10.7490/f1000research.1111314.1Eva M. ChildreyCristina E. StanciuChristopher J. EhrhardtQuantification of residual fatty acids in forensic microbiological samples using Direct Analysis in Real Time Mass Spectrometry (DART-MS)F1000Research2016The goal of this research was to develop a rapid, quantitative assay for determining the structure and relative abundance of residual fatty acids from forensically-relevant microbiological samples using Direct Analysis in Real Time Mass Spectrometry (DART-MS). Although cellular fatty acids have been used to characterize the taxonomy and growth conditions for an unknown organism, the analytical sensitivity of ambient mass spectrometry techniques and their compatibility with standard forensic collection practices has not been explored. To address this, we developed a fatty acid quantitation assay for Bacillus cereus and Bacillus thuringiensis spores using pentadecanoic acid to analyze membrane-derived, as well as free fatty acids found in different types of forensic samples. These included liquid suspensions of whole cells, dried spore material, spent medium and the supernatant fraction from water wash purification steps of spores.<br /><br /> DART-MS profiles of Bc14579, BcT-strain, BtHD1 and BtHD522 spores showed five distinct fatty acid biomarkers: 213 m/z, 227 m/z, 241 m/z, 267 m/z, and 269 m/z, which correspond to 13:0/13:0iso, 14:0/14:0iso, 15:0/15:0iso, 17:1 w5c, and 17:0/17:0 iso structures, respectively. The limit of detection for the DART-MS method was ~ 1x10-5 μg of fatty acid, equivalent to ~100 spore cells. The biomarkers from whole cell suspensions were observed in the culture medium after the spore cells had been removed, as well as in the supernatant of subsequent water washing purification steps. Fatty acid concentrations ranged between 0.002 μg/mL to 6 μg/mL for spent water supernatant samples, suggesting that Bacillus fatty acids are released into the culture medium and/or the water wash fraction during spore production and persist after the cells have been harvested. This has the potential to enhance forensic collection methods for microbiological samples since residual fatty acids may be used to identify surfaces or locations where biothreat agents were grown, even when cells are not present.