[en] This paper reports detailed studies on the internal energy of ions formed in matrix-assisted laser desorption/ionization (MALDI) using delayed extraction MALDI-time-of-flight (TOF) and atmospheric pressure (AP) MALDI mass spectrometric (MS) methods. We use benzylpyridinium cations as internal energy probes. Our study reveals three distinct contributions to internal energy build-up in vacuum-MALDI (classical MALDI-TOF), each having different effects on ion fragmentation. Some fragments are formed before ion extraction (i.e. no more than 100 ns after the laser impact), and they are therefore well resolved and recorded as sharp signals in the MALDI-TOFMS scan. This prompt fragmentation can have two origins: (i) in-plume thermal activation, presumably always present, and (ii) in-plume chemical activation, in the course of reactions with hydrogen radicals. In addition to early internal energy build-up associated with these well-resolved promptly formed fragments, a broad peak slightly offset to higher masses could be detected corresponding to fragments formed after the extraction has started. This second signal corresponds to a third source of internal energy in MALDI ions, (iii) the extraction-induced collisional activation of the ions with the neutral components of the plume. These three contributions are difficult to quantify in vacuum-MALDI, because of the combined influence of several parameters (nature of the matrix, spot-to-spot variability, total laser exposure, delay time, acceleration voltage) on extraction-induced fragmentation. AP-MALDI, on the other hand, has two advantages for comparative studies of analyte fragmentation. First, extraction-induced fragmentation is absent, and only the contributions of early plume activation remain. Second, the reproducibility is far better than in vacuum-MALDI. AP-MALDI is therefore expected to shed new light on the early steps of the MALDI process. Copyright (C) 2004 John Wiley Sons, Ltd.
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