Tandem accurate mass measurement

Geometries composing of an oaTOF as MS2 bear the advantage that advanced TOFs offer accurate mass measurements close the the accuracy of magnetic sector instruments. Currently, QqTOF systems can be regarded as the commercially most successful hybrid. While the linear quadrupole serves as MSI in MS/MS experiments, it is operated in RF-only mode when tandem MS is not intended, because... 

In tandem MS mode, because the product ions are recorded with the same TOF mass analyzers as in full scan mode, the same high resolution and mass accuracy is obtained. Isolation of the precursor ion can be performed either at unit mass resolution or at 2-3 m/z units for multiply charged ions. Accurate mass measurements of the elemental composition of product ions greatly facilitate spectra interpretation and the main applications are peptide analysis and metabolite identification using electrospray iomzation [68]. In TOF mass analyzers accurate mass determination can be affected by various parameters such as (i) ion intensities, (ii) room temperature or (iii) detector dead time. Interestingly, the mass spectrum can be recalibrated post-acquisition using the mass of a known ion (lock mass). The lock mass can be a cluster ion in full scan mode or the residual precursor ion in the product ion mode. For LC-MS analysis a dual spray (LockSpray) source has been described, which allows the continuous introduction of a reference analyte into the mass spectrometer for improved accurate mass measurements [69]. The versatile precursor ion scan, another specific feature of the triple quadrupole, is maintained in the QqTOF instrument. However, in pre-... 

The TOF mass analyzer has a low duty cycle, and the combination with an ion accumulation device such as an ion trap is therefore very advantageous. It offers also MS capabilities with accurate mass measurement. In all acquisition modes, the ions are accelerated into the time of flight for mass analysis. Various other hybrid mass spectrometers with TOF have been described, including quadrupole ion trap [70] and linear ion trap [58]. High energy tandem mass spectrometry can be performed on TOF-TOF mass spectrometers [71, 72]. 

Superior sensitivity, efficiency, and specificity have made high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS), the predominant analytical technique for characterization and quantitative analysis of metabolites (Kostiainen et al., 2003 Ma et al., 2006 Prakash et al., 2007). Ion trap, triple-quadrupole, and quadmpole time-of-flight (Q-TOF) mass spectrometers are routinely used to profile and characterize metabolites in plasma and excreta (Ma et al., 2006). The combination of scan types and features available on mass spectrometers of different design (product ion, MS", neutral loss, precursor ion scans, accurate mass measurements) allows identification and characterization of putative and unexpected metabolites with or without little prior knowledge of biotransformation pathways of a given dmg molecule. 

Gross and coworkers129 also studied the unimolecular dissociation of protonated acy-lanilines, viz. A-[2-(benzoyloxy)phenyl]benzamides formed via both fast-atom bombardment (FAB) and electrospray ionization (ESI). They found that cyclization occurs upon the loss of a molecule of benzoic acid, and that a similar process occurs for the molecular ion under El. This gas-phase reaction is analogous to a solution reaction leading to phenyl-benzoxazoles. The proposed cyclization process, for which concurrent mechanisms were proposed (Scheme 38 depicts only the displacement reaction route), was corroborated by accurate mass measurements, tandem mass spectrometric experiments with comparison with reference ions, isotopic labeling and theoretical calculations. 

A useful first step is to analyze the sample using LC interfaced to ESI or APCI on a tandem mass spectrometer capable of accurate mass measurement at high resolution. The LC will serve to separate the unknown away from the matrix components and will reduce the potential for ion suppression. A short linear gradient of acetonitrile against 0.05 mol l-1 ammonium acetate on a reverse-phase C-18 column represents a good starting point (see, e.g., Eckers et al. 21 Arthur et al. 22 Tozuka et al. 22 and Wolff et al 2 1). 

By examining this brief history of the development of instrumentation and methods, it should be clear what parameters define the instrument of choice for analysis of pharmaceutically important molecules. Such an instrument is one capable of performing LC-MS, typically using a reverse-phase HPLC separation and one of the API techniques. MS-MS capability is desirable—using an ion trap, a triple quadrupole, or other tandem mass analyzer instrument. Accurate mass measurement capability is also desirable. These attributes make up a prioritized list of capabilities for the ideal full-purpose instrument to be used in a pharmaceutical research environment. As one progresses through this list, the expense of the instrument increases, the sophistication of the instrument increases, and the intellectual and technological commitment required to do these experiments increases. 

Nielen, M.W., Vissers, J.P., Fuchs, R.E., van Velde, J.W. and Lommen, A. (2001). Screening for anabolic steroids and related compounds in illegal cocktails by liquid chromatography/time-of-flight mass spectrometry and liquid chromatography/quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement. Rapid Commun. Mass Spectrom., 15, 1577-1585. 

In more complex or less well-characterized systems, the FI/FD molecular weights alone may not be sufficient to elucidate the proper chemical structures. In these cases, the FI/FD results can suggest appropriate methods (spectroscopic and/or chromatographic) for further study. For example, other ionization methods can be used to obtain additional mass spectra. Tandem mass spectrometry (MS/MS) can be used to obtain fragmentation patterns, and high resolution accurate mass measurements can be used to obtain atomic compositions (AC-MS) for various components. 

Among the advantages claimed for this approach were (i) qualitative identification of molecular classes (ii) exclusion of chemical noise (iii) potential for accurate mass measurement and (iv) possibility (not shown) for tandem mass spectrometry (MS/MS). 

While the development of ESI and MALDI provided techniques that ionize polar compounds effectively the spectra provide little or no structural information because molecular species are the only ions formed. The need to investigate not only the mass but also the structure of ions was an important factor in the development of multianalyzer mass spectrometers. There are several formats of MS/MS systems based on combinations of quadrupole, ion trap (Q and FT), and TOF analyzers. Combinations that use the same type of analyzers are tandem instruments, whereas different separation methods are used in sequence in hybrid systems. In MS/MS instruments, the first analyzer is used (usually) to select an ion of interest that is then passed, with or without fragmentation, into a second analyzer, which is often of higher resolving power and can be used for accurate mass measurement. 

Ballistreri and co-workers [59] examined the primary thermal decomposition mechanism of this polymer by Py-MS. Several MS techniques were used to identify compounds present in the pyrolysis mixture comparison of electron impact and chemical ionisation spectra, high resolution accurate mass measurements and tandem mass spectroscopy (daughter and parent ion spectra). The results obtained indicate that intramolecular exchange reactions predominate in the primary thermal fragmentation processes yielding cyclic oligomers up to tetramer. No other pyrolysis products were detectable. 

Liquid chromatography—mass spectroscopy has become one of the most powerful analytical techniques in the drug discovery and development proeess. LC tandem MS is obviously the technique of choice for the identification of metabolites as illustrated in this chapter. Exact mass measurements and elemental composition assignment are essential for the characterization of the metabolites. Accurate mass measurements of the product ions, formed in an MS/MS experiment, greatly facilitate the structure elucidation of the metabolites. With the ever evolving technological advances in mass spectrometry and separation science, LC MS will continue to play an important role in metabolite identification in the future. 

Regarding MS instruments, time-of-flight detectors provide elevated mass resolution and accuracy over a broad mass of range, and thus help structural elucidation of nontargeted compounds based on accurate mass measurements and isotopic patterns. Triple quadrupole instmments are particularly well suited for targeted analysis and provide excellent sensitive and selectivity by performing tandem MS/MS analysis [71]. 

The main advantages of TOF instruments are i) that in principle, the m/z range is unlimited [29,30] ii) from each ionizing event, i.e., a single laser shot in MALDI, a complete mass spectrum is obtained within several tens of microseconds Hi) improved transmission of TOF analyzers, with high sensitivity iv) TOF instrument design and construction is comparatively simple and inexpensive and v) recent instruments allow for accurate mass measurements and tandem MS experiments [31]. 

Example The NICI mass spectrum of tetraiodoethene, I2C=CI2, has been obtained using isobutane reagent gas (Fig. 7.12). The negative molecular ion, NT, at m/z 531.6 has a relative intensity of just 0.15%, while the product of nucleophilic addition, [M+I]", tn/z 658.5, yields the base peak [77], Losses of T and I2 from M"" are also observed. The series of peaks at m/z 126.9, 253.8, and 380.7 corresponds to traces of iodine present as impurity of tetraiodoethene. The iodine is also ionized by both electron capture (EC, next paragraph) and iodide addition. The spectrum nicely exemplifies the superimposition of mass spectra of two components of a mixture. It is not always simple to tell the corresponding peaks apart accurate mass measurements or tandem mass spectrometry may be required. It is worth noting the mass defect introduced by the iodine and the C isotopic peak of merely 2% due to only two carbon atoms present. 

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