Base peak profile

The capillary HPLC separation from a selected protein spot provides a base-peak profile shown in Figure 6.2A. The base-peak profile is similar to a total ion current (TIC) profile, but it contains only the most abundant mass spectral peak in each scan. The chromatogram is simplified and the contributions from background ion abundances are eliminated, resulting in an enhanced signal-to-ion ratio for an improved visualization of data. The molecular mass for each component is labeled along with corresponding amino acid residues. This format provides a comprehensive approach for peak selection and peptide identification. 

Figure 6.2 Identification of proteins separated by 2-DGE. (A) Base-peak profile from the capillary HPLC separation from a selected protein spot. (.B) Mass spectrum of the second major chromatographic peak (residue 60-70). (C) LC/MS/MS product ion spectrum of m/z 451.3. (Reprinted with permission from Amott et al., 1995. Copyright 1995 American Chemical Society.)...

Figure 4. LC-MS analysis of Cytochrome C Lys-C digestion mixtures in the absence (a) and presence (b) of SDS. The base peak profile of the maw spectral analyses is displayed. The insets I and II show the corresponding m/z spectra acquired over peak I and n.

As already stated, the mass spectrum is a two-dimensional graph that reports the m/z ratio of ions (abscissa) and their relative intensity (ordinate). The most abundant ions are assigned as 100%. A mass spectrum can be displayed as peak profiles or as bar graphs corresponding to the peak centroids, i.e. the weighted centre of mass of the peak, or as a table (Figure 2.16). The most abundant ions in a mass spectrum constitute the base peak whose intensity is assumed equal to 100%. 

The success of the analysis is dependent on the selection of a suitable internal standard. Some important criteria for such selection are as follows (a) that it is not chemically reactive towards any of the components of the mixture (b) that it has a retention time which gives a base line separation from the other components, including any impurities (c) that the retention time is comparable with that of the components of the mixture (d) that the peak profile is symmetrical and therefore does not exhibit either fronting or tailing and (e) that the detector response to the internal standard is such that neither an excessive, nor a minute, weight of standard compared to the weight of mixture needs to be used. 

Figure 6.3. Metabolite profile of razaxaban (C24H2oNg02F4) in dog bile (a) base peak chromatogram of unprocessed LC-MS data (b) base peak chromatogram of processed LC-MS data (c) corresponding radioactivity chromatogram. The arrows annotate the retention time of the peak shown in Fig. 6.2. Bile was collected from dogs orally administered with 14C-labeled razaxaban (20 mg/kg). HPLC solvents were 10mM NH4HC03 (pH 9.0) and acetonitrile. A portion of the HPLC effluent was collected in 15-s fractions for the radioactivity chromatogram. Another portion of the HPLC effluent was directed to a Q-TOF Ultima mass spectrometer.

Figure 6.6. Metabolite profiles of omeprazole in human plasma (a) base peak ion chromatogram of unprocessed data and (b) base peak ion chromatogram of MDF-processed data exhibiting all metabolite peaks present and some endogenous peaks. High-resolution LC-MS data were obtained for a human plasma sample spiked with omeprazole metabolites generated by microsomal incubation (the equivalent of a 1.0-mL plasma injection). A MDF ivas set at 50 mDa around the apparent mass defect of the omeprazole ion.

FAB-MS profile. This technique has been used to a limited extent to establish certain structural characteristics of this lysophosphoglyceride (Hana-han et al., 1990). Using the same procedure as discussed earlier in this chapter, the following major ions were noted for a highly purified (naturally occurring) sample [MH]+, molecular mass ions at m/z 480, m/z 510, and mlz 508. These ions were representative of vinyl ether homologs of side chain 16 0, 18 0, and 18 1, respectively. A base peak at m/z 184 was indicative of O-phosphocholine. 

In some advanced implementations of the modified pseudo-Voigt function, an asymmetric peak can be constructed as a convolution of a symmetric peak shape and a certain asymmetrization function, which can be either empirical or based on the real instrumental parameters. For example, as described in section 2.9.1, and using the Simpson s multi-term integration rule this convolution can be approximated using a sum of several (usually 3 or 5) symmetric Bragg peak profiles ... 

Most Rietveld software can perform size-strain analysis, even though this may require some manual calculations based on the software output. Some powder diffraction peak profiling programs also provide simple options for size-strain analysis. Table 17.19 gives a miscellaneous non-comprehensive list of programs. 

We have just seen how measuring these breadths enables us to quantify the defects. This analysis method implies the fitting of the peaks and therefore requires us to define a priori the shape of the diffraction peaks. Berlaut [BER 49] followed by Warren and Averbach [WAR 50, WAR 55, WAR 69] showed that with the help of a Fotrrier series decomposition of the peak profiles, any material with voltrme structrrral defects can be analyzed without having to make hypotheses on the shape of the peaks. This analysis, which we will now describe, when it can be implemented, remairts even today one of the most exterrsive ways to study microstructrtral effects based on the profiles of the diffraction peaks. 

Three different methods have been designed to quantitatively study structural volume defects. The integral breadth method, based on the theoretical considerations we discussed in Chapter 5, was introduced in 1918 by Scherrer [SCH 18] and generalized by Stokes and Wilson [STO 42], among others. Later on, Toumarie [TOU 56a, TOU 56b] followed by Wilson [WIL 62b, WIL 63] suggested a different analysis based on the variance of the intensity distribution. We described how Bertaut [BER 49] showed in 1949 that the Fourier series decomposition of the peak profile makes it possible to obtain the mean value and the distribution of the different effects that cause the increase in peak width. This method was further elaborated by Warren and Averbach [WAR 50, WAR 55, WAR 69]. 

Methods based on the Fourier analysis of the peak profiles have an intrinsic flaw, since it is necessary for each studied peak to be clearly isolated. If several peaks partially overlap, the resulting experimental signal corresponds to the sum of the elementary contributions, in which case it is impossible to extract the Fourier coefficients of each peak. This is why, in practice, the method suggested by Bertaut, and then by Warren and Averbach was essentially applied to crystals with a cubic... 

Microstructural study based on the modeling of the diffraction peak profiles... 

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