Chemical Composition Overview on Two Organic Residues from the Inner Part of an Archaeological Bronze Vessel from Cumae (Italy) by GC–MS and FTICR MS Analyses

Eur. Phys. J. Plus (2021) 136:661 https://doi.org/10.1140/epjp/s13360-021-01627-1

Regular Article

Chemical composition overview on two organic residues from the inner part of an archaeological bronze vessel from Cumae (Italy) by GC–MS and FTICR MS analyses

Jasmine Hertzog1,2,a , Hitomi Fujii3,b , Andrea Babbi4,5,6 , Agnès Lattuati-Derieux3,7, Philippe Schmitt-Kopplin1,2 1 Analytical Food Chemistry, TUM Technische Universität München, 85354 Freising, Germany 2 Analytical BioGeoChemistry, Helmholtz Zentrum Muenchen, 85764 Oberschleisheim, Germany 3 Research Department, Centre de Recherche et de Restauration des Musées de France, C2RMF, Ministère de la Culture, 14 quai François Mitterrand, 75001 Paris, France 4 Istituto di Scienze del Patrimonio Culturale del Consiglio Nazionale delle Ricerche, Monterotondo St., 00015 Roma, Italy 5 Graduiertenkolleg 1876 ”Frühe Konzepte von Mensch und Natur”, Institut Für Altertumswissenschaften/Ägyptologie, Johannes Gutenberg-Universität Mainz, 55122 Mainz, Germany 6 Leibniz-Forschungsinstitut Für Archäologie des Römisch-Germanischen Zentralmuseums (RGZM), 55116 Mainz, Germany 7 CNRS – IRCP, Chimie-ParisTech, Institut de Recherche de Chimie-Paris, PSL University, 11 rue Pierre et Marie Curie, 75005 Paris, France

Received: 11 October 2020 / Accepted: 31 May 2021 © The Author(s), under exclusive licence to Società Italiana di Fisica and Springer-Verlag GmbH Germany, part of Springer Nature 2021

Abstract A bronze vessel, containing solid black material, was found in a grave dated to the late eighth century BC and located in the Middle Tyrrhenian region. Two residue samples of this black material were subject to molecular characterization in order to assess first its composition and then the function of this precious object. Two different technique analyses, namely gas chromatography–mass spectrometry (GC–MS) and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, in negative-ion mode (ESI(−) FTICR MS), were employed in that way. If GC–MS analysis is commonly used for the characterization of archaeological samples by identifying biomarkers related to organic material, FTICR MS was used to achieve a fast global molecular description with up to thousands of assignments. In addition, this technique enabled to hypothesize about the different materials involved in the black material composition. As a result, lipids, beeswax, conifer resin, and pitch from birch bark were supposed, which was thereafter confirmed by GC–MS. Conse- quently, FTICR MS can be regarded as an efficient tool for the fast profiling of the organic archaeological compounds.

1 Introduction

Composition characterization of archaeological samples is essential to increase our knowledge of ancient civilizations regarding their cultural habits, the contemporary trade routes

a e-mail: [email protected] (corresponding author) b e-mail: [email protected] (corresponding author)

0123456789().: V,-vol 123 661 Page 2 of 22 Eur. Phys. J. Plus (2021) 136:661 allowing importing specific materials, or the status of the buried person. Up to now, one of the preferred analytical methods is gas chromatography coupled to mass spectrometry (GC–MS) [1]. This technique is generally performed to achieve the molecular characterization of archaeological samples by identifying archaeological organic biomarkers that are directly related to a specific substance. For instance, abietic acid derivatives and betulin derivatives are, respectively, related to pine resin and pitch from birchbark [2, 3]. Another and less common technique in the field of archaeometry was employed, namely Fourier transform ion cyclotron resonance mass spectrometry with electrospray ionization, in negative-ion mode (ESI(−) FTICR MS). This non-targeted approach requires a high- resolution mass spectrometer in order to acquire mass spectra of thousands features, which are assigned with a unique molecular formula thanks to the high-mass accuracy. The previous works reported on the study of archaeological samples by FTICR MS are essentially focused on one biochemical class such as lipids or proteins [4–6]. To obtain a global view of the sample composition, the thousands of assignments are plotted according to their hydrogen-to-carbon versus oxygen-to-carbon ratios to generate a van Krevelen diagram [7, 8]. Depending on the plot location on the diagram, it is possible to distinguish fatty acids, terpenoids or sugars. Furthermore, comparison of molecular formula of achieved assignments with those of known archaeological biomarkers can be performed for putative assignments [9, 10]. In fact, as no structural information can be obtained by FTICR MS, only putative species can be attributed to a MS signal based on their molecular formula. However, these candidate compounds are assigned based on our knowledge on the organic composition of archaeological material [10, 11]. Thus, based on these candidates, hypotheses can be done on the used material. This study is focused on two black material residue samples, Tb104-1 and Tb104-2, from within a bronze vessel dating to the late eighth century BC (Fig. 1). In order to determine its chemical composition, these two analyses were performed. The obtained assumed chemical compounds by EST FTICR MS are then compared to GC–MS results.

2 Archaeological context

Grave Artiaco 104 represents a key context of the beginning of the Early Orientalizing Period in the Middle Tyrrhenian region (ca 730–680 BC). This burial was uncovered by accident in Don Alfonso Artiaco’s field about 100 m N of the NE Gate of the historical defensive walls of Cumae (Naples) on 27 March 1902 (Fig. 2). Actually, the digging activities which led to the discovery of the grave aimed mainly to free the majestic tholos tomb of the Sannitic era (fourth century BC) from the earth which had already been turned to light on 7 February [12–17]. Grave 104, a male cremation placed in a large rectangular tufa-stone container, was not the only burial in the Artiaco field. Actually, two inhumations with wooden coffins turned up in the area surrounding the tholos tomb. It is the case of graves 103bis and 111 that are almost concurrent with the context of 104 (104: end of the eighth century BC; 103bis, 111: end of the eighth century, early seventh century BC) and can be paralleled to it for both, the depth and the orientation (104: NE–SW; 103bis, 111: E–W) [12]. These tantalizing similarities lend weight to the hypothesis of a funerary cluster, possibly kin-based [14]. Nonetheless, the differences in the burial structure, ritual, and particularly in the wealth and composition of the grave-goods, point to an unequivocal difference in social status. In fact, if the cremated male individual was buried with an extremely rich, varied, and quite exotic 123 Eur. Phys. J. Plus (2021) 136:661 Page 3 of 22 661

Fig. 1 Pictures of the bronze vessel (a) from the Grave 104 Artiaco, vessel residues (b), and studied black material (c). Pictures from Andrea Babbi

Fig. 2 Location of Cumae. Map from Andrea Babbi

inventory clearly echoing the privileged and almost unique social status of the deceased, the two inhumed female individuals had been offered a much simpler assemblage furnished with vases displaying either an ‘Aegean’ or a local pedigree. Grave Artiaco 104 comprised a large and deep quadrangular pit (2.20×2.10×4.0 m), at the centre of which a monolithic rectangular tufa-stone container with a lid made up of two tufa slabs had been laid on a layer of fern leaves covering the bedrock. Before commenting on the burial accoutrements, it must be underlined that the inventory could be incomplete 123 661 Page 4 of 22 Eur. Phys. J. Plus (2021) 136:661 due to the accidental and partial removal of one side of the pit that led to the discovery of the grave itself [12]. Within the lithic container and at its centre, there was a Chinese boxes sequence of sorts consisting of metal containers: a silver urn buried in a slightly flattened bronze cauldron which in turn had been laid within a larger globular bronze one, and that again had been covered by a large, richly decorated, bronze shield made of very thin metal foil. A varied set made up of gold and silver jewels and vases for drinking and possibly offering had been buried outside and around the globular cauldron, i.e. around the deceased. This location and the fact that all these offerings had been originally laid on the funeral pyre, as hinted at by the clear signs of exposure to fire, make it possible to interpret them as belonging of the deceased and the space itself as a sort of ‘private’ area/dimension. Outside the container, therefore in a sort of ‘public’ area because more accessible and ’visible’ than the previous one, the funerary offerings laid on the fern leaves likely conveyed and displayed distinctive social traits of the deceased due to their connection with the accom- plishment of specific practices [12, 17]. Along the south-east side, there was a cluster of iron objects, mainly weaponry (e.g. swords and spears), but also two horse bits and a few remnants possibly of a chariot, as well as a set of quite large spits. At the east corner of the lithic container, the occurrence of pottery fragments usually referred to as stemming from a large amphora with an ‘Aegean’ aura (‘SOS’ type) [12] was recorded. Along the short north- eastern side two rather thick bronze discs, recently interpreted as a pair of pans belonging to a balance scale, likely used for weighing not only small amounts of precious metals, but also larger quantities of raw materials (e.g. amber, bronze, and lead) [14], were found. Finally, two small cauldrons with lotus flower-shaped handles, likely a Middle Tyrrhenian re-elaboration of a Cypriot shape [13], had been stacked on a bronze stand interred along the north-western side of the lithic container [12](Fig.1a). When focusing on the social traits conveyed and displayed by these offerings, the following can be asserted: the group of iron objects could have represented the ability to wield weapons and/or the right to use force as an instrument of coercion, the availability of a pair of horses and a chariot would have been helpful not only for battling but also for riding across and keeping under control the land owned by the deceased and his kin, while the spits could have represented a full supply of meat to share with the relatives, as well as with a following of men and other members of the highest echelons of the community; the large amphora hinted perhaps at the availability of a rather large amount of olive oil or, more probably, wine; the pair of pans would point to the involvement of the deceased in the exchange networks through which raw materials and goods flowed across the Tyrrhenian Sea and more generally the Mediterranean region; finally, the bronze stand and the two elegant small cauldrons would be necessary to display the availability of beverage, as well as the know-how of practices and rituals needed to blend, share, and consume it in the most appropriate way [13, 14]. The black material residue taken into consideration in this paper stems from within the cauldron located directly on the top of the bronze stand (Fig. 1b, c). In fact, in 1903 this cauldron is described as follows: ‘the inner surface of the vase is all covered with a thick layer of resin or pitch, evidently to make it more suitable to contain liquids and at the same time to strengthen the walls’ [12]. As a matter of fact, both the cauldrons had been made by at least two bronze foils overlapping at the maximal expansion of the vase and adhering by a means of a continuous series of sturdy rivets. On the one hand, this detail could perhaps explain the use of the resin or pitch to optimize the insulation of the cauldrons; on the other hand, it is unclear why the black substance was preserved only in the specimen situated on the stand. The accurate inspection of both the black material remains and the stand leaves open the possibility that the small cauldron under discussion had been fixed to the top of 123 Eur. Phys. J. Plus (2021) 136:661 Page 5 of 22 661 the stand using four more rivets. At this regard, it is worth remembering that in the 1903 publication, it says that ‘the bottom of the vase on the top of the stand adheres to the stand’ [12]. If so, it seems possible that only the cauldron with rivets hammered through the bottom needed a thorough inner lining insulation to be used as a container of beverages. This feature recalls a procedure of waterproofing documented in other bronze foil vases with a closed shape (i.e. suitable for containing liquids) documented in Crete and Euboea in prestigious contexts with weaponry dating back to the transition of the II–I millennium BC, as well as in much later burials from central and southern Etruria dating back to the advanced eighth century BC [18].

3 Materials and methods

3.1 Sample preparation and methods for FTICR MS analysis

Five milligrams of the two solid samples were first separately powdered in an agate mortar. For each, the achieved powder was dissolved with 1 mL methanol in a vial placed in an ultrasonic bath for 5 min. The mixture was centrifuged, and the supernatant was collected. This solution was then diluted to 10 with methanol. The achieved solutions were analysed by Fourier transform ion cyclotron resonance mass spectrometry, coupled to electrospray in negative-ion mode (ESI(−) FTICR MS). Negative- ion mode was chosen as most of the sought archaeological biomarkers contain acidic function, which are better detected under these conditions. Three hundred scans were acquired to achieve the final mass spectrum on an m/z 147.4–1000 range, with a 4 M time domain. Sample was directly infused in the source with a flow rate of 2 μL.min−1. The capillary voltage was set at 4 kV. The temperature of the drying gas was set at 200 °C with a flow rate of 4 L min−1. Calibration was performed with known annotations of archaeological biomarkers. The peak annotation was performed with Composer software (Sierra Analytics, Canada) with C, H, O, N, and S elements within a mass accuracy window of 0.75 ppm. Thousands of features were finally assigned, which belong to CHO, CHON, CHONS, and CHOS molecular series (Figs. 3 and 4). The achieved formulae were plotted based on their hydrogen-to-carbon and oxygen-to-carbon ratios to generate a van Krevelen diagram [7]. On this kind of graph, it is possible to evidence biochemical families such as lipids, carbohydrates, amino acids, polyphenols as well as some chemical reactions such as hydrogenation, condensation, or hydration.

3.2 Analytical methods based on gas chromatography–mass spectroscopy

Few milligrams of each sample was directly derivatized with 100 μL of N, O-bis (trimethylsi- lyl) triﬂuoroacetamide (BSTFA, Thermo, USA) containing 1% trimethylchlorosilane and addition of 10 μL of pyridine at 75 °C for 1 h. After evaporation until dryness, the extract was dissolved in 1 mL of dichloromethane (VWR International, USA). Two complementary conditions of GC–MS analysis were applied for detecting various types of organic molecular from low molecular weight acids to substances with a higher molecular weight such as palmitate esters. The analysis for low molecular weight acids was carried out on a GC-2010 gas chromatograph (Shimadzu) with a GC–MS-QP2010 (Shimadzu). The silica capillary, column length (30 m), CP5860 CP-Sil 8 CB low bleed/MS (Agilent) has an internal diameter 0.25 mm 123 661 Page 6 of 22 Eur. Phys. J. Plus (2021) 136:661

Fig. 3 ESI(−) FTICR mass spectrum of sample Tb104-1 with the corresponding van Krevelen diagram (bubble size refers to peak intensity) and pie chart representing the distribution of annotated features according to the heteroatom class

and 0.25 μm film thickness. The temperature programme of the oven was 50 °C for 1 min, 50–150 °C at 10 °C/min, 150–325 °C at 5 °C/min, and kept at 325 °C during 10 min. Tem- peratures of the ion source and the interface were fixed at 200 °C and 320 °C, respectively. Analytical data were treated with GC–MS solution software. Another condition for higher molecular weight analysis was performed using a 7890B gas chromatograph (Agilent Technologies, USA) with a 5977B GC MSD (Agilent Technologies). The silica capillary, column length (15 m), CP-Sil 5 CB low bleed/MS (Agilent J&W) has an internal diameter 0.25 mm and 0.1 μm film thickness. The temperature programme of the oven was 50 °C for 1 min, 50–270 °C at 10 °C/min, 270–320 °C at 5 °C/min, and kept at 320 °C during 5 min. Temperatures of the ion source and the interface were fixed at 230 °C and 320 °C, respectively. Analytical data were treated with Agilent Mass Hunter Qualitative Analysis Navigator B.08.00 software. Helium was used as carrier gas at a constant flow of 3 mL/min. The mass spectrometer was performed by electron impact (EI) mode at 70 eV ionization energy. Injection mode used is splitless, and mass spectrometer acquisition was done over m/z 50–650 mass range.

4Results

Table 1 shows the biomarkers, with their molecular formulae, either identiﬁed or putatively assigned by GC–MS or FTICR MS, respectively, for both samples. Table 2 shows the FTICR MS signals, for which archaeological biomarkers were found in both samples. It comprises 123 Eur. Phys. J. Plus (2021) 136:661 Page 7 of 22 661

Fig. 4 ESI(−) FTICR mass spectrum of sample Tb104-2 with the corresponding van Krevelen diagram (bubble size refers to peak intensity) and pie chart representing the distribution of annotated features according to the heteroatom class the experimental and calculated m/z, molecular assignments, relative intensity, and mass error (in ppm).

4.1 FTICR MS

Mass spectra achieved by ESI(-) FTICR MS for the two samples are shown in Figs. 3 and 4, as well as the corresponding van Krevelen diagrams and heteroatom class distributions. For sample Tb104-1, close to 88% of the annotations are related to CHO compound class. The remaining formulae are CHON class. The achieved van Krevelen diagram indicates the presence of lipids, terpenoids, and some phenolics within the CHO components. For Tb104- 2, all the features belong to CHO molecular series, with some lipids, terpenes, and a few phenolics. A deeper insight into these species was done by comparing the elemental formulae with those of biomarkers known to be often observed in organic archaeological samples [10, 11](Tables1 and 2). Thus, several hints with saturated and unsaturated fatty acids were found, as well as with some hydroxy- and diacids [19, 20]. These latter compounds are known biomarkers of unsaturated fatty acids that underwent oxidative process relative to ageing or burning [20, 21]. Possible monoacylglycerols (MAG) were found and one diacylglycerol (DAG). These different compounds suggest the presence of animal and/or vegetal fat. Among the putative fatty acids, even-numbered carbon species, from C16 to C28,were detected. The relative intensities for these respective species, given in Table 2, show that 123 661 Page 8 of 22 Eur. Phys. J. Plus (2021) 136:661 ) FTICRMS, respectively − Tb104-1FTICRMSGCMSFTICRMSGCMS Tb104-2 a m/z Candidate biomarker Formula Nonanoic acid—FA(C9:0)Decanoic acid—FA(C10:0)Dodecanoic acid—FA(C12:0)Tetradecanoic acid—FA(C14:0)Pentadecanoic acid—FA(C15:0)Hexadecanoic acid—FA(C16:0) C9H18O2Heptadecanoic acid—FA(C17:0) C10H20O2 C12H22O2Octadecanoic C14H28O2 acid—FA(C18:0) C15H30O2Nonadecanoic acid—FA(C19:0) 117 117Eicosanoic C16H32O2 acid—FA(C20:0) 117 117 C17H34O2Docosanoic acid—FA(C22:0) 117Tricosanoic acid—FA(C23:0) C18H36O2 117Tetracosanoic C19H38O2 acid—FA(C24:0) 117Hexacosanoic acid—FA(C26:0) 117 C20H40O2Octacosanoic acid—FA(C28:0) X – C22H44O2Triacontanoic acid—FA(C30:0) X C23H46O2Dotriacontanoic C24H48O2 acid—FA(C32:0) 117 X XTetratriacontanoic C26H52O2 acid—FA(C34:0) X 117 X XHexadecenoic X acid C28H56O2 – 117 X(9Z)-Octadecenoic C30H60O2 acid—FA(C18:1) X X C32H64O2 117 C34H68O2Octadecatrienoic X acid 117Octadecatetraenoic acid X X 117 X C18H34O3Eicosenoic 117 acid X 117 X X 117 X X X C16H30O3 X X X X C18H30O3 X C18H28O2 X X – X X X X X – X – X C20H38O3 X X X X X X X X – X X X X X X X X X X X X X Archaeological biomarker list identified or putatively assigned, based on the raw formulae, by GC–MS and ESI( Table 1 Fatty acids Octanoic acid—FA(C8:0) C8H16O2 117 X X 123 Eur. Phys. J. Plus (2021) 136:661 Page 9 of 22 661 Tb104-1FTICRMSGCMSFTICRMSGCMS Tb104-2 a m/z Candidate biomarkerDocosenoic acidTetracosenoic acidDihydroxyoctadecanoic acidDihydroxyeicosanoic acidDihydroxydocosanoic acid Formula Dihydroxytetracosanoic acidHydroxydecanoic acidHydroxyhexadecanoic C18H36O4 acidHydroxyoctadecanoic acid C22H42O3 C24H46O3 C20H40O4Hydroxyoctadecenoic acid C22H44O4 317Hydroxyeicosanoic C24H48O4 acid –Hydroxydocosanoic acid – – – C16H32O3 X – C10H20O3Succinic acid—DA(C4:0) C18H36O3Methyl succinic acid C18H34O3 X –Glutaric acid—DA(C5:0) – XMethyl X – glutaric C20H40O3 acidHexanedioic C22H44O3 – acid—DA(C6:0) XHeptanedioic acid—DA(C7:0) X X C4H6O4 –Octanedioic acid—DA(C8:0) – XNonanedioic acid—DA(C9:0) X C5H8O4Decanedioic acid—DA(C10:0) C5H8O4 X C6H10O4 147Hydroxynonanedioic acid X C7H12O4Hexadecanedioic X acid C6H10O4 C8H14O4 147 X X 147 73 C9H16O4 X X C10H18O4 73 X 147 73 73 C9H16O5 73 X X X X C16H30O4 X X X – X X X – X X X X X X X X X X X X X X X X X X X X X X X X continued Diacids Fumaric acid C4H4O4 245 X X Table 1 123 661 Page 10 of 22 Eur. Phys. J. Plus (2021) 136:661 Tb104-1FTICRMSGCMSFTICRMSGCMS Tb104-2 a m/z C40H80O2C42H84O2 257C44H88O2 257C46H92O2 257 257 X X X X X X X X ester—E42 ester—E44 ester—E46 ester—E40 Hexadecanoic acid, hexacosyl Hexadecanoic acid, octacosyl Hexadecanoic acid, triacontyl 1-TetracosanolTricosane—C23Pentacosane—C25Heptacosane—C27Nonacosane—C29Hentriacontane—C31Tritriacontane—C33 C24H50O C23H48 C25H52 C27H56 414 C29H60 C31H64 71 71 C33H68 71 71 71 71 X X X X X X X X X X X X X Candidate biomarkerOctadecenedioic acidOctadecanedioic acidEicosanedioic acidDocosanedioic acid Formula MAG—C16:0MAG—C16:1 C18H32O4MAG—C18:0 C18H34O4MAG—C18:1Diacylglycerol—C14:0 C16:0 – C20H38O4 C22H42O4 – – – X C19H38O4 C33H64O5 X C19H36O4 C21H42O4 X – – X C21H40O4 – – – X X X X X X X X X X X X X continued Glycerides Monoacylglycerol (MAG)—C14:0Waxes C17H34O4 – Hexadecanoic acid, tetracosyl X X Table 1 123 Eur. Phys. J. Plus (2021) 136:661 Page 11 of 22 661 Tb104-1FTICRMSGCMSFTICRMSGCMS Tb104-2 a m/z C10H10O3 – X X acid*/coniferaldehyde* Candidate biomarker Formula Hydroxycinnamic acidEugenolHomovanillinVanillic acidMethoxycinnamic Coniferyl alcohol*/vinyl syringol* C9H8O4Syringaldehyde*/veratric acid*Ferulic acidDihydroferulic acid C10H12O3Syringic – acid C9H10O3 C9H10O4 C10H12O2Norsimonelite – C8H8O4Dehydroferruginol – –Dehydro-7-dehydroabietic acid –Dehydroabietic acid 297 XSandaracopimaric acid C10H12O4Pimaric acid X C10H10O4 X XAbietic acid C20H26O2 X –Dihydroisopimaric C9H10O5 acid –7-Oxodehydroabietic acid C20H28O C18H22 237Methyldehydroabietate – C20H28O2 X C20H30O2 – X X X X 223 239 121 X C20H32O2 X C20H26O3 C20H30O2 X X C21H30O2 C20H30O2 X X X 243 X 253 X 121 239 – X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X continued Table 1 Aromatics Vanillin C8H8O3 – X X Diterpenes 18,19-Norabietane C20H36 159 X X 123 661 Page 12 of 22 Eur. Phys. J. Plus (2021) 136:661 Tb104-1FTICRMSGCMSFTICRMSGCMS Tb104-2 a m/z Specific fragment ions sought and obtained by GC–MS to identify biomarkers a C20H26O4 459 X X X X Hydroxydehydroabietic acid C20H28O3 237 X X X X Hydroxydehydroabietic acid C20H28O3 191 X X X X β α acid PhenanthreneMethylphenanthreneDimethylphenanthreneReteneTetrahydroretene C15H12 C16H14 C14H10 192 206 C18H23 178 C18H18 238 219 X X X X X X X X X Methoxydehydroabietic acidAgathic acidPinifolic acidAgatholic acetateBetulone*/allobetulone* C21H30O3LupeolBetulin –AllobetulinBetulinic acid C20H30O4 C30H48O2 C22H34O4 C20H32O4 X – – – – X C30H50O X X C30H50O2 C30H50O2 C30H48O3 189 189 189 – X X X X X X X X X X X X X X 15-Hydroxydehydroabietic acidTetradehydroabietic acidAgathalic acidAgatholic acid3-Hydroxyeperuic acid C20H28O37-Oxodehydroabietic acid (Me)15-Hydroxy-7-oxodehydroabietic 445 C22H22O3 C21H28O3 – X C20H34O3 C20H30O3 C20H32O3 253 – – – X X X X X X X X X X X X X 7- Candidate biomarker7- Formula continued hydrocar- bons Triterpenes Lupa-2,20(29)-dien-28-olPolyaromatic C30H48O 189 X X Table 1 Species labelled with * are isomers, which cannot be distinguished in FTICRMS. 123 Eur. Phys. J. Plus (2021) 136:661 Page 13 of 22 661 values, R.I. (%) z / m tical 0.10 4.04 0.10 0.14 0.06 0.33 0.04 0.13 0.02 1.35 Mass error (ppm) − − − − − − experimental [M-H] R.I. (%) m/z 0.10 1.57 255.23298 0.02 1.33 395.38941 0.11 1.64 0.13 6.61 367.35803 0.34 10.88 0.08 0.97 339.32676 0.28 2.03 0.06 0.16 269.24862 0.15 0.26 311.29555 0.01 0.54 0.02 28.08 315.25404 0.14 8.47 0.09 0.26 275.20164 0.05 1.69 Mass error (ppm) − − − − − − − − [M- − m/z experimental H] − theoretical [M-H] Formula [M] m/z C18H36O4 315.25408 315.25409 C18H28O2 275.20165 275.20168 acid acid Candidate compound Tb104-1 Tb104-2 Octacosanoic acidTriacontanoic acid C28H56O2 C30H60O2 423.42075 451.45205 423.42072 451.45203 0.08 0.05 0.60 0.17 423.42069 451.45210 0.15 0.62 Hexacosanoic acid C26H52O2 395.38945 395.38946 Tricosanoic acidTetracosanoic acid C23H46O2 C24H48O2 353.34250 367.35815 353.34248 367.35820 0.07 0.34 353.34245 0.15 0.65 Docosanoic acid C22H44O2 339.32685 339.32688 Heptadecanoic acid C17H34O2 269.24860 269.24862 Octadecanoic acidNonadecanoic acidEicosanoic C18H36O2 acid C19H38O2 283.26425 297.27990 C20H40O2 283.26425 311.29555 297.27989 311.29560 0.01 0.05 1.18 0.14 283.26421 ND 0.16 3.05 Dihydroxyoctadecanoic Eicosenoic acidDocosenoic acidTetracosenoic acid C20H38O3 C22H42O3 C24H46O3 309.27990 337.31120 365.34250 ND ND ND 309.27990 337.31116 365.34252 0.01 0.13 0.20 0.52 Octadecatrienoic acidOctadecatetraenoic C18H30O3 277.21730 ND 277.21729 0.05 0.59 Hexadecenoic acidOctadecenoic acid C16H30O3 C18H34O3 253.21730 281.24860 ND ND 253.21729 281.24861 0.06 0.12 List of archaeological biomarker candidates found by ESI(-) FTICR MS in samples Tb104-1 and Tb104-2, with their raw formula, experimental and theore mass error (in ppm), and relative intensity Table 2 Fatty acids Hexadecanoic acid C16H32O2 255.23295 255.23298 123 661 Page 14 of 22 Eur. Phys. J. Plus (2021) 136:661 R.I. (%) 0.02 0.39 0.16 0.32 0.02 0.27 0.03 1.56 0.02 0.33 Mass error (ppm) − − − − − − experimental [M-H] R.I. (%) m/z 0.05 1.17 343.28539 0.60 0.26 159.06631 0.06 1.10 327.29047 0.00 0.85 0.17 10.55 297.24349 0.10 4.38 0.07 2.79 299.25916 0.03 1.45 0.15 6.70 271.22786 0.03 5.59 0.03 1.13 173.08194 0.06 0.50 257.17584 Mass error (ppm) − − − − − − − − [M- − m/z experimental H] − theoretical [M-H] Formula [M] m/z C22H44O4 371.31668C24H48O4 399.34798 371.31664 399.34785 0.12 0.34 1.09 1.44 371.31667 399.34799 0.04 0.36 C20H40O4 343.28538 343.28540 C22H44O3 355.32177 355.32176 0.03 2.16 355.32173 0.11 1.51 C20H40O3 327.29047 327.29049 C18H34O3 297.24352 297.24357 C18H36O3 299.25917 299.25919 C16H32O3 271.22787 271.22791 C9H16O5 203.09250 203.09249 0.04 0.51 203.09250 0.00 0.25 acid acid acid acid acid acid acid acid acid Dihydroxydocosanoic Dihydroxytetracosanoic Candidate compound Tb104-1Dihydroxyeicosanoic Tb104-2 Hydroxydocosanoic Hydroxyeicosanoic Hydroxyoctadecenoic Hydroxyoctadecanoic Hydroxydecanoic acidHydroxyhexadecanoic C10H20O3 187.13397 187.13395 0.10 0.19 187.13396 0.05 0.29 Octanedioic acid C8H14O4 173.08193 173.08194 Nonanedioic acidDecanedioic acidHydroxynonanedioic C9H16O4 C10H18O4Tetradecanedioic acid 187.09758 201.11323 C14H26O4 187.09757 257.17583 201.11323 257.17585 0.07 0.02 2.63 1.03 187.09758 201.11323 0.02 0.02 2.14 0.94 continued Table 2 Diacids Heptanedioic acid C7H12O4 159.06628 159.06638 123 Eur. Phys. J. Plus (2021) 136:661 Page 15 of 22 661 R.I. (%) 0.07 0.12 0.02 0.54 0.04 0.22 0.13 0.15 0.10 0.28 0.13 0.19 0.10 0.19 0.01 0.23 Mass error (ppm) − − − − − − − − − experimental [M-H] R.I. (%) m/z 0.02 0.77 357.30105 0.02 4.98 341.26973 0.01 2.59 0.02 18.49 313.23835 0.27 8.00 0.08 7.22 311.22274 0.14 2.62 0.06 2.06 285.20712 0.05 1.15 0.04 0.18 163.07647 0.01 0.20 165.05574 0.10 0.30 167.03500 Mass error (ppm) − − − − − − − − [M- − m/z experimental H] − theoretical [M-H] Formula [M] m/z C9H8O4 163.04007 ND 163.04009 C33H64O5 539.46810 539.46800 0.18 0.19 ND C17H34O4 301.23843 301.23840 0.11 0.57 301.23843 0.01 0.19 C10H10O3 177.05572 177.05571 0.05 0.29 177.05572 acid C16:0 (MAG)—C14:0 acid/coniferaldehyde Hydroxycinnamic Diacylglycerol—C14:0 Docosanedioic acid C22H42O4 369.30103 369.30093 0.28 19.36 369.30096 0.20 7.36 MAG—C16:0MAG—C16:1MAG—C18:0 C19H38O4 C19H36O4 329.26973 C21H42O4 327.25408 357.30103 329.26966 327.25407 357.30104 0.22 0.04 0.61 1.34 329.26972 327.25406 0.04 0.07 0.15 0.52 MAG—C18:1 C21H40O4 355.28538 355.28536 0.07 1.33 355.28539 Eicosanedioic acid C20H38O4 341.26973 341.26974 Octadecanedioic acid C18H34O4 313.23843 313.23844 Octadecenedioic acid C18H32O4 311.22278 311.22281 Candidate compound Tb104-1Hexadecanedioic acid C16H30O4 285.20713 285.20715 Tb104-2 Eugenol C10H12O2 163.07645 163.07646 Homovanillin C9H10O3 165.05572 165.05572 Vanillic acid C8H8O4 167.03498 167.03500 Methoxycinnamic continued Table 2 Aromatics Vanillin C8H8O3 151.04007 151.04006 0.06 0.12 151.04008 Glycerides Monoacylglycerol 123 661 Page 16 of 22 Eur. Phys. J. Plus (2021) 136:661 R.I. (%) 0.01 0.57 Mass error (ppm) − − 0.02 10.31 experimental [M-H] − R.I. (%) m/z 0.21 0.44 313.21729 0.04 0.81 0.10 54.64 313.18084 0.25 100.00 0.18 0.85 303.23293 0.08 1.00 0.15 31.08 299.20160 0.18 44.83 0.26 1.46 297.18598 0.08 2.59 0.01 0.38 179.07137 − − − Mass error (ppm) − − − [M- − 0.48 6.47 301.21731 m/z experimental − H] − theoretical [M-H] C20H26O3 313.18092 313.18095 C20H32O2 303.23295 303.23301 Formula [M] m/z C20H28O2 299.20165 299.20170 C9H10O4 181.05063 181.05063C20H26O2 0.02 297.18600 297.18608 0.58 181.05063 0.02 0.44 C10H12O3 179.07137 179.07137 Hydroxydehydroabietic acid acid acid acid/sugiol acid dehydroabietic acid alcohol/vinyl syringol 15- Methyldehydrobietate C21H30O2 313.21730 313.21737 7-Oxodehydroabietic Dihydroisopimaric Sandaracopimaric/isopimaric C20H30O2 301.21730 301.21745 Dehydroabietic Syringaldehyde/veratric Ferulic acidDihydroferulic acidSyringic acid C10H12O4 C10H10O4Dehydro-7- 195.06628 193.05063 C9H10O5 195.06626 197.04555 193.05062 0.12 197.04553 0.07 0.10 0.50 0.91 195.06627 193.05063 0.42 0.07 197.04554 0.02 0.05 0.45 0.52 0.24 Candidate compound Tb104-1Coniferyl Tb104-2 continued acid Table 2 Communic/abietic Diterpenes Dehydroferruginol C20H28O 283.20674 ND 283.20672 0.07 0.43 123 Eur. Phys. J. Plus (2021) 136:661 Page 17 of 22 661 R.I. (%) 0.03 0.43 0.09 1.41 0.09 0.12 Mass error (ppm) − − − − experimental [M-H] R.I. (%) m/z 0.09 1.17 329.21212 0.30 1.62 0.17 39.23 329.17581 0.07 71.73 0.22 0.95 327.19653 0.12 1.64 0.13 2.48 319.22779 0.25 3.68 0.10 14.67 317.21214 0.25 21.27 Mass error (ppm) − − − − − [M- − 0.10 67.99 315.19650 0.22 90.13 m/z experimental − H] − theoretical [M-H] Formula [M] m/z C21H30O3 329.21222 329.21225 C20H26O4 329.17583 329.17589 C20H34O3 321.24352C21H28O3 327.19657 321.24350 327.19664 0.06 0.36 321.24352 0.00 0.40 C22H22O3 317.15470 ND 317.15467 0.11 0.36 acid oxodehydroabietic acid acid acid (Me) acid Agathic acidPinifolic acidAgatholic acetate C20H30O4 C22H34O4 C20H32O4 333.20713 361.23843 335.22278 333.20713 361.23841 335.22278 0.01 0.07 0.01 43.78 48.78 1.49 333.20704 335.22271 361.23840 0.28 0.22 0.09 40.61 39.93 0.71 Methoxydehydroabietic 15-Hydroxy-7- 3-Hydroxyeperuic 7-Oxodehydroabietic Agatholic acid C20H32O3 319.22787 319.22791 Tetradehydroabietic Agathalic acid C20H30O3 317.21222 317.21225 C20H28O3 315.19657 315.19660 Candidate compound Tb104-1 Tb104-2 Tetrahydroretene C18H23 237.16487 ND 237.16487 0.02 0.15 Betulinic acid C30H48O3 455.35307 455.35304 0.06 0.20 455.35307 0.00 0.20 Retene C18H18 233.13357 ND 233.13359 Betulin*/allobetulin* C30H50O2 441.37380 ND 441.37384 continued β not detected ,7- α Hydroxyde- hydroabietic acid hydrocar- bons Triterpenes Betulone*/allobetulone* C30H48O2 439.35815 ND 439.35817 Table 2 7- ND Polyaromatic 123 661 Page 18 of 22 Eur. Phys. J. Plus (2021) 136:661 the putative C24:0 acid has the highest intensity among the distribution extending from C20:0 to C30:0. These compounds are notably observed in beeswax [22, 23]. Moreover, similar observations were obtained by GC–MS, by Regert et al., on altered beeswax [24], where they also observed some phenolic compounds such as vanillic and ferulic acids attributed to chemical degradation of flavonoids initially present in raw beeswax. Thus, this suggests the presence of beeswax in both samples of study. Furthermore, several formulae corresponding to conifer resin biomarkers were assigned. These putative species correspond to abietic and pimaric acid derivatives [25–27]. From Table 2, the relative intensities of these diterpenic species are particularly high. For instance, in sample Tb104-2, the putative 7- oxodehydroabietic acid is the most intense peak, followed by the hydroxydehydroabietic acid, the 15-hydroxy-7-oxodehydroabietic acid, and the dehydroabietic acid. These species are known to be biomarkers of pine resin and result from the degradation of the abietic acid [26, 27]. This latter compound is prominent in fresh pine resin, whereas in these samples, its relative intensity is lower than its degradation product ones (Table 2). These observations agree with the works made by Colombini et al. [25] who also observed the increase of 7-oxodehydroabietic acid and the decrease of abietic acid, between recent and ancient resin. Moreover, some hints with betulin derivatives, in addition to diacids and hydroxy fatty acids, imply the presence of pitch from birch bark [28]. This latter substance is also characterized by long-chain α-ω-dicarboxylic acids and ω-hydroxycarboxylic acids. Indeed, these compounds are characteristic monomers of the suberin of birch bark [2]. It was demon- strated that in archaeological environment, and particularly in the presence of water, suberin can undergo depolymerization and oxidation reactions leading to the recovery of the free monomers [28]. Several formulae were found by FTICR MS and that can be attributed to these degradation products. The presence of woody products, such as the conifer resin and the pitch, is also observed with putative lignin derivatives, namely aromatic compounds, also observed on the van Krevelen diagram in the phenolics area. Moreover, putative retene and tetrahydroretene were also found, which are markers of a strong heat of the material [29]. The heating process could have been done as part of the waterproofing material production [30] of the bronze vessel or during ritual practices. The material assumptions done by the ESI(−) FTICR MS are then verified by GC–MS analyses.

4.2 Comparison with GC–MS results

Various organic archaeological biomarkers were identiﬁed on the total ion chromatograms, respectively, obtained from samples Tb104-1 and Tb104-2 (Fig. 5 and Table 1). Diterpenic acids such as dehydroabietic and pimaric acids were identiﬁed in large amount. They are biomarkers of pine tree resin belonging to the Pinaceae species [31]. One of the main peaks is 7-oxo-dehydroabietic acid brought with 15-hydroxy- dehydroabiatic and 15-hydroxy-dehydroabiatic acids. These compounds are well-known oxidation products originated from dehydroabietic acid [32] by ageing and environmental factors. Tetrahydroretene, 18,19-norabietane, and dehydroabietic acid methyl ester were also characterized only by GC–MS. The presence of these compounds can be explained by a thermal degradation corresponding to the heating of the resin with the distillation of softwood [33]. GC–MS analysis allowed identifying palmitate esters (E40, 42, 44, 46), which are beeswax biomarkers [22]. In addition, saturated fatty acids, such as palmitic acid (C16:0), even- numbered linear fatty acids (C24:0-C34:0), and alcohol C24, were observed. These compounds are from hydrolysis of palmitate esters [34]. Moreover, odd-numbered n-alkanes 123 Eur. Phys. J. Plus (2021) 136:661 Page 19 of 22 661

Counts

Counts Analyse condion 1 acid

(Me) Analyse condion 2 E40 Dehydroabiec E42 E44 E46 (Me) 7-oxo-dehydroabiec acid Dehydroabiec acid acid hydroxydehydroabiec acid 7β- Retene (C27) α-hydroxydehydroabiec acid FA (C16:0) 7 FA (C24:0) Time (min) Alkane -oxo-dehydroabiec 7 15-hydroxy-7-oxo-dehydroabiec acid (C25) hydroxydehydroabiec acid FA (C18:0) Sandaracopimaric acid - 15 Alkane (C29) Alkane (C31) Alkane FA (C28:0) FA (C18:1) phenanthrene Dehydro-7-dehydroabiec acid Dehydro-7-dehydroabiec FA (C26:0) (C33) FA (C22:0) Pimaric acid DA (4:0) DA (C9:0) FA (C30:0) acid Dihydroisopimaric acid 9,10-dihydroxyoctadecanoic acid Dimethyl Alkane (C23) FA (C9:0) Alkane FA (C32:0) Monostearine DA (C10:0) Betulin Tetrahydroretene DA (C8:0) acid DA (C7:0) DA (C6:0) 19-norabietane FA (C14:0) Norsimonelite norabietane 1-Tetracosanol 1-Tetracosanol FA (C10:0) - FA (C15:0) FA (C34:0) FA (C12:0) Allobetulin Lupeol 18 Vanillic Phenanthrene Methyl glutaric Lupa-2,20(29)-dien-28ol 2-methyl-Phenanthrene - Fumaric acid 2 Methyl succinic acid

Time (min)

Fig. 5 Total ion chromatogram of sample Tb104-1 from Cumae Grave 104 by GC–MS (FA fatty acid, DA dicarboxylic acid, E palmitate ester)

(C23-C33) were identified with n-heptacosane (C27) at very high level [24]. The identification of these species strongly suggests the presence of beeswax [34]. Lupeol and betulin were characterized in a very low amount. These degradation products such as lupa-2,20(29)-dien-28ol and allobetulin were also identified [35]. These triterpenoid- based compounds are known as birch bark markers [36], which were also suggested by FTICR MS. The series of linear α-ω-dicarboxylic acids (C16-C22) and ω-hydroxycarboxylic acids (C16-C22) were not identified by GC–MS. To detect such species, the sample would require hydrolysis to release these monomers [28]. Consequently, the hydroxy fatty acids and diacids putatively observed by FTICR MS, and to a lesser extent by GC–MS, are more probably related to unsaturated fatty acids, from animal and vegetal fatty material, that underwent ageing oxidative reactions.

5 Conclusion and perspective

Two analyses were applied on two organic residues from within a bronze vessel from Cumae ‘Grave 104 Artiaco’ in Italy. The non-targeted approach by FTICR MS appears to be as a complementary technique to the GC–MS. Indeed, it enables to detect more compounds with higher molecular weight and more polarity, which are not observable by GC–MS. Some matchings between measured FTICR MS signals and known archaeological biomarkers have been found, which allowed making hypotheses. Thus, the samples com- prise beeswax, conifer resin, birchbark, and fatty acids from vegetal oil and/or animal fat. These latter suggestions were veriﬁed and conﬁrmed by GC–MS. Moreover, FTICR MS technique allows achieving a global and rapid overview on sample composition without tedious sample preparation. As for the presence of dicarboxylic and hydroxycarboxylic acids compounds possibly originating from suberin, the analysis by FTCR MS provides more information than GC–MS. However, the analysis of GC–MS is necessary to ascertain the chemical composition of the sample. To suggest a possible function of the black substance under discussion, it is necessary to ponder the bronze vase whence the organic remains come from and a couple of technological features. 123 661 Page 20 of 22 Eur. Phys. J. Plus (2021) 136:661

As for the vase, it is the case of a small cauldron (closed shape) embellished with lotus flower-shaped handles and linked to a rather sophisticated stand (holmos). Both objects can be regarded as tokens reminding of the way of life of the deceased, sort of status-enhancing objects imbued with specific meanings. In particular, they form a set aiming at displaying and emphasizing the disposal of a considerable amount of beverage and, consequently, the relational capacity of their owner, who could afford to gather a large number of people, with whom he shared the consumption of liquids possibly during feasting activities. The multi-foils structure of the cauldron, and most of all the possibility that it had been joined at the top of the stand using rivets, could have entailed the desire to improve the waterproofing of the vase. It is likely that the bronzes with blended beeswax and pine tree resin, extracted by heating the pine wood (hence the occurrence of tetrahydroretene, 18,19- norabietane and dehydroabietic acid methyl ester), and produced a sticky malleable substance that could be evenly spread on the inner surface of the cauldron as a waterproof coating without having to fear much degradation even when filling it with alcoholic substances [37]. As commented on above, these speculations seem to be substantiated by some other bronze vases that clearly attest a quite widespread diffusion of this kind of technique in both time (end of the second millennium BC and late eighth century BC) and space (East Mediterranean and Italian Peninsula). This study allows suggesting the non-targeted FTCR MS analysis as a new approach for the global characterization of cultural heritage samples. This approach is worth pursuing on other archaeological samples in order to establish the effective result.

Acknowledgements The principal debts of gratitude are to the Director of the National Archaeological Museum in Naples Dr. P. Giulierini and his predecessor Dr. V. Sampaolo, who allowed Dr. Andrea Babbi to inspect the ﬁndings and take the micro-samples needed to carry out the archaeometrical analyses. The inspection of the funerary assemblage would not have been so fruitful without the support of many colleagues working at the Museum in Naples (G. Albano, G. Bifulco, R. Danise, C. Esposito, Dr. M.L. Giacco, Dr. T. Giove, Dr. M. Lista, A. Manillo, M.G. Martucci, Dr. L. Melillo, Dr. E. Santaniello, A. Scognamiglio, F. Stefanizzi, S. Venanzoni, and Dr. A. Villone). Furthermore, the study of the historical documents concerning the discovery has been possible thanks to the kindness of the Soprintendente of the Soprintendenza Speciale per i Beni Archeologici di Napoli e Pompei, Dr. T.E. Cinquantaquattro, and the people in charge for the archives (Dr. F. Lamberti Viscafè, M. Staiano, A. Capuccio).

Author contributions JH and PSK performed FTICR MS analysis. HF and ALD performed GC–MS analysis. AB performed the archaeological study. JH, HF and AB wrote the paper: JH (1.,3.1.,4.1.,5.), H.F. (1.,3.2.,4.2.,5.), AB (1., 2.,5.). All authors have given approval to the ﬁnal version of the manuscript.

Funding The study of the rich and very informative assemblage from Grave Artiaco 104 at Cumae would not have been possible without the generous ﬁnancial support granted to Dr. Andrea Babbi by the Gerda Henkel Stiftung, the Leibniz Forschungsinstitut für Archäologie des Römisch-Germanischen Zentralmuse- ums in Mainz, and the Gesellschaft der Freunde des Römisch-Germanischen Zentralmuseums in Mainz, respectively, in 2012 and 2013, 2014, and 2019.

Data availability statement This manuscript has associated data in a data repository. [Authors’ comment: All data in this manuscript are available upon request by contacting with the corresponding author.]

Declarations

Conﬂict of interest The authors declare no conﬂict of interest.

123 Eur. Phys. J. Plus (2021) 136:661 Page 21 of 22 661

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