Microchemical Journal 122 (2015) 127136

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Microchemical Journal

journal homepage: www.elsevier.com/locate/microc

An integrated multianalytical approach to the reconstruction of daily

activities at the Bronze Age settlement in Pealosa (Jan, Spain)
E. Manzano a,, A. Garca b, E. Alarcn b, S. Cantarero a, F. Contreras b, J.L. Vlchez a
a
Department of Analytical Chemistry, University of Granada, E-18071 Granada, Spain
b
Department of Prehistory and Archaeology, University of Granada, E-18071 Granada, Spain

a r t i c l e i n f o a b s t r a c t

Article history: This paper seeks to reconstruct everyday human activities in a Bronze Age Argaric site in southeastern Spain. We
Received 4 April 2015 used an integrated multi-analytical approach to study residues extracted from 5 ceramic pieces and 3 soil sam-
Accepted 26 April 2015 ples. The results of these analyses were complemented with archaeological interpretations to produce the rst
Available online 3 May 2015
ndings on domestic activities four millennia ago at the Pealosa settlement in Jan, Spain. The bands observed
in FTIR and ATRFTIR spectra of the archaeological residues showed a peak assignment that conrmed the silicate
Keywords:
GCMS
components of the clay as raw material and the presence of organic matter. The amorphous organic materials
GCCIRMS were analyzed by GCMS, HRMS and GCCIRMS, and the mineral residues by non-invasive portable X-ray uo-
HRMS rescence (pXRF). We identied ,-dicarboxylic and -(o-alkylphenyl) alkanoic acids produced by oxidative
Organic residue degradation of unsaturated fatty acids, as well as monocarboxylic acids, n-alkanes, diterpenoid acids, cholesterol,
Bronze Age dyes and chemical grape markers. Assignments were based on lipid prole, fatty acid ratios and the 13C values of
Pealosa site C16:0 and C18:0. The results are consistent with the presence of tissues from ruminant (ovine or bovine) and non-
ruminant (swine and equine) animals, vegetable oils, waxes, conifer resin and grape seeds. The presence of sh-
based fats was suggested in one of the residues. Our evidence also suggests that grape juice or wine may have
been consumed at this Bronze Age site. As far as we know this is the rst investigation of the organic residues
absorbed into an Argaric goblet used in a domestic situation.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Food preparation and consumption are essential daily activities for
the survival of any social group. These timeless practices generate resi-
dues and every remain artifact and ecofact in the archaeological record
of a site is not only a reection of the decisions taken by a group of
humans and the relations between them [1,2] but is also often the prod-
uct of a particular phase in the food production chain. Thanks to these
remains we know where (space) and in what (ceramic vessels) people
stored, cooked and ate food four thousand years ago, but answering
questions as to what they cooked or what they ate remains difcult.
In order to nd out which foods were cooked or consumed in an an-
cient potsherd, it is necessary to analyze the organic residues absorbed
by the pores in the pottery or those adhering to its inner surface. In addi-
tion to foods, these vessels may also have contained beverages, medi-
cines, dyestuffs, essential oil, wine, etc. For archaeologists, residues
containing lipids are perhaps of most interest, because the altered prod-
ucts formed as a result of burial or other human activities (for example
by heating) are relatively stable. They can therefore remain intact for a
Corresponding author. Tel.: +34 958 243388; fax: +34 958 243328.
E-mail address: emanzano@ugr.es (E. Manzano).
long time in a ceramic matrix, although this depends on their ability to
withstand biodegradation. The chemical characterization of solid resi-
dues is a difcult task due to their complex composition and their de-
graded state. In order to identify these residues, specic methodologies
for extraction, purication and further analysis of the total lipid extract
(TLE) must be developed. The analytical techniques used most common-
ly for this purpose are Fourier transformed infrared spectroscopy (FTIR)
[3], Raman spectroscopy (RS) [4], gas chromatographymass spectrom-
etry (GCMS) [58], high-performance liquid chromatographymass
spectrometry (HPLCMS) [9,10] and pyrolysisgas chromatography
mass spectrometry (PyGC/MS) [11]. More recently the determination
of the ratios of the stable isotopes of carbon (12C and 13C) and nitrogen
(14N and 15N) using gas chromatographycombustionisotope ratio
mass spectrometry (GCCIRMS) [12] has provided insights into the
type of food consumed and the source of the original lipids giving rise
to these residues. A better understanding of the analytical data can be ob-
tained by establishing the relationship between the molecular constitu-
ents that remain in the organic residues and the source from which
they originate. To this end, we used various criteria, the simplest of
which was the comparison of chemical ngerprints of molecules that
have remained in the ceramics for many millennia with the reference
materials. The ratios of some common fatty acids, the presence of specic

http://dx.doi.org/10.1016/j.microc.2015.04.021
0026-265X/ 2015 Elsevier B.V. All rights reserved.
128 E. Manzano et al. / Microchemical Journal 122 (2015) 127136
archaeological biomarkers and the stable carbon isotope values of specif-
ic lipid components have also proved useful for this characterization [13,
14].
This paper focuses on the analysis of residues discovered in ve ce-
ramic vessels recovered from the Pealosa site (Baos de la Encina,
Jan, Spain) and the associated soil residues found inside three of
these vessels. These vessels date from the Bronze Age and in chronolog-
ical and cultural terms are associated with the Argar people, an ancient
culture that ourished in south-east Spain around the period 2250
1550 BC [15]. Since the 19th century, this ancient culture has been a
subject of great interest among the scientic community, as exemplied
by the abundant bibliography [16,17]. The analysis and study of their fu-
nerary rituals have been a recurrent theme, to the extent that it has
eclipsed other possible lines of investigation such as everyday activities,
which in many ways offer a broader view of life in ancient civilizations
[2]. In fact, in research into the Bronze Age in Southeast Spain, only
two publications have analyzed the organic remains found in ceramic
vessels and both of these were from funerary nds [18,19]. The novel as-
pect of our research is that it is the rst study in which domestic ce-
ramics, rather than funerary objects, have been analyzed. To this end,
we embarked on interdisciplinary research using a combination of cur-
rent analytical techniques (pXRF, FTIR, ATRFTIR, HRMS, GC/MS and
GCCIRMS) with two ambitious goals: (1) to identify the compounds
present in the organic residues extracted from the vessels, (2) to discuss
the possible biomolecular origin of these organic residues in order to
better understand what the people from the Argaric site in Pealosa
cooked and ate. The results obtained using this combination of analyti-
cal techniques showed unambiguous identications of constituents of
chemically complex residues and offer a rigorous scientic insight into
everyday activities in this part of southeastern Spain four millennia ago.
2. Experimental
2.1. Chemicals and reagents
Dichloromethane and methanol (Analytical Grade) purchased
from Fluka (St. Louis, MO, USA) were used as extraction solvents. Tolu-
ene (Analytical Grade) and Meth-Prep II (m-triuoromethylphenyl
trimethylammonium hydroxide) were respectively selected as the sol-
vent and the reagent for the derivatization process prior to gas chroma-
tography analysis. Both were purchased from Sigma-Aldrich (St. Louis,
MO, USA). In order to prevent degradation and obtain high levels of re-
producibility, Meth-Prep II was stored at 4 C in the freezer. Further-
more, LC-MS grade Formic Acid and Methanol (Sigma-Aldrich) were
used in High Resolution Mass Spectrometry assays.
Analytical grade standards of fatty acids (C10, C12, C13, C14, C15, C16,
C16:1, C18, C18:1, C18:2, C19, C20, C22, C26, C30) and Cinnamic acid, Azelaic
acid, Suberic acid, Adipic acid, Tartaric acid, Syringic acid, Cholesterol
and -Sitosterol were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Fatty acid C13 was used as an Internal Standard. Individual stan-
dard solutions of compounds (1000 mg mL1) were prepared in meth-
anol and stored at 20 C. These solutions were prepared fresh monthly.
Working standard mixtures were prepared by diluting the individual
stock solution in methanol. They were stored at 4 C and prepared
fresh weekly. All solutions were stored in dark glass bottles to prevent
photodegradation.
2.2. Instrumentation
2.2.1. Attenuated total reectanceFourier transform infrared spectroscopy
(ATRFT-IR) and Fourier transform infrared spectroscopy (FT-IR)
The FT-IR spectra were collected using a JASCO Spectrometer 6200,
working in transmission mode and diamond micro-ATR accessory. The
instrument was connected to a Pentium 200 and the instrument soft-
ware was SPECTRA MANAGER v2. The FT-IR spectra were registered
from 400 cm 1 to 4000 cm 1 and the ATR spectra from 600 to
4000 cm1, with a resolution of 2 cm1 and 200 scan. The spectrometer
has a TGS Detector. The spectrum of pure KBr was used as background.
2.2.2. Gas chromatographymass spectrometry (GCMS)
The chromatographic system consisted of an Agilent 6890 N series
(Agilent Technologies, Palo Alto, CA, USA) equipped with an automatic
injector (model 7683) and automatic sample tray (model 7683). An
HP-5 (5% Phenylsiloxane 30 m 0.25 mm, 0.25 m of particle size)
was used as a capillary column. In addition, an Agilent 5973N mass
spectrometry detector (Agilent Technologies, Palo Alto, CA, USA) was
coupled to the chromatograph. The detector consisted of an ionic im-
pact (70 eV) and a quadrupole as an ionization source and analyzer re-
spectively. Table 1 shows the parameters used for gas chromatography
mass spectrometry analysis. Each parameter was optimized before
commencing the analysis. In order to achieve the most efcient chro-
matographic separation, ve different temperature gradients were
assayed. As regards gas ow, several ows were tested with a ow of
1 mL min1 producing the best results. Four different injection volumes
were tested and the best chromatogram was obtained using a volume of
1.5 L. After assaying four voltages, we found that the best result was ob-
tained with the 3 EV gain factor. Finally, we optimized the m/z range,
selecting the 50520 uma range as the m/z working range. Peak assign-
ments were performed on the basis of the analysis of available standard
compounds and by comparing their mass spectra to those from the
Wiley mass spectra database.
2.2.3. Gas chromatographycombustionisotope ratio mass spectrometry
(GCCIRMS)
The GCCIRMS equipment was based on a Thermo Trace GC ultra
and Thermo Delta V Advantage as an IRMS detector (Thermo Fisher
Scientic, Waltham, MA). Cono III was selected as an interface and
reactor temperature (CuNiPt) was set at 940 C. The mass spectrom-
eter source pressure was 1.9 10 06 mBar. HP-1 (30 m 0.25 mm
ID 0.25 m) was used as a GC column for assessing chromatographic
conditions. The carrier gas was helium with the GC oven programmed
from 60 (hold 1 min) to 320 C at 6 C min1 for 20 min. Carbon isotope
ratios are reported in the standard delta notation relative to the Pee Dee
Belemnite (PDB) standard. The results were expressed as 13C (%) =
[(Rsample Rstandard) / Rstandard], where R is 13C/12C in per mil.
2.2.4. High resolution mass spectrometry (HRMS)
HRMS analysis was performed to identify other residues apart from
fatty acids and lipids, which cannot be detected by gas chromatogra-
phymass spectrometry, focusing specically on the identication of
peptides and proteins.
An LCT Premier (Waters, Manchester, UK) spectrometer was used
for the HRMS analysis and was operated with electrospray ionization
(ESI) in positive mode. Mass spectrometer parameters were as follows:
capillary voltage, 2.60 kV; source temperature, 100 C; desolvation tem-
perature, 400 C; cone gas ow, 40 L h1; desolvation gas ow,
400 L h1; nitrogen (99.995%) was used as cone and desolvation gas
and cone gas ow. It is important to mention that before injection the
samples were diluted with a mixture of methanol (0.1% acid formic)
to ionize the compounds, so enabling them to be detected by HRMS.
2.2.5. Portable X-ray uorescence (pXRF)
Portable X-ray uorescence analyzer, with a 40 kV X-ray tube with
Ag anode target excitation source, and a silicon PIN-diode with a Peltier
cooled detector. The analyzer was initially calibrated using the silver
and tungsten shielding on the inside of the shutter, and the source
count time for analysis was xed in 90s.
Direct measurements can be performed with portable instruments
that do not affect the integrity of the sample.
 E. Manzano et al. / Microchemical Journal 122 (2015) 127136 129

Table 1
Gas chromatography-mass spectrometry parameters.

Gas chromatography Mass spectrometry

Injector temperature 250 C Interface temperature 230 C

Initial column temperature 70 C (2 minute hold) m/z range 50520 uma
Gas ow 1.0 mL min1 (helium) Voltage 2235 V
Mode Splitless Quadrupole temperature 150 C
Column HP-5 Mode Scan
Temperature gradient 12 C min1 until 250 C Scan velocity 2.12 scans/s
20 C min1 until 290 C
Injection volume 1.5 L Gain factor 3 EV

2.3. Archaeological samples. Criteria for selecting the artefacts
Archaeological samples were taken from ceramics found in situ at
the occupation level of House 25 of the Pealosa site. This domestic
space is located on the western side of the oriental acropolis of the set-
tlement, near a further three domestic and productive spaces. Together
they present a complex constructive and urban system, located in the
highest part of the settlement. Archaeological research has dened
House 25 as a large social space, measuring approximately 20 m2. It is
completely open and has no subdivisions. Its rectangular layout has an
EW orientation, as do most of the documented houses in Pealosa.
We studied ve ceramic vessels (Fig. 1) and the associated soil resi-
dues preserved inside them. These vessels were selected in accordance
with a series of archaeological criteria, including their good state of

Fig. 1. The ve ceramic vessels studied (25567, 25617, 25638, 25705 and 25789).
130 E. Manzano et al. / Microchemical Journal 122 (2015) 127136
preservation (complete vessels), the context they were found in (all the
ceramics were found in primary position on the paved oor of the
room), their possible uses and functions and their position in the food
production chain (relations morphology-use/function). These vessels
included a medium-sized spherical pot with a convex base, three
bowls (one spherical, one semi-spherical and one with open sides),
and a goblet with a tall, thin stem, the most characteristic and emblem-
atic element of the El Argar Culture. All these pieces belong to different
phases in the food preparation process and provide a fascinating insight
into daily life 4000 years ago.
The goblet is perhaps the most interesting of all these pieces. Archae-
ologists believe that in the Argaric Culture, these kinds of goblets were
associated with high social status and hierarchical organization, because
of their design and technical complexity. This goblet is especially inter-
esting because although similar vessels have been found among Argaric
grave goods, this is almost the only goblet thought to have been used for
everyday domestic purposes. Chemical residue analysis on this goblet
can provide an insight into the daily life of this culture from the recent
prehistory of southern Spain, since it would be the rst Argaric goblet
to be analyzed from a domestic context from the recent prehistory of
southern Spain.
2.4. Sampling and sample handling
In-situ measurements were carried out using a portable X-ray uo-
rescence (pXRF) spectrometer for trace elements from samples 25567
and 25638. XRF analysis does not require sampling.
Small amounts of ne powder were collected for FTIR, ATRFTIR,
GCMS, HRMS and GCCIRMS analysis by:
- scraping the inner surface and the bottom of the 5 pieces of pottery
(samples 25567, 25617, 25638, 25705 and 25789),
- taking the associated soil residues (samples 25568, 25706 and
25791) found inside 3 of the above pieces25567, 25705 and
25789, respectively.
The samples analyzed using the attenuated total reectance (ATR)
sampling accessory was taken nely ground and did not require prepa-
ration. For Fourier transform infrared spectroscopy (FTIR), in transmis-
sion mode, the samples were mixed with KBr (sample:KBr, 1:100) and
pressed into discs. For GCMS a small amount of ne powder from each
of the 8 samples was weighed and analyzed (samples 25567 (0.9264 g),
25617 (0.3675 g), 25638 (0.2771 g), 25705 (0.5235 g), 25789 (0.3678 g),
25568 (0.9772 g), 25706 (0.9961 g) and 25791 (0.9919 g)). Each sample
was crushed and ground in an agate mortar for chromatographic analy-
sis. For GCCIRMS similar amounts of samples were processed. This
technique was only applied to samples 25706 and 25791 due to the un-
availability of samples. To avoid contamination, researchers wore kid
gloves and a mask when handling the vessels and the samples.
2.5. Sample treatment
2.5.1. Extraction and derivatization
The samples were examined for lipids and proteins. Organic resi-
dues were extracted from the pottery using the procedure rst present-
ed by Evershed et al. [5], which we optimized for these purposes,
assaying various solvents, mixed in different proportions, namely meth-
anol, dichloromethane, chloroform and toluene. The best results were
achieved with mixtures of methanol:dichloromethane (1:2) and
methanol:chloroform (1:2). Due to the high toxicity of chloroform,
the methanol:dichloromethane mixture was selected as our extraction
solvent. Proteins were extracted using a procedure based on Chertov
et al. [20]. The extracts we obtained were ltered through 0.22 m
nylon Millipore lters before injection into the high resolution mass
spectrometer (100 L).
So as to analyze lipid matter and fatty acids by gas chromatography
coupled mass spectrometry, a derivatization reaction was required prior
to injection into the chromatograph. Meth-Prep II was selected as the
derivatization reagent because it can derivatize both fatty acids and
lipid materials simply in a single step without further collateral reac-
tions. The derivatization procedure was based on a procedure for char-
acterizing drying oil in paintings that we developed and successfully
tested in previous research [21].
2.5.2. Analytical procedure
The analytical procedure for GC comprises the following steps:
1) 0.31 g of sample were weighed and placed in an ultrasound vessel;
2) 15 mL of the dichloromethane and methanol (2:1 v/v) mixture was
added; 3) lipids, fatty acids and other compounds were extracted
by immersing the vessel in an ultrasonic bath (Mod 5133 JP Selecta,
Barcelona, Spain) for 15 min (twice); 4) the extracts were collected
and centrifuged at 3500 rpm for 5 min; 5) the two extracted liquids
were collected and dried in a nitrogen atmosphere at 5060 C; 6)
500 L of Toluene and 37.5 L of Meth-Prep II were added to carry out
the derivatization reaction in the ultrasonic bath, which took 30 min
at ambient temperature; and 7) nally, the derivatized samples were
injected into the chromatograph (1 L).
The procedure for analyzing the proteins by HRMS was based on
Chertov et al. [20]. The nal extracts were diluted in a mixture of meth-
anol with 0.1% of formic acid and were directly injected (100 L) into the
high resolution mass spectrometer.
3. Results and discussion
Since the origin of the organic residues was unknown, we began by
analyzing the samples using infrared spectroscopy. The preliminary
FTIR and FTIR-ATR analysis provided a ngerprint which suggested pos-
sible initial hypotheses about the substances present in the residues and
pottery characterization. Due to the complex nature of the organic ma-
terials, a combination of analytical approaches (GCMS, HRMS and GC
CIRMS) was used to ensure comprehensive analysis. Since few studies
have focused on HRMS, in this paper we support the lipid residues
obtained from GCMS with residues of peptide chains provided by
HRMS. Finally, the mineral residue was analyzed by portable non-
invasive X-ray uorescence (pXRF).
3.1. FTIR and ATR analysis
The FTIR and ATRFTIR spectra for the eight residues analyzed are
very similar. Fig. 2 shows the FT-IR spectrum for one of the samples
(sample 25789) with the main wavenumbers and their corresponding
intensities. All samples analyzed are rich in clay minerals showing char-
acteristic bands in the region 37003600 cm1 (OH stretching bands
at 3694 and more intense at 3619 cm1), 11001000 cm1 (asymmet-
ric SiOSi stretching bands) and 910830 cm 1 (SiO stretching
bands) [22]. This band contains contributions from various silicate min-
erals typically found in clay. Quartz is identied by a characteristic dou-
blet around 779 and 797 cm1 and the SiO stretch around 1000 cm1.
The weak broad band around 3440 cm1 and less intensive absorption
at 1635 cm1 in all FTIR spectra can be attributed to the OH stretching
mode and HOH bending mode respectively from water from crystal-
lization and/or hydroxyl groups from the clay. A CO2 3 band is observed
at 14201440 cm 1 in the IR spectra of sample 25789 and its inner
sediment 25791 due to calcite remains probably originated from soil
depositions. The weak band at 1538 cm 1 in samples 25789 and
25791 attributed to (NH) and supported by a mild band (NH) at
3127 cm 1 may be attributed to traces of proteinaceous material.
Organic materials (wax traces) could be inferred from the features
around 2900 cm 1 characteristic of CH stretching in 25789, 25638,
25705 and 25791, a nding later conrmed by GCMS.
 E. Manzano et al. / Microchemical Journal 122 (2015) 127136 131

Fig. 2. FT-IR spectrum of organic residues extracted from bowl sample 25789.

3.2. GCMS and GCCIRMS analysis compounds, most of which are fatty acid methyl esters (FAMEs)
which result from the transesterication of the acyl glycerides and the
The gas chromatograms obtained from the organic residues extract- esterication of fatty acids. Table 2 reports the peak assignment of the
ed from the ceramic vessels show the presence of different classes of compounds identied in all samples (assignment percentage of over

Table 2
Peak assignment to compounds identied (% assignment greater than 90%), retention time (tR) and m/z selected.

# tR m/z Compound identied

1 11.229 153.0,184.0, 125.0 Aconitic acid

2 11.418 138.0, 171.0 Octanodioic acid (suberic acid)
3 11.630 143.0, 101.0, 175.0 Trimethyl citrato (citric acid)
4 12.439 143.0, 171.1, 183.1 Dodecanoicacid (lauric acid)
5 12.753 167.1, 182.0 2-Allyl-1-methylnaphthalene
6 13.963 153.1 Methyldihydrojasmonate
7 14.780 143.1, 199.1 Tetradecanoic acid (myristic acid)
8 15.086 115.0, 129.0, 145.1 Hexylcinnamicaldehyde
9 15.864 143.1, 213.1 Pentadecanoic acid
10 16.359 111.1, 239.2 Methyl 1-anthraquinonesulfenate
11 16.665 236.1, 194.1, 111.1, 152.1 9-Hexadecenoic acid (palmitoleic acid)
12 16.940 143.1, 227.2, 270.2 Hexadecanoic acid (palmitic acid)
13 17.003 291.1 7-Ethoxy-2-hydroxy-8-methoxy-3-methyl-5,6-
methylenedioxy-1,4-naphthoquinone
14 17.882 143.1, 241.2, 284.2 Heptadecanoic acid (margaric acid)
15 18.652 264.2, 111.0 9-Octadecenoic acid (oleic acid)
16 18.841 143.1, 298.2, 256.2 Octadecanoic acid (stearic acid)
17 19.132 157.0, 112.0 Tributylaconitate
18 19.352 185.0, 129.0, 259.1 Tributylcitrate
19 19.791 109.1 Retinaldehyde
20 19.886 186.0, 259.1 Tributylacetylcitrate
21 20. 561 178.0, 161.0 2-Ethylhexyl p-methoxycinnamate
22 20.616 326.3, 143.0, 283.2 Eicosanoic acid (arachidic acid)
23 20.781 239.1 Dehydroabietic acid
24 20.844 109.0 Sanguinone a
25 20.938 126.1 9-Octadecenamide
26 21.119 129.0 Hexanodioicacid (adipic acid)
27 22.030 113.1 Pentacosane
28 23.766 382.3 Tetracosanoic acid (lignoceric acid)
29 25.341 410.4 Hexacosanoic acid (cerotic acid)
30 25.832 386.3 Cholestan-6-one
31 27.395 207.0, 105.0, 386.4 Cholesterol
32 27.477 438.4 2,3-Didecyl-1,4-naphthoquinone
33 29.756 207.0 Gibberellin a3
34 30.600 466.5 Triacontanoic acid (melisic acid)

Sample 25567: 1; 3, 4; 5; 6; 8; 11; 12; 15; 16; 17; 18; 20; 21; 22; 25; 26; 28.
Sample 25568: 12.
Sample 25617: 2; 4; 7; 9; 11; 12; 14; 15; 16; 20; 21; 22; 28; 29; 31.
Sample 25638: 2; 4; 7; 12; 14; 15; 16; 22; 23; 27; 34.
Sample 25705: 7; 8; 10; 12; 13; 19; 23; 24.
Sample 25706: 11; 12; 15; 30.
Sample 25789: 7; 15; 16.
Sample 25791: 4; 12; 15; 16; 22; 26; 29; 31; 32; 33.
132 E. Manzano et al. / Microchemical Journal 122 (2015) 127136

90%), the retention time (tR) and m/z selected. The compounds identi-
ed in each of the 8 samples are specied at the bottom of the
table. Fig. 3 shows a chromatogram for the residues extracted from
the inner surface of the goblet (sample 25567) and the bowl (sample
25638).
3.2.1. Ceramic goblet and the associated soil residue inside the goblet
(samples 25567 and 25568)
The general chromatographic pattern of the fatty acid prole in the or-
ganic residue extracted from goblet 25567 shows that it contained animal
fat as the original raw materials (Table 2). This nding is supported by the
relatively high proportion of saturated fatty acids (C16:0 and C18:0) com-
pared to unsaturated fatty acids (C16:1 and C18:1) in the residues analyzed.
The presence of 9-octadecenamide conrms the presence of animal fat
since this compound is considered an important marker of animal lipids
[23]. A lipid prole exhibiting a C16:0/C18:0 ratio of 1.75 was interpreted
as a non-ruminant animal according to the ratio value proposed by
Romanus et al. [24]. This conclusion is also supported by the absence of
C15:0, C17:0, C19:0 and branched-chain fatty acids from the GCMS analysis,
compounds characteristic of ruminants [25]. The consumption of red
meat by the Pealosa people has been reported in previous paleopatho-
logical studies of human bones [2]. Biochemical paleonutritional indica-
tors (Sr/Ca(c) and Zn/Ca ratios) have improved previous knowledge
about human diets in the Argaric culture, which seems to have been
mainly based on non-ruminants (swine or equine) and ruminants
(ovine or bovine) animals, as well as cereals and legumes. Lignoceric
and arachidic acids identied by GCMS in the scraped sample from gob-
let 25567 are compounds that are naturally abundant in the surface of
leaves and the skins of berries and fruits, which in this case could be at-
tributed to the presence of plant waxes [26].

Fig. 3. Chromatograms for the organic residues extracted from vessels 25567 and 25638.
 E. Manzano et al. / Microchemical Journal 122 (2015) 127136
The important presence of C16:0, and C16:1 and C18:1 acids together
with a low C18:0/C16:0 ratio could infer a possible sh residue [27].
There is little evidence for sh consumption at Pealosa. Nevertheless,
the chemical analysis of bone samples from an adult woman found in
this settlement (Log Ba/Sr = 1.55) conrmed repeated ingestion of
sh proteins (crustaceans, river carbs, molluscs, prawns, etc.) possibly
from the River Rumblar [2]. Fish lipid residues are very difcult to distin-
guish from plant lipid residues mainly because the n-alkanoic acid dis-
tribution of both is strongly dominated by the C16:0. Consequently the
attribution of sh residues in goblet 25567 still remains ambiguous
but GCMS analyses provide an indicator to justify further study.
The high content of C16:0 in the sample in addition to the relatively
higher levels of C16:0 compared to C18:0 (C16:0/C18:0 = 1. 75) could sug-
gest that the goblet once contained vegetable oils. The archaeological
documentation describing the presence of cereal, ax seeds, lavender
(culinary and aromatic plants such as rosemary, thyme, basil, etc.) and
carbonized olive stones at the Pealosa site reinforces the evidence for
vegetable oils [28]. The identication of adipic acid indicates original un-
saturated fatty acids in prehistoric pottery which also supports the use
of vegetable oils [29,30]. The absence of other dicarboxylic acids, such
as azelaic acid, that usually result from the oxidation of the original un-
saturated fatty acids, may be justied by their susceptibility to leaching
into groundwater, given the proximity of the Rumblar river to the
Pealosa settlement (in the High Guadalquivir, Spain). These dicarbox-
ylic acids and other polar compounds are only encountered in archaeo-
logical samples from especially arid environments [31].
It seems likely that goblet 25567 was used for a variety of purposes.
Chromatographic analysis has detected other compounds containing
phenolic acids often related to wine or grape products, such as cinnamic
acids [3234], while citric acid, a natural preservative used in
winemaking, and the aconitic acids derivatives (tributyl aconitate)
identied in the residue, are a strong indication of wine or other grape
derivatives. The unsuccessful search for other potential wine bio-
markers such as tartaric acid and other phenolic compounds could be
due to their higher water solubility. As discussed above, these com-
pounds may well have leached away, given the damp conditions of
the Pealosa settlement, next to the river. Although the identication
of the aforementioned grape markers suggests the presence of wine or
grapes in 25567 residues, the consumption of grape juice or wine in
the Bronze Age in the southeast of Spain has not yet been proved by ar-
chaeological research (dates) at Argaric sites, although tartrate band
identication by IR spectrometry [18] and the discovery of carbonized
grape seeds at Castelln Alto [35], Fuente Amarga and Fuente lamo
[36] and Pealosa [28] all point in this direction. This paper represents
a step forward in that for the rst time an Argaric goblet has been ana-
lyzed using an integrated analytical approach. The compounds pre-
served within ceramic lead us to the conclusion that grape juice/wine
was consumed in this area 4000 years ago.
Polycyclic aromatic hydrocarbons (PAHs) are common in smoke
condensates from wood res and offer a useful record of past human ac-
tivities. Thus the presence of 2-allyl-methyl naphthalene is considered
in the literature to be the result of cooking on an open re [37]. The pres-
ence of contaminants of pyrolitic origin in the goblet would be a rele-
vant nding because it would indicate possible use of culinary
techniques such as roasting, boiling or cooking. This implies that the
people who cooked with it had some degree of technical knowledge.
Finally, the content of residue 25568 from the inside of bowl 25567
did not provide any signicant information and there was no evidence
of migration of fatty acids to the soil.
3.2.2. Ceramic pot 25617
The biomolecular origin of the lipid prole could indicate an animal
lipid. Residues extracted from ceramic pot 25617 (Table 2) are charac-
terized by high amounts of saturated fatty acid (C12:0, C14:0, C16:0,
C18:0, C20:0, C24:0, C26:0). Most prominent among these is lignoceric
acid (C24:0), a characteristic compound of beeswax. The relatively high
133
level of stearic acid and the traces of cholesterol support the assumption
that it once contained animal fat, an assumption conrmed by archaeo-
logical data from the Pealosa area [2].
Signicant amounts of odd number fatty acids (C15:0 and C17:0) could
indicate bacterial action and suggest that ruminant animals were prob-
ably cooked in this pot [25]. This hypothesis is supported by the fact that
the value for the C15:0 + C17:0/C12:0 + C14:0 + C16:0 + C18:0 ratio is great-
er than 0.04 as proposed by Malainey [38]. The residue from sample
25617 ts the ruminant fat prole. The presence of bovine and ovine an-
imals was previously demonstrated by experimental studies on animal
and human bones found at the Pealosa settlement [2].
Moreover, the presence of unsaturated fatty acids (C16:1 and C18:1),
dicarboxylic acid (suberic acid) and traces of short chain fatty acids
(C12:0 and C14:0) in residue indicates that pot 25617 once contained veg-
etable oils. Eerkens diagram [39] has been used with the necessary cau-
tion because of possible changes in chemical composition arising from
the repeated heating of cooking vessels or oxidative and microbial pro-
cesses occurring as a result of use and burial. Thus, in both of the graphs
proposed by Eerkens (C12:0/C14:0 against the C16:0/C18:0 ratios and
C15:0 + C17:0/C18:0 against C16:1/C18:1 ratios), ceramic pot 25617 tends
to fall into elliptical groupings identied as green plants and next to
the Eerkens region for terrestrial mammals. Consequently, both raw
materials proposed (vegetable and animal oil) are consistent with the
Eerkens results and also with previous archaeological data [2].
3.2.3. Ceramic bowl 25638
The list of compounds identied from GCMS analysis of scrapings
from the inside surfaces of bowl 25638 is reported in Table 2. The chro-
matogram shows a large amount of saturated fatty acid (C12:0, C14:0,
C16:0, C18:0, C20:0, C30:0) originating from both animal and plant lipids.
The signicant content of C20 and C30 fatty acids and the saturated hy-
drocarbon (pentacosane, C25H52) identied in the residues suggests
some kind of waxy material in the vessel. The absence of lignoceric
acid means that beeswax can be ruled out, although vegetable wax
may be possible. The high content of oleic acid together with suberic,
lauric and myristic acids indicate the use of vegetable oil in the bowl.
In addition, the biplot of fatty acids C12:0/C14:0 against the C16:0/C18:0
falls next to the seeds ellipse in Eerkens graphs [39]. Finally
anthracological researches suggest that the dehydroabietic acid could
be attributable to pine resin from pinus carrascus [28].
3.2.4. Ceramic bowl and associated soil residue inside the bowl (25705 and
25706 samples)
The content of the extract obtained from ceramic bowl 25705 and
from the soil found inside (25706) is quite poor in organic components
as can be seen in Table 2. Two saturated fatty acids were identied (C14:0
and C16:0) together with dehydroabietic acid and cinnamaldehyde,
which seem to suggest raw materials of plant origin. The essential oils
could be used mixed with polycyclic aromatic hydrocarbon (PAH), as
anthraquinone and naphthoquinone derivatives, and Sanguinone A, all
used as dyes for making ointments [40]. In addition, unsaturated fatty
acids identied in the residue support the idea of a vegetable oil.
In order to provide a robust framework for interpreting the com-
pounds we identied, it is essential to make a clear distinction between
the lipids absorbed into the pottery and the lipids from the associated
soil residue that resulted from the decay of plants, animals and microor-
ganisms. Thus, the dehydroabietic acid identied in residue 25705 could
be attributed to the leaching of terpenic compounds from pine trees.
Nevertheless, the absence of this marker compound in the associated
soil residue found inside the bowl (25706 sample) proves that this
acid was not a product of a pine environment, as had been hypothe-
sized, and may instead have been used as an ointment. By contrast,
the identication of cholestan-6-one compound and the high content
of C18:0 vs C16:0, provides evidence of animal fat remains in the sur-
rounding soil.
134 E. Manzano et al. / Microchemical Journal 122 (2015) 127136

Table 3 Table 5
Mean 13C and standard deviation (SD) values of C16:0 to C18:0 fatty acids to 25706 and Quantitative results (g g1) obtained by non-invasive pXRF analysis from the 25567 and
25791 samples. Triplicate sample analysis. 25638 potsherds. The average concentration for n = 2 measurements is given in the third
column and the standard deviations (SD) in the last column.
C16:0 C18:0
Sample Mean SD
Sample 13C value SD 13C value SD
Sb 25567 81.000 27.920
25706 31.06 0.11 31.06 0.09
25638 87.100 27.425
25791 30.26 0.19 30.64 0.25
Sn 25567 66.845 27.075
25638 86.490 26.720
Sr 25567 114.940 6.205
The origin of this animal fat in sample 25706 (or a possible mixture 25638 79.640 5.295
of fats from different species) was identied by GCcombustioniso- Rb 25567 92.770 6.320
tope ratio MS (GCCIRMS) (Table 3). 13C data (dened as 13C/12C, iso- 25638 124.010 7.260
Pb 25567 26.185 8.445
topic composition of the palmitic and stearic acids ratio) and 13C value 25638 2221.400 57.725
(dened as 13C18:0-13C16:0) plotted against 13C16:0 provide a robust Zn 25567 66.005 17.025
criterion to differentiate between fats from ruminant and non- 25638 51.460 15.895
ruminant animals [41]. The comparison of the experimental values we Cu 25567 56.795 19.430
25638 155.770 25.450
obtained 13C (31.06%0; 31.06%0) and 13C (0) with those for mod-
Ni 25567 bLD 70.635
ern reference fats using data from a diagram by Mottram [30,41,42], 25638 85.135 48.140
allowed us to conclude that the 25706 residue falls within the ruminant Fe 25567 24,145.160 409.930
adipose fats cluster. The 13C value obtained for this residue plotted 25638 18,747.440 355.460
against 13C16:0 falls between the reference area for porcine adipose Mn 25567 335.445 83.400
25638 209.915 74.035
fats (non-ruminant) and the area for ruminant adipose fats [43]. The re-
Cr 25567 121.575 29.950
sults indicate a mixture of fats in the original pottery, a fact supported by 25638 317.665 34.805
the GCMS and archaeological data [2]. In sample 25706, the 13C values Ti 25567 3332.600 231.105
obtained for C16:0 and C18:0 fatty acid ( 28.3 2.0) were consistent 25638 3092.840 241.310
Ca 25567 14,447.430 452.870
with animals that subsisted mainly on C3 plants, according to data pre-
25638 13,063.480 424.105
sented by Bender [44]. K 25567 27,744.530 862.275
25638 26,203.900 821.875

Cd, Ag, Se, As, Hg, Co, V, Sc b LD.

3.2.5. Ceramic bowl and the associated soil residue found inside the bowl
(samples 25789 and 25791)
The residues extracted from ceramic 25789 (C14:0, C18:0 and C18:1)
(Table 2) shed little light on the diet of the prehistoric people from
Pealosa. Nevertheless, the chromatogram for the associated soil (sam-
ple 25791) is more complex and characterized by certain fatty acids
(C12:0, C16:0, C18:0, C20:0, C26:0) and cholesterol (Table 2) that may be re-
lated to animal fat. Some quinone derivatives, Gibberellin A3, and oleic
and adipic acids identied are typically attributed to leafy greens. Mea-
surements of the relative abundances of 13C/12C in organic residues
from 25791 [30,41] allowed us to conclude that the bowl had once
contained meat from ruminant cattle. The 13C value obtained from
the residue plotted against 13C16:0 falls between the reference area
for porcine adipose fats (non-ruminant) and ruminant adipose fats, in-
dicating animals that subsisted mainly on C3 plants [44]. Since interpre-
tation of chromatographic and isotope analyses remains challenging,
the results help us to distinguish between the ruminant and non-
ruminants fats that the vessels once contained. Both animal species
are supported by archaeological reports from Pealosa [2].
As can be seen in Table 2, the analysis of residues from ceramic bowl
25789 and the associated soil residues 25791 produced no signs of pos-
sible exchange of signicant amounts of organic material between
them. It is possible that the residues once absorbed into the pottery
are no longer preserved.
In all the samples, we found evidence for contamination with
phthalates and derivatives. These compounds have been excluded
from Table 2 because they are not intrinsic materials from the residues.
This suggests unfortunately that the material reported here was not
treated with sufcient precaution during excavation and subsequent
handling and washing.
3.3. HRMS analysis
Although there is extensive bibliography about lipids retained in
pottery, there are surprisingly few reports on the occurrence of other
types of compounds, e.g. proteins and carbohydrates, the major molec-
ular constituents of plants and animals. In this study, results for some re-
sidual peptides have already been added together to lipid analyses and
the benet of additional information has been considered.
The HRMS technique allowed us to detect the monoisotopic molecu-
lar weight of substances to four decimal places. Compounds can there-
fore be identied with high accuracy. In this study, the assumed error
was established at 10 ppm. Taking this into account, only the compounds
below this cut-off level were considered and presented as results.
According to the results shown in Table 4, four peptide sequences
have been undoubtedly identied in 4 of the 7 samples analyzed using
high resolution mass spectrometry (HRMS). It is worth to mention that
neither peptides nor amino acids were detected in the blank sample. It
is therefore assumed that the samples contained animal or plant proteins.
3.4. pXRF analysis
The XRF results obtained for each ceramic investigated without sam-
pling are summarized in Table 5. Since all the vessels originate from the

Table 4
HRMS analysis.

Sample Molecular mass (high resolution) Deviation (ppm) Molecular formula Peptide sequence

25638 452.1808 1,5 C22H24N6O5 L-Asparticacid, N-(4-(((2,4-diamino-5-ethyl-6-quinazolinyl) methyl) amino)benzoyl)

25617 626.2417 7 C24H38N10O6S2 Cys-cys-his-lys-his
25789 542.2740 0,6 C28H38N4O7 Phe-tyr-leu-thr
25789 709.3184 7,4 C33H43N9O9 Glu-ala-his-try-ala-pro
 E. Manzano et al. / Microchemical Journal 122 (2015) 127136
Fig. 4. Isotopic peak prole based on organic residues (peptides) extracted from bowl sample 25638.
same house in Pealosa, one would not expect big differences in
the concentrations of the chemical elements that make them up. It is in-
teresting to note therefore that Ni is only present in bowl 25638, whose
Cr concentration is twice that of goblet 25567. Similarly its Cu concen-
tration is three times higher than that of the goblet and its Pb concentra-
tion is eighty times higher. These quantitative data are very interesting
for provenance studies. The major elements identied in the potteries
between 13,000 and 28,000 g g1 were Ca, Fe and K. These elements
were associated with the minerals (such as calcite, potassium feldspar,
iron oxides, hydroxides and silicates) typically found in potter's clay.
Other elements found between 22003400 g g1 were Ti and Pb;
while Sb, Sn, Sr, Rb, Pb, Zn, Cu, Ni, Mn and Cr were found between 50
and 320 g g1.
4. Conclusions
An integrated multi-analytical investigation of organic residues pre-
served in Bronze Age vessels from the southeast of Spain has been pro-
posed. As far as we know, this is the rst attempt to investigate this issue
using domestic rather than funerary objects. The study began by screen-
ing the samples using infrared spectroscopy (FTIR and FTIRATR). The
organic materials were then analyzed by GCMS, HRMS and GCC
IRMS and the mineral residues by non-invasive portable X-ray uores-
cence (pXRF). The sample treatment for chromatographic analyses
was successfully optimized in order to minimize the amount of sample
required (0.31 g). This was a signicant improvement on earlier stud-
ies that used at least 2 g of sample. The results of isotopic and molecular
analysis of residues preserved on the ceramic were compared with
anthracological and paleopathological studies of animal and human
skeletal remains. This interdisciplinary study provides key information
about the diet of these ancient people which allows us to reconstruct
food habits in Argaric society 4000 years ago. GCMS and isotopic anal-
yses of lipid residues conrm the presence of animal fat (from non-
ruminant and ruminant animals) and mixtures of vegetable oil (essen-
tial oil) as the main foodstuffs detected in the archaeological potsherds
from Pealosa site. The presence of animal fat is supported by the rela-
tively high abundance of saturated fatty acids (C16:0 and C18:0) versus
unsaturated fatty acids (C16:1 and C18:1) and the occurrence of 9-
octadecenamide or cholesterol derivatives in the residues. The presence
of phthalates (exogenous contaminants) highlights the need to estab-
lish a new protocol for Good Archaeological Practices (GAP), which
covers all stages of the process from the moment the pieces uncovered
at the archaeological site until it is sent to the laboratory. Pieces must al-
ways be handled with gloves and must not be washed before being an-
alyzed. They must also be packed and transported in suitable containers
to prevent the risk of contamination by exogenous compounds (Fig. 4).
Among all the pieces analyzed, the goblet is perhaps the most inter-
esting. This is the rst analysis of a domestic goblet produced by the
Argar people, a group that ourished in the recent prehistory of south-
eastern Spain. The components of residues identied by GCMS include
some specic biomarkers for grapes, such as cinnamic acids and their
derivatives, citric acid and aconitic acids derivatives. Since the absence
of other phenolic acids may be justied by their susceptibility to
135
leaching into groundwater, given the proximity of the Rumblar river
to the Pealosa settlement, the occurrence of the aforementioned com-
pounds could indicate that this ancient people drank grape juice or
wine. The chemical evidence for wine is an important nding when
tracing the prehistoric origins of this drink in Spain, which by Roman
times had become one of the three basic pillars of Mediterranean food
production along with wheat and olive oil.
Acknowledgments
This research has been supported by Junta de Andaluca (FQM 338)
and the R & D Project HAR2011-30131-CO2-01 funded by the Spanish
Ministry of Economy and Competitiveness. A. Garca acknowledges sup-
port from a FPU 13/02389 scholarship from the Spanish Ministry of Ed-
ucation. ERDF Funds and Junta de Andaluca's support to acquire the FT-
IR spectrophotometer Jasco 6300 is acknowledged. The authors would
like to thank to F. Martin (UGR) for handling the pXRF.
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