Liquid Chromatography with Mass Spec trometry in

Tandem Mode Applied for the Identific ation of

Wine Markers in Residues from Anc ient Egyptian
Vessels
Maria Rosa Guasc h-J ane,

Maite Ibern-Gomez,

Cristina Andres-Lac ueva,

Olga J auregui,

and
Rosa Maria Lamuela-Raventos*
,
Nutrition and Food Science Department, Pharmacy Faculty and Scientific and Technical Services, University of Barcelona,
08028 Barcelona, Spain
Presentedinthispaper isanewmethodfor theidentifica-
tion of tartaric acid as a wine marker in archaeological
residues fromEgyptian vessels usingliquid chromatog-
raphywithmassspectrometryintandemmode(LC/ MS/
MS). Owingtothespecial characteristicsofthesesamples,
such as thedryness and thesmall quantityavailablefor
analysis, it was necessary to have a very sensitive and
highlyspecific analytical methodtodetecttartaric acidat
trace levels in the residues. Furthermore, an alkaline
fusion was carried out to identify syringic acid derived
frommalvidinasaredwinemarker, inadepositresidue
fromawinejar foundat thetombof kingTutankhamun.
Malvidin-3-glucoside, the main anthocyanin that gives
youngwines their red color, polymerizes with aginginto
morestablepigments. However, thepresenceofmalvidin
in ancient residues can be proved by alkaline fusion of
the residue to release syringic acid from the pigment,
whichhasbeenidentified, herefor thefirsttime, byusing
theLC/ MS/ MSmethod revealingthered grapeorigin of
an ancient Egyptian wineresidue.
In ancient Egypt, vines were grown throughout the country,
although the best wines came from the Nile River Delta and the
Western oasis.
1
In fact, wine was a product of great importance,
offered in funerary rituals and in temples to worship gods and
consumed daily by the upper classes during meals and parties.
1
Since the Early Dynastic period (2920-2575 BC
2
) wine jars were
placed in tombs as funerary meals, some with engraved inscrip-
tions. From the Old Kingdom (2575-2134 BC
2
) to the New
Kingdom (1550-1070 BC
2
) periods, tomb walls of the nobles were
decorated with scenes including viticulture and wine-making.
Egyptian mythology even related the red color of the Nile during
flooding to the color of wine.
1
The New Kingdom wine jars were
labeled with product, year, source, and even the name of the vine
grower,
3,4
but they did not mention the type (color) of wines
contained. Most of the labeled jars are nowonly broken fragments,
but some are completely preserved. Despite that, residues from
archaeological vessels have been barely investigated.
Considering the special characteristics of archaeological samples,
our aim was to develop avery sensitive and highly specific method
for identification of wine markers that might be present in trace
quantities. In particular, tartaric acid, rarely found in nature in
sources other than grapes, has been reported as a wine marker
in ancient residues and could be preserved in the pottery because
it can be strongly absorbed on silicates by hydrogen bonding.
5
Analysis of ancient samples requires a very sensitive method in
order to minimize the amount of sample used.
6
Four analytical
methods (thin-layer chromatography,
7
gas chromatography,
7
dif-
fuse-reflectance Fourier transform infrared spectroscopy,
5,8,9
and
high-performance liquid chromatography with UV detection
9
) have
been applied for tartaric acid determination in archaeological
samples. However, all of them lack enough selectivity and
sensitivity for the size of samples available, so an improved method
was required. Liquid chromatography/ mass spectrometry in
tandem mode (LC/ MS/ MS) has become an ideal technique due
to its speed, sensitivity, and selectivity and a powerful tool for
identification based on retention times and fragmentation patterns
of the compounds during MS/ MS analysis apart from low
detection limits. Another important feature of triple quadrupole
MS instruments is that they provide the highest sensitivity in
multiple reaction monitoring (MRM) mode. When the compounds
are present at trace levels or the amount of sample available is
limited, an MRM assay is the method of choice because it provides
* To whom correspondence should be addressed. E-mail: lamuela@ub.edu.

Nutrition and Food Science Department.

Scientific and Technical Services.

(1) Lexicon der A gyptologye. 1986, Band VI, 1169-1192.
(2) Baines, J.; Malek, J. Atlasof Ancient Egypt; Phaidon: Oxford, 1980; pp 36-
37.
(3) Cerny, J. HieraticInscriptionsfromthetombof Tutankhamun; Tutankhamuns
Tomb Series II; Griffith Institute: Oxford, 1965; pp 1-4.
(4) Martin, G. T. TheHidden Tombsof Memphis; Thames & Hudson: London,
1991; p 98.
(5) Michel, R. H.; McGovern, P. E.; Badler, V. R. Anal. Chem. 1993, 65, 408A-
413A.
(6) Garnier, N.; Cren-Olive, C.; Rolando, C.; Regert, M. Anal. Chem. 2002, 74,
4868-4877.
(7) Condamin, J.; Formenti, F. Figlina 1 1976, 143-158.
(8) Badler, V. R.; McGovern P. E.; Michel, R. H. Drink andbeMerry!; MASCA
Research Papers in Science and Archaeology; University of Pennsylvania:
Philadelphia, PA, 1990; Vol. 7, pp 25-36.
(9) McGovern, P. E.; Glusker, D. L.; Exner, L. J.; Voig, M. M. Nature1996,
381, 480-481.
Anal. Chem. 2004, 76, 1672-1677
1672 Analytical Chemistry, Vol. 76, No. 6, March 15, 2004 10.1021/ac035082z CCC: $27.50 2004 American Chemical Society
Published on Web 02/18/2004
the highest sensitivity in MS/ MSmode. To our knowledge, LC/
MS/ MS has not been used before for the analysis of tartaric or
syringic acids in archaeological residues or in any other kind of
sample. In addition, we optimized this LC/ MS/ MS method for
syringic acid detection in order to study the color of a wine
residue. Malvidin is the major red wine anthocyanin,
10
and it
polymerizes with aging. By alkaline fusion of ared wine, malvidin
releases syringic acid,
11
a red wine marker. In that case, syringic
acid identification would indicate that a dark brown wine residue
had red grape origin. Syringic acid has never been detected in
any archaeological samples, even though a previous attempt was
made using alkaline fusion in a Roman wine residue by HPLC-
UV
12
with no success.
EXPERIMENTAL SECTION
Archaeological Samples. As residues from archaeological
remains are very precious, a small quantity of sample was taken
from the inside of Egyptian pottery vessels for analysis, by special
permissions of the Egyptian Supreme Council for Antiquities
(SCA) and the Egyptian Museum in Cairo as well as the British
Museum in London. The residues were collected from three
Egyptian pottery jars at the British Museum (named BM samples)
and two pottery jars at the Egyptian Museum in Cairo (named
CM samples). A short description of the objects, dating periods,
and sites, if known, is included in Table 1. Sample CM1 was a
dry deposit of dark brown color from the bottom of a wine jar;
samples BM1 and CM2 were thin encrustations on the inside of
pottery jars; samples BM2 and BM3 were obtained by scraping
the inside surface of the jar (not having visible deposits). Alkaline
fusion was only performed on the CM1 sample because it is a
deposit residue of dark-brown color. Due to the nature of the other
samples, alkaline fusion was not possible.
A blank as a sample control was also included in this study to
identify possible interference in ceramics not due to tartaric acid.
It was apottery handle of ancient Egyptian origin not having come
into contact with vessel contents.
Standards and Reagents. A standard of L-tartaric acid (99%
purity) was purchased from Aldrich Chemical Co. (Steinheim,
Germany) and prepared at a concentration of 100 mg/ L in water.
A standard of syringic acid (98%) from FlukaChemie AG(Buchs,
Switzerland) was prepared at a concentration of 100 mg/ L in
methanol/ water (20:80, v/ v). The working solution of 100 g/ L
was made by diluting the standard solutions with the LC mobile
phase (0.1%formic acid in water/ acetonitrile, 90:10). Acetonitrile
and methanol of HPLCgrade were purchased from SDS(Peypin,
France), formic acid and ethyl acetate (99%) from Panreac
(Barcelona, Spain), and potassium hydroxide pellets (85%) from
Carlo Erba (Milano, Italy), and ultrapure water (Milli-Q) was
obtained from Millipore System (Bedford, MA).
SamplePreparation. An amount of 2 mg of the pulverized
residue was extracted with 5 mL of 0.1%formic acid in water/
methanol (80:20, v/ v) with magnetic stirring and ultrasound. The
liquid was centrifuged for 15 min at 1620g, the supernatant was
concentrated under nitrogen to one-fifth volume for CM samples
and 0.1 volume for BM samples, and finally, they were filtered
with an Acrodisc 13 CRPTFE 0.45 m (Waters). Alkaline fusion
was performed by addition of potassium hydroxide pellets (0.2
g) to the sample described above under heating for 5 min,
acidification, and finally extraction with ethyl acetate, following
the previous published literature.
11,13
Instrumentation. Analyses were carried out using a liquid
chromatograph with a mass spectrometer in tandem mode. LC
equipment was an Agilent 1100 (Waldbronn, Germany) with a
quaternary pump. A Waters Atlantis C
18
column (2.1 150 mm
i.d., 5 m) was used at ambient temperature, and the injected
volume was 15 L. A constant flow rate of 200 L/ min was used
with two elution solvents: 0.1%formic acid in water (solvent A)
and acetonitrile (solvent B). The gradient was initially isocratic
until minute 5 with 100%of solvent A; at minute 10 solvents were
A/ B (80:20) and a second isocratic step was performed from
minute 15 to 30 with solvents A/ B (50:50). The mass spectrometer
was an API 3000 triple quadrupole MS/ MS system (PE Sciex,
Concord, ON, Canada) equipped with a Turbo ion spray source
operating in negative-ion modefor monitoring ions of deprotonated
molecules [ M - H]
-
.
MethodOptimization. Method selectivity was based on the
requirement to find a column with a better retention of polar
(10) Macheix, J. J.; Fleuriet, A.; Billot, J. Fruit phenolics; CRCPress: BocaRaton,
FL, 1990; pp 41-57.
(11) Singleton, V. L. In Theoriginsandancient historyof wine; Gordon & Breach
Publishers: Philadelphia, PA, 1996; pp 67-77.
(12) Para, M. H.; Riviere, H. Notre histoire dans le fond des amphores:
determination des acides amines et des acides phenoliques en chro-
matografie liquide haute performance. Dissertacion, Institut de Chimie et
Physique Industrielles de Lyon, 1982. (13) Zugla, M.; Kiss, A. Acta Chim. Hung. 1987, 124, 458-489.
Table 1. Arc haeologic al Samples Collec ted from Anc ient Egyptian Pottery J ars
samples
pottery
objects
object
n. date provenance inscription
BM1 fragment of
a wine jar
EA 32684
a
Dynasty I Abydos, tomb of king
Semerkhet
name of a royal vineyard of Semerkhet
BM2 decorated
wine jar
EA 59774 late XVIII-
early XIX Dyn.
unknown Delta wine for the Osiris Nedjmet
BM3 pink jar EA 51187 Early Dynastic Faras (Nubia), cemetery 3,
grave 5
CM1 wine jar JE 62313
b
Dynasty XVIII Western Thebes,
Tutankhamuns tomb
Year 5. Wine of the House-of-
Tutankhamun Ruler-of-the-Southern-On,
l.p.h, [ in] the Western River. By the chief
vintner Khaa
CM2 wine jar JE 57356 Dynasty XVIII El Amarna
a
EA, Egyptian Archaeology number of the British Museum in London.
b
JE, Journal d'Entreenumber of the Egyptian Museum in Cairo. Dates
included here are Dynasty I (2920-2770 BC), Dynasty XVIII (1550-1307 BC), and Dynasty XIX (1307-1196 BC)
2
.
Analytical Chemistry, Vol. 76, No. 6, March 15, 2004 1673
compounds, in a water mobile phase, than normally achieved by
a conventional C
18
column. Preliminary studies (data not shown)
led us to select an Atlantis (Waters) column. Ammonium formate
and formic acid/ water mobile phases were tested for peak shape
optimization, maximum of retention, and ionization of the com-
pounds. MS/ MS conditions were optimized for tartaric and
syringic acids: capillary voltage -4500 V, curtain gas (N
2
) 12
(arbitrary units), nebulizer gas (N
2
) 10 (arbitrary units), collision
gas (N
2
) 4 (arbitrary units), focusing potential -200 V, entrance
potential (N
2
) 10 V, collision cell exit potential -15 V, and drying
gas (N
2
) heated to 400 C and introduced at a flow rate of 6500
cm
3
/ min. Declustering potential (DP) and collision energy (CE)
were optimized (see Table 2) in infusions of individual standard
solutions (1 mg/ L) at a constant flow rate of 5 L/ min into the
mass spectrometer using a syringe pump model 11 (Harvard
Apparatus, Holliston, MA). The DP was varied from 0 to 200 V
and the CE from -5 to -45 V, which were optimized for maximum
sensitivity of the multiple reaction monitoring (MRM) signal. To
choose fragmentation patterns of m/ z (Q1) f m/ z (Q3) ions for
the MRM transitions, product ion scan mass spectra were
produced by collision-activated dissociation of selected precursor
ions in thecollision cell of thetriplequadrupolemass spectrometer
and analyzed using the second analyzer of the instrument. Full-
scan dataacquisition was performed scanning from m/ z80 to 800
in profile mode, using a scan time of 2 s with a step size of 0.1 u
and a pause between each scan of 2 ms. To enhance sensitivity,
shorter ranges were scanned (from 100 to 250 u for tartaric acid).
Confirmation of the presence of tartaric acid was also done, when
it was possible, by injection of samples in product ion scan of 149,
scanning from 70 to 160 u using a scan time of 2 s. When low
concentrations are present, the injections in single ion monitoring
(SIM) mode or multiple reaction monitoring (MRM) mode were
performed.
The spectragenerated for tartaric acid (M
w
150) using full scan
in the negative ion mode showed the deprotonated molecule [ M
- H]
-
with an optimum DP of -25 V. The product ion scan of
m/ z149 gives m/ z87, due to aloss of the COOH and OH groups.
In this manner, the MRM method is established between ions
m/ z 149 and 87 at a CE of -20 V. The spectra generated for
syringic acid (M
w
198) in the negative ion mode showed the
deprotonated molecule [ M - H]
-
with an optimum DPof -30 V.
The product ion scan observed for syringic acid (m/ z 197) gave
Table 2. LC/MS/MS Optimum Conditions for Tartaric
Ac id and Syringic Ac id MS/MS Detec tion in the
Negative Mode
compound Mw
MS/ MSions
(m/ z(rel abund, %))
DP
(V)
CE
(V)
tartaric acid 150 149 (100), 87 (22) -25 -20
syringic acid 198 197 (100), 182 (28) -30 -20
Figure 1. LC/MS/MS chromatograms for tartaric acid (TA) in sample CM1. (A) Total ion chromatogram (TIC) in full-scan mode from 100 to
250 u. (B) LC/MS/MS chromatogram in SIM mode. (C) LC/MS/MS chromatogram in MRM mode.
1674 Analytical Chemistry, Vol. 76, No. 6, March 15, 2004
a loss of one CH
3
group, providing the [ M - H - CH
3
]
-
anion
radical at m/ z 182. In this manner, the MRM method was
established between ions m/ z 197 and 182 at a CE of -20 V.
Limit ordinary of detection in MRM mode was calculated by
repeated injections (n ) 10) of the working solution (15 L
injected) at asignal-to-noise ratio of 3, and the value obtained was
0.05 g/ L for both compounds.
RESULTS AND DISCUSSION
IdentificationofTartaricAcid. The presence of tartaric acid
in the archaeological residues was first investigated in the deposit
residue (CM1 sample). Injection of this sample in full-scan mode
(Figure 1A) produced a peak in the m/ z 149 trace at a retention
time of 2.64 min. The product ion scan of m/ z149, scanning from
80 to 152 u, gave the mass spectra of m/ z87 in the CM1 sample
and in the standard of tartaric acid. This sample was also injected
in SIM mode (Figure 1B) of m/ z 149 and also in MRM of the
transition m/ z 149 f 87 (Figure 1C), thus giving a peak in the
chromatograms at the same retention time of the standard injected
in the same conditions.
All the other samples, obtained from jar incrustations and
scrapings, were also analyzed in full-scan mode, but unfortunately,
there was no result for tartaric acid, probably due to the low
concentration present in these kinds of samples. As the lowest
detection limits using LC/ MS/ MScan be achieved in MRM mode,
this was the method of choice for the rest of samples.
Figure 2 shows the MS/ MS chromatograms corresponding
to the three samples (BM1, BM2, CM2) where tartaric acid (TA)
was positively identified, as well as the chromatogram of the
pottery blank, which was scraped from an ancient Egyptian pottery
handle. Due to the fact that the MRM chromatogram of the blank
shows a peak at a retention time close to that of tartaric acid, the
positive identification was done on the basis of the retention time
compared with TA standard and by spiking the sample with
tartaric solution. The chromatogram in MRM mode corresponding
to BM3 sample showed no peak at the retention time of tartaric
standard, but a peak at 2.76 min, the same as in the blank, so the
sample was spiked with standard which demonstrated that no
tartaric acid was present. The retention time of the spiked tartaric
(2.55 min) did not correspond with the unknown peak (2.76 min).
In view of the fact that analysis of the pottery blank also showed
this peak at 2.78 min, this led us to the conclusion that it was due
to the ceramic. Note that a peak at 2.79 min is also present in the
CM2 sample, which was a jar incrustation. The positive results
for tartaric acid obtained in samples BM1, BM2, CM1, and CM2
from Egyptian jars confirm they were used as wine containers,
being wine jars of type.
1
Tartaric absence does not necessarily mean a container was
not used for wine.
11
However, the jar from which the BM3 sample
was collected does not correspond to a wine jar type.
1
IdentificationofSyringicAcid. To study the color of ancient
Egyptian wines, we focused on determining the presence of
malvidin, because no other juice or liquid from the ancient wine
areas of the Near East and Mediterranean region is high in
malvidin
11
apart from those coming from red grapes. Malvidin-3-
glucoside is the major anthocyanin that gives the red color to
young red wines. Aging of wine changes the red-purple color of
wine into a more red-brown hue, which has been attributed to
the formation of anthocyanin-derived pigments. These pigments
are more stable due to chemical reactions involving malvidin and
Figure 3. Production of syringic acid. Syringic acid is released from
the flavylium structure of malvidin-3-glucoside in the polymerized
pigment by alkaline fusion through the formation of a hydrated
hemichemical form in which the pyran (C ring) is broken in two steps.
Figure 2. LC/MS/MS chromatogram in MRM mode using m/z 149 f 87 transition showing the tartaric acid (TA) peak in the BM1, BM2, and
CM2 sample residues. No tartaric acid is present in BM3 sample, the same as in the blank, and confirmed by the spiked of the sample with
standard.
Analytical Chemistry, Vol. 76, No. 6, March 15, 2004 1675
phenolic
14,15
or nonphenolic compounds
16,17
present in wines.
However, the stability and evolution of wine pigments over
thousands of years are still unknown. Polymerized pigment
isolation and identification in aged wines was reported to be
difficult, especially because their levels are much lower than those
of the original anthocyanins,
12
and malvidin being a predominant
structural component of wine pigments.
14
We carried out alkaline
fusion with the CM1 sample, which was a dark brown deposit
found inside a wine jar at the tomb of king Tutankhamun. By
alkaline fusion of the residue, and according to the reaction
described for chromonoid compounds,
13
malvidin in the structure
of the polymerized pigment would react as shown in Figure 3,
breaking the C ring in two steps and releasing syringic acid. The
deposit sample, before (Figure 4A) and after (Figure 4B) alkaline
fusion, was analyzed by LC/ MS/ MSusing the MRM acquisition
mode, as the previous full scan gave no signal for the m/ z 197
ion. Thus, the MS/ MS chromatogram at m/ z 197 f 182 ions of
fragmentation pattern of CM1 sample after alkaline fusion (Figure
4B) showed a peak at 18.61 min. Retention time was confirmed
by comparison with that of the standard (Figure 4C). On the
(14) Mateus, N.; de Pascual-Teresa, S.; Rivas-Gonzalo, J. C.; Santos-Buelga, C.;
de Freitas, V. Food Chem. 2002, 76, 335-342.
(15) Remy, S.; Fulcrand, H.; Labarbe, B.; Cheynier, V.; Moutounet, M. J. Sci.
Food Agric. 2000, 80, 745-751.
(16) Atanasova, V.; Fulcrand, H.; Le Guerneve, C.; Cheynier, V.; Moutounet, M.
Tetrahedron Lett. 2002, 43, 6151-6153.
(17) Fulcrand, H.; Benabdeljalil, C.; Rigaud, J.; Cheynier, V.; Moutounet, M.
Phytochemistry 1998, 47 (7), 1401-1407.
Figure 4. LC/MS/MS chromatograms in MRM mode for syringic acid (Syr) at m/z 197 f 182 transition. (A) CM1 deposit sample of dark color
before performing alkaline fusion. (B) CM1 sample after alkaline fusion showing syringic acid peak at the same retention time of the standard.
(C) Syringic acid standard.
1676 Analytical Chemistry, Vol. 76, No. 6, March 15, 2004
contrary, in CM1 sample before alkaline fusion (Figure 4A) there
was no peak at the retention time of the standard at 18.61 min.
These results confirm that syringic acid obtained after sample
oxidation came from polymerized malvidin, having a red grape
origin. Due to these new data, we can now add to the jar label
(see Table 1) information of the kind of wine it contained. In this
case, a red wine.
CONCLUSION
An LC/ MS/ MS method for the identification of tartaric and
syringic acids using a triple quadrupole mass spectrometer is
presented in this paper. The LC/ MS/ MS method proposed is
particularly suitable in terms of both selectivity and sensitivity for
archaeological pottery analysis of tartaric acid, whether there are
visible residues or not. Moreover, this method led us for the first
time not only to identify the presence of wine but also to reveal
the red grape origin of the wine contained in a jar belonging to
the tomb of king Tutankhamun, through LC/ MS/ MS detection
of syringic acid from polymerized malvidin. Here we have the key
to uncovering the origins of enology, as well as opening future
investigations into the color of ancient wines.
ACKNOWLEDGMENT
We are grateful to the Egyptian Supreme Council for Antiqui-
ties (SCA) and the Egyptian Museum in Cairo, for sampling
authorization. We especially thank Dr. M. Eldamaty, and we
greatly appreciate the assistance of Dr. S. Hassan, Dr. A.
Mahmoud, Dr. H. Hassan, Dr. N. I. Lokma, Mr. W. E. Guirgis
and people working at the Egyptian Museum for their collabora-
tion. We are grateful to the Department of Ancient Egypt and
Sudan at the British Museum in London, for sampling authoriza-
tion, we especially thank Mr. W. V. Davis and Dr. J. H. Taylor,
whose assistance is greatly appreciated. We thank Dr. A. Romero
for fruitful discussions. Finally, we express our gratitude to
Codorn u S.A winery and Fundacion para la Cultura del Vino for
financial support.
Received for review September 15, 2003. Accepted
January 12, 2004.
AC035082Z
Analytical Chemistry, Vol. 76, No. 6, March 15, 2004 1677