The Detection of Clandestine Human Burials using Bio-amine Decomposition Products

1. The Detection of Clandestine Human Burials using Bio-amine Decomposition Products Giorgio Blom John Cassella Alison Davidson Jamie Pringle 08-01-14

2. Do you think that the current methods to detect clandestine graves (hidden human remains) are good enough? • How do you think clandestine graves are discovered most commonly?

3. Introduction 1 2 5 3 4

4. Introduction • 663 homicides reported in 2012 • 200,000 people reported missing annually • 5,500 of those cases have a fatal outcome • Number of people missing due to a homicide is indeterminate

5. Introduction • Current procedures to detect clandestine graves • Limitations of these procedures • Ideal procedure to detect clandestine graves

6. Would the use of chemistry be able to aid in the detection of human remains? • Soil Matrix Interactions in a Taphonomic Environment: Forensic Considerations

7. Introduction • Putrescine and cadaverine are significant decomposition products • Putrescine and cadaverine are considered biomarkers for analytical instrument detection of clandestine graves • Prominent researchers did not detect these compounds in grave headspace or soil

8. Theory Table 1: Stages of decomposition.

9. Theory Table 2: Physical properties. • Biogenic amines: • Putrescine • Cadaverine Figure 2: Decarboxylation reaction of ornithine to produce putrescine. Figure 3: Decarboxylation reaction of lysine to produce cadaverine.

10. Theory • The use of gas chromatography • The use of a derivatisation agent Figure 5: Putrescine PFB derivate. Figure 6: Step by step derivatisation procedure

11. Sampling Procedure • A domestic pig (Sus domesticus scrofa) was as a proxy for a human cadaver due to the Human Tissue Act (2004) [Pringle et al. 2010]. • Soil moisture and decomposition fluids pass into lysimeter through buried micro pore end. • Lysimeter was emptied two days prior to sample collection, then resealed and placed under partial vacuum pressure [see Pringle et al. 2010]. • The samples were collected, placed in labelled plastic bottles and frozen. • Control samples were taken from a lysimeter 10m from the grave site. • Samples were collected from a grave site at Keele University from 07-12-07. Figure 7: Photograph of the grave site.

12. Comparison of Different Extraction Methods Table 3: Limit of detection of putrescine and cadaverine using different methods.

13. Analysis of Leachate Samples Figure 7: Detection of putrescine and cadaverine in the leachate samples over time.

14. High Performance Liquid Chromatography • Failure to detect certain compounds by GC could be due to: • Compounds are not volatile enough • Compounds are not easily extracted from aqueous environment due to their high polarity • GC data showed that volatile compounds such as bio-amines are not volatile when dissolved in water • HPLC is able to separate non volatile compounds • HPLC will be able to analyse aqueous samples therefore no extraction and/or derivatisation is needed which can affect quantification • HPLC data will aid in the comparison of non volatile compounds and optimisation of GC method

15. +/- 1 Month post burial +/- 6 Months post burial +/- 12 Months post burial +/- 24 Months post burial

16. +/- 2 weeks post burial 19-12-07 +/- 1.5 years post burial 18-06-09 +/- 3.3 years post burial 18-04-11

17. Conclusion • Putrescine and cadaverine were detected in the leachate samples from 181 days up to 902 days post burial by GC-FID. Methylamine was also detected around this time interval. • There is no linear relationship for the concentration of putrescine and cadaverine over time. • Putrescine and cadaverine are less volatile if dissolved in water. • Water soluble compounds are present in the leachate which absorb UV light at a wavelength of 260nm. These compounds are present in the gravesamples but not in the control samples and have different chemical compositions over time since burial. • More chemicals are detected using HPLC-MS in comparison to HPLC-UV/Vis.

18. Further work • Further analysis of leachate samples using LC-QTOF-MS (at the University of Huddersfield) to obtain more accurate results. • Optimisation of extraction and derivatisation methods. • Screening of leachatewith GC-MS for comparison with HPLC data.

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20. Thank you for listening Any Questions?

21. References • Vass et al. (2004). DecompositionalOdor Analysis Database. Journal of Forensic Sciences, 49, 760-769. • Dekeirsschieter et al. (2009). Cadaveric volatile organic compounds released by decaying pig carcasses (Susdomesticus L.) in different biotopes. Forensic Science International, 189,46-53. • Dent et al. (2004). Review of human decomposition in soil. Environmental geology,45, 576-585. • Pringle et al. (2010) Preliminary soilwater conductivity analysis to date clandestine burials of homicide victims. Forensic Science International,198, 126-133. • Ngim et al. (2000). Optimized procedures for analyzing primary alkylamines in wines by pentafluorobenzaldehyde derivatisation and GC-MS. Journal of agric. Food Chemistry, 48, 3311-3316.