The Value of Accurate Mass Measurement

A mass spectrometer can measure integer relative mass with high accuracy, but the result is not nearly so informative as measurement of accurate relative mass. An example illustrates the reason. 

There is a more important use. Suppose a mass spectrometer has accurately measured the molecular mass of an unknown substance as 58.04189. Reference to tables of molecular mass vs. elemental composition will reveal that the molecular formula is CjH O (see Table 38.2). The molecular formula for an unknown substance can be determined which is enormously helpful in identifying it. 

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Until the early 1980s, accurate mass measurements were nearly restricted to electron ionization, and for a while, the technique even seemed to become abandoned. New options available through FT-ICR instrumentation then revived the value of accurate mass measurements. The newly developed orbitrap and a new generation of oaTOF analyzers contributed to an increased demand for accurate mass data. Nowadays, formula elucidation can be performed using any ionization method [35], their widespread application thus demanding a thorough understanding of their potential and limitations [33]. 

Even mass spectroscopists use the terms exact mass and accurate mass interchangeably when they probably should not. An exact molecular mass is the mass calculated from the accepted exact atomic masses of the isotopes for a specified empirical formula. It is the value that one would expect to observe if one could measure a molecular mass exactly. An accurate mass is a value measured carefully, with high precision, on an instrument capable of making such precise measurements, typically expressed to at least four decimal places—the nearest 0.1 mDa. An accurate mass measurement is compared to the exact masses of empirical formulae being considered. Sufficiently accurate measurements can be used to assign empirical formulae to peaks in a mass spectrum. Knowing that these two terms are commonly used interchangeably, but that there may be subtle differences in the way practitioners use them, is sufficient for our discussions here. We will comment more on the utility of accurate mass measurements later. 

What, then, are the options currently available to the scientist, and is aeeurate mass measurement a help The latter question is largely rhetorieal, especially for anyone who has tried to make sense of product ion spectra obtained on instrumentation capable only of generating nominal mass data. Trivial neutral losses can usually be assigned on the latter instruments, but not always, for example, distinguishing between a neutral loss of methane from that of an oxygen atom is not possible. The presence or absence of a halogen isotope pattern can still be used to limit possible explanations for a product ion. On the other hand, the benefit of accurate mass measurement is dramatic, as noted earlier, in that it affords a dramatic reduction in elemental compositions that are possible explanations of the measured w/z value—and thus limits the number of possibilities that must be considered by the analyst. 

ICP-MS atomic mass spectra of the generated ions accurate mass measurement based on m/z values, and peak intensity as counts per second (cps). 

What is ionic mass The ionic mass of an ion takes into account the mass of an electron (0.000548 Da = 0.548 mDa mDa is also referred to as millimass units, mmu) that is removed or added during the formation of the ion (remember that a proton is a hydrogen atom, H isotope, after removal of an electron). This small mass effect is frequently ignored when masses are determined experimentally and compared with calculated values. However, as the accuracy and precision of accurate mass measurements have improved, the mass attributable to an electron has become of relevance to empirical formula determinations. The mass of an electron represents 1 part per million at 500 Da (-0.5 mDa), which is a significant error in miz determinations for instruments designed to obtain mass accuracies at the mDa level or less. 

Although a spectral feature reveals the mass of an ion, the value is not necessarily accurate enough for rehable identification of the corresponding molecule. In low-resolution mass spectra, the isotopic pattern of an ion may be buried within a single spectral feature. The measurement of m/z may result in an observation closer to the average mass than the exact mass of the ion (as discussed in Section 5.3.2). Even in the case of a well-resolved isotopic pattern, mass measurement may still be affected by the width and shape of the spectral feature. Deconvolution of spectral features contaminated with multiple ions of similar mass is important for accurate molecular identification but beyond the scope of this book. Here we will focus on the determination of accurate masses of single-component samples. 

In general terms, the main function of the magnetic/electric-sector section of the hybrid is to be able to resolve m/z values differing by only a few parts per million. Such accuracy allows highly accurate measurement of m/z values and therefore affords excellent elemental compositions of ions if these are molecular ions, the resulting compositions are in fact molecular formulae, which is the usual MS mode. Apart from accurate mass measurement, full mass spectra can also be obtained. The high-resolution separation of ions also allows ions having only small mass differences to be carefully selected for MS/MS studies. 

When multicharged ions are formed, the simple rule of thumb used widely in mass spectrometry that m/z = m because, usually, z = 1 no longer applies for z > 1 then m/z < m, and the apparent mass of an ion is much smaller than its true mass. Accurate mass measurement is much easier at low mass than at high, and the small m/z values, corresponding to high mass with multiple charges, yield accurate values for the high mass. 

Other methods attempt to probe the stmcture of the foam indirectly, without directly imaging it. Eor example, since the Hquid portion of the foam typically contains electrolytes, it conducts electrical current, and much work has been done on relating the electrical conductivity of a foam to its Hquid content, both experimentally (15) and theoretically (16). The value of the conductivity depends in a very complex fashion on not only the Hquid content and its distribution between films and borders, but the geometrical stmcture of the bubble packing arrangement. Thus electrical measurements offer only a rather cmde probe of the gas Hquid ratio, a quantity that can be accurately estimated from the foam s mass density. 

The results for bacterial whole-cell analysis described here establish the utility of MALDI-FTMS for mass spectral analysis of whole-cell bacteria and (potentially) more complex single-celled organisms. The use of MALDI-measured accurate mass values combined with mass defect plots is rapid, accurate, and simpler in sample preparation then conventional liquid chromatographic methods for bacterial lipid analysis. Intact cell MALDI-FTMS bacterial lipid characterization complements the use of proteomics profiling by mass spectrometry because it relies on accurate mass measurements of chemical species that are not subject to posttranslational modification or proteolytic degradation. 

The Most Intense Peaks. It is not so easy to extract valuable information dealing with the most intense peaks in mass spectra. In contrast to other physicochemical methods (IR, NMR, UV), registration of an ion peak of an integer m/z value does not provide an unequivocal identification of its structure or even composition. Even accurate mass measurements (high resolution mass spectrometry) defining the elemental composition of an ion does not establish its structure, as it could be formed directly from the M+, with minimal distortion of the authentic structure, or as a result of numerous rearrangement processes. 

It has been stated that measured accurate masses when used to assign molecular formulae should always be accompanied by their mass accuracies. [34] Ideally, this can be done by giving the mean mass value and the corresponding error in terms of standard deviation as obtained from several repeated measurements of the same ion. [35] This is definitely not identical to the error which is usually provided with the listing from mass spectrometer data systems, where an error is given as the difference of calculated and measured mass value. 

So far, the concepts of exact mass, mass accuracy and resolution have been introduced without considering the means by which accurate mass measurements can be realized. The key to this problem is mass calibration. Resolution alone can separate ions of different m/z value, but it does not automatically include the information where on the m/z axis the respective signals precisely are located. 

Example Cesium iodide is frequently used for mass calibration in fast atom bombardment (FAB) mass spectrometry (Chap. 9) because it yields cluster ions of the general formula [Cs(CsI)n] in positive-ion and [I(CsI)J in negative-ion mode. For the [Cs(CsI)io] cluster ion, m/z 2730.9 is calculated instead of the correct value m/z 2731.00405 by using only one decimal place instead of the exact values Mi33Cs = 132.905447 and M1271 = 126.904468. T e error of 0.104 u is acceptable for LR work, but definitely not acceptable if accurate mass measurements have to be performed. 

As the monitored m/z values are selected to best represent the target compound, SIM exhibits high selectivity that can be further increased by high resolution SIM (HR-SIM) because this reduces isobaric interferences. [41-44] As HR-SIM requires precise and drift-free positioning on narrow peaks, one or several lock masses are generally employed although rarely explicitly mentioned. [44,45] The role of the lock mass is to serve as internal mass reference for accurate mass measurement. (Examples are given below.)... 

Figure 5A, B shows the isotopic distribution, of protonated bosentan (C27H30N5O6S, Mr 552.6) with a mass resolution of 0.5 and 0.1 at FWHM, respectively. It is worthwhile to observe the mass shift of the most abundant ion from m/z 552.2006 to m/z 552.1911. This value does not change with a mass resolving power of 15 000 (Fig. 1.5C) or even 500000 (Fig. 1.5D). Accurate mass measurements are essential to obtain the elemental composition of unknown compounds or for confirmatory analysis. An important aspect in the calculation of the exact mass of a charged ion is to count for the loss of the electron for the protonated molecule [M+H]+. The mass of the electron is about 2000 times lower than of the proton and corresponds to 9.10956 x 10 kg. The exact mass of protonated bosentan without counting the electron loss is 552.1917 units, while it is 552.1911 units with counting the loss of the electron. This represents an error of about 1 ppm.

Accurate mass measurement and the associated empirical formulae allow routine calculation of the double bond equivalent (DBF) by the spectrometer s computer system. This is a measure of the number of double bonds and/or the number of rings in a molecule, and is derived from a consideration of the valences of the various elements in a given composition. This gives information about the aromaticity or conjugation of the unknown. The values given by the MS data system are based on a simple calculation ... 

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