS)^, pimaranes show a fragment respectively at 229/227 (ESM, appendix H), absent in abietanes. In GC-MS, pimaranes have a lower retention time than abietanes. In this way, we observed that all the molecules identified as nor-pimaranes have a lower retention time than nor-abietanes which support our identification criterion. 7α-OH,18-norDHA and 7β-OH,18-norDHA were detected according to the presence of ions (o) m/z = 234 and (p) m/z = 191 and a molecular ion at m/z = 344 (Fig. 12). We could also identify (l′)OH7α and (l′)OH7β 7 -OH, 18-nor-DHA

OTMS

OTMS

7 -OH, 18-nor-DHA

OTMS

OTMS

Fig. 12 Mass spectra of 7β-OH,18-norDHA and 7α-OH,18 norDHA

Analysis of diterpenic compounds by GC-MS/MS

Fig. 13 Mass spectrum of agathic acid and proposed fragmentation mechanism for labdanes

at m/z = 301 formed by a pathway similar to Fig. 6b, with similar structure than ion (l)OH7 without the double bond formed by the decarboxylation step. The fragmentation leading to (b2)–(e2) and associated cations (as in Fig. 3) was validated by MS/MS study on m/z = 254 and 211 ions (data not shown). Table 2 Summarise of characteristic marker fragments

Ion 256, 241, 213, 199, 185 256 121 254, 239, 211, 197, 183 252, 237, 209, 195, 181 299 234, 191 Intense base peak at Mw-133 Intense base peak at Mw-15 Peak at Mw-187

Dicyclic diterpenes: labdanes The following compounds were identified in the sandarac resin thanks to the literature and the study of the fragmentation. Labdanes are bicyclic diterpenes with an alkyl chain in C9 that can be hydroxylated or carboxylated. Those compounds

MS/MS

241, 227, 213, 199, 185 241, 213, 199, 186 105, 91

243, 227, 215

Comment Abietanes or pimaranes with 2 insaturations Pimaranes Abietanes If intense: no double bond on the cycle B DHA DH-DHA Hydroxy-DHA Position of OH group on C7 Position of OH group on C12 Position of OH group on C15 Labdanes (loss of the alkyl chain)

C. Azemard et al.

present a more complex fragmentation than previous diterpenes. The usual losses of -118 or -90 from the functional groups are observed in addition with new fragments. In sandarac, we found m/z = 293 for the 3-hydroxy-eperuic acid and the fragment m/z = 307 for agathic and pinifolic acids corresponding to a loss of -187. Those ions can be explained by the loss of a part of the alkyl chain and of its carboxyl group as presented Fig. 13. The mass spectra of these molecules also presented a high 121 ion, in agreement with the presence of a double bond in Δ7.

2. 3.

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Conclusion MS/MS spectrometry was used in order to explore and validate fragmentation mechanisms of diterpenes in various abietanes, pimaranes or labdanes. Such approach gives great structural information permitting to identify unknown molecules. In pimaranes and abietanes, the most common reaction pathway undergoes a retro-Diels Alder reaction and was detected in all diterpenes tested (Fig. 3). Furthermore, the position and the number of the double bonds are closely linked to the formation of specific fragments. MS/MS also allowed finding helpful discrimination criteria to distinguish abietane and pimarane molecules. For hydroxylated abietane compounds, we showed that the alcohol group in positions 7, 12 or 15 undergoes specific fragmentation leading to rapid identification. Finally, we used these identification criteria in order to identify unknown degradation products: the 18-norabietanes and pimaranes. For labdanes, the main fragmentation notably involved a cleavage of the alkyl chain. All the characteristic marker fragments are summarised in Table 2. To conclude, MS/MS is an efficient and adapted tool, giving fine structural information. It requires a very few amount of molecule, and is fast, very sensitive and possibly quantitative. Such advantages make it very useful when dealing with some problematic of archaeometry. As there is no study on such aspects in the literature, it requires further studies and the establishment of a diterpene MS/MS databank in order to facilitate the identification of unknown compounds.

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Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

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