GCMS ANALYSIS OF TRITERPENOID RESINS: IN SITU DERIVATIZATION
PROCEDURES USING QUATERNARY AMMONIUM HYDROXIDES
Siobhan Watts and E. René de la Rie
Summary—Derivatization procedures using quaternary ammonium hydroxides are now frequently
applied to the gas chromatography/mass spectrometry (GCMS) analysis of paint media and
varnishes. This paper compares the results obtained when triterpenoid resins are analysed using
different reagents and sample preparation methods. Depending on the reagent and derivatization
method used, the quaternary ammonium hydroxides produced multiple derivatives and induced
isomerization in a number of the components of triterpenoid resins. This has implications for
selecting an appropriate derivatization technique for the analysis of triterpenoid resins such as
dammar and mastic.
Introduction
The development of a rapid, sensitive technique is important for the analysis of natural organic
materials from works of art, where the available sample size is usually very small. For several
years, pyrolysisgas chromatography/mass spectrometry (Py-GCMS) has been used routinely for the
identification and characterization of modern synthetic materials used by artists and conservators.
The molecular markers for characterizing natural resins and oils found on paintings are often polar
compounds, containing carboxylic acid and other groups that require derivatization before they can
be analysed by gas chromatography/mass spectrometry (GCMS). Py-GCMS of underivatized
samples therefore generally does not yield usable results.
Derivatization procedures using quaternary ammonium hydroxides (also known as thermally
assisted methylation, and thermally assisted hydrolysis and methylation) have recently been applied
to the GCMS analysis of paint media and varnishes by several different research groups within the
field of conservation science [1-5]. Several different quaternary ammonium hydroxide reagents are
available, and two in particular have been used for the analysis of natural materials from works of
art: tetramethylammonium hydroxide (TMAH) [1-4] and trimethyl(α,α,α-trifluoro-mtolyl(ammonium hydroxide (TMTFTH) [5].
The mechanism by which acid groups are derivatized is outlined by Kossa et al. [6]: thermally
assisted methylation of free acidic compounds involves deprotonation of the acid by the reagent to
form the quaternary ammonium salt of the carboxylate anion. When injected into the gas
chromatograph. this quaternary ammonium salt is thermally decomposed to form the methyl
derivative of the acid and a tertiary amine, e.g., for stearic acid and TMAH [6]:
(∆ = thermal decomposition)
Quaternary ammonium hydroxide reagents can also perform a thermally assisted hydrolysis and
methylation of ester linkages in polymers and glycerides, which makes them appropriate for the
analysis of polymerized oil paint media, e.g., for octadecyl octadecanoate and TMAH [7]:
(∆ = thermal decomposition)
It has also been shown that TMAH will methylate, or partly methylate. OH groups as follows [7]:
(∆ = thermal decomposition)
The development of thermally assisted hydrolysis and methylation techniques has recently been
reviewed for a range of applications [8]. The main advantage of employing these derivatization
procedures is that they can be performed in a single stage, whereas the traditional sample work-up
methods for GCMS often involve several stages of extraction, hydrolysis and methylation. A singlestage method reduces the risk of sample contamination, and can also increase the sensitivity of the
technique so that the sampling requirements are minimized.
The published procedures for effecting this one-stage derivatization vary considerably, both in the
quaternary ammonium hydroxide reagent used and in the technique used to introduce the sample
into the gas chromatograph. Although the mechanism for the formation of methyl esters from
polymeric materials using quaternary ammonium hydroxides is considered to be a thermally
assisted hydrolysis and methylation procedure [7-9] rather than simultaneous pyrolysis/methylation,
the pyrolysis interface is a convenient vehicle for introducing the sample into the GC injector and it
can also allow the analysis of any non-hydrolysable polymeric material in the sample at the same
time. A pyrolysis interface is not essential, however, and the method currently used for the analysis
of paint media at the Scientific Department of the National Gallery in London involves injecting a
solution of the sample and the reagent TMTFTH into the injection port of the gas chromatograph
[5].
Two recent studies [4. 10] have shown that, in some cases, thermally assisted methylation can result
in more than one derivative being formed from a single compound, so that the results are more
complex than those produced by traditional derivatization methods and require careful interpretation. This is the case particularly for compounds with more than one reactive group, such as the
oxidation products formed in the natural resins used as varnishes or components of paint media.
Van den Berg and co-workers [4] illustrated the formation of multiple derivatives through thermally
assisted methylation of oxidized diterpenoid acids in aged Pinaceae resins from paintings.
Factors that may influence the formation of multiple derivatives include the alkalinity of the quaternary ammonium hydroxide reagent, the method by which the sample and reagent are introduced
into the gas chromatograph, the geometry of the GC injector and flow of carrier gas, and other
reaction parameters including the temperature at which thermally assisted hydrolysis and/or
methylation takes place and the duration and degree of contact between sample and reagent [6, 9].
In addition, hydroxyl groups can undergo thermal dehydration reactions which are dependent on the
temperature and nature of the injector or pyrolysis interface, and occur even in the absence of the
quaternary ammonium hydroxide reagent [10, 11].
In order to determine the most suitable derivatization method for use in the Scientific Research
Department of the National Gallery of Art in Washington DC, a comparative study of the effects of
different reagents and analysis conditions for thermally assisted methylation was carried out on
triterpenoid standards and on both fresh and aged samples of the triterpenoid resin, dammar. The
different reagents and experimental conditions were compared to assess whether there was evidence
for the formation of multiple derivatives or isomerization of particular compounds. The relative
sensitivities (and hence sample size requirements) were also compared.
The most common procedure for thermally assisted methylation, which involves analysis of a solid
sample placed together with the reagent into a pyrolysis interface (e.g., [10]) was compared for the
reagents TMAH and TMTFTH. The TMTFTH method developed by White and Pile for the analysis of paint media [5], involving extraction of the sample in methanolic TMTFTH and injection of
the solution in the GC injector port, was also tested. The results were compared to the traditional
methylation procedure using diazomethane [12], and the samples methylated by both diazomethane
and TMTFTH were also injected through the pyrolysis interface via an injector nut attachment to
investigate thermal dehydration reactions not necessarily related to the use of a quaternary
ammonium hvdroxide reasent.
Materials
The following four standards were analysed using the methylation procedures outlined below:
•
ursolic acid from the reference collection at the National Gallery of Art, originally supplied by
Raymond White
•
oleanolic acid from Gisela van der Doelen, originally supplied by Aldrich Chemicals
• methyl ursonate from the reference collection
at the National Gallery of Art, originally supplied by Raymond White
• hydroxydammarenone II (dipterocarpol) from Gisela van der Doelen, originally supplied by
Aldrich Chemicals
These were selected because they are triterpenoids which have been identified as, or are closely
related to, constituents of dammar and mastic resin [13, 14], and because they have different
functionalities which could be affected by the derivatization procedures.
On the basis of the results obtained from the standards, fresh and aged triterpenoid resins were
analysed using selected derivatization conditions. Fresh dammar resin from the species Shorea
javanica (supplied by Adam Messer) was analysed. Two aged varnishes were analysed: one from
the painting Nude on a blue cushion by Modigliani (National Gallery of Art, Washington DC, ace.
no. 1963.10.46) and varnish sample no. A5 (supplied by Gisela van der Doelen. from a painting by
Jean Jacque in a private collection). The sample from the Modigliani was a varnish scraping.
Sample no. A5 was removed using a swab with isopropyl alcohol (propan-2-ol) and then extracted
from the swab with isopropyl alcohol.
Experimental
GCMS
The analyses were carried out using a Varian 3500 gas chromatograph (GC) with a split/splitless
injector which was operated in split mode. The split flow was adjusted within the range of 30100cm3min-1, according to the analysis conditions (the on-line methylation of solid samples
generally-required an increased split flow). The carrier gas was helium, and the GC was fitted with
an RTX-1 capillary column (30m X 0-32mm i.d., 0-25μm film thickness, 62kPa column head
pressure). The temperature of the interface/injector was 300°C. The GC oven was programmed with
an initial temperature of 100°C, which was held for five minutes. The temperature was increased at
a rate of 4°C per minute to 300°C and held for 20 minutes. The GC was interfaced to a Finnigan
MAT ion trap detector (ITD 800), the transfer line being held at 250°C. Typical operating
conditions for the ITD were: ITD manifold at 213°C. electron multiplier 1600V. scan range 50-600
atomic mass units, scan time one second, data analysis Finnigan ITDS 4.10.
Diazomethane methylation
The sample was dissolved in a 10:1 mixture of ethenmethanol, and diazomethane gas (CH2N2) was
bubbled through the solution until an excess of the reagent was visible. The sample was
concentrated for GCMS analysis and an aliquot («2μl) was injected into the GC. A similar quantity
of sample, prepared in exactly the same way, was injected through a CDS Pyroprobe 2000 pyrolysis
interface mounted directly onto the GC injector, fitted with a quartz liner and a Pyrex filler
(designed to reduce the dead volume of the interface, so that it could function as an interface for the
injection of samples in solution). A third portion of the sample was injected through the interface
with the Pyrex filler removed, to investigate the effect of a large dead volume on the analysis.
On-line thermally assisted methylation of solid samples via the pyrolysis interface
A small amount (=0.l-0.5mg) of solid sample was placed in a quartz boat, and 3μl of TMAH
(25wt% in methanol) was added. When the solvent had evaporated, the quartz boat was placed into
the platinum pyrolysis coil which was then inserted into the pyrolysis interface. The pyrolysis probe
was not fired, since the aim of this research was to compare the chromatograms of the lower
molecular weight triterpenoid components with the chromatograms resulting from derivatization
with diazomethane injected through a conventional GC split/splitless injector. The polymeric
component of these resins is small, and the triterpenoid fraction forms the bulk of the samples. The
interface and GC injector were kept at 300°C, so that the thermally assisted methylation reaction
took place immediately, and the products were swept into the GC injector by the carrier gas. This
procedure was repeated using TMTFTH (5% in methanol) as the reagent, instead of TMAH. Further
experiments to investigate the effect of a lower interface temperature with TMTFTH were carried
out for two of the standards (ursolic acid and dipterocarpol). In these experiments the interface and
GC injector temperatures were reduced to 200°C and the solid sample was analysed with TMTFTH
as described above.
Thermally assisted methylation of samples in TMTFTH solution
This procedure was adapted from the method outlined by White and Pile [5]. Several experiments
were run to determine a suitable reaction time and temperature. The sample was dissolved in 20|j.l
of a methanolic solution of TMTFTH (5wt%) and allowed to stand at room temperature for 16
hours. The solution was then centrifuged for five minutes and left to stand for a minimum of a
further two hours. Shorter reaction times resulted in incomplete methylation of the carboxylic acids.
The effects of
longer reaction times and/or higher temperatures were investigated for two of the standards, and the
results are discussed in the relevant section below. An aliquot (=2μl) of this solution was injected
into the GC. As for the samples methylated with diazomethane, a similar quantity of the sample was
injected through the pyrolysis interface, fitted with a quartz liner and a Pyrex filler. A third portion
of the sample was injected through the interface with the Pyrex filler removed.
Identification of the constituents of the triterenoid resins was based on published chromato-graphic
and mass spectral data [13-16]. The additional derivatization products were identified by
comparison with published work on the effect of TMAH on diterpenoid resins [4. 11] and with reference to the retention times of standard compounds.
Results and discussion
The one-stage derivatization methods produced multiple derivatives and appear to have induced
isomerization in a number of the standards analysed. Relative concentrations of the derivatives and
isomers formed varied according to the analysis conditions. Injection of the standards through the
pyrolysis interface without the presence of a quaternary ammonium hydroxide reagent did not result
in any isomerization or additional products—the isomers and multiple derivatives result specifically
from the use of these reagents. The effect of the different reagents and procedures used is discussed
below for each of the standards analysed. Table 1 summarizes the derivatization products produced
for each of the standards using the four different derivatization methods. The mass spectra of these
products are given in the Appendix. The mass spectra obtained on the ion trap detector often show
(M + l)+ ions, and relative intensities may differ from those in spectra obtained on a quadrupole
mass spectrometer.
The results from the analysis of the standards are then applied to the interpretation of the results
from the fresh and aged resins, in order to assess the application of one-stage derivatization procedures to the analysis of triterpenoid varnishes.
Ursolic acid and oleanolic acid
Triterpenoids with the oleanane/ursane skeleton have been identified in fresh dammar and mastic
resin and aged triterpenoid varnishes [13-17]. Using the derivatization procedures described above.
GCMS analysis of two isomeric pentacyclic hydrocarbons, ursolic acid and oleanolic acid, yielded
similar results. Both have a carboxylic group in the 28-position and a hydroxyl group in the 3position. The different derivatization products of ursolic acid are illustrated in Figure la. together
with diagnostic fragments in the mass spectra. Methylation with diazomethane produces the 28carbomethoxy product (Figure 2a). When TMAH is used to methylate a solid sample in the
pyrolysis interface, the major product is the compound with both the OH and the COOH methylated
(Figure 2d), and this elutes earlier in the chromatogram since it is less polar.
The methylation with TMTFTH produced a number of additional products. Figure 2b shows the
chromatogram from a sample of ursolic acid reacted in solution with TMTFTH and then injected
through the GC split/splitless injector. The major product formed is methyl ursolate (peak 1), some
of the twice methylated product is observed (peak 2), and two additional products elute after the
methyl ursolate peak. The lack of molecular ions and high molecular weight fragment ions in their
mass spectra means that positive identification of these compounds is difficult, although they are
similar to the mass spectrum from methyl ursolate, and therefore are interpreted as isomers.
Experiments with a lower GC injector temperature did not reduce the levels of later eluting isomers.
The relative amounts of the twice methylated product, and the later eluting isomers. did not increase
consistently with an increase in the reaction temperature and duration, suggesting that the formation
of these products is not dependent simply on reaction time and temperature.
Finally, when TMTFTH was used to methylate a solid sample of oleanolic or ursolic acid in the
pyrolysis interface, the resultant chromatogram contained at least 11 different peaks (Figure 2c).
Not all of these could be assigned, but the four major peaks eluting before both the twice methylated product and methyl ursolate are interpreted as isomers of the dehydrated methyl ester (peaks
labelled 3). These were not noted when samples of oleanolic acid and ursolic acid methylated in
diazomethane were injected through the pyrolysis interface, so this dehydration appears to be
caused by the reagent rather than simply the high temperature or presence of reactive surfaces in the
pyrolysis interface.
Methyl ursonate
Ursonic acid has been identified using GCMS as a constituent of both fresh and aged dammar resin
[13, 14]. Methyl ursonate has the same skeletal structure as methyl ursolate, but with a ketone group
in the 3-position instead of the hydroxyl group (see Figure 1b for the structure of methyl
Table 1
Summary of derivatization products formed with one-stage derivatization methods
ursonate and its additional derivatization products). When the sample is dissolved in ether and
injected through the GC interface, only the peak for methyl ursonate is observed (Figure 3a).
Pastorova [11] showed that when TMAH is used to methylate diterpenoid resin acids containing
ketone groups, the enol tautomer is formed and methylated in a proportion of the sample. The same
process occurs to some extent with methyl ursonate. when the solid sample is reacted with TMAH
in the pyrolysis interface (peak 5. Figure 3d). A small amount of a compound with a third methyl
group is also observed (peak 6, Figure 3d) as well as a fourth unidentified pentacyclic
ursane/oleanane derivative (peak U, Figure 3d).
When the sample of methyl ursonate is reacted in solution with TMTFTH and injected through the
GC interface, the results are similar to those obtained with TMAH, with a significant peak from the
methylated enol tautomer (Figure 3b). When TMTFTH is added to the solid sample in the
pyrolysis interface, only a small amount of the methylated enol tautomer is formed (Figure 3c).
Hydroxydammarenone II (dipterocarpol)
Hydroxydammarenones I and II are major constituents of dammar resin [13], although their characteristic side-chains are readily oxidized to form molecules with ocotillone and -y-lactone
structures [14]. Hydroxydammarenone has a ketone in the 3-position, as well as an OH group
located at the sterically hindered tertiary position in the characteristic dammarane side-chain (Figure
Ic). The molecular ion (m/z 442) is not observed in the mass spectrum, since it is easily dehydrated
to give the m/z 424 fragment ion [13].
When either of the reagents TMAH or TMTFTH is used for on-line methylation of the sample in
the pyrolysis interface, at least nine products elute before hydroxydammarenone (peak 7, Figures 4c
and 4d), and not all of these were identi-
Figure 1
Derivatization products identified and their diagnostic mass spectral fragments.
Figure 1
Derivatization products identified and their diagnostic mass spectral fragments.
fied. A small amount of the methylated enol tautomer is formed with TMTFTH (Figure 4c, peak 9);
with the more alkaline TMAH, the enol tautomer is more readily methylated (Figure 4d. peak 9).
The OH in the 20-position of the dammarane side-chain is sterically hindered, and therefore is
methylated to a limited degree (peak 11 in both 4c and 4d). As with ursolic acid and TMTFTH
(Figure 2c), dehydration of the molecule occurs during this derivatization procedure when the OH
group is not methylated. Peaks labelled 8 are interpreted as isomers of dehydrated hydroxydammarenone, in which a molecule of H2O is lost before separation on the GC column. Three
isomers appear to be formed—probably due to migration of the double bond formed on dehydration
of the side-chain. Three isomers of the dehydrated, methylated enol tautomer are also formed (peaks
labelled 10). Reducing the temperature of the pyrolysis interface to 200°C did not prevent the
formation of these iso-
Figure 2 A comparison of the total ion chromatograms from four derivatization procedures applied
to the GCMS analysis of ursolic acid: (a) diazomethane methylation, GC injector; (b) TMTFTH
methylation, GC injector; (c) TMTFTH on-line methylation, pyrolysis interface; (d) TMAH on-line
methylation, pyrolysis interface.
Figure 3 A comparison of the total ion chromatograms from four derivatization procedures applied
to the GCMS analysis of methyl ursonate: (a) in ether, GC injector; (b) TMTFTH methylation, GC
injector; fcj TMTFTH on-line methylation, pyrolysis interface; (d) TMAH on-line methylation,
pyrolysis interface.
mers, but merely prevented the transfer of hydroxydammarenone and reaction products to the GC
column. These were found to elute in a later, blank run, when the interface temperature was raised
again to 300°C.
This isomerization did not occur when the sample was dissolved in TMTFTH and injected through
the GC interface (Figure 4b). The major peak in the chromatogram is from hydroxydammarenone
(peak 7), with one additional peak resulting from methylation of the enol tautomer (peak 9). There
was no evidence for methylation of the OH group in the 20-position. When the duration and
temperature of the reaction between the sample and reagent were increased, only trace amounts of
the product with the OH methylated were produced. However, small peaks from ocotillone-type
structures were observed in the chromatogram, indicating that oxidation of the side-chain took place
at increased temperatures.
Figure 4 A comparison of the total ion chromatograms from four derivatization procedures applied
to the GCMS analysis of hydroxydammarenone II (dipterocarpol): (a) diazomethane methylation,
GC injector; (b) TMTFTH methylation, GC injector; (c) TMTFTH on-line methylation, pyrolysis
interface; (d) TMAH on-line methylation, pyrolysis interface.
Fresh dammar resin
The triterpenoid fraction of fresh dammar resin has been the subject of detailed studies using
GCMS analysis, and many of the components have been elucidated [13-15, and references therein].
GCMS analysis of the resin sample methylated with diazomethane (Figure 5a) for this study
produced results comparable to previous work: major components include hydroxydammarenone
(peak 7), dammarenolic acid (methyl ester, .peak 12). dammaradienone (peak 8), dammaradienol
(peak 17), dammarendiol (peak 19) and ursonic acid (methyl ester, peak 4). Minor components
include small peaks from the oxidation products shoreic/eichlerianic acid (peak 20) and ocotillone
(peak 14) [13-15].
All the derivatization methods using quaternary ammonium hydroxides produced more complex
chromatograms from the fresh resins. Some of the
additional peaks could not be assigned, and these have been labelled “U” in the chromatograms for
the three one-stage methylation procedures.
Analysis of the fresh resin after dissolving the sample in TMTFTH and injecting it through the GC
interface produced a chromatogram which was similar to the sample methylated using diazomethane: the chromatogram shows the predominant triterpenoids in fresh dammar resin (Figure 5b).
There are a number of extra products, some co-eluting with other components, making the chromatogram more complex. Multiple derivatization products were identified for
hydroxydammarenone and ursonic acid, both of which produced an extra peak from methylation of
the enol tautomer in the 3-position (peaks 9 and 5).
The results from both on-line methylation procedures (Figure 5c and 5d) were significantly
different from those obtained from standard methylation of fresh dammar resin. The same series of
three iso-
Figure 5 A comparison of the total ion chromatograms from four derivatization procedures applied
to the GCMS analysis of fresh dammar resin: (a) diazomethane methylation, GC injector; (bj
TMTFTH methylation, GC injector; (c) TMTFTH on-line methylation, pyrolysis interface; (d)
TMAH on-line methylation, pyrolysis interface.
mers of dehydrated hydroxydammarenone (peaks labelled 8), observed from the analysis of the pure
standard, were noted. The second of these isomers co-elutes with dammaradienone (peak 8 in the
sample of fresh dammar methylated with diazomethane, Figure 5a) and is presumed to be identical
in structure to this constituent of fresh dammar; hydroxydammarenone would produce dammaradienone on loss of water. A similar series of three peaks, interpreted as isomers (labelled 13) of
methyl dammarenolate also dehydrated through loss of the OH at the 20-position, elute before the
peak for methyl dammarenolate itself. The relative concentrations of hydroxydammarenone and
methyl dammarenolate are reduced, particularly in the sample methylated on-line with TMAH
(Figure 5d). There are surprisingly low amounts of additional methylation products, such as the
methylated enol tautomers of hydroxydammarenone and corresponding dehydration products,
compared to the analysis of the pure standards. This may be due to the nature of the sample, which
was not in the same finely divided powder form as the pure standards. However, a sample from the
same lump of resin was analysed a year later, and the results were closer to those from the
standards, with higher concentrations of additional methylation products formed (Figure 6).
Analysis conditions were exactly the same, except that a new batch of TMAH was used, and a new
GC column (with an identical stationary phase) was installed. Clearly, the relative amounts of
additional methylation products formed can vary considerably, depending on column conditions and
any deterioration of the reagent.
Figure 6 Reproducibility tests for the GCMS analysis of fresh dammar resin, methylated on-line
using TMAH:(a) sample of fresh dammar analysed August 1997; (b) sample of fresh dammar
analysed May 1998;(c) repeat of (b).
Aged triterpenoid varnishes
The components of aged triterpenoid varnishes are the subject of recent research. which has elucidated the structures of many of the oxidized derivatives of the dammarane- and ursane/oleananetype molecules [14—16]. The two samples analysed here were both identified as aged dammar
resin on the basis of the abundance of ocotillone-type molecules and the lack of a major peak from
methyl moronate (Figures 7a and 8a). Both samples produced very similar chromatograms when
methylated with diazomethane, with the only major difference being the peak from methyl
dammarenolate in sample A5 (Figure 8a. peak 12), suggesting a slightly reduced degree of
oxidation for this sample.
When the varnish samples were methylated by dissolving them in TMTFTH and injecting them
through the GC injector, the results were comparable to those from the diazomethane procedure
(Figures 7b and 8b). The major peaks are
from the oxidized triterpenoids: ocotillone (peak 14), methyl ester of shoreic/eichlerianic acid (peak
20), and ocotillol (peak 21). Methyl ursonate (peak 4) and methyl dammarenolate (Figure 8b, peak
12) could still be identified, and no major peaks from additional methylation products were noted.
The on-line methylation procedures produced rather different results, and they were not consistent
for the two varnishes. In Figure 7c and 7d, the major peak (14) from ocotillone is still identifiable.
The methyl ester of shoreic/eichlerianic acid is no longer a significant peak in the sample
methylated on line with TMTFTH (Figure 7c). Several additional early eluting peaks are present in
this sample: the second of these (peak 15) may be from ocotillone which has been dehydrated
through loss of the tertiary OH in the side-chain. The mass spectrum has a base peak at m/z 125,
without the corresponding m/z 143 characteristic of the ocotillone side-chain.
When the second varnish sample, A5, was
Figure 7 A comparison of the total ion chromatograms from four derivatiation procedures applied
to the GCMS analysis of an aged varnish from Modigliani's 'Nude on a blue cushion': (a) diazomethane methylation, GC injector; (b) TMTFTH methylation, GC injector; (c) TMTFTH on-line
methylation, pyrolysis interface; (d) TMAH on-line methylation, pyrolysis interface. * indicates
peaks characteristic of dammar.
analysed using the TMAH on-line methylation method, ocotillone was not identifiable as a major
peak in the chromatogram (Figure 8d). Three early eluting peaks which were not present in samples
methylated using different procedures could not be identified. A peak resulting from ocotillone in
which the OH in the side-chain had been methylated was noted (peak 16). A small peak (5) from
the methylated enol tautomer of methyl ursonate was also identified.
It may be significant that the chromatograms from the two aged varnishes were very similar when
methylated with diazomethane, but different when methylated using an on-line technique. This suggests that the on-line methods do not produce reproducible results for the analysis of aged resins,
although further work is required to establish if this is the case.
Implications for the application of one-stage derivatization procedures
From these results it is clear that reactions of triterpenoid compounds containing more than one
functional group with a quaternary ammonium hydroxide reagent can produce a number of different
derivatives. It is essential that this is taken into consideration when selecting a reagent or sample
preparation method. Some of the observations noted here may be specific to this particular pyrolysis
unit and GC injector, since injector geometry and carrier gas flow can affect the efficiency of the
derivatization procedure [6].
On-line derivatization within the pyrolysis interface appears to be problematic for samples with OH
groups that are not methylated by the reagent. This was the case for the methylation of oleanolic
acid with TMTFTH in the pyrolysis interface, but also for hydroxydammarenone with both TMAH
Figure 8 A comparison of the total ion chromatograms from four derivatization procedures applied
to the GCMS analysis of an aged varnish sample no. A5: (a) diazomethane methylation, GC
injector; (b) TMTFTH methylation, GC injector; (c) TMTFTH on-line methylation, pyrolysis interface; (d) TMAH on-line methylation, pyrolysis interface. * indicates peaks characteristic of
dammar.
and TMTFTH. The higher alkalinity of TMAH results in methylation of the hydroxyl group in the
case of oleanolic acid, and so prevents the dehydration and isomerization. The OH group in
hydroxydammarenone, however, is not methylated by TMAH because it is sterically hindered,
resulting in dehydration and isomerization similar to that seen with on-line methylation using
TMTFTH.
These sterically hindered hydroxyl groups are present in many of the characteristic markers of both
fresh and aged triterpenoid resins, so that on-line methylation of these resins produces complex
chromatograms in which individual components are poorly resolved and difficult to interpret. This
is a problem particularly for aged resins which produce complex chromatograms by standard
derivatization methods, so that additional methylation products from these methods are even more
difficult to interpret. The results presented here show that it may not be possible to identify the
diagnostic molecular markers, such as ocotillone-type molecules, which are characteristic of aged
dammar resin. Further work, to compare the effect of these one-step derivatization procedures on
pure samples of the oxidized triterpenoids such as ocotillone, is necessary in order to understand
fully the products of one-step methylation procedures for aged resins. The analysis of pure oxidized
triterpenoids would also be useful in assessing whether the experimental effects of these
derivatization methods could result in the confusion of aged dammar and mastic resins. Ocotillone
is very abundant in dammar resin and is also present in mastic resin. Moronic acid is only present in
mastic resin [15, 17], so that the analysis of pure moronic acid would be useful to investigate
whether aged dammar and mastic resin can be distinguished using these procedures.
The TMTFTH method developed by White and Pile [5], involving reaction of the sample in
solution with the reagent and injection through the GC injector, appears to be more appropriate for
the analysis of triterpenoid resins, which have a high concentration of compounds with tertiary OH
groups, than on-line derivatization methods using a pyrolysis interface.
Acknowledgements
This project was supported by the J. Paul Getty Trust, the Andrew W. Mellon Foundation and the
National Gallery of Art, Washington DC. We are also grateful to Gisela van der Doelen of the FOM
Institute. Amsterdam for providing samples and for discussion of the results, and to Chris Maines of
the National Gallery of Art, Washington DC for help with the instrumentation and producing the
mass spectra. Thanks are due to the following for helpful discussions of the project: Raymond
White of the National Gallery in London, Klaas Jan van den Berg of the Netherlands Institute for
Cultural Heritage, Suzanne Quillen Lomax of the National Gallery of Art, Washington DC, and
Carl Heron and Ben Stern of the Department of Archaeological Sciences, University of Bradford.
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18
Authors
SIOBHAN WATTS received a PhD on the geochemical characterization of jet and jet-like materials in
archaeology from the University of Bradford in 1996, and an MA in the conservation of historic
objects from the University of Durham in 1992. In 1997 she joined the National Museums and
Galleries on Merseyside, where she is the conservation scientist at the Conservation Centre.
Previous to this she held a Getty internship at the Department of Scientific Research, National
Gallery of Art. Washington DC. Address: The Conservation Centre, Whitechapel, Liverpool LI
6HZ, UK.
E. RENE DE LA RIE received MS and PhD degrees in chemistry from the University of Amsterdam.
Since 1989 he has been head of scientific research at the National Gallery of Art in Washington DC,
where he directs a staff of researchers who study artists' methods and materials and test and develop
conservation materials. Previously, he held positions at the Metropolitan Museum of Art, New
York, and at the Training Programme for Conservators and the Central Research Laboratory for
Objects of Art and Science, both in Amsterdam. Address: Scientific Research Department, National
Gallery of Art. Washington, DC 20565, USA.
Resume—On utilise aujourd'hui frequemment pour I'analyse des peintures et vernis par
chromatographie en phase gazeuse/spectrometrie de masse (CG/SM) des procedures de
derivatisation a base d'hydroxydes d'ammonium quaternaires. Cet article compare les resultats
obtenus lors de I'analyse de resines triterpenoi'des en utilisant differents reactifs et methodes de
preparation des echantillons. Selon le reactif et la methode de derivatisation utilises, les hydroxides
d'ammonium quaternaires produisent plusieurs derives et induisent I'isomeri-sation de nombre de
composants des resines. Ceci a des implications dans la selection des methode de derivatisation
appropriees pour I'analyse des resines triterpenoi'des comme le dammar ou le mastic.
Zusammenfassung—Derivatisierungsmethoden, bei denen quaterncire Ammoniumhydroxide
verwendet werden, werden hdufig :ur Analyse van Bindemitteln und Firnissen mittels
Gaschromatograhie/Massenspektrometrie (GCMSj eingesetzt. In der vorliegenden Studie werden
die Ergebnisse verschiedener Reagenze und Praparationsmethoden fur die Untersuchung van
Triterpen-Harzen miteinander verglichen. In Abhdngigkeit vom Reagen: und der
Derivatisierungsmethode, werden durch die quaternaren Ammoniumhydroxide sehr vielfaltige
Derivate und Isomerisierungsprodukte der Triterpene erzeugt. Dies hat eine grofie Bedeutung fur
die
Auswahl der richtigen Derivatisierungsmethode fur die Untersuchung von Triterpen-Harzen wie
beispielsweise Dammar oder Mastix.
Resumen—Los procesos de derivation usando hidroxidos de amonio cuaternario son
frecuentemente aplicados hoy en dia para el andlisis par cromatografia de gases-espectrometria de
masas (GC-MS) de medios agluti-nantes y barnices. Este articulo compara los resultados obtenidos
cuando resinas triterpenicas son analizadas usando diferentes reactivos y diferentes metodos de
preparation de muestras. Dependiendo del reactivo y del metodo de derivation, los hidroxidos de
amonio cuaternario produjeron multiples derivados e indujeron iso-merizacion en un numero de
componentes de las resinas triterpenicas. Esto tiene implicaciones a la hora de seleccionar una
derivation apropiada para el andlisis de resinas triterpenicas como la dammar o el almdciga.
Appendix: mass spectra of derivatization products of standards