Accurate mass data

The electrospray source can be coupled directly to a liquid chromatographic (LC) column so that, as components of a mixture emerge from the column, they are passed through the source to give accurate mass data. As an example, a mixture of the peptides shown in Figure 40.8(a) was separated by LC and accurately mass-analyzed by ES. 

Once the mass spectral information has been acquired, various software programs can be employed to print out a complete or partial spectrum, a raw or normalized spectrum, a total ion current (TIC) chromatogram, a mass chromatogram, accurate mass data, and metastable or MS/MS spectra. 

Care must be taken in the interpretation of accurate mass data. In this case, the experimentally determined values of 280.0628 and 280.0633 Da have a mass... 

Isotopic patterns provide a prime source of such additional information. Combining the information from accurate mass data and experimental peak intensities with calculated isotopic patterns allows to significantly reduce the number of potential elemental compositions of a particular ion. [31] Otherwise, even at an extremely high mass accuracy of 1 ppm the elemental composition of peptides, for example, can only be uniquely identified up to about 800 u, i.e., an error of less than 0.8 mmu is required even if only C, H, N, O and S are allowed. [27,32,33]... 

Is accurate mass data available for some of the peaks ... 

The LTQ-FT mass spectrometer was introduced in late 2003 and, as expected, the main application discussed in the literature is for the analysis of proteins and peptides (Johnson et al., 2004 Syka et al., 2004). A recent book chapter (van der Greef et al., 2004) and a review article (Brown et al., 2005) discussed the application of the LTQ-FT to metabolomics. FTMS applications to dmg metabolism are still very new and dmg discovery research laboratories which have recently purchased the instmment are still in the process of developing and validating methods and approaches. A recent publication describes the depth and flexibility of the experimental setup utilizing accurate mass data-dependent exclusion MS" measurements with a LTQ-FT (Tozuka et al., 2005). We have reported several integrated approaches for determination of metabolic stability, characterization of metabolites and metabolic... 

Relative to GC-MS, the application of LC-MS within the metabolomics held is still at a preliminary stage. Reproducibility is a major concern, and true quantification can be hindered by ion suppression effects whereby one co-eluting metabolite affects the ionization of another (26). The lack of electrospray ionization mass spectral libraries also makes identification by LC-MS a particularly challenging problem. Nevertheless, the technique is developing fast and has benefited from several technological advancements such as the acquisition of accurate mass data by the use of ion cyclotron resonance Fourier transform MS (26). 

AlOg Is the least abundant of the five Al-containing species identified In the vapor over AlgOg. Ho and Burns (J ), unlike Parber et al. (4-5), identified AlOg as the source of the mass=59 ion by accurate mass data combined with intensities and... 

In a series of papers, the group of Volmer [130-132] studied the analysis of azaspiracid biotoxins. Ultrafast and/or high-resolution LC of azaspiracids on monohthic LC columns was evaluated [130]. Chromatograms of five azaspiracids on a 100-mm and a 700-mm monolithic column are shown in Figure 14.11. Fragmentation of azaspiracids in MS-MS on ion-trap and triple-quadrupole instruments was studied as well [131]. The interpretation was confirmed using accurate-mass data from a Q-TOF instrument. Validation of a quantitative method for AZA-1 was also reported [132]. The LOQ was 5 and 50 pg/ml extract using a triple-quadrapole in SRM mode and an ion-trap instrument, respectively. 

Assuming that the mass spectrometer has sufficient mass resolution, the computer can prepare accurate mass data on the m/z values from an unknown substance. To prepare that data, the system must acquire the mass spectrum of a known reference substance for which accurate masses for its ions are already known, and the computer must have a stored table of these reference masses. The computer is programmed first to inspect the newly acquired data from the reference compound in comparison with its stored reference spectrum if all is well, the system then acquires data from the unknown substance. By comparison and interpolation techniques using the known reference... 

Other monovalent elements (F, Cl, Br, and I) are counted as hydrogens, trivalent elements (P) are counted as nitrogen, and tetravalent elements (Si) are included with carbon. For chemically possible formulae, r+ db> — 1.5. Odd-electron ions (M+ ) will have an integer value and even-electron ions will have 0.5 r + db more than expected, so round up to next lowest integer.32,33 By way of example, Kind and Fiehn139 have described an integrated application of accurate mass data to metabolite identification, constrained by isotope abundance information and valence rules, in addition to the KI (Section 9.10.4.3.2). 

Fourier transform ion cyclotron mass spectrometry (FT-ICR-MS) instrumentation offers excellent sensitivity, accuracy (<1 ppm), and high mass resolution (>1,000,000) (Table 10.2). However, because of being too expensive, difficult to use, and not compatible with conventional HPLC columns and flow rates, FTMS has not been frequently used in pharmaceutical research. This changed with an introduction of a hybrid instrument consisting of a linear ion-trap mass spectrometer compatible with LC and an ion-cyclotron-resonance (ICR) detector. Such a hybrid instrument is compatible with conventional HPLC and allows for acquisition of accurate mass data-dependent MS" spectra. Sanders et al. [128] recently reviewed the utility of hybrid LTQ-FTMS for drug metabolism and metabonomics applications while Brown et al. [129] reviewed the metabolomics applications of FT-ICR-MS. 

In addition to ion trap and quadrupole mass spectrometers, OA group toxins can be analyzed on other types of MS analyzers. For example, time of flight (ToF) mass spectrometers provide accurate mass data and have been used to isolate and characterize new compounds (Suzuki et al., 2004). However, it is more common for ion trap (Cruz et al., 2006) and quadrupole instruments (Vale, 2006a) to be used for OA group toxin qualitative analysis. Various MS experiments can be used to gain information about the composition of samples and some examples are given in Figure 10.6. 

With the advent of accurate mass instruments, access to a reference database or library of compounds with the exact masses of precursor and product ions is becoming increasingly important. Many instrument manufacturers, as well as independent companies or institutions, are developing software that allows chemists to manage this type of information for specific applications such as proteomics or chemical contaminants. Examples of online resources that may be useful for searching for unknown residues based on molecular formulas obtained from accurate mass data inciude Metlin from the Scripps Center for Mass Spectrometry and ChemSpider from the Royal Society of Chemistry. " ... 

Mass defect filter is solely dependent on the availability of accurate mass data. Therefore, it can detect unusual metabolites resulting from novel biotransformation pathways and is especially useful in metabolite profiling studies when radiolabeled drug is not available. 

You should have been able to solve this problem without referring to the accurate mass data. The presence of sulfur is indicated by the P + 2 isotope peak of the ion at m/z 180, which appears to be the molecular ion. 

Quadrupole analyzers have unit resolution throughout their mass range i.e one mass is discriminated from the next, be that 28 from 29 or 2,000 from 2,001. This classifies quadrupoles as low-resolution instruments which are poorly suited to obtain accurate mass data. A consequence of the poor resolution is that two ions with the same nominal mass but with different empirical formulae, and therefore different exact masses (isobaric ions), will not be separated, but instead, their masses will be averaged into the mJz observed value. Furthermore, the peaks are broad (-1 mIz wide), and thus it is difficult to obtain precise measurements of the centroids. The mass range of quadrupoles extends to 4 kDa for singly charged ions. These analyzers are compatible with all ionization methods except MALDI (because of the mass range limitation). 

The inherent sensitivity, resolution, and rapid data acquisition of TOP analyzers are major assets of LC-MS/MS analysis. The TOP format provides accurate mass data on both molecular species and fragments. QTOP instruments feature multiple ionization modes, including convenient switching between ESI and MALDI. 

This chapter describes and discusses the measures of instrument performance, the types of information that can be acquired, and the methodologies and strategies available. The discussions are with respect to both the multiple types of instruments manufactured and the various classes of compounds that can be analyzed. The range of compounds makes defining the types of data that can be obtained somewhat problematic. Therefore, small molecules are dealt with in Sections 3.2 and 3.3, while biopolymers are covered in Section 3.5, although the differentiation is somewhat artificial. For instance, the determination of accurate mass data described in Section 3.1.3 is relevant to both small molecules and the analysis of the peptides derived from proteins. 

---