Isobaric species

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...

However, tandem mass spectrometry, as a separation technique, does have limitations. It cannot easily differentiate between isomeric and isobaric species, and, in complex matrices, the presence of components with a high surface activity can suppress the ionization of components with a lower surface activity, leading to the nondetection of analytes (66). Therefore, the combination of MS-MS with a readily available chromatographic separation method such as TLC affords analysts real benefits. 

The term used to describe two compounds with the same overall relative molecular mass but with different molecular formulae is isobaric species... 

Often a mass spectrometer is interfaced with an HPLC-PDA system. This technique is especially useful because isobaric species can be chromatographically separated before entering the MS. Interestingly, there are a large number of isobaric species in the field of carotenoids, such as lycopene, [3-carotene, oc-carotene, and y-carotene which all have a parent mass of 536 mu, or (3-cryptoxanthin, oc-cryptoxanthin, zeinox-anthin, and rubixanthin which all have a parent mass of 552 mu. 

MALDI has been used to ionize multiple carotenoids and carotenoid ester standards (Kaufrnann et ah, 1996) and carotenoids in a variety of plant tissue samples (Fraser et ah, 2007). Fraser et ah (2007) found that the use of a nitrocellulose matrix produced the least variability in analyte detection, and also observed that MALDI was able to detect large differences in carotenoid phenotypes, but not small differences in carotenoid levels. Likewise, MALDI was not able to differentiate between isobaric species (like (3-carotene and lycopene, for example). 

In addition, due to lack of MS/MS capability, SIM has been more commonly performed on single quadrupole MS, while SRM has been broadly adapted on triple quadrupole (Figure 13-4) and ion-trap mass spectrometers. The increase in sensitivity and selectivity of SRM stem from the ion-chromatogram (i.e., LC-MS/MS) obtained by specific precursor-to-product ion transition for an analyte of interest (Figure 13-4). Conversely, in an SIM mode, the relative background noise due to the presence of other isobaric species (i.e., ions with a same m/z as the analyte of interest) can result in a lower signal-to-noise ratio for the analyte. Due to the widespread acceptance of SRM in quantitative analysis, the remaining part of this section focuses on a description of tandem-mass spectrometry (MS/MS), which is utilized in SRM (or MRM) experiments. 

If the peak shape is approximately Gaussian, the resolution can be obtained by a single peak. In fact, as shown by Fig. 2.1, the mass difference, AM, is equal to the peak width at 5% of its height and, accordingly to the gaussian definition, it is about two times the fwhm. Consequently, with this approach it is possible to estimate the resolution of a mass analyzer simply by looking at a single peak, without introduction of two isobaric species of different accurate mass. 

Isobaric Interferences. Isobaric species are two elements that have isotopes of csscnually the same mass. For atomic mass spectrometry with a quadrupole mass spectrometer, isobaric species arc isotopes that differ in mass by less than one unit. With higher-resolution instruments, smaller differences can be tolerated. 

As Foe for each ion always depends on by Equation 4.51, the values of F e and Foe for any gap geometry will be ion-specific and the FAIMS spectmm will not transpose as a whole, even for isomeric or isobaric species. In this regime, ions will be filtered based on a combination of Fc and mF. While, resolution of a particular set of species may improve or worsen depending on those two quantities for each. 

In unit resolution, the measured peak may actually be an average of a several imresolved isobaric species... 

In the same fashion, IMS can be used to discriminate against potential isobaric species to improve analyte specificity in MSI experiments. Trim et al. (2008) demonstrated the utility of IMS to enhance detection of vinblastine in whole-body tissue sections. Sprague—Dawley rats were dosed intravenously with vinblastine, after which they were sacrificed 1 h postdose. After preparing the tissue section, the samples were analyzed on a QqTOF in MS/MS mode. CID occurred after the ions were separated by IMS, thus product ions can be... 

The methods presented here describe the ability to characterize phosphatidylcholines (PCs), sphingomyelins (SPMs), phos-phatidylserines (PSs), and phosphatidylethanolamines (PEs) in the positive ion mode from rat brain tissue sections using imaging tandem MS and the creation of compound-specific images from full-scan MS and MS, while using MS for the final identification. The use of MS images for the separation of isobaric ions coupled with the ability to perform MS provides the means to identify isobaric species present in the tissue section. 

The ability to separate isobaric ions using tandem MS in imaging tissue has been shown previously (6), but the characterization of the many isobaric ions present has not been fully explored. For example, Cha and Yeung (18) showed the characterization of cerebrosides and PCs using graphite-assisted laser desorption and the separation of isobars including cerebrosides, but not across the entire tissue. In the current study, tandem MS data were collected across the entire tissue from over 40 different ions. The full-scan tandem mass spectra were then analyzed for possible isobaric species present see Table 12.2 for a list of all the tandem MS experiments. In addition, to help characterize the isobars, MS was performed on selected MS/MS daughter ions to elucidate the structure of the PL species present see Table 12.2). 

Fig. 12.5. MS/MS and MS data showing the identification of one of the isobars at m/z 856. A is the MS/WIS spectrum from tissue for m/z 856.6. (a) NL of 59 is indicative of a PC or SPM, whiie a NL of 87 or 43 is indicative of PS or PE, respectiveiy. (a) NL of 154 or 155 corresponds to a DHB duster ion. (b) The average MS spectrum for 856- -769- -. For this MS experiment, the entire tissue was scanned and thus the spectrum is the average of 10,528 spectra. The fragment ion at m/z 769 was identified as resuiting from a PS ion because of the NL of 87. MS spectra from tissue (b) and a PS standard mixture from bovine brain (c). The assignment of the fatty acid chains is Ri =stearic acid (18 0) and f 2=oieic acid (18 1). The TiC vaiues shown correspond to the signal from the standard (a) and from the tissue (b). MS was required for the correct assignment of the fatty acid fails of this isobaric species and identifies this PS species as PS (18 0,18 1) [M-2H+3Na]+.

The mass spectrometric images of the various PLs in rat brain showed a remarkable variation in distribution. The use of imaging tandem MS provided a means to properly identify the ions detected and to show the many isobaric species present in the lipid mass region. In addition, it was determined that there were sometimes np to five different ions found under MALDI conditions... 

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