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Validation of a novel extraction method for studying hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) biodegradation by ruminal microbiota

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  • A simple, fast liquid-liquid extraction method was developed for studying hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) biodegradation using small sample volumes. The method was tested in vitro with anaerobic incubations of RDX with whole rumen fluid (WRF) and a commercial S. acetigenes strain in methanogenic media for RDX. Additionally, validation experiments were conducted in deionized water in order to show applicability towards various aqueous matrices. Conditions for extraction were as follows: 300 μL of sample were mixed with an equal volume of a 0.34 M ammonium hydroxide solution to reach a basic pH, extracted with a hexane/ethyl acetate 1:1 (v/v) solution (1 mL) and shaken vigorously for 10 secs. The resulting organic phase was transferred, then dried under a constant flow of N₂ and reconstituted with acetonitrile (300μL) for HPLC- UV and LC-MS/MS analysis. Percent recovery values were obtained (83-101%) in all matrices for RDX. In WRF (n =3 animals), RDX degradation was observed with almost 100% elimination of RDX after 4 h. The five nitroso and ring cleavage metabolites (were observed by mass spectrometry. Liquid cultures of S. acetigenes did not show significant RDX biodegradation activity. Deionized water extractions indicated acceptable recoveries with low variability, suggesting suitability of the method for aqueous matrices. Overall, the new method demonstrated acceptable efficiency and reproducibility across three matrices, providing an advantageous alternative for studies where complex matrices and small volume samples are in use.
  • Keywords: HPLC-UV, Biodegradation, RDX, Organic extraction, Ovine ruminal microbiota
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  • Giarrizzo, J., Murty, L., Tanaree, D., Walker, K., & Craig, A. (2013). Validation of a novel extraction method for studying hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) biodegradation by ruminal microbiota. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 925, 70-75. doi:10.1016/j.jchromb.2013.02.033
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  • 925
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  • This research was supported in part the Oregon Agricultural Experiment Station project no. ORE00871 and the US Department of Agriculture, Agriculture Research Service project no. 50-1265-6-076.
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