In the field of nucleic acid analysis, pyrolysis mass spectrometry techniques have produced intriguing, sometimes paradoxical results. The work of Wiebers et al. (refs. 15 - 17) shows that direct probe pyrolysis produces large characteristic fragments, apparently representing intact bases or even more or less severely dehydrated nucleosides. As a result, they successfully applied this technique to the characterisation of unusual base moieties in nucleic acids. However, when applying Curie-point pyrolysis techniques to the analysis of nucleic acids, Meuzelaar et al. (ref. 48) obtained pyrolysis mass spectra showing only ribose and/or deoxy-ribose fragments (see Figure 4). Although this enabled a clear differentiation to be made between RNA and DNA samples, the complete lack of signals derived from the base moieties was both disappointing and puzzling. Later studies by Posthumus et al. (ref. 72) and Schulten et al. (ref. 62), employing high-resolution field ionisation and field desorption techniques, respectively, as well as laser Py-MS studies by Kistemaker et al. (ref. 29) (see Figure 5) and direct probe CID measurements by Levsen and Schulten (ref. 75) succeeded in detailing the degradation behaviour of nucleic acids.
The main mechanism taking place at temperatures as low as 180°C appears to be the expulsion of the sugar moiety with the simultaneous formation of base-phosphate condensates (ref. 72). Under standard Curie-point Py-MS conditions only the sugar moiety is detected, since the base-phosphate complex is trapped intact on the relatively cold wall of the reaction chamber. In direct probe pyrolysis, however, the base-phosphate complex is apparently further pyrolysed, yielding the base fragments. Alternatively, some of the base-phosphate complexes may conceivably reach the ion source without further wall collisions and then may fragment during electron impact ionisation, again producing ions corresponding to the base moieties. A second important reaction mechanism appears to be the formation of polyphosphates (ref. 62) with the expulsion of intact nucleosides. Again, these nucleosides can only contribute to the mass spectrum by secondary pyrolysis into more volatile fragments, or by direct diffusion into the ion source without further wall collisions.
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