Stone-age sediments’ secrets seen by GC-MS

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  • Published: Dec 1, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Gas Chromatography
thumbnail image: Stone-age sediments’ secrets seen by GC-MS

Early manure piles give information on domestic animals

In a number of prehistoric societies in the Mediterranean region, manure was allowed to accumulate in heaps in animal shelters; these piles were burnt at intervals, giving rise to characteristic sediments known as ‘fumiers’. Some chemical compounds can remain in these sediments over the millennia, especially lipids, whose hydrophobic properties mean they are not readily washed out.

The University of the Basque Country researchers examined samples from fumier sediments from a rock shelter in northern Spain. Twenty five samples, dating from the Neolithic to the Bronze Age, were taken. The scientists aimed to extract 5β-sterols and bile acids from the samples, since the relative amounts of some of these compounds can be used to determine whether the source of the manure was ruminants or pigs/humans. Existing extraction methods for these compounds are lengthy, so a new microwave-assisted extraction (MAE) technique was devised, which was optimised using a central composite design with Statgraphics Centurion XV software.

Lipids from ancient manure examined by GC-MS

Samples were placed in microwave vessels, then spiked with a mixture of three deuterated lipids (used to estimate recoveries/matrix effects). Microwave extraction was carried out using a 2:1 mixture of dichloromethane and methanol at 150 °C for 10 minutes. Solids were removed by centrifugation and the solution was evaporated. Saponification was carried out using KOH in aqueous methanol in order to free any conjugated lipids. After saponification, the solution was acidified and extracted with dichloromethane. The dichloromethane was then evaporated and the residue was taken up into ethyl acetate. The compounds of interest were absorbed onto a silica solid-phase extraction (SPE) cartridge. They were extracted from the cartridge with acetone, which was then evaporated. The lipids were derivatised to give their trimethylsilyl derivatives using BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) in pyridine.

GC was carried out with an Agilent 7890 system fitted with an HP 5MS column. Helium was employed as the carrier gas with a flow rate of 1.5 mL/min. The GC was kept at 180 °C for 5 minutes, then heated to 300 °C at 2 °C/minute and lastly kept at 300 °C for 7 minutes. An Agilent 5973 mass-selective detector was used, with data being acquired in both the scan and selective ion monitoring (SIM) modes. Appropriate quantification and qualification ions were found for all the compounds examined.

Spiking experiments with standards showed that the method gave good linearity and recoveries for the ten compounds of interest. The recoveries of the deuterated compounds were used to compensate for the relatively small matrix effects. The limits of quantification (LOQs) for the ten lipids ranged from 1.5 to 33.0 ng/g.

Looking at the ratios of 5β-sterols, it was clear that five of the samples were of ruminant origin and some other samples were possibly of ruminant origin. Principal component analysis (PCA) was used to differentiate between samples of ruminant origin and those that were unclassifiable. Based on the PCA results, a further nine samples could be classified as being derived from ruminants, making fourteen in all. The remaining unclassifiable samples were mainly those (white samples) where burning had been the most intense. No samples could be ascribed to pigs/humans. It was clear that the site had been used as a pen for ruminant animals over a period of 2000 years, from the Neolithic to the Bronze Age, with some gaps when it was not in use.

New extraction GC-MS protocol useful for archaeology

The carefully optimised extraction technique, along with GC-MS and PCA, gave useful information on lipid compounds and hence about prehistoric livestock. The use of biomarkers in archaeology is a valuable tool for the discipline, allowing guesswork to be replaced by reliable data. It would be interesting to apply this method to other sites.

Related Links

Journal of Separation Science, 2017, Early View Paper. Gea et al. Characterization of ancient lipids in prehistoric organic residues: Chemical evidence of livestock-pens in rock-shelters since early neolithic to bronze age.

World Archaeology, 2009, 41, 191-214. Angelucci et al. Shepherds and Karst: The use of caves and rock-shelters in the Mediterranean region during the Neolithic.

Archaeometry, 2008, 50, 895-924. Evershed et al. Organic residue analysis in archaeology: The archaeological biomarker revolution.

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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