Structure elucidation by LC-MS. Foreword

Dossier Structure elucidation by LC-MS ■ Structure elucidation by LC-MS. Foreword W.M.A. Niessen hyphen MassSpec Consultancy, De Wetstraat 8, 2332 XT Leiden, The Netherlands that many authors tried and still try to cover this by incorrectly calling the (de)protonated molecule a ‘(de)protonated molecular ion’ [4], this difference is obvious and important when one starts to interpret the MS–MS product-ion mass spectra. In addition, the interpretation of the product-ion mass spectra must actually be performed, because no spectral libraries were available which could be used to assist in the structure elucidation. Introduction The topic of this Analusis Dossier is structure elucidation by liquid chromatography – mass spectrometry (LC-MS). When LC-MS first came along in the mid 1970’s [1], one of the incentives and objectives was to develop a technique similar to the very successful gas chromatography – mass spectrometry (GC-MS), but capable of identifying especially those components which were not amenable to GC-MS. To that end, interfaces were developed, aiming at the application of electron ionisation (EI), like in GC-MS. Although these interfaces like the moving-belt and the particle-beam interface actually demonstrated that the applicability range of EI could be significantly extended, ironically enough these two interfaces did not achieved the major breakthroughs in the development of LC-MS. Fragmentation of even-electron ions In the past 14 years, ever since my practical introduction to MS–MS in 1986, the editor of this Dossier has developed a great interest in the interpretation of MS–MS product-ion mass spectra generated from the dissociation of even-electron ions. Like many colleagues in the field, I was often puzzled by the fragmentation observed. Sometimes, a useful strategy appeared to be: just cut the molecule at the bonds which are likely to be cleaved, play a little with proton shifts in order to get the correct m/z for the fragment, but do not pay much attention to the actual fragmentation mechanism. From a pragmatic point of view, this can be quite a successful approach, but certainly not always, and also often both frustrating and unsatisfactory. Major steps in the history of LC-MS were made using interfaces that only allow soft ionisation techniques, resulting in (de)protonated molecules with little or no fragmentation. Thermospray interfacing from the mid 1980 onwards for the first time gave a glimpse on what LC-MS could really do. With the broad implementation of the atmospheric-pressure ionization and interfacing, viz. (pneumatically-assisted) electrospray ionisation and interfacing (ESI) and the heated nebuliser in combination with atmosphericpressure chemical ionisation (APCI), LC-MS became a powerful technique which could even be used by less experienced people. However, in addition, the most frequent use of LC-MS today is quite different from the initial objective indicated above. LC-MS has significantly changed the impact of MS in a laboratory, because with LC-MS the mass spectrometer has entered both laboratories and application areas in a way which was certainly not foreseen at the beginning of its history in the mid 1970’s [2]. Especially the large interest in routine quantitative analysis and peptide and protein analysis was not anticipated. By studying many MS–MS product-ion mass spectra of a wide variety of molecules, one starts to get a better insight. With LC-MS being based on soft ionisation techniques, the development of LC-MS to some extent stimulated the developments in tandem mass spectrometry, especially in triple-quadrupole and ion-trap instruments. With the ability to fragment the even-electron protonated or deprotonated ions, generated in ESI and APCI, by means of collisioninduced dissociation (CID), the first steps in the direction to structure elucidation could be made. However, soon it was realised that in fact there is far less knowledge on the fragmentation of these even-electron ions compared to that of odd-electron ions [3], as generated in EI. Despite the fact ANALUSIS, 2000, 28, N° 10 Figure 1. Four-center fragmentation mechanism, illustrated for ethers, amines and amides. 885 © EDP Sciences, Wiley-VCH 2000 Article available at http://analusis.edpsciences.org or http://dx.doi.org/10.1051/analusis:2000280885 Dossier Structure elucidation by LC-MS Some major fragmentation reactions of a variety of compound classes can be rationalised by means of a four-center mechanism, as demonstrated in figure 1. However, especially in the last few years, several detailed studies were published on the actual fragmentation mechanisms of some protonated molecules, using derivatisation, isotope labelling, precursorion experiments, accurate mass determination, and other advanced MS methods. Some examples are related to the fragmentation of testosterone [5], propanolol [6], and polyamine spider toxins [7]. In fact, one of these studies [7] indicated that the four-center mechanism, though useful in the rationalisation of some fragmentation reactions, is actually not the correct mechanism for N-(4aminobutyl)-3-(4hydroxyphenyl)prop-2-enamide. Deuterium labelling experiments showed that the fragmentation in this case can better be explained from neighbouring-group participation (see Fig. 2). complex, can actually be applied to explain the most important dissociations in negative-ion MS-MS, as is for instance demonstrated in studies related to the fragmentation of ethylene glycol diacetates [9] and poly-hydroxy acids [10]. Fragmentation of even-electron negative-ions is generally considered to be even more complex than that of positive ions. However, the main fragmentation routes in the fragmentation of negative-ions are well classified by Bowie [8]. These fragmentation routes, some of which are indeed A powerful tool in elucidating fragmentation mechanisms is the ion trap, which allows step-wise and controlled fragmentation in multiple-stage MS-MS. A nice and recent example of its potential is demonstrated in the elucidation of the fragmentation pattern of dextromethorphan and some of its metabolites [12]. Progress in mass spectrometry instrumentation Developments in instrumentation also facilitate more advanced studies in fragmentation and mechanisms. An example is the current availability of routinely applicable micro- and nano-LC instrumentation, which more readily enables the use of deuterated solvents, for instance, to perform H/D exchange studies, e.g., for discrimination between N-oxides and hydroxylated compounds [11]. The ability of a quadrupole–time-of-flight hybrid instrument (Q-TOF) to provide accurate mass determination of the product ions in MS-MS is yet another powerful tool in structure elucidation, not so much in elucidation of the mechanism, but in understanding the fragments that are actually generated. An elegant example of the application of the Q-TOF is described by Hopfgartner et al. [13] for bosentan (...truncated)