Neutral loss experiment

Not all tandem MS experiments are possible (no neutral loss experiments)... 

Figure 4.18. Example of neutral loss experiment showing loss of pyroglutamic acid moiety.

Figure 4.20. Schematic of neutral loss experiments with triple-quadrupole mass spectrometer.

Figure 4.22. Schematic of exact mass neutral loss experiments with Q-TOFmass spectrometer.

Use of a triple quadrupole mass spectrometer with ES-source opens up additional possibilities for the characterization of libraries. The parent ion scan can be used if all compounds of a library form the same fragment ion. In addition, the neutral loss experiment allows the detection of all molecules undergoing the same mass loss. In both cases the adequate fragmentation of library compounds must be ensured by the appropriate choice of... 

Figure 17.15. Characterization of library PYL-1 by the neutral loss experiment (Am = -43 amu).

Neutral loss experiment. The two mass analysers are set to detect a constant neutral loss. For example, the first mass analyser is scanned from m/z 37 to 300, at the same time as the second mass analyser is scanned from m/z 20 to 283. In this example the neutral loss of 17 mass units may represent a series of nitrocompounds losing OH. 

The main MS/MS techniques are precursor ion, product ion, and neutral loss. In addition, it is possible to carry out MSn experiments using an ion trap (Kang and others 2007). In this context, de Rijke and others (2003) carried out a study with 15 flavonoids, comparing different ionization sources and different analyzers. Among the results, the authors showed that the main fragmentations observed in the MS spectra on the ion trap, or the tandem MS spectra on the triple-quadrupole, were generally the same. 

Various instruments allow working in special regimes to detect only metastable ions (MI spectra). The conditions of experiments in this case are the same as for the MS/MS experiments, but without collision activation. Any sort of spectrum (daughter ions, parent ions, constant neutral losses) may be generated this way. These spectra are used to establish the pathways of fragmentation, to resolve structural problems. However, the abundance of the metastable signals and even their presence or absence in the spectrum depends on the energy of the parent ions. Therefore, in contrast to CID (see Chapter 3) spectra the difference in MI spectra of two parent ions does not confirm their different structures. 

Fig. 11.16. Representation of three tandem mass spectrometry (MS/MS) scan modes illustrated for a triple quadrupole instrument configuration. The top panel shows the attributes of the popular and prevalent product ion CID experiment. The first mass filter is held at a constant m/z value transmitting only ions of a single mlz value into the collision region. Conversion of a portion of translational energy into internal energy in the collision event results in excitation of the mass-selected ions, followed by unimolecular dissociation. The spectrum of product ions is recorded by scanning the second mass filter (commonly referred to as Q3 ). The center panel illustrates the precursor ion CID experiment. Ions of all mlz values are transmitted sequentially into the collision region as the first analyzer (Ql) is scanned. Only dissociation processes that generate product ions of a specific mlz ratio are transmitted by Q3 to the detector. The lower panel shows the constant neutral loss CID experiment. Both mass analyzers are scanned simultaneously, at the same rate, and at a constant mlz offset. The mlz offset is selected on the basis of known neutral elimination products (e.g., H20, NH3, CH3COOH, etc.) that may be particularly diagnostic of one or more compound classes that may be present in a sample mixture. The utility of the two compound class-specific scans (precursor ion and neutral loss) is illustrated in Fig. 11.17.

Several years later, the next step in the application of MS-MS for mixture analysis was developed by Hunt et al. [3-5] who described a master scheme for the direct analysis of organic compounds in environmental samples using soft chemical ionisation (Cl) to perform product, parent and neutral loss MS-MS experiments for identification [6,7]. The breakthrough in LC-MS was the development of soft ionisation techniques, e.g. desorption ionisation (continuous flow-fast atom bombardment (CF-FAB), secondary ion mass spectrometry (SIMS) or laser desorption (LD)), and nebulisation ionisation techniques such as thermospray ionisation (TSI), and atmospheric pressure ionisation (API) techniques such as atmospheric pressure chemical ionisation (APCI), and electrospray ionisation (ESI). 

Tandem mass spectrometry (MS-MS) is a term which covers a number of techniques in which one stage of mass spectrometry, not necessarily the first, is used to isolate an ion of interest and a second stage is then used to probe the relationship of this ion with others from which it may have been generated or which it may generate on decomposition. The two stages of mass spectrometry are related in specific ways in order to provide the desired analytical information. There are a large number of different MS-MS experiments that can be carried out [9, 10] but the four most widely used are (i) the product-ion scan, (ii) the precursor-ion scan, (iii) the constant-neutral-loss scan, and (iv) selected decomposition monitoring. 

Ion traps are tandem-in-time instruments, i.e., they perform the steps of precursor ion selection, ion activation and acquisition of fragment ion spectra in the very same place. This advantageous property allows the multiple use of a single QTT to perform not only MS but also MS and higher order MS experiments - indeed a very economic concept. Depending on the abundance of the initial precursor ion, its fragmentation behavior - and of course, on the performance of the QIT - MS experiments are possible. [138] However, in contrast to tandem-in-space instruments, tandem-in-time instruments do not support constant neutral loss and precursor ion scans. 

Different MS MS experiments of product ion scan, precursor ion scan, and neutral loss scan modes of selected flavonoids can be carried out in order to confirm the structure of flavonoids previously detected by the full-scan mode. In the product ion scan experiments, MS MS product ions can be produced by CID of selected precursor ions in the collision cell of the triple-quadrupole mass spectrometer (Q2) and mass analyzed using the second analyzer of the instrument (Q3). However, in the precursor ion scan experiments, Q1 scans over all possible precursors of the selected ion in Q3 of the triple quadrupole. Finally, in neutral loss... 

A constant neutral loss (CNL) MS experiment was then used to identify components in the crude ethyl acetate fraction of P. pensylvanicum with structures likely to be homologous to the vanicosides.32 Since the ions derived from a loss of 146 amu (the ketene species derived from the p-coumaryl esters) were the most intense fragment ions in the spectra of... 

Constant Neutral Loss Scan (MS/MS) Determination of precursor/product ion combinations that exhibit a specific, characteristic loss of a portion of a molecular ion. Particularly useful when the characteristic species (loss) is neutral and cannot be detected directly by the mass spectrometer. Analysis of glutathione conjugates via neutral loss of 129 is an example. For the purposes of this book, NLS is used to describe these types of MS/MS experiments. 

A new generation of linear ion trap mass spectrometers has been developed and exhibits increased performance compared to traditional three-dimensional (3D) ion traps (Hopfgartner et al., 2003 Douglas et al., 2005). A further evolution of the triple-quadrupole family and ion trap class of instruments is the production of the hybrid triple-quadrupole/linear ion trap (QQQ/LIT) platform. Hybrid instruments of this nature allow for operation in space and not just in time when performing MS/MS analysis. This feature allows for increased performance compared to classical ion traps. A powerful combination possible on a hybrid LIT/QQQ instrument is the ability to use highly sensitive and selective precursor ion, constant neutral loss, and multi-MRM as a survey scan for dependent LIT MS/MS. Compared to a simple MS experiment, these comprehensive triple-quadrupole and LIT modes can be more complex to setup. 

With the strong sensitivity of multiple MRMs as a method of metabolite detection, one can assume that this method is superior to all others. However, like any analytical technique, a number of limitations exist for this type of experiment. First, any metabolites that are not predicted will not be detected. Single MS, precursor, and neutral loss scans are not subject to this limitation. Second, the more modifications that take place on the parent, the more number permutations of MRMs are required. For example, to detect an oxidation and methylation metabolite, as shown in the following table, four theoretical MRMs would be required ... 

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