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Crime buffs will be familiar with the term adipocere which turns up regularly in our TV dramas. This strange word literally means fat wax, from the Latin words adipis (fat) and cera (wax) and it refers to the waxy substance that is produced by some corpses during decomposition. Also know as grave wax, mortuary wax or corpse wax, it is a result of the decomposition of adipose tissue but is not produced in every case. Its formation depends on several environmental factors, such as temperature, burial conditions and the materials around the body. Adipocere can be produced quite quickly following death, even before the body has undergone much decomposition and this waxy coating can lead to mummification, so that the main features of the body are preserved. We have to thank it for giving us the famous iceman, named Otzi, discovered in the Italian Alps, as well as ancient bog bodies. In the world of criminal forensic science, bodies that have been submerged in water often have adipocere present and its discovery can give vital clues to medical examiners about the body and its surrounds. It is produced when triacylglycerides in the adipose tissue are converted enzymatically to unsaturated and saturated fatty acids, the former being reduced further to saturated acids. The proportion of the various acids in this mixture changes as the adipocere is formed. The major fatty acids present in order of abundance are palmitic (C16:0), stearic (C18:0) and myristic acid (C14:0) and their ratios are characteristic. To determine these ratios, the acids have to be extracted from the adipocere before analysis. The traditional methods use thin-layer chromatography (TLC), liquid extraction and column chromatography but these are known to suffer from low recovery and relatively large solvent volumes. In addition, TLC is fairly time consuming and this can lead to air oxidation of the polyunsaturated acids present. To counter these shortcomings, scientists from the University of Technology, Sydney, have devised a rapid solid-phase extraction (SPE) method which produces fatty acids ready for analysis by GC/MS. The procedure was optimised with standard solutions of unsaturated fatty acids, using an aminopropyl SPE cartridge. Neutral lipids were eluted first, followed by the fatty acids. Extraction was not entirely straightforward. When blank samples of hexane were processed, fatty acid contamination by palmitic and stearic acids was found in all washings and eluates. This was narrowed down to the use of polypropylene collection tubes. Replacement with glass reduced the level to about 15 µg/mL, which is acceptable compared with the typically adipocere concentrations of 500-1000 µg/mL. The fatty acids were derivatised to the trimethylsilyl esters which were analysed by GC/MS with electron ionisation. Saturated and unsaturated fatty acids were identified from their retention times and by comparing the mass spectra with those in a library. The relative proportions of the fatty acids were determined from the relative response factors. Recoveries were higher than 90% with coefficients of variation less than 10%, which is acceptable. Linear standard curves were obtained with limits of quantification at the ppm level. The process was applied to homemade adipocere prepared in the lab by smearing pig adipose tissue with internal pig gut flora as faeces and immersing in water in an airtight container for 15 months. Samples were collected every 3 months and the lipids were extracted by liquid extraction after application of heptadecanoic acid (C17:0) as an internal standard. SPE was then applied to obtain the free fatty acid fraction. The mono-unsaturated oleic acid (C18:1) was the most abundant fatty acid in fresh pig adipose tissue, with smaller amounts of the C16:0, C17:0 and C18:0 fatty acids. However, this situation was gradually reversed as the adipocere was formed over 15 months, with the concentrations moving over the range 33.2-8.7% (C18:1), 2.8-4.1% (C14:0), 23.4-55.2% (C16:0) and 13.2-23.4% (C18:0). This is consistent with published findings. In addition, 10-hydroxystearic acid, which is absent from fresh tissue, increased to 9.9% after 15 months. This is a minor but characteristic component of adipocere and its detection confirms the suitability of the SPE procedure and the GC/MS analysis. The method has good recovery and reproducibility and is more rapid and less time consuming than published methods for the extraction and analysis of fatty acids in adipocere, lending itself to routine and high throughput analysis in forensic environments. Related links:
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 Otzi, the iceman |
