1 Unravelling the Egyptian embalming materials by a multi-method approach

2 comprising high-resolution mass spectrometry

3
4 Jasmine Hertzog*,1,2,3, Hitomi Fujii4, Rugilė Žostautaitė1, Agnès Lattuati-Derieux4,5, Pascale
5 Richardin4,6, Vincent Carré1, Frédéric Aubriet1, Philippe Schmitt-Kopplin2,3
6
7 1 Université de Lorraine, LCP-A2MC, 57000 Metz, France.
8 2 Analytical BioGeoChemistry, Helmholtz Zentrum Muenchen, 85764 Neuherberg, Germany.
9 3 Analytical Food Chemistry, Technische Universität Muenchen, 85354 Freising, Germany.
10 4 Centre de Recherche et de Restauration des Musées de France (C2RMF), Palais du Louvre,
11 Porte des Lions, 14 quai François Mitterrand, 75001 Paris, France.
12 5 CNRS – IRCP, Institut de Recherche de Chimie-Paris, Chimie-ParisTech, PSL University,
13 11 rue Pierre et Marie Curie, 75005 Paris, France.
14 6 CNRS - UMR 7055 – Préhistoire & Technologie, Maison Archéologie & Ethnologie René
15 Ginouves, 21, allée de l'Université, 92023 Nanterre Cedex, France.
16
17 * Corresponding author. E-mail: jasmine.hertzog@univ-lorraine.fr
18
19 ORCID
20 Jasmine HERTZOG: 0000-0002-7946-1904
21 Hitomi FUJII: 0000-0003-2419-4180
22 Pascale RICHARDIN: 0000-0001-6741-0352
23 Vincent CARRE: 0000-0002-0719-7366
24 Frédéric AUBRIET: 0000-0003-2457-7974
25 Philippe SCHMITT-KOPPLIN: 0000-0003-0824-2664
26
27

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28 ABSTRACT
29 Mummification balms are considered among the best-preserved organic archaeological
30 samples. Therefore, deciphering their composition can contribute to increasing our knowledge
31 about the status of the buried person and the habits of the corresponding civilization. It also
32 makes it possible to estimate the state of degradation of the sample. The determination of the
33 organic substances involved in the embalming material is based on identifying biomarkers,
34 which is commonly carried out by gas chromatography-mass spectrometry (GC-MS). Here
35 high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS)
36 was used, along with gas chromatography-mass spectrometry and infrared spectroscopy, to
37 investigate the organics of embalming material of Egyptian crocodile mummies. FT-ICR MS
38 differentiated and assigned thousands of molecular contributions comprising potential new
39 biomarkers. Thus, pine resin, beeswax, and vegetal and animal fat were suggested, which
40 was confirmed by GC-MS. Eventually, the FT-ICR MS molecular fingerprint obtained for the
41 archaeological samples was compared with those of modern pine resin, which allowed for
42 assessing the ageing and heating process.
43
44 Keyword
45 FT-ICR MS, archaeometry, complex organic matrix, non-targeted analysis, GC-MS, FTIR.
46
47
48

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49 1. Introduction
50 Identification of organics in archaeological objects is paramount for archaeologists, art
51 curators, and historians. When they are well-preserved, samples can reveal valuable
52 information about the ancient civilizations. One example is mummification balms that are
53 amongst the best well-preserved archaeological organic material as they are aimed at
54 conserving the deceased’s body over time [1]. Studies performed on these samples evidenced
55 the use of resin from pine or pistachio trees, beeswax, vegetal oils, and animal fats [1,2]. This
56 increases our knowledge on the cultural and social habits, the fauna and the flora of a specific
57 period in a given area, and the contemporary trade routes [3,4]. Besides, it also advises on
58 the use and content of some archaeological objects like jars containing balms [5,6], beverages
59 [7,8], foods [9], and perfumes [10], or the material used to waterproof pottery [7,11].
60 Different analytical techniques have been developed and applied to identify the various
61 materials comprising archeological samples [3,12]. Thus, nuclear magnetic resonance (NMR)
62 spectroscopy gives information on the structures and chemical functions of the molecules
63 present in the sample [13]. Infrared spectroscopy (IR) is more widely applied for the fast and
64 global non-invasive analysis of archaeological samples by characterizing functional groups of
65 organic and identifying inorganic substances [14–17]. Both spectroscopy techniques can be
66 employed in a non-destructive way. However, they are not resolutive enough and only give
67 global chemical information, especially as part of complex substances. To address this issue,
68 mass spectrometry (MS), used in direct infusion mode or coupled to chromatography, can be
69 used. Some studies were performed by liquid chromatography hyphenated to mass
70 spectrometry (LC-MS) [5,18], but gas chromatography coupled to MS (GC-MS) is more
71 commonly employed for archaeochemistry studies [4,7]. Molecular characterization of organic
72 substances is based on the identification of specific molecules called biomarkers [19], which
73 are compounds whose carbon skeleton is specific enough to be related to its origin precursor
74 and is specific to a material. Indeed, original components can undergo some chemical
75 reactions, such as oxidation, due to the heating process related to the manufacturing and/or
76 the natural degradation of the sample. Some studies were also reported using direct injection
77 MS for the characterization of organic substances from cultural heritage objects [20–22]. Most
78 of these studies were performed by direct exposure electron impact MS (DE-MS). Some
79 electrospray ionization-MS (ESI-MS) analyses were also achieved on material extracts
80 [23,24]. This approach presents several advantages such as the high sensitivity, a soft
81 ionization process, and an easy sample preparation [3]. Moreover, low sample amount is
82 required for such analyses, which is an asset in the archaeometry field where often only a
83 limited quantity is available due to sample degradation or to preserve object integrity.
84 However, the mass resolution power encountered in these studies led to the characterization
85 of a limited number of species. In fact, archaeological samples can be regarded as complex

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86 organic mixtures due to the several substances involved, such as bitumen [2], and the
87 chemical changes that occurred during the manufacturing process and the ageing. Therefore,
88 non-targeted analysis, using ultra-high-resolution mass spectrometers, can be considered as
89 an interesting way to distinguish numerous species over a large mass and polarity range.
90 High-resolution power (R > 106) and high-mass accuracy (< 100 ppb) provided by Fourier
91 transform ion cyclotron resonance mass spectrometry (FT-ICR MS) allow the detection of
92 hundreds of thousands of m/z signals and the assignment of a unique molecular formula to
93 each of them, respectively [25–27]. A few studies have been conducted with FT-ICR MS for
94 the analysis of archeological samples with the characterization of lipids [28], triacylglycerols
95 [29], or proteins [30]. Our group also employed FT-ICR MS for the chemical composition
96 overview of organic residues contained in the inner part of prehistorical and archaeological
97 vessels [31,32].
98 Here, FT-ICR MS was used to investigate the composition of mummification balms from two
99 skulls of Egyptian crocodiles, from the collection of Osteology of the Musée des Confluences
100 (Lyon, France). Some objects of this collection were already subject to Fourier transform IR
101 (FTIR) and GC-MS analyses [16], 14C dating by accelerator mass spectroscopy [33,34], and
102 synchrotron imaging [35]. The present study is aimed at demonstrating to what extent FT-ICR
103 MS can be a powerful tool for the fast screening of organic constituents and, by extension, the
104 substances involved in the composition of the archaeological object. Moreover, the fingerprint
105 achieved by this technique compared to organic standard, here pine resin, known to be
106 involved in embalming material, advises on the oxidation state of the sample. Part of the
107 composition hypotheses formulated from the FT-ICR MS analysis, and to a lesser extent by
108 FTIR, were consolidated by GC-MS. The complementarity of these techniques was discussed,
109 with their advantages and limitations, GC-MS and FT-ICR MS respectively allowing the
110 structural identification and assignment of more and higher-mass compounds, can be
111 considered as potential new biomarkers.

112 2. Materials and methods

113 Three embalming material samples, from two mummified crocodile skulls belonging to the
114 collection of Osteology of the Musée des Confluences (Lyon, France), were analyzed. Two
115 samples, 1840 a and 1840 b, were collected on the skull referenced 90001840, while the other
116 balm sample, 1841, comes from the skull 90001841 (Figure S1). These mummified skulls
117 were discovered in Upper Egypt and brought to France, among other osteological remains of
118 human and animal mummies, by Charles-Louis Lortet (1836-1909) and Ernest Chantre (1843-
119 1924) during the late 19th century [36]. Binocular pictures of these samples (Figure S1) already
120 evidence mixture of bone and embalming materials for samples 1840 a and 1841, while
121 sample 1840 b appears more homogeneous with essentially embalming material.

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122 Fresh pine tree resin (from Pinaceae tree) was collected and analyzed as standard by FT-ICR
123 MS. One bitumen from Judea was used as a standard to assess the presence of this material
124 in the three samples by GC-MS, according to protocol 2 detailed in the sub-section 2.3.

125 2.1. Infrared analysis

126 FTIR spectroscopy analysis was performed on the raw samples with a PerkinElmer Spectrum
127 One instrument, in attenuated total reflection (ATR) mode. Ten scans were accumulated to
128 obtain a spectrum over a 400-4000 cm-1 range with a 4 cm-1 resolution.

129 2.2. FT-ICR MS analysis and data processing

130 Both the FT-ICR MS analysis and data processing are similar as what published in two recent
131 studies [31,32]. Three milligrams of each sample (archaeological samples and pine tree resin)
132 were dissolved in 1 mL methanol (LC-MS grade, Fluka, Germany) and put in an ultrasonic
133 bath for 10 min. After centrifugation, the solution was diluted to 1/100 (v/v) in methanol. This
134 solvent was used as it well dissolves the sample and is also well-adapted to electrospray
135 ionization (ESI). In addition, most of the biomarkers that are sought present an acidic function
136 (Table S1), which ensures their ionization by ESI in negative-ion mode. The methanolic
137 solutions were directly infused, at a 2 μL/min flow rate, in a 12 T Bruker Solarix mass
138 spectrometer (Bruker Daltonics, Germany) equipped with an APOLLO II electrospray source.
139 Acquisition parameters were optimized by FTMS-Control V2.2.0 (Bruker Daltonics) software.
140 The ESI source settings were as following: capillary voltage 3600 V and drying gas
141 temperature 180 °C at 4 L/min flow rate. The ions were accumulated for 0.350 s. The mass
142 spectra were acquired over the 92 – 1000 m/z range, with a 4 M time-domain, and 500 scans
143 were accumulated to obtain the final mass spectrum. At m/z 300, the achieved resolving power
144 was 400 000. Prior sample analysis, the mass spectrometer was externally calibrated by using
145 well-known fatty acid ions.
146 FT-ICR mass spectra were processed by Compass DataAnalysis 5.0 (Bruker Daltonics,
147 Germany). First, they were internally calibrated with a list of known archaeological biomarkers
148 and fatty acids. A list of peaks with a signal-to-noise ratio (S/N) greater than 3 was generated
149 and filtered from each mass spectrum. Thus, peaks related to magnetron and satellite signals
150 were removed by means of an algorithm [37]. The assignment of the m/z peaks was carried
151 out with Composer software (Sierra Analytics, Modesto, CA) with C5-50H5-75O1-15N0-4S0-2
152 elements within a mass accuracy of 0.6 ppm. As a result, thousands of features were finally
153 assigned for the archaeological samples, which belong to CHO, CHON, CHONS, and CHOS
154 heteroatom classes. The achieved formulae were plotted based on their hydrogen-to-carbon
155 and oxygen-to-carbon ratios to generate a van Krevelen diagram [38]. This graph evidences
156 biochemical families such as lipids, carbohydrates, amino acids, polyphenols, as well as some

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157 chemical reactions such as hydrogenation, condensation or hydration (Figure S2) [38]. Other
158 graphs representing H/C vs. m/z and DBE vs. carbon number were done (Figure S3). The
159 Double Bound Equivalent (DBE) represents the unsaturation degree of a molecule and is
160 given by this equation: DBE = C - (H/2) + (N/2) + 1.
161 A deeper insight into sample composition was tentatively done by comparing the achieved
162 elemental molecular formulae with those of known archaeological biomarkers. The obtained
163 hits allow doing some hypotheses on the natural substances used in the balm production.

164 2.3. Analysis by Gas Chromatography coupled to Mass Spectrometry

165 Two different and complementary protocols were applied to the three embalming materials
166 depending on the targeted compounds. For all GC-MS experiments, ionization was performed
167 by 70 eV electron impact (EI). The temperature of the ion source and interface transfer line
168 were respectively set at 200°C and 320 °C. Molecular components were eluted using helium
169 as carrier gas at a constant flow of 3 mL/min. Mass spectra were acquired over a m/z 50–650
170 range. GC-MS data were treated with Agilent Mass Hunter Qualitative Analysis Navigator
171 B.08.00 software.
172
173 2.3.1. Protocol 1
174 The first protocol is suitable for the GC-MS analysis of a wide range of organic compounds
175 that can be used as embalming material [31,39].
176 The powdered sample (< 1 mg) was directly derivatized by 100 μL of N, O- Bis (trimethylsilyl)
177 trifluoroacetamide (BSTFA, Thermo, USA) containing 1% trimethylchlorosilane and 10 μL of
178 pyridine at 75 °C for 1 hour. The GC-MS analysis was carried out using a Shimadzu GC-2010
179 GC system with a Shimadzu GCMS-QP2010. The GC separation was performed on a fused
180 silica capillary column CP5860 CP-Sil 8 CB low bleed/MS (30 m length, 0.25 mm i.d., 0.25 μm
181 film thickness). Injection was done in split-less mode and the data were treated with GC-MS
182 solution software. The temperature program of the oven was 50 °C for 1 min, 50-150 °C at 10
183 °C/min−1, 150-325 °C at 5 °C/min−1, and let at 325 °C during 10 min.
184 2.3.2. Protocol 2
185 The second protocol is aimed at investigating bitumen compounds and was described by
186 Burger et al. [40].
187 Four milligrams of the sample were first extracted twice with 2 mL of a 9:1 (v/v)
188 dichloromethane/methanol mixture and then with 2 mL of a 1:1 (v/v) dichloromethane/ethyl
189 acetate mixture using ultrasonic bath (Branson 2510), for 5 min. The achieved extract was
190 concentrated until 100 μL under a gentle stream of nitrogen. Then it was separated into two
191 different fractions using a silica column (SIGMA, 230-400 mesh, 60 Å diameter). The first one
192 was achieved with cyclohexane, which yields the fraction 1 containing hydrocarbon

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193 compounds. The fraction 2 was obtained with a 1:1 (v/v) dichloromethane/ethyl acetate
194 mixture and comprises compounds with an intermediate polarity such as fatty acids and
195 esters.
196 The GC-MS analyses were carried out with an Agilent Technologies 7890B GC system by
197 using an Agilent Technologies 5977B GC/MSD. Samples were injected in split-less mode and
198 molecular components were eluted using two different chromatographic parameters so called
199 conditions 1 and 2.
200 In condition 1, applied to fractions 1 and 2, GC separation was performed on a fused silica
201 capillary column Agilent HP-5ms 19091S-433UI (30 m length, 0.25 mm i.d., 0.25 μm film
202 thickness). The temperature program of the oven was 50 °C for 1 min, 50-150 °C at 10
203 °C/min−1, 150–324 °C at 5 °C/min−1, and let at 324 °C during 10 min.
204 In condition 2, dedicated to higher molecular weight compounds such as palmitate esters from
205 the fraction 2, GC separation was performed on a fused silica capillary column CP-Sil 5 CB
206 low bleed/MS (Agilent J&W) (15 m length, 0.25 mm i.d., 0.1 μm film thickness). The
207 temperature program of the oven was 50 °C for 1 min, 50-270 °C at 10 °C/min, 270–320 °C
208 at 15 °C/min, and kept at 320 °C during 5 min.
209 Known biomarkers of diverse materials were specifically considered, which are characterized
210 by specific fragment ions (Tables S2 and S3). Thus, these ions were tracked in the achieved
211 total ion chromatograms (TIC) by selected ion monitoring (SIM) method and their detection
212 enabled to identify some compound classes.

213 3. Results and discussion

214 3.1. Infrared spectroscopy

215 FTIR spectra achieved for samples 1840 b and 1841 present similar profiles (Figure 1). The
216 bound vibration assignments are gathered in Table 1, as well as tentative match with
217 embalming materials. The broad band around 3430 cm-1 is due to stretching vibrations of the
218 OH bound, ν(O-H), which can be due to oils, fats, and tree resin [16,41,42]. There are bands
219 related to proteins at 3280 cm-1 and 3050 cm-1, and more precisely to ν(N-H) vibration of
220 amides A and B, respectively [43]. They are also observed with a lower intensity, in the 1840
221 a sample. Other bands characteristic to proteins at 1630 cm-1, relative to amide I ν(C=O), and
222 at 1550 cm-1, relative to amide II [43] are also detected. The bands in the 2960-2850 cm-1
223 range correspond to νsaturated(C-H) vibrations, which can be attributed to fatty matter, beeswax,
224 and resin [16]. In the spectrum of the 1840 b sample, the intense band close to 1700 cm-1
225 corresponds to the stretching of the carbonyl bound, and the shoulder at high wavenumber is
226 specific to ester bound [44]. Such chemical function is present in beeswax and fatty matter.
227 Regarding ester C-O bound, its stretching band is observed at 1170 cm-1 [44]. There is a small

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228 peak at 1604 cm-1, especially in the spectrum of the 1840 b sample, corresponding to the
229 stretching vibration of the C=C aromatic bounds, which is observed in resin material. The
230 bands in the 1460-1380 cm-1 wavenumber range is relative to the C-H bound bending (δ),
231 which can be associated to most of the organic compounds and materials, whereas the band
232 at 820 cm-1 refers to the bending of substituted C-H bounds, such as in resin [11]. The doublet
233 at 730 cm-1 is characteristic of semi-crystalline structures like beeswax and fatty matter [16].
234 On the other hand, the IR spectrum of the sample 1840 a presents some differences compared
235 to the two other spectra. Indeed, bands in the 2960-2850 cm-1 and 1500-1100 cm-1 regions
236 are significantly less intense than in the two other samples. Interestingly, this spectrum shows
237 an intense band at 1010 cm-1, which is less pronounced or absent in the other samples, that
238 can be attributed to stretching of phosphate ion, linked to calcium from bone material [41,45].
239 Such observation agrees with the sample pictures (Figure S1), which clearly shows that
240 sample 1840 a, and to a lesser extent, sample 1841 comprise osseus material from the skull.
241 The infrared spectroscopy allows obtaining an overview of the chemical functions of the
242 organics of the embalming material but also the presence of bones with the detection of bands
243 related to proteins and phosphate. Even if IR spectroscopy is a non-destructive analytical
244 method, it does not enable to precisely ascertain the composition of the archaeological
245 material, especially when different materials are used. Therefore, more powerful analytical
246 techniques are required such as GC-MS and FT-ICR MS, to obtain an information at the
247 molecular level.

248
249 Figure 1: ATR-Infrared spectra of sample 1840 a, 1840 b, and 1841. Band assignments are given in
250 Table 1.
251

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 Wavenumber (cm-1) Assignment bound Material
3425 ν (O-H) Oils/Fats, Conifer resin
3288 Amide A Proteins
3053 Amide B Proteins
2957 ν (CH3) Beeswax, Oils/Fats, Conifer resins
2920-2850 ν (CH2) Beeswax, Oils/Fats, Conifer resins
1730 ν (C=O ester) Beeswax, Oils/Fats
1700 ν (C=O) Conifer resins
1629 Amide I Proteins
1604 ν (C=C aromatic) Conifer resins
1550 Amide II Proteins
1460 δ (C-H) Beeswax, Oils/Fats
1382 δ (C-H) Beeswax, Oils/Fats
1230-1170 ν (C-O) Beeswax, Oils/Fats
1010 ν (phosphate) Mineral part of the bone
820 δ (C-H aromatic) Conifer resins
730 Doublet of semi-crystalline structure Beeswax, Oils/Fats
252 Table 1: Infrared wavenumbers (in cm-1) of the bands observed for the embalming material samples,
253 with their assigned bound and corresponding organic materials.

254 3.2. ESI (‒) FT-ICR MS

255 FT-ICR MS analysis of samples 1840 a, 1840 b, and 1841 enabled to respectively obtain
256 6944, 3169, and 3939 monoisotopic molecular formulae (Figure 2). Composition description
257 of samples 1840 b and 1841 are more similar with CHO class respectively representing 95%
258 and 77% of the assignments. Concerning sample 1840 a, CHO compounds correspond to
259 44% of the assignments while 55% are related to CHON species. Representation of the
260 features on van Krevelen diagram already ensures to differentiate some biochemical classes.
261 Thus, within the CHO molecular series, one observes some fatty acids (H/C ≈ 2 and 0 < O/C
262 < 0.4), terpenoids (0.5 < H/C < 1.5 and 0.1 < O/C < 0.4), and phenolics (0.5 < H/C < 1.5 and
263 0.3 < O/C < 0.8). Concerning nitrogen-containing species, van Krevelen diagrams (Figure 2)
264 and DBE vs. carbon number representations (Figure S3) indicate that these compounds
265 present a significant number of unsaturations, with DBE ranging from 5 to 25. Their origin is
266 not ascertained yet but these compounds may come from the degradation
267 (dehydrated/deaminated) of proteins from the bone material as they are mainly observed in
268 samples 1840 a and 1841 (Figure S1) [46,47]. This also agrees with what previously observed
269 on the FTIR mass spectra and on the binocular pictures.
270 Although FT-ICR MS does not provide structural information, deeper investigation on the
271 molecular composition, and therefore substances composing the embalming material, was
272 attempted by comparing achieved FT-ICR MS assignments with those of known
273 archaeological biomarkers observed in mummification balms [3]. Thus, a focus was done on
274 sample 1840 b, and more especially on the CHO series, as it presented better homogeneity
275 compared to the others, i.e., no bone contribution. Nevertheless, putative assignment of some
276 biomarkers was also proposed for the other samples (Table S1). Sought biomarkers are those
277 corresponding to the following materials: pine tree [1,6,48] and mastic resins [5,16]; beeswax

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278 [6,49]; animal and vegetal sterols [16,50]; vegetal and pine oils [3,5]; saturated and
279 unsaturated, dicarboxylic, and hydroxyl fatty acids; and pitch of birch bark [21,51]. Compounds
280 for which a hit was obtained are gathered in Table S1.

281
282 Figure 2: ESI (‒) FT-ICR mass spectra, van Krevelen diagrams with bubble size relative to peak
283 intensity, and pie charts representing the heteroatom class distribution with feature number,
284 obtained for the three embalming materials (1840 a, 1840 b, 1841) and the fresh pine tree

285 As shown on Figure 2, some fatty acids were detected in sample 1840 b. Some were
286 putatively assigned to saturated (C12-34) and unsaturated linear fatty acids, such as oleic and
287 palmitic ones. Other molecular formulae were found, which can correspond to esters, but due
288 to the ionization and detection parameters, ESI (‒), diacid (C8-22) putative assignments were
289 preferred and also because these compounds are known to be oxidation products of
290 unsaturated fatty acids [52,53]. Several matchings with hydroxyl fatty acids were also
291 obtained, that are the result of oxidation processes of unsaturated fatty acids or lipids due to
292 ageing or burning phenomenon [53,54]. The presence of fatty acids and corresponding
293 oxidation products is typical of vegetal oils and/or animal fat.
294 Some other features observed by ESI (‒) FT-ICR MS can be linked to beeswax degradation,
295 such as fatty acids and alcohols. Beeswax was reported as a material used for mummification
296 balm or other archaeological objects [16,23,41]. It is mostly made of palmitate esters, whose
297 ageing by hydrolysis, leads to the formation of free palmitic acid and long chain alcohols [55].
298 Here, the C16H31O2- ion may be reasonably assigned to the deprotonated form of the palmitic
299 acid. Nevertheless, its presence in several other organic matrixes makes it not specific enough

10

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300 to assess the beeswax presence in the investigated mummification balm. However, the
301 C24H47O2- ion corresponding to the deprotonated ion of a long-chain C24:0 fatty acid was
302 observed at m/z 367.358150 and can be assigned to the tetracosanoic acid, already identified
303 in beeswax [23,56].
304 To determine the animal or vegetal origin of fatty acid contribution, palmitic acid-to-stearic acid
305 (C16:0/C18:0) ratio can be used [5,54]. A higher content of C16:0 indicates vegetal oils whereas
306 lower amount suggests animal fat. However, C16:0 from beeswax degradation biases the
307 calculation of this ratio, which cannot be used to assess fatty acids’ origin. Another parameter
308 frequently used to determine fatty acids’ origin is the presence of fatty acids with an odd
309 number of carbon atoms (C15, C17, and C19) [5,57]. Formulae corresponding to these
310 compounds have been assigned for sample 1840 b suggesting the presence of animal fat.
311 However, this latter can be part of the original recipe or originate from the mummified animal.
312 Identification of conifer tree resin is done by searching characteristic biomarkers that are
313 diterpenoid compounds deriving from abietic and pimaric acids [48]. Here, 15 potential
314 biomarkers were found in the 1840 b mass spectrum (Table S1). In Figure 2, the most intense
315 signal was observed at m/z 313.180802 and corresponded to the C20H26O3- formulae, which
316 can be assigned to the deprotonated ion of the 7-oxodehydroabietic acid. This compound is
317 one of the major oxidative product of abietic acid in pine tree resin [1,58,59]. Some aromatics
318 were also detected whose origin is not known (Table S1). They can be regarded as lignin
319 degradation products from wood, such as conifer tree where the resin was collected or from
320 flax textile fiber employed for embalming [60,61].
321 No markers linked to mastic resin, pitch from birchbark, pine oil, and sterol-based compounds,
322 such as cholesterol and sitosterol, were found. Thus, FT-ICR MS analysis allowed
323 hypothesizing that beeswax, pine resin, vegetal oils, and/or animal fat were used to produce
324 this balm. All these materials are commonly used and are widely observed in mummification
325 balms [6,16,49]. However, no structural information can be obtained by non-targeted analysis.
326 Only overall sample composition can be given based on van Krevelen diagram (lipids,
327 terpenes, aromatics…) and hypotheses are made based on molecular formulae and putative
328 assignments. To confirm the previous hypotheses GC-MS analyses were achieved as detailed
329 in the GC-MS section.

330 On the other hand, FT-ICR MS was also used to assess oxidation state of balm components.
331 Such information is relevant regarding conservation over the ages (presence or absence of
332 oxygen) and manufacturing conditions (heating) of an archaeological object. In that respect,
333 modern pine tree resin was analyzed within the same conditions and its molecular fingerprint
334 compared to the 1840 b sample one. This substance is known to be frequently used in
335 mummification balms [16,41]. A total of 936 assignments were achieved by ESI (‒) FT-ICR

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336 MS for the modern resin (Figure 2), which are mainly CHO compounds located in the area of
337 terpenoid species of the van Krevelen diagram. The most intense peak detected at m/z
338 301.217242 was assigned with the molecular formula C20H30O2 and can be regarded as abietic
339 acid that is characteristic of fresh diterpenoid resin. Other putative biomarkers of pine resin
340 were also found (Table S1), such as dehydroabietic, 7-oxodehydroabietic,
341 hydroxydehydroabietic, and 15-hydroxy-7-oxodehydroabietic acids. Comparison of known
342 pine resin biomarkers putatively found in both modern resin and embalming material are very
343 informative on the chemical evolution of the embalming material over time. Indeed, it is
344 possible to observe on the van Krevelen diagram (Figure 3) some chemical relationships
345 between putative pine tree biomarkers, such as dehydrogenation and oxidation. These
346 phenomenon result in a shift of the components to lower H/C values and higher O/C values,
347 respectively. Therefore, putative pine tree biomakers in sample 1840 b present higher
348 oxidation and unsaturation degrees and are observed with higher intensity compared to
349 modern pine resin. For instance, feature putatively assigned to 7-oxodehydroabietic acid at
350 m/z 313.180802 is the most intensely detected peak whereas the signal putatively attributed
351 to abietic acid is very small. The 15-hydroxy-7-oxodehydroabietic (m/z 315.19657) and
352 tetrahydroabietic (m/z 317.15470) acids were respectively more intensely and specifically
353 observed on the mass spectrum of the ancient material. The former compound is the most
354 oxidized while the second one is the most unsaturated. These observations agree with the
355 ageing of pine tree resin under oxidative processes, which leads to the formation of
356 dehydroabietic acid from abietic acid, follows by its oxidation into 7-oxodehydroabietic acid
357 and 15-hydroxy-7-oxodehydroabietic acid [1,58,59,62]. Similar observations were obtained by
358 Schmitt-Kopplin et al. and Colombini et al. [1,63]. The latter authors performed analysis of two
359 fresh pine tree resins (Pinus pinea and Pinus sylvester) and an ancient pine tree resin by GC-
360 MS [1]. They observed that abietic acid is more abundant in the fresh samples (16% and 31%
361 or total chromatogram peak area) than in the ancient material (1%). Dehydroabietic acid was
362 observed in the fresh samples with a percentage of 10% and 58%, respectively, and 17% in
363 the ancient material. The 7-oxodehydroabietic acid was not detected in the fresh samples but
364 corresponds to the main contribution (78% of the peak area) of the chromatograms of the
365 ancient pine tree resin.

366 Other known products are the results of abietic acid oxidation linked to ageing or heating
367 process, such as retene (C18H18) and tetrahydroretene (C18H22) [20,62,64]. They were not
368 observed in the present study due to the poor ionization efficiency of such species by ESI.

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369
370 Figure 3: van Krevelen diagram of the CHO species achieved by ESI (‒) FT-ICR MS for the fresh pine
371 resin (in grey), with some pine biomarkers found in pine resin (blue) and in sample 1840 b (red). Some
372 known pine resin biomarkers are given in the expansion: (1) C20H30O2, Abietic acid; (2) C20H28O2,
373 Dehydroabietic acid; (3) C20H26O3, 7-Oxodehydroabietic acid; (4) C21H30O3, Methoxy-
374 dehydroabietic acid; (5) C20H28O3, Hydroxydehydroabietic acid; (6) C20H26O4, 15-Hydroxy-7-
375 oxodehydroabietic acid; (7) C22H22O2, Tetradehydroabietic acid.

376 3.3. GC-MS

377 The GC-MS experiments allowed the identification of several biomarkers (Tables S2 and S3)
378 whatever the used sample preparation protocols and analytical conditions (Figures 4, S4, S5,
379 and S6). Thus, the presence of pine tree resin in the 1840 b sample was assessed by the
380 identification of biomarkers such as dehydroabietic acid, pimaric acid, dehydro-7-
381 dehydroabietic acid, retene, and 7-oxodehydroabietic acid (Figures 4 and S4) [1,58,59].
382 Retene was observed in low amount, as well as the methyl 7-oxodehydroabietate, whereas 7-
383 oxodehydroabietic acid was intensively detected, indicating a low heating of the resin for the
384 sample preparation [64]. These observations are in agreement with those obtained by FT-ICR
385 MS. Some phenolic species were identified, namely, vanillic acid and several benzoic acid
386 derivatives, whose origins can be diverse. It could be either part of the original balm
387 composition with the degradation of the wood material where pine resin was extracted. But it
388 may also be the result of global ageing of the balm organics and textile [60,61].
389 As observed by FT-ICR MS, GC-MS analysis of the sample 1840 b, treated with the protocol
390 1, evidenced several diacids (C6-10 and C14) and linear carboxylic acids (C8-20) including
391 saturated and mono-unsaturated fatty acids (Figures 4 and S4; Table S2). Identification of
392 straight chain C15:0 and C17:0 fatty acids could suggest the presence of animal fat whose origin
393 is ascertained [5,57].

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 Dehydro-7-dehydroabietic acid
FA (C16:0)
Pine resin

Dehydroabietic acid
DA (dicarboxylic acid), FA (fatty acid)
TIC Alkane
×10 8
Fermentation product

FA (C18:0)

7-oxo-dehydroabietic acid methyl ester

4-Trimethylsilyloxybenzoate

C31
benzoic acid

3-Trimethylsilyloxybenzoate
Fumaric acid

C27

C29
DA (C9:0)

Pimaric acid
FA (C9:0)

Vanillic acid
Succinic acid

7-oxo-dehydroabietic acid
2-methyl

Retene
DA (C8:0)

FA (C 20:0)

C33
Alcohol C 24
DA (C14:0)
FA (C14:0)

FA (C17:0)
DA (C7:0)
2-Methyl glutaric acid

FA (C15:0)

FA (C18:1)
Methyl succinic acid

FA (C10:0)

DA (C10:0)
DA (C6:0)
FA (C8:0)

Glutaric acid

394 time (min)

395 Figure 4: Total ion chromatogram of the sample 1840 b prepared and analysed according to the
396 protocol 1. Some biomarkers are annotated and coloured based on their biochemical families.

397 The fraction 2 of the sample was analysed under two GC-MS conditions. With condition 1
398 even carbon number long-chain fatty acids C20-30 were identified (Figure S4). Condition 2
399 ensured to evidence the even carbon palmitate esters (E), E40, E42, E44, E46, E48 (Figure
400 S4). In addition, Figure S5 shows a distribution of odd carbon number n-alkanes (C15-35), of
401 which the C27 one is the most abundant. This pattern is specific to beeswax [1, 23, 65, 66].
402 The GC-MS analysis of the fraction 1 obtained with the protocol 2 highlighted bitumen
403 biomarkers (Table S3 and Figure S6) [67,68]. This material is known to be involved in several
404 mummification balms [1,6]. The SIM for m/z 191 and m/z 217 ions, which are respectively
405 relative to the well-known hopane and sterane type diagnostic biomarkers, are given in Figure
406 S6 [69]. As a result, several terpane, hopane, and sterane derivatives were observed and 32
407 species were identified (Table S3). By comparing biomarkers found in the archaeological
408 samples with those evidenced in the bitumen standard, it appears that all archaeological
409 samples and Judean bitumen contained n-alkanes, constituting the paraffinic fraction of the
410 bitumen.
411 Accordingly, the GC-MS analyses performed on the three samples made it possible to identify
412 biomarkers relative to pine resin, beeswax, vegetal and animal fats, and bitumen. With the
413 exception of bitumen, all the above balm constituents were evidenced by FT-ICR MS.

414 The Van Krevelen diagram of the CHO species respectively identified or assigned by GC-MS
415 and FT-ICR MS (Figure S7) evidences the complementarity of both techniques, as already

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416 reported with other complex matrixes [71,72]. More species related to lipids were detected by
417 ESI (‒) FT-ICR MS than by GC-MS. Furthermore, the H/C vs. m/z graph (Figure S7)
418 demonstrates that the fatty acids additionally observed by FT-ICR MS are mainly high-mass
419 compounds. Regarding the polarity, these techniques are complementary as GC-MS ensured
420 to study low to non-polar species while ESI FT-ICR MS is more dedicated to mid-to-polar
421 compounds. Eventually, structural information, which is paramount for compound
422 identification, is here only brought by GC-MS. Nevertheless, FT-ICR MS allows the detection
423 and the assignment of thousands of features, whose some may be potential new biomarkers.
424 Indeed, results obtained by non-targeted FT-ICR MS agreed with previous observations on
425 modern and old resins, but only on a limited number of compounds. In a future study, it would
426 be interesting to focus on the molecular evolution of this substance over time, to highlight new
427 biomarkers specific to resin ageing that will be characterized by higher O/C and lower H/C
428 values.

429 4. Conclusion and perspectives

430 Fourier transform ion cyclotron resonance mass spectrometry was applied to assess the
431 organic composition of three embalming material samples from Egyptian crocodile mummies.
432 Even if this technique does not allow structure identification of all detected species, it enables
433 to rapidly obtain a molecular profile of the sample. The putative molecular markers observed
434 by ESI (–) FT-ICR MS allowed hypothesizing organic materials used for balm production,
435 which were confirmed by GC-MS. Furthermore, FT-ICR MS was shown to be a powerful
436 technique for the evaluation of the degradation pathways of cultural heritage object containing
437 organic substances. Comparison of van Krevelen diagrams of archaeological and modern
438 samples ensured to estimate if the sample underwent heating process and/or was preserved
439 from oxidation or not. It is also noteworthy to mention that more compounds were detected by
440 FT-ICR MS than by GC-MS, which is an asset as part of complex mixture analysis. Another
441 advantage of this analytical method is the simple sample preparation and data evaluation (no
442 derivatization, poor fragmentation) compared to GC-MS (derivatization, intensive
443 fragmentation patterns by EI). GC-MS remains the method of choice for the identification of
444 the materials involved in archaeological samples. Both techniques ensured achieving
445 complementary information for the extensive description of the sample composition.
446 Perspective studies would consist in analyzing low- to non-polar organic species comprising
447 embalming material by using ionization sources more suited to less polar balm components.

448 Declaration of Competing Interest

449 The authors declare that they have no known competing financial interests or personal
450 relationships that could have appeared to influence the work reported in this paper.

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451 Acknowledgments
452 The authors thank Didier Berthet from the Musée des Confluences, Lyon, France, for its
453 collaboration for sample collection.

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