Mass spectrometer structure elucidation

Figure 5.45 Structures of (1) Bosentan (C27H29N5O6S [M + H]+ 552.1917) and three of its metabolites, formed by (2) oxidation (C27H29N5O7S [M + H]+ 568.1866), (3) demethylation (C26H27N5O6S [M- -H]+ 538.1760), and (4) demethylation-oxidation (C26H27N5O7S [M + M]+ 554.1709). Reprinted by permission of Elsevier Science from Exact mass measurement of product ions for the structural elucidation of drug metabolites with a tandem quadrupole orthogonal-acceleration time-of-flight mass spectrometer , by Hopfgartner, G., Chemushevich, I. V., Covey, T., Plomley, 1. B. and Bonner, R., Journal of the American Society for Mass Spectrometry, Vol. 10, pp. 1305-1314, Copyright 1999 by the American Society for Mass Spectrometry.

To summarise, a fractionation step allows the isolation of the compounds of interest from the other molecular constituents, particularly from the fatty acids that are well-ionised. To compensate for the low ionisation yield of some compounds, such as TAGs, the solutions may be doped with a cation. Samples are then directly infused into the ion electrospray source of the mass spectrometer. A first spectrum provides an overview of the main molecular compounds present in the solution based on the peaks related to molecular cations. The MS/MS experiment is then performed to elucidate the structure of each high molecular compound. Table 4.2 shows the different methods of sample preparation and analysis of nonvolatile compounds as esters and TAGs from reference beeswax, animal fats and archaeological samples. 

Multiple mass analyzers exist that can perform tandem mass spectrometry. Some use a tandem-in-space configuration, such as the triple quadrupole mass analyzers illustrated (Fig.3.9). Others use a tandem-in-time configuration and include instruments such as ion-traps (ITMS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS or FTMS). A triple quadrupole mass spectrometer can only perform the tandem process once for an isolated precursor ion (e.g., MS/MS), but trapping or tandem-in-time instruments can perform repetitive tandem mass spectrometry (MS ), thus adding n 1 degrees of structural characterization and elucidation. When an ion-trap is combined with HPLC and photodiode array detection, the net result is a profiling tool that is a powerful tool for both metabolite profiling and metabolite identification. 

Full scan experiments set the mass spectrometer in a cycle of repeatedly scanning a selected range of m/z values. The experiment is done primarily for structure elucidation, and the richness of structural... 

HPTLC is a very fast and convenient assay to separate samples components and is often used in Organic Chemistry and in Synthetic approach. Unknown substances, after different display assay, were generally scraped off from the TLC/HPTLC plate, diluted into a tube and transferred into the MS system for structural elucidation and characterization. Now, a TLC-MS interface was developed by CAMAG, which can semi-automatically extract zones of interest and on-line direct them into any brand of a HPLC-MS system. The TLC-MS interface is connected by two fittings to any HPLC instrument coupled with mass spectrometer, without other system configuration adjustments or mass spectrometer modifications. By this way, the unknown substances can be directly extracted from a TLC/HPTLC plate, eluted and resolved by HPLC system and sensitive and selective mass spectrometric signals are obtained within a minute per substance zone [33],... 

Hopfgartner, G. Chemushevich, I.V. Covey, T. Plomley, J.B. Bonner, R. Exact Mass Measurement of Product Ions for the Structural Elucidation of Drug Metabolites With a Tandem Quad-rupole oaTOF Mass Spectrometer. J. Am. Soc. Mass Spectrom. 1999, 10, 1305-1314. 

The coupling of a mass spectrometer to CE and CEC provides a powerful system for the analysis of pharmaceuticals and complex biological mixtures. This can replace or complement other conventional detection methods such as UV, electrochemical, or LIE that provide less structural information. The use of mass spectrometer as a detector enhances the usefulness of the CE and CEC and allows an efficient separation and identification of complex mixtures, obtaining structure and/or molecular mass information. The choice of mass analyzers used in CE/CEC-MS depends on factors such as sensitivity, mass resolution, requirement for structural elucidation, and the type of application (Table 5). The analyzers that have been used in CEC analysis include time-of-flight (TOE), quadrupole (Q), ion-trap (IT), fourier... 

The emerging of CEC and the increased scientific work on the preparation of different phases, characterization, and applications of the CEC columns have given much credence to their future potentials in microseparations. The fabrication and availability of different phases for analysis with both particle-packed and monolithic columns give the technique a great future. This is because a variety of mechanisms can be exploited in the analysis and separation of compounds that could otherwise be difficult to analyze with HPLC or CE alone. The ease of coupling CEC to sensitive detectors such as mass spectrometers for enhanced sensitivity, structural elucidation, and characterization bestows the technique with great versatility. 

Mass spectrometers for structure elucidation can be classified according to the method of separating the charged particles ... 

Several scan modes are unique to the triple-quadrupole instrument, and most of these modes are superior in duty cycle versus an ion trap, Fourier transform (FT), or time-of-flight (TOF) mass spectrometers. Different elements of the triple-quadrupole perform different operations for each scan mode. These scan modes, each of which will be described in detail, are single-reaction monitoring (SRM) or multiple-reaction monitoring (MRM), precursor ion scanning (PIS), and constant-neutral-loss scanning (NLS). These scan modes and applications for structural elucidation have been described in detail (Yost and Enke, 1978, 1979). 

As mentioned in the previous section, triple-quadrupole instruments are very good at finding low levels and structurally related compounds in the presence of biological matrices as well as being the gold standard technique for quantitation. Ion trap mass spectrometers, on the other hand, have the capabilities to obtain high-sensitivity full-scan MS and MS/MS spectra therefore, they are widely used for qualitative analysis, such as structural elucidation and unknown identification. For complete metabolite identification, it is important to have both the sensitivity and selectivity of triple-quadrupole instruments and the full-scan data quality of ion traps. 

As shown in Fig. 4.8, the mass difference between the two metabolites is 36.4 mDa and a mass spectrometer with a resolving power of at least 9500 (M/AM = 344/ 0.0364 = 9450) is required to separate between these two metabolites. MS/MS fragmentation combined with accurate mass measurement is the preferred method for structural elucidation of metabolites, especially when it can help to correlate the elemental composition determined in the MS mode. In the example shown in Fig. 4.8, exact mass measurements of the precursor ions are used as lock mass to measure the exact mass of each fragment ion. Exact mass measurements of the fragment ion at m/z 226 help to narrow down the sites of modifications and allows one to distinguish between rabeprazole-sulphide and rabeprazole-aldehyde. 

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