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The reconstruction of the pharmacological and cosmetic practices of our ancestors is an important topic in the archaeological investigation. Drugs and cosmetics are important sources of information not only because they are a matter of daily life, but also because their social, cultural and economical implications. Under this light, the organic residues preserved in potsherds and various vessels may deliver key elements in elucidating our history. Due to chemical complexity of such organic residues, accurate and powerful analytical chemical techniques are required.
The present work focuses on the application of different mass spectrometry approaches, mainly based on soft ionization techniques, aiming to reconstruct the original material used for the formulation of drugs, ointments and cosmetics in the past, through the identification of lipid fractions of organic residues found in archaeological artefacts.
In order to cover in detail this analytical challenge, samples from different ages and location were studied. In particular, we investigated the organic residues found in vessels having the well known shape of kohl container from the ancient cities of Assiut (Egypt, Middle Kingdom), beehive remains from the Etruscan site of Forcello (Italy, 500-480 B.C) and various ointments of late monastic provenance (Spain, XIV- XVII A.C.)
The first goal of the research was to asses and validate a soft ionization - mass spectrometry approach for recognizing the original materials in archaeological residues by triacylglycerols (TAGs) identification. In fact, despite the chemotaxonomic potential of TAGs in determining the origin of lipid substances, few are the related applications reported in literature.
Therefore, we performed a comparative study on the TAGs distributions found in the archaeological residues and in different natural lipid-containing materials used as references. Thanks to the use of recently introduced non-porous core-particles stationary phases , we have been able to develop a new liquid chromatographic – mass spectrometry method capable to resolve complex lipid mixtures in very short time. The very good chromatographic performances prevented co-elution and enabled the use of database containing more than 500 TAGs molecular formula for the research of TAGs in the samples. Two different ionization sources were employed (APCI and ESI) and their performance were compared. In particular, their use appeared complementary, due to their different sensitivity against unsaturated TAGs.
Analysis were carried out also by GC-MS (with an electron impact ionization) which is currently the most employed technique for the characterization of lipids in archaeological residues. A new derivatization procedure based on the use of switchable polarity solvents (SPS) was tested with the aim to identify specific conditions for ancient residues treatment. In fact, due to general scarcity of archaeological residues for the analysis and the need of performing a broad range of different characterizations on the same material, the development of new methods capable to separate all the different components in few steps, minimizing the loss of material, is particularly important.
The analysis carried on the historical ointments residues gave us the possibility to evaluate the performance of the methods in a unprecedented and controlled aging experiment. This was possible thanks to the presence of latin label on the vessels, indicating the original recipes adopted to prepare the ointments, retrieved in extraordinary detail from historical bibliographic sources. This study gave us also the possibility to gain important indication for reconstructing the pathways of degradation of lipid materials in complex compositions. Hydrolysis and oxidation products were found as predominant fraction in the residues. Nonetheless, the original lipid material were still recognizable thanks to the use of LC-MS, because the surviving TAGs were very selectively separated from the other components and identified. Artificial aging treatments on TAG standards and the reference material showed the absence of inter-conversion phenomena from saturated to unsaturated TAGs confirming the validity of TAGs as biomarkers.
Noteworthy, the characterizations of the lipid components of the historical ointments obtained by LC-MS were in agreement with those obtained by the application of GC-MS and other analytical techniques generally adopted in the study of the archaeological residues (such as FTIR). Consistency of the TAG profiling approach by LC-MS in recognizing the original lipid materials was also acknowledged through a large scale round robin test involving eleven laboratory with consolidate experience in the analysis of archeological objects. In particular, we showed as LC-MS is the most powerful technique in determining the origin of oily and fatty substances. At the same time, the characterisation of these ointments enabled us to give precious contributions in the study of an important historical period, in which the monastic pharmaceutical practices were changing from a classical “humoral medicine” to the application of the experimental approach.
Nice results were obtained also by analyzing the khol remains from the ancient Egyptian city of Assiut. Thanks to the complementary use of information regarding TAG distributions and the structural feature of some specific TAGs, we have been able to distinguish vegetable oil from animal fat. In some cases, we have been also able to identify very precisely the provenance of the fatty materials. Very difficult was instead the characterisation of beehive remains found in the Etruscan site of Forcello, which is thought to be one of the first archaeological evidences of beekeeping practice prior to the classical age. The problems were related probably to contaminations of the surface, due to the firing process encountered by the archaeological site after his apogee (fifth century BC).