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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
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 Nâ‚‚ 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 microbiot