Identification of spontaneous conversion products of unstable 2,4,6-trinitrotoluene metabolites, hydroxylamino-dinitrotoluenes, by combination of thin-layer chromatography and laser time-of-flight mass spectrometry

Abstract

Two kinds of hydroxylamino-dinitrotoluenes, 2-hydroxylamino-4, 6-dinitrotoluene (2HADNT) and 4-hydroxylamino-2, 6-dinitrotoluene (4HADNT), are known to be major metabolites produced from 2,4,6-trinitrotoluene (TNT) by bacteria. The amounts of 2HADNT and 4HADNT in distilled water are found to spontaneously decrease with time, albeit abiotic conditions, and many white precipitates are educed in the processes of this phenomenon; however, how these compounds are converted is unclear. We evinced the mystery of this phenomenon by using thin-layer chromatography (TLC) and laser time-of-flight mass spectrometry (TOFMS). TLC analyses in the spontaneous conversion processes of 2HADNT, 4HADNT, and 2HADNT plus 4HADNT demonstrats that three novel spots emerge on the TLC plate, respectively. These products are individually extracted into acetonitrile by collecting each spot. The purity of these extracts, which have retention times of 14.0, 17.7, and 15.4 min, is approximately 98%, judging from the results of high-performance liquid chromatographic analyses. The spontaneous conversion products of 2HADNT, 4HADNT, and 2HADNT plus 4HADNT are identified as 4,4′,6,6′-tetranitro-2,2′-azoxytoluene (2,2′AZT), 2,2′,6,6′-tetranitro-4,4′-azoxytoluene (4,4′AZT), and 4,2′,6,6′-tetranitro-2,4′-azoxytoluene (2,4′AZT) by obtaining their mass spectra with laser TOFMS. It is confirmed that most of the spontaneous conversion products are 2,2′AZT, 4,4′AZT, or 2,4′AZT, judging from the results of mass balance in the spontaneous conversion processes of 2HADNT, 4HADNT, and 2HADNT plus 4HADNT.