Isobaric species, resolving

Figure 2.7 Mass spectra recorded at different resolutions. Mass spectrum obtained by a two dimensional ion trap at low resolution (a) and by an Orbitrap at resolving power 50000 (b). Mass spectrum of a mixture of three isobaric species [C19H7N]+, [C20H9]+, [C13H19N302]+ obtained at low resolution (black line) and at resolving power 50000 (grey line) (c). It is noteworthy that at low resolution the three peaks are completely unresolved...

The other, LC-MS, approach makes use of on-line preseparation of the lipids before MS analysis. This method allows analysis of species for which no specific scan modes exist. Also, many isobaric species, not readily resolved by MS/MS, can be analyzed. Finally, this method, particularly when employing multiple reaction monitoring (MRM), provides the highest sensitivity of detection of many minor lipid classes as the suppression effects are minimized. 

Resolution (or resolving power) plays an important role in mass spectrometry for applications requiring the characterization of very similar chemical species. The ability to detect and accurately measure the m/z ratio of a particular ion depends directly on the resolving power of the mass analyzer. For example, if a sample contains two isobaric compounds (i.e., having the same nominal molecular mass but different elemental formulae) the difference in the exact masses of the molecular ions will be much less than 1 m/z unit. Any mass analyzer possessing a nominal resolving power (e.g., RP< 1000) will register only one peak in the mass spectrum of such a binary mixture. Attempts to measure the... 

Argon plasmas also contain polyatomic Ions at very low concentrations, which arise from the air, from argon or from the sample matrix. These species can be Isobaric with the analytes Ions and then could be a source of possible confusion when MS have a low resolving power. Thus If we consider N2 (28 u), O2 (32 u), ArH (78 u), CIO (51 u), ArO (56 u), CaO (56 u), the corresponding molecular Ions have the same miz ratio as elements such as SI, S, V and Fe, which could be the species under Investigation. In this case, two methods for clarification are necessary selection of another Isotope of the element considered ( " Fe rather than Fe) or choice of another spectrometer with a collision chamber (section 16.12) to dissociate the polyatomic Ions. 

The accuracy with which the mass of an ion can be determined is a combination of the ability to resolve one ion from another and how accurately the mass scale has been calibrated. There are multiple elemental compositions possible for any given nominal mass, e.g., (as noted above) there are three possible formulae, CO, Nj, and C2H4, at ndz 28, and the number increases rapidly as one moves up the mass scale. To obtain accurate measurements for each component of a set of isobaric ions, it is essential to resolve them from each other. As resolution increases and peaks become narrower, it becomes easier to be certain that an observed peak is a unique species and that the outline of the peak is Gaussian. Consequently, the center of gravity of the peak can be determined and the accurate mass of the ion calculated (Figure 3.3). 

Easier at high resolution because isobaric (same nominal mass) species are resolved... 

High Resolution. The separation of isobaric ionic species (i.e., high resolution, with / > 10000), is a relative requirement. It depends on various factors, such as the type of heteroatoms. but with / = 10 000 most common species can be resolved. Exceptions can be ions containing, e.g., sulfur. Increasing the resolution requires reducing the width of the ion beam in the mass spectrometer. Thus, in theory, the height of the signal is inversely proportional to the resolution. In reality, this relation is not linear over the full... 

To achieve identification of the quantified species, an approach with data-dependent product-ion analysis would be useful. However, an increased duty cycle of the instrument employed is required as the number of the analyzing lipids is increased. Alternatively, a high mass accuracy/resolution mass spectrometer would help to resolve the isobaric molecular ions from different lipid classes although isomeric species resulting from the regiospecificity and/or the double-bond location still cannot be resolved. 

As described in Chapter 15, correction for different stable isotopologue distribution due to differences in the number of carbon atoms between the species of interest and the selected internal standard(s) and correction for baseline should be performed prior to the comparison of the intensities. It should be recognized that in the majority of the studies in plant lipidomics after direct infusion, MDMS-type analysis is generally not conducted thus, the isobaric or isomeric species are not resolved as regrettable. A detailed protocol for profihng of polar hpids in plants after direct infusion can be found [9]. 

Figure 1 HRMS of mlz 95 ions from 70 eV electron ionization of CeH50CH2CD20H, illustrating the resolution of isobaric ions on a double focusing reverse Nier->Johnson (B-E) instrument with resolving power of 70 000. Each of the peaks that contains a rare isotope is a radical ion (odd number of electrons), which is a mixture of isomeric structures, all the possibilities for which are drawn. Asterisks designate C-containing positions of the ring. The unlabelled ion is an even-electron species. Reproduced a from Nguyen V, Bennett JS and Morton TH (1997) Journal of the American Chemical Society 119 8342 with permission of the American Chemical Society with modifications.

Most commercial quadrupole mass spectrometers operate with a typical resolving power capable of unit mass resolution R = 300). This resolution is capable of resolving most analytical species used in the I CP. However, to resolve isobaric interferences (i.e., species occurring at the same unit mass), a resolution exceeding R = 3000 is necessary. Therefore, although the quadrupole is an exceptional spectrometer for accompHshing 90% of common analytical requirements, there are some appHcations for which simple direct ion current measurements are not sufficient to achieve accurate analytical data. For these applications, either another type of mass spectrometer is required, mathematical corrections must be appHed to the data, or in the case of interferences, a chemical separation prior to measurement must be made. 

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