Anal Bioanal Chem (2011) 401:1727–1738
DOI 10.1007/s00216-011-5112-2
REVIEW
Discovering the composition of ancient cosmetics
and remedies: analytical techniques and materials
Erika Ribechini & Francesca Modugno &
Josefina Pérez-Arantegui & Maria Perla Colombini
Received: 28 March 2011 / Revised: 12 May 2011 / Accepted: 13 May 2011 / Published online: 3 June 2011
# Springer-Verlag 2011
Abstract This article reviews the analytical techniques and
procedures used in the study of ancient cosmetics,
therapeutic chemicals, and remedies found in historical
and archaeological sites. Well consolidated techniques
based on molecular and atomic spectroscopy, for example
FT-IR, Raman, SEM-EDX, and XRD, and analytical
procedures based on high-performance chromatography
and mass spectrometry, for example GC–MS and HPLC–
MS are reviewed. The advantages of recently introduced
techniques based on synchrotron radiation and on direct
mass spectrometric techniques are also discussed. The
possibility of extracting information about composition,
preparation techniques, and the degradation processes of
ancient cosmetics, pharmaceutics, and ritual balms is
analysed by use of several case studies.
Keywords Ancient cosmetics and remedies . Molecular
and atomic spectroscopy . Synchrotron radiation . High
performance chromatography . Mass spectrometry
Published in the special issue Analytical Chemistry to Illuminate the
Past with guest editor Maria Perla Colombini.
E. Ribechini (*) : F. Modugno : M. P. Colombini
Dipartimento di Chimica e Chimica Industriale,
University of Pisa,
Via Risorgimento 35,
56126 Pisa, Italy
e-mail: erika@dcci.unipi.it
J. Pérez-Arantegui
Instituto Universitario de Investigación en Ciencias Ambientales
de Aragon (IUCA), Universidad de Zaragoza,
50009 Zaragoza, Spain
Introduction
Recent developments in archaeometry have been of
increasing interest in scientific studies, not only regarding
precious or artistic heritage objects, for example paintings,
coins, statues, but also residues of substances found in
archaeological or historical vessels pertinent to various
aspects of everyday life in ancient societies. The chemical
investigation of residues of ancient pharmaceutical and
cosmetic preparations, which are found in stone, ceramic,
or glass containers, can be an extraordinary source of
information that sheds light on how they were made and on
chemical and pharmaceutical knowledge and practices in
antiquity. For example, the funerary objects sometimes
found in an exceptionally good state of conservation in
Egyptian tombs often include instruments for cleaning and
grooming, and make-up receptacles; these have recently
been studied as an important source of archaeological
information about the customs, beliefs, and rituals in
Ancient Egypt.
Although these kinds of residues have been of interest
since the first archaeometric studies [1], over the last
10 years an increasingly wider array of analytical tools has
been applied to a large series of case studies. These studies
have confirmed that cosmetics and make-up were used in
ancient times not only for aesthetic purposes, but also for
religious and healing purposes playing an important role
during rituals and burial ceremonies [2]. This is also
supported by scenes depicted in paintings, by statues, and
by written texts, for example papyrus documents [3] and
the works of Greco–Roman authors [4, 5], which can in
some cases be exploited to reconstruct ancient recipes and
formulations. It has been hypothesized that in ancient
1728
Egypt, cosmetics were purposely formulated to have
hygienic and therapeutic properties. In addition they were
believed to magically protect the bearers against several
illnesses, and they also contained real active chemicals for
the prevention of specific diseases. This is so for the
cosmetic materials conserved at the Louvre Museum, whose
components had the property of protecting the make-up
wearer from ophthalmic bacterial infections, because of the
non-specific immune-stimulating action of micromolar
amounts of Pb2+ released by the constituents [6].
Despite the value of the historical and conservation
information that can be obtained, the analysis of such
cultural heritage samples suffers in some critical aspects
because of the need to preserve the material intact,
minimising sampling and destructive analysis, and because
of the presence of complex mixtures of organic and
inorganic phases. For these reasons, an investigation needs
non-destructive or micro-destructive techniques with high
selectivity and high chemical sensitivity, in order to obtain
atomic, molecular, and morphological information.
We review several analytical procedures and techniques,
and discuss their potential and the main analytical problems
involved in the study of cosmetics and therapeutic
chemicals in historical and archaeological findings. Well
consolidated analytical techniques based on molecular and
atomic spectroscopy, for example Fourier transform-
infrared spectroscopy (FT-IR), Raman, scanning electron
microscopy–energy dispersive X-ray analysis (SEM–EDX),
and X-ray diffraction (XRD), and procedures based on
chromatography and mass spectrometry, for example gas
chromatography–mass spectrometry (GC–MS) and high-
performance liquid chromatography–mass spectrometry
(HPLC–MS) are reviewed. The advantages and prospects
of techniques based on synchrotron radiation and on direct
mass spectrometric techniques are also discussed. In
addition, we present several case studies relating to the
analysis of ancient cosmetics, pharmaceutical preparations,
and archaeological ritual balms. These studies highlight the
possibility of extracting information on composition,
preparation procedures, and degradation pathways.
Infrared spectroscopy
Infrared spectroscopy (IR) is one of the most widely
applied spectroscopic techniques. It is often used as a
preliminary analytical screening method, because it is non-
destructive and furnishes information on both organic and
mineral components simultaneously. Fourier transform-
infrared spectroscopy (FT-IR) has been used for analysis
of the cosmetics found in two Roman glass unguentaria,
revealing the presence of gypsum, calcite, and haematite,
and the presence of organic components [7]. FT-IR is a
E. Ribechini et al.
rapid fingerprinting tool that can highlight different classes
of organic material, for example in a study of the
components of an Etruscan ointment in which GC–MS
was used to identify the organic substances (resins and oils)
and their degradation products [8] after wet-chemical
treatment of the samples.
Attenuated total reflectance (ATR) FT-IR spectroscopy,
which does not require any chemical preparation, has been
successfully used to understand the formation of lead soaps
in ancient cosmetics and to follow the transformation of
lipids during the preparation of pharmaceutical formula-
tions, as shown in Fig. 1 for triolein [9].
Knowledge of the behaviour of these lead salts improves
our knowledge of the original preparation and conservation.
The methodology also enables rapid measurement and the
evolution of time spectra to be studied. However, because the
absorbance value in ATR mode does not have quantitative
significance, indirect calibration is required. Previously, FT-IR
had already highlighted the presence of lead soaps in ancient
cosmetics inside a three millennium-old Egyptian container
[10]. ATR FT-IR has also been used as a complementary
technique to identify the organic components of a 16th-
century ointment, and applied to a dried subsample,
providing strong evidence for carboxylates [11].
Combined with micro-Raman spectroscopy, FT-IR has
been useful for characterising a large number of black to
grey powders found inside Pompeian unguentaria [12]. Not
only black compounds were identified (amorphous carbon)
among the pigments, but also white minerals, for example
apatite, aragonite, calcite, dolomite, and cerussite. FT-IR
spectra also led to reconstruction of the original binders
from the bands arising from degradation products, and to
determination of whether the pigments were prepared as
cosmetic products or for inks or paints.
Fig. 1 Evolution of the FT-IR spectra acquired during the saponifi-
cation of triolein by PbO. Reproduced from Ref.[9], copyright 2006,
with kind permission from Elsevier
Discovering the composition of ancient cosmetics and remedies
However, the analysis of samples by FT-IR spectroscopy
alone is not always conclusive and requires the complemen-
tary use of many other analytical techniques to complete the
characterisation. Mazzocchin et al. [13] used four other
techniques (optical microscopy, scanning electron microsco-
py, XRD, and cyclic voltammetry) with FT-IR to identify a
Roman “bulk pigment”, a synthetic product derived from
alunite (KAl(SO4)2.12H2O) dissolved or suspended in water,
from which white aluminium hydroxide was precipitated,
and to which an alkaline madder root extract solution was
probably added to colour the powder. FT-IR is also among
the analytical techniques involved in the characterisation of
an ancient Roman cosmetic to verify the presence of
cellulose/starch, mixed with animal fats [14].
Raman spectroscopy
Raman spectroscopy can give valuable information about
the organic and inorganic components of coloured matter.
Direct identification of organic colorants in heterogeneous
matrices by micro-Raman spectroscopy was used to detect
the composition of Greco–Roman pink cosmetics, testing
different excitations and also surface-enhanced Raman
scattering (SERS) [15]. The analytical method proposed
does not require sampling or solvent extraction. The
complementary use of micro-Raman and FT-IR, as already
reported in the previous section [12], is especially interest-
ing for identification of well-crystallised components. The
combination of both methods provides the information
needed for accurate characterization of the contents of
holders and for complete and unambiguous identification of
the archaeological samples investigated. In analysis of
Punic make-up materials by combined use of several
techniques, the presence of inorganic compounds, for
example α-quartz, mercury(II) sulfide, iron(III) oxide, and
calcite was verified by use of Raman microscopy [16].
X-ray diffraction
X-ray diffraction (XRD) is crucial for identification of
crystalline phases. XRD patterns, supported by scanning
electron microscopy (SEM), have enabled identification of
non-natural lead chlorides, namely phosgenite (Pb2Cl2CO3)
and laurionite (PbOHCl), mixed with galena (PbS) and
cerussite (PbCO3) in cosmetic powders dating from
between 2000 and 1200 BC. These powders are preserved
in their original containers and housed in the Egyptian
department of the Louvre Museum in Paris [17–19]. The
ancient procedure has been replicated in a laboratory;
synthesis of these compounds required very simple but
extremely delicate wet chemistry involving the reaction of
1729
litharge (PbO) with rock salt (NaCl) for several weeks,
under pH control. Use of the chlorinated products for
ophthalmologic and skin care is found in the recipes of
medicinal products reported in papyrus documents and by
classical authors [3–5]. Investigation of the immuno-
stimulating effects of submicromolar Pb2+ on the oxidative
stress response of keratinocytes by use of amperometry
with ultramicroeletrodes (platinized carbon fibre) confirmed
that it can protect from ophthalmic bacterial infections [6].
Conventional XRD and SEM/EDX analysis has also
been performed to characterise white pigments from the
Hellenistic period, revealing the presence of hydrocerussite
(2PbCO3.Pb(OH)2) and cerussite (PbCO3) [20]. SEM
analysis enabled the authors to describe the choice of
materials, and their preparation by grinding or chemical
synthesis, to obtain white pigments, on the basis of grains
shapes and sizes (Fig. 2).
Results from SEM/EDX and XRD can be combined to
confirm the presence of several minerals observed by
microscopy, for instance in the analysis of ancient kohls [21]
or for comparison of modern and ancient Egyptian kohls
[22]. This study revealed that the modern products were made
using galena as the major component, or that the cosmetics
could be amorphous-carbon based, or the powders could be
calcite (CaCO3) as the major phase. The ancient contents, on
the other hand, were always lead-based products.
The mineral fraction of a 16th-century ointment was also
studied by XRD revealing calcium sulfate (gypsum) and
lead sulphate as the main inorganic crystalline components,
despite the high background observed in the diffractogram,
because of to the presence of large amounts of non-
crystalline material in the ointment, and the consequent
low sensitivity of XRD [11].
In the investigation of a Roman cosmetic product found
inside a small tin canister in London, XRD confirmed the
presence of cassiterite (SnO2), already suspected from X-
ray fluorescence analysis [14]. Addition of SnO2 to the
starch/fat base conferred white opacity to the cosmetic
cream; SnO2 is thought to have been used as a substitute of
lead acetate (cerussa).
Similar agreement between the XRF results and XRD
patterns was achieved in the study of raw materials and
tools for preparation or use of lead pigments at Akrotiri in
Thera, Greece, during the Early, Middle and Late Cycladic
Bronze Ages (c. 3000–1600 BC); varying amounts of
calcite were detected and quantified [23].
Synchrotron-based techniques
Use of synchrotron-based micro-analytical techniques
which exploit the high photon flux and coherence of
synchrotron radiation, has resulted in increasing success in
1730
Fig. 2 SEM observations
of different grains of lead white
cosmetic powders: (a) Derveni
E32A, cerussite grains; (b)
Volos 396, cerussite and
hydrocerussite grains; (c)
Eleusis 1, cerussite grains;
(d) Paestum PAE1, cerussite
grains. Reproduced from Ref.
[20], copyright 2006, with kind
permission from Springer
cultural heritage research. A series of case studies on
ancient cosmetic preparations has been reported in the
literature. In fact, because of the small amount of sample
that can be used for the analysis and because of the micro-
heterogeneity of this type of product, techniques based on
synchrotron radiation sources are a powerful and efficient
mean of characterising archaeological materials.
The combined application of μ-XRF,μ-XRD, and μ-
XANES (X-ray absorption near edge structure) synchrotron
micro-beam techniques on the same samples of Egyptian
cosmetics discussed above [24, 25] enabled trace elements
to be identified, revealing different origins of the galena
ores and confirming that the chlorinated Pb salts were
deliberately manufactured and used in the composition of
the make-up. These results highlight the great variety in the
composition of lead-based cosmetics in ancient Egypt, and
advanced know-how in chemical synthesis and cosmetic
technology—it seems that even 4000 years ago people
already wanted more from their cosmetics than simply
highlighting their eyes.
SR-XRD, supplemented by scanning electron microsco-
py (SEM) and transmission electron microscopy (TEM),
have been used to study the microstructure of galena (PbS),
a major constituent of Egyptian black eye make-up. The
results enabled the authors to describe the procedures used
for manufacture of eye make-up in the Middle and New
Kingdoms of Egypt approximately 4000 years ago [26].
The coupling of IR microscopy with a synchrotron
source has also been used to investigate remains of
E. Ribechini et al.
Egyptian cosmetics in a receptacle from the 18th dynasty
[10]. Use of synchrotron radiation brightness enhances
lateral resolution and signal-to-noise ratio in IR microscopy,
enabling detailed location of the various ingredients within
the particles.
Mineralogical analysis of Punic make-up samples has
been performed by use of synchrotron radiation X-ray
diffraction (XRD) in two modes: moderate-resolution XRD
with a charge-coupled detector (CCD) to obtain two-
dimensional powder patterns and for rapid identification
of the major mineral phases present within each sample;
and high-resolution powder diffraction in stepping mode to
obtain high-quality data for quantitative determination of
phase abundance [16]. In this way, two compounds,
cinnabar and haematite, were found to be responsible for
the red colour of the make-up. Other examples, already
noted in this paper, also used SR-XRD to perform quantitative
analysis of the different phases [20]. SR-XRD analysis
revealed inhomogeneous distribution of the minor phases
(calcite and cerussite) in hydrocerussite (Fig. 3a), and the
textures of both phases could be observed (Fig. 3b, c).
Chromatographic techniques and chromatographic/
mass spectrometric hyphenated methods
The characterisation of biomaterials (including lipids,
proteins, plant resins, polysaccharides, organic dyes, and a
variety of plant extracts) that have been fundamental
Discovering the composition of ancient cosmetics and remedies 1731

Fig. 3 SR–XRD analyses

of sample Volos 471,
ESRF-ID18F, 28 keV: (a)
XRD diagram on eight points.
(b, c) XRD image after
straightening out the diffraction
rings of a point, with a large
amount of cerussite (b) and
calcite (c). Reproduced from
Ref.[20], copyright 2006, with
kind permission from Springer
1732
components of cosmetics and pharmaceuticals since remote
antiquity is generally performed by analytical procedures
based on high-performance chromatography, both GC and
HPLC, and/or mass spectrometric techniques.
Biomaterials are made up of complex mixtures of many
chemical species, with a wide range of molecular weights,
from highly volatile monoterpenes and sesquiterpenes to
highly insoluble macromolecules, for example denatured
proteins, and highly polymerised resins, for example amber.
The high chemical complexity of these natural substan-
ces and their transformation products, as a result of man-
induced transformation and ageing, makes it extremely
difficult to identify them in archaeological residues. The
chemical composition of natural organic materials varies
according to the animal or plant species of origin, and
because of ageing processes. All this means that only a
detailed molecular fingerprint enables us to form a
hypothesis on the sources of the organic ingredients present
in pharmaceuticals and make-up.
To provide detailed compositional information about
mixtures of organic compounds, gas chromatography
coupled with mass spectrometry (GC–MS) and high-
temperature gas chromatography coupled with mass spec-
trometry (HTGC–MS) have been of primary importance
because of their unsurpassed capacity to resolve the
molecular composition of the organic materials. Although
such techniques usually involve a relatively long analysis
time, requiring wet chemical pre-treatment of the samples,
they have been successfully used for more than two decades
for characterization of archaeological organic residues
containing glycerolipids, natural waxes and terpenic resins.
Sample pre-treatment is usually performed to: extract or
solubilise the analytes of interest; to decompose high-
molecular-weight compounds, for example triglycerides,
cerides, and polyesters, into smaller molecules (fatty acids
and alcohols) by cleavage of ester bonds; and to transform
polar compounds, for example fatty acids, into volatile
molecules, by performing derivatising reactions [27, 28].
Analysis of a Roman cosmetic product found in a tin
canister discovered in London during the excavation of
Roman architectural remains and dated to the middle of the
second century AD [14] was a landmark in the study of this
class of artefacts. GC–MS analysis provided a fatty acid
profile featuring the prevalence of saturated octadecanoic
acid (stearic acid), and the presence of odd-number carbon
fatty acids which included iso and anteiso branched isomers
(Fig. 4). This fatty acid profile, with measured 13C values
for the C16:0 and C18:0 fatty acids, led the authors to
hypothesise the presence of a ruminant – for example cattle
or sheep – adipose-fat source for the glycerolipid material.
The other components of the formulation were starch and
stannic oxide. Starch was highlighted by Py–GC–MS, on
the basis of the formation of levoglucosan as a pyrolysis
E. Ribechini et al.
Fig. 4 Partial gas chromatogram obtained from lipids extracted from
the contents of a Roman canister. Peaks: C14:0 to C18:0, saturated
straight-chain fatty acids (FA) with 14 to 18 carbon atoms; C15br and
C17br, iso and anteiso branched-chain fatty acids; C18:1FA, mono-
unsaturated; C18:0OH-FA, hydroxylated form derived from oleic acid.
Reproduced from Ref.[14], copyright 2004, with kind permission
from Nature Publishing Group
product, FT-IR, an iodine test, and GC–MS analysis of the
alditol/acetate derivatives of the hydrolysed residue.
Identification of the origin of glycerolipids on the basis
of the fatty acid profile or triacylglycerol (TAG) profile is
complicated by the occurrence of mixtures, the unspecific
fatty acid composition of many lipid materials, and the
changes induced by ageing. In such cases, gas chromatog-
raphy–combustion–isotope ratio mass spectrometry (GC–
C–IRMS) is an important complementary technique, which
can furnish accurate information on the sources of the
archaeological lipids [14, 29]. It can distinguish between
ruminant and porcine adipose fats, on the basis of the
differences in the δ13C values of various fatty acids.
For plant oils, often only a hypothetical source can be
suggested, by interpreting the fatty acid profile data in
conjunction with historical documents from which infor-
mation can sometimes be obtained on the botanical sources
that were exploited in the geographical area and historical
period under study [11, 27].
In contrast, the molecular fingerprints of most natural
waxes, for example beeswax, are very specific and clearly
detectable by GC–MS. The technique has proved to be
successful in revealing the presence of natural waxes that
have been used in cosmetic preparation and balms since
Egyptian and Roman times. A significant example is
analysis of the contents of a series of Roman glass
containers (unguentaria) recovered in a Roman villa in
Oplontis (Naples, Italy) [30]—chemical investigation based
both on HTGC–MS and GC–MS furnished detailed
Discovering the composition of ancient cosmetics and remedies
molecular compositions. Awide range of compound classes
were found, including medium and long-chain fatty acids,
long-chain hydroxy acids, n-alkanols, alkandiols, n-alkanes,
long-chain monoesters, phytosterols, and diterpenoid acids.
The results were consistent with beeswax being used in the
preparation of the cosmetics preserved in the unguentaria,
whereas the other lipids are most likely to be the residue of
plant extracts, possibly derived from flowers and/or leaves.
The composition of the residues is consistent with the
ancient practices of maceration and/or “enfleurage”, in
which lipid-based materials, for example beeswax, animal
fats, or vegetables oils, were used to extract aromatic and
fragrant substances from resin, flowers, spices, and scented
wood, in order to produce unguents and balms.
A multi-analytical approach based on the use of
chromatographic, mass spectrometric, and spectrophoto-
metric techniques (GC–C–IRMS, GC–MS, LC–APCI–MS,
FT-IR, XRD) was recently used for characterization of the
material collected from a ceramic vessel discovered in
Belgium and dating back to the late 16th and early 17th
century AD [11]. The analytical procedure used for
characterisation of the organic compounds involved extrac-
tion of the sample (0.5 g) with a mixture of chloroform and
methanol (2:1v/v). The first dried aliquot was subjected to
transesterification by treatment with methanolic KOH and
analyzed by GC and by GC–C–IRMS. A second dried
aliquot was used to prepare fatty acid methyl esters
(FAMEs) from both triglycerides and free fatty acids by
use of boron trifluoride and MeOH; these were then
analysed by GC. A third aliquot was silylated with N-
methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and
analysed by GC–MS. Finally, a fourth aliquot was used
for LC–MS analysis. The multi-analytical approach
revealed a complex chemical composition (lithargirium,
gypsum, beeswax, animal fat, and vegetable oil) and
enabled the authors to hypothesize that the mixture was
intended for use as a medicinal plaster.
Analytical investigations based on chromatographic tech-
niques coupled with mass spectrometry (GC–MS, LC–MS,
GC–CIRMS) were also the key tools used to study the
ancient ritual of mummification and which enabled deter-
mination of the complex organic composition of these ritual/
medical preparations, which included bitumen, animal fat,
beeswax, resin, vegetal tar, and plant oils [31–35].
In organic archaeology and cultural heritage chemistry,
chromatographic identification is often based on detection
of biomarkers. Biomarkers are regarded as diagnostically
stable molecules present in the original material which are
preserved intact in archaeological environments or formed
over centuries because of ageing. They have a carbon
skeleton that is sufficiently specific to be related to their
biological precursors, thus enabling identification of the
natural substances from which they originated. Thus the
1733
presence of bitumen or asphalt was proved by identification
of characteristic saturated hydrocarbons. The C15 alkanes,
pristane and phytane, steranes, and terpanes are biomarkers
which can be used to identify bitumen and its geographical
origin [36].
GC–MS analysis and biomarker detection have also been
successfully used for chemical characterisation of the contents
of an Etruscan alabaster unguentarium (alabastron) from
Chiusi (Italy). The analytical procedure based on GC–MS
after alkaline hydrolysis, solvent extraction, and trimethylsi-
lylation revealed a mixture of lipid and resinous substances—
a vegetable oil, probably moringa, pine resin exuded from
Pinaceae, and mastic resin from Anacardiaceae trees [8].
Pinaceae resin was recognized because of the presence of the
characteristic diterpenoid compounds dehydroabietic and 7-
oxo-dehydroabietic acids, and identification of mastic resin
was based on the detection of characteristic triterpenoids, for
example moronic, oleanonic, masticadienonic, and iso-masti-
cadienonic acids (Fig. 5).
Analysis of volatile components that could be attributed
to perfume components, for example monoterpenes and
sesquiterpenes, can be performed by head-space SPME gas
chromatography with mass spectrometry (GC–MS). An
interesting application of SPME–GC–MS was the study of
mummification balms and unguents from ancient Egypt
[37]. α-Cedrene and α-cedrol were linked to the use of a
substance from the Juniperus species; longifolone was
regarded as a biomarker of a substance from a conifer tree;
and cembrene A and isoincensole acetate were indicative of
the presence of olibanum in one of the samples [37].
High-performance liquid chromatography-based techni-
ques are widely used in art and archaeometry, because of
their ability to analyse a wide range of organic compounds.
Analytes that are thermally labile, are highly polar, or have
a high molecular weight and are consequently not suitable
for GC analysis, can all be analysed by HPLC. The
technique has the advantage of short analytical run times
and easy sample preparation. In addition, when MS is used
as detector, the possibility of using both atmospheric
pressure chemical ionisation (APCI) and electrospray
ionisation (ESI) can give information both on polar and
apolar compounds. ESI is more suitable for polar com-
pounds and APCI for apolar compounds. HPLC–MS has
very high analytical specificity and sensitivity, especially
when MS–MS (tandem mass spectrometry) or SIM
(selected ion monitoring) acquisition modes are used.
HPLC with UV–visible or diode-array detection (DAD)
and HPLC–MS have been mainly used for identification of
organic dyes in textiles and paintings, lipids in archaeological
pottery and works of art, and proteins in pottery and paintings
[38–43]. The techniques have only rarely been used for
study of ancient cosmetics or pharmaceutical preparations
[11, 31, 44–46]. In the study of a pharmaceutical preparation
1734 E. Ribechini et al.

C18:1
100 9 and 10OHC18:1

9,10diOHC18
90

80

70
9,10diOHC18
Relative Abundance

60

diC9
50

40
C18:0
diC8 Dehydroabietic acid Moronic acid
30 C16:0 7-oxo-dehydroabietic acid
IS2 9 and 10OHC18:0 Oleanonic acid
20 C20:0 (iso)Masticadienonic acid
C22:0
Masticadienonic acid
C24:0
10 IS1 diC10

0
10 15 20 25 30 35 40 45 50 55 60 65 70
Time (min)

Fig. 5 TIC of the trimethylsilylated acid fraction of the contents of an acids of chain length x and with hydroxy group at position X;
Etruscan alabaster unguentarium from Chiusi (Italy). AX:Y are fatty 9,10OHA18:0 are 9,10-dihydroxyoctadecanoic acids; IS1 and IS2 are
acids of chain length x and degree of unsaturation y; diAX are α,ω- the n-hexadecane and tridecanoic acid internal standards [8]
dicarboxylic fatty acids of chain length x; XOHAX are hydroxy fatty

[11], HPLC–MS was used to reveal beeswax by the presence
of monoesters, diesters, hydroxymonoester, and hydroxy-
diesters. The compounds were identified because of the
presence of specific ion fragments of beeswax components
and use of the APCI source. Monoesters led to the formation
of [M−H]+, [RCOOH−H]+, and [RCOO−H2O]+, diesters to
[M+H]+, [M+H−RCOOH]+, and [RCOOH−H]+, hydrox-
ymonoesters to [M−H]+, [ROHCOOH−H]+, and hydrox-
ydiesters to [M+H]+, [M+H−RCOOH]+.
Other mass spectrometric techniques
Direct exposure mass spectrometry (DE-MS) and direct
ionization-mass spectrometry (DIMS) can be performed
rapidly (a few minutes for analysis and data acquisition)
and are sufficiently sensitive for identification of many
organic substances in archaeological objects [47, 48]. In
particular, they can be used as preliminary screening
techniques. DE-MS has recently provided interesting data
on the presence of resinous, fatty and waxy substances in
Roman glass containers from Southern Italy [30, 49].
Figure 6 shows the DE mass spectrum obtained from
analysis of the residue collected from one of the unguen-
taria [30]. A complex mass spectrum was obtained
featuring a high abundance of m/z 253, together with m/z
239. These ions are the base peaks in the spectra of 7-
oxodehydroabietic and dehydroabietic acids, respectively,
and suggest the presence of a diterpenoid resin of Pinaceae
origin. A series of six regularly spaced ions separated by
28 amu form a cluster in the range m/z 592–732 and are
attributable to M+. of long-chain fatty acyl esters, which are
present in high abundance in beeswax. The presence of
beeswax is further confirmed by fragment ions at m/z 256
and 257, which are derived from the characteristic long-
chain palmitate wax esters of beeswax caused by fission of
the alkyl–oxygen bond. Further pairs of fragment ions
present at m/z 284 and 285, 312 and 313, 340 and 341, and
368 and 369, indicate the presence of long-chain mono-
esters other than palmitate esters. The aforementioned pairs
of ions suggest the presence of C18, C20, C22, and C24 fatty
acyl moieties, respectively. Their presence implies the
presence of another waxy lipid composed of aliphatic
long-chain wax esters with a chain length ranging between
C40 and C54 carbon atoms, which is, coincidentally, the
same carbon number range as beeswax, but whose fatty
acyl moieties contain between 18 and 22 carbon atoms.
Direct analysis of micro-samples by laser desorption
ionization–mass spectrometry (LDI–MS) or matrix-assisted
laser desorption ionization–mass spectrometry (MALDI–MS)
Discovering the composition of ancient cosmetics and remedies 1735

81
100
95 67
253
90 256
85 91 95
80
185 257
75
70
65 105121171 213
Relative Abundance

239
60 159 284
55
50 312
313
45
40
35 300
30 341
25
369
20 648 676
15 412 620
465
10 704
508
5 732
0
100 200 300 400 500 600 700 800 900 1000
m/z

Fig. 6 Mass spectrum obtained by DE–MS of the contents of a glass unguentarium from Oplontis [30]

is a potential source of chemical information about the
components in cosmetics and remedies. These techniques
are appropriate when we need to analyse very small samples,
because the soft ionisation means that very small quantities
of compounds can be detected. These are also excellent
techniques for characterising colorants [50] and lipids [51].
Anthraquinone-type colorant compounds have been identi-
fied by mass spectrometry of samples from ancient Greco–
Roman [44] and Roman pink cosmetics [51] by deposition of
only discrete particles of sample on the surface of the
MALDI plate. The positive-ion LDI mass spectra also
revealed the presence of traces of triacylglycerols and
diacylglycerols derived from plant oils in the cosmetics [51].
Another mass spectrometric technique with increasing
potential for new applications in cultural heritage analysis is
TOF-SIMS [52]. It is one of the few techniques enabling
spatial mapping the presence of specific organic compounds
(e.g. fatty acids in painting layers) in heterogeneous samples
or cross sections. TOF-SIMS, used to study paintings and
conservation materials, natural fibres, and biological sam-
ples, can be used to identify organic molecules in a sample,
with high sensitivity and selectivity. This ability plus its
excellent lateral resolution could make it the reference
technique for chemical imaging by mass spectrometry.
Other analytical techniques
Elemental analysis of ancient make-up and pharmaceutical
products is included in most studies, because it furnishes
complementary information on the components of the
residues. In such cases, several analytical techniques have
been used, for example X-ray fluorescence (XRF) [11, 14,
23], inductively coupled plasma–atomic emission spec-
trometry (ICP–AES), inductively coupled plasma–mass
spectrometry (ICP-MS), or neutron activation. Major and
minor elements (14 elements in total) were determined by
ICP–AES in red cosmetics excavated from ancient burials
in Japan [53]. Of these elements, Hg, Fe, Cu, and Zn were
indicative of different types of sample, especially Hg and
Fe, because cinnabar (mercury sulfide) and iron oxides
were the main constituents of the cosmetics, and the
cosmetic preparations were classified into four groups with
local variations. The determination of trace elements in
cosmetics can be helpful when searching for the original
source of these materials, especially mineral products, for
example iron oxides. Specular hematite, used by some
peoples of southern Africa for cosmetic purposes, was
analysed by neutron activation to fingerprint the mineral
and trace it to specific mines [54]. Surprisingly, ICP–MS
has been less used in for elemental analysis of cosmetics
and pharmaceutical products. Elemental analysis was
performed by electrothermal-vaporisation ICP–TOF-MS,
and portable XRF, on a 16th-century ointment. It revealed
Si and Ca as the major components of the inorganic
fraction, as well as Pb and Fe and other trace elements,
revealing that lithargyrum was probably one of the original
ingredients of a lead plaster [11]. ICP–MS has also been
used to measure the isotopic composition and concentration
of Pb in cosmetic pigments, in order to compare them with
1736 E. Ribechini et al.

data from soils and human bones from the same archaeo-
logical environment [55]. Several papers have reported that
kohl was one of several lead-based ancient Egyptian
artefacts subjected to lead isotopic analysis by other mass
spectrometric techniques [56–58].
Although total reflection X-ray fluorescence (TXRF) is
one of the most important developments in X-ray spec-
trometry in the recent years, it has not yet achieved the
same reputation as, and extensive application of, ICP–OES
and ICP–MS. Nevertheless, TXRF is an ideal tool for
qualitative and the quantitative analyses of elements in tiny
and precious micro-samples from cultural heritage [59] and
its use could have some advantages in the analysis of
ancient make up and remedies.
Because of the different textures, morphologies, and very
small size of possible sampling of this type of residue,
electron microscopy techniques (SEM and TEM) have
provided very valuable information on the microstructure
(Fig. 2) and composition (when coupled with-energy
dispersive X-ray analysis), especially combined with other
analytical techniques [13, 17, 20, 22, 26, 45, 51].
Knowledge of the processing techniques used in the
preparation of ancient Egyptian cosmetic powders has
improved because of TEM; the microstructure and charac-
teristic defect structures of these materials were induced by
the powder processing of the lead compounds [26, 60, 61].
The application of an ancient hair-dye containing lead salts
to human hair and observation of cross sections by
scanning confocal electron microscopy (SCEM) and of
longitudinal sections by high-resolution transmission elec-
tron microscopy (HRTEM), together with optical micros-
copy and SR–XRD, revealed the shape and distribution of
PbS nanocrystals that form within the hair during blacken-
ing [62].

Table 1 Ingredients chemically identified in historical-archaeological residues of cosmetics and pharmaceutical preparations, and analytical
techniques applied for their recognition

Ingredient Sample matrix Analytical techniques

Lead chlorides Egyptian make-up, Greek cosmetics, Greek XRD and SEM–EDX [17–19, 23], XRF [23], SR–XRD
(laurionite and phosgenite) wood and stone make-up brushes etc. and SR–XRF [24, 25]
Galena Egyptian make-up, Roman cosmetics XRD and SEM–EDX [17–19, 21], SR–XRD and SR–XRF
[24, 25]
Hydrocerussite and cerussite Roman cosmetics, Egyptian make-up, Greek FT-IR and Raman [12], XRD and SEM–EDX [17–20, 23],
cosmetics, Greek wood and stone tools XRF [23]
Haematite Roman cosmetics, Punic make-up, Greek FT-IR [7, 12], Raman [12, 16], SR–XRD [16], XRD
cosmetics, Greek wood and stone tools [7, 23], SEM–EDX and XRF [23]
Cinnabar Punic make-up Raman and SR–XRD [16]
Cassiterite Roman cosmetics XRF and XRD [14]
Gypsum Roman cosmetics XRD and FT-IR [7]
Calcite Roman cosmetics, Greek cosmetics, Greek XRD [7, 23], FT-IR [7], SEM–EDX and XRF [23]
wood and stone tools
Beeswax XVII pharmaceutical ointment, Roman GC–MS [11, 30–32, 35], HTGC–MS [11, 30–32], LC–MS
unguents, Egyptian mummification balms [11], FT-IR [12], DE–MS [30], Py–GC–MS [31, 32]
Plant waxes Roman unguents GC–MS, HTGC–MS, and DE–MS [30]
Mastic resin Etruscan ointment, Egyptian mummification HTGC [31, 32], Py–GC–MS [31, 32], GC–MS
balms [8, 31, 32, 35], FT-IR [8]
Pine resin Etruscan ointment, Egyptian mummification HTGC [31, 32], Py–GC–MS [31, 32], GC–MS
balms [8, 31, 32, 35], FT-IR [8]
Frankincense resin Egyptian unguents SPME–GC–MS [37]
Bitumen Egyptian mummification balms HTGC [31, 32], Py–GC–MS [31, 32], GC–MS [31, 32, 34]
Unidentified plant oil Etruscan ointment, Egyptian mummification GC–MS [8, 31, 32, 34, 51], FT-IR [8], HTGC and
balms, Roman cosmetics Py–GC–MS [31, 32], LDI–MS [51]
Castor oil Egyptian mummification balms GC–MS [35]
Animal fat Roman cosmetics, Egyptian mummification HTGC [31, 32], Py–GC–MS [31, 32], GC–MS
balms [14, 31, 32], GC–C–IR/MS [14]
Animal fat/vegetable oil XVII pharmaceutical ointment, Egyptian HTGC [31, 32], Py–GC–MS [31, 32], GC–MS
mummification balms [11, 31, 32, 35], LC–MS [11], GC–C–IR/MS [11]
Polysaccharide materials Roman cosmetics, Egyptian mummification FT-IR [14], GC–MS [14, 31, 32, 35], HTGC, and
(starch, plant gums) balms Py–GC–MS [14, 31, 32]
Madder Roman and Greco–Roman cosmetics Raman [15], HPLC–MS [44], LDI–MS [44, 51]
Discovering the composition of ancient cosmetics and remedies
Various electroanalytical techniques have also been used
to study cosmetics, for example, identification of colorants
by cyclic voltammetry [13] or by voltammetry of immobi-
lised microparticles (VIM) [51]. In addition, these techni-
ques have been used to obtain new insights into the
biochemical interactions between Pb(II) ions and cells; for
example, ultramicroelectrodes in amperometry have been
used to confirm the ancient medical use of poorly soluble
lead compounds [6].
Although nuclear magnetic resonance spectroscopy
(NMR) is recognised as extremely significant in character-
ising molecular structures, and it is widely used in several
fields of organic and analytical chemistry, it has rarely been
used in the study of cultural heritage. The somewhat limited
use NMR in studying archaeological and historical samples
may be because of the frequent presence of complex
mixtures of organic compounds. With the present state of
the art, the most significant applications of NMR have been
in analysing highly polymerised materials, for example fossil
resins [63] and archaeological wood [64]. NMR is suitable
not only for identification, but also for studying alterations: it
could be an excellent complementary technique in the study
of organic compounds from ancient make up and remedies.
Conclusions
Applying state-of-the-art analytical techniques in a comple-
mentary fashion provides a comprehensive view of the
multiplicity of inorganic and organic materials that may be
present in a sample from a residue of a cosmetic or
pharmaceutical preparation. In order to contribute to improve-
ment of analytical strategies and to validate analytical
procedures for study of ancient cosmetics and remedies, use
of replica materials prepared according to historically docu-
mented recipes can play an important role. In addition, the
study of replicas submitted to artificial aging treatments can
help by simulating the degradation processes.
Table 1 summarises the types of material that have been
encountered and scientifically investigated in historical and
archaeological remains of ancient cosmetics, pharmaceut-
icals, or ritual preparations, and reports the most significant
analytical techniques that have been used to characterise
these materials. For inorganic products, the key techniques
include elemental analysis by XRF, SEM–EDX, and mass
spectrometry, phase identification by XRD, and spectro-
scopic fingerprinting by FT-IR and Raman spectroscopy.
Synchrotron radiation-based techniques, mainly XRD and
FT-IR coupled with high resolution microscopy, are now
increasingly used in the study of cultural heritage. This is
mainly because of their high lateral resolution and high
chemical sensitivity that enhance the advantages of the
corresponding classical laboratory instrumentation.
1737
When natural organic substances, for example lipid
materials, natural resins or plant extracts, are investigated,
the analytical approaches take into account that such
substances are complex mixtures of many chemical species,
which are often very similar to each other, and that their
chemical composition can be profoundly altered by treatment
by people before or during use, and by ageing. All this means
that techniques based on molecular extraction (liquid or gas
chromatographic separation) and mass spectrometric detec-
tion are paramount in order to identify specific biomarkers or
characteristic profiles at a molecular level. With the present
state of the art, GC–MS and LC–MS seem to be the only
analytical approaches that provide the necessary high selec-
tivity and sensitivity, especially when multidimensional mass
spectrometry (MSn) or SIM acquisition modes are used.
Although for plant resins, beeswax, bitumen, and organic
colorants, the identification of a single or a few compounds
may be enough to determine their exact origin, the
composition of acyl-lipids is, on the other hand, rather
unspecific, and usually prevents their precise botanical or
animal origin from being identified. Gas chromatography–
combustion–isotope ratio mass spectrometry (GC–C–IRMS)
has been used to obtain the isotopic signature [29], and ESI–
MS and ESI–MS–MS [65] have been used to determine the
fatty acyl moiety distributions in TAGs of several fatty
materials (fish oils, dairy fats, ruminant and non-ruminant
adipose fats), enabling us to recognise them in archaeolog-
ical findings. However, distinguishing between plant oils and
animal fats remains an open issue and a challenge for the
future.
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