Optimizing the release and methylation of bacterial endospore dipicolinic acid.

摘要

Rapid, portable detection of biological threat agents such as Bacillus anthracis endospores ("spores") is extremely important given the real and perceived threats of bioterrorism. Gas chromatography-mass spectrometry (GC-MS) is an excellent general means for chemical detection, although special sample preparation and specialized equipment are required to employ GC-MS for detecting biological agents such as spores in the field.;By this process, Me2DPA yields from spores were quantitatively assessed for different combinations of tetramethylammonium hydroxide (TMA +OH-), sodium hydroxide (Na+OH -), and hydrogen monomethyl sulfate (H+MeSO 4-). The best reagent mixture was found by varying the combinations of the ions within the neutral or basic system containing TMA+, Na+, OH-, and MeSO 4- according to a novel scheme for design of experiments termed ionic mixtures design of experiments (IMDOE). A combination of the above ions was found that is near-quantitative in its methylation of DPA to Me2DPA; this mixture contained a 1:3:1:3 mole ratio of TMA +:Na+:OH-:MeSO4 -. This yield of Me2DPA was approximately a ten-fold increase over the best performance observed at the same conditions with tetramethylammonium hydroxide alone, the TCM reagent widely-used for GC.;The reactions involving MeSO4- and TMA + as methylating reagents, plus relevant hydrolysis and methylation reactions involving acid and base plus water and methanol, were investigated. An overall model is presented and mechanisms are proposed for reasons why basic mixtures of MeSO4- salts are more effective in methylating DPA compared to TMA+ salts at the conditions employed.;A GC sample introduction probe consisting of a helical wire that can be retracted inside a syringe needle, called a coiled wire filament (CWF), was employed as a simple, passively-heated means to introduce the mixture of spores plus reagents for thermochemolysis methylation (TCM) into the pre-heated GC inlet. There, reactions between spore biomarkers and the TCM reagent mixture occur between 250--290°C. At these conditions, monomethyl sulfate salt mixtures are convenient and efficient TCM reagents for the rapid conversion of a key unique spore biomarker, dipicolinic acid (DPA), to its dimethyl ester (Me2DPA).