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peak represents the exact mass of an ion or a molecule calculated from the most abundant isope of each element. The relative intensity of this ion compared to the others ions is 100%. A weaker isotopic peak (M+' + 1) is observed at m/z 85 with an abundance of 6.5% corresponding to one 13C, five 12C and 12 JH atoms. An even weaker peak (0.2% abundance) is visible at m/z 86 (M+' + 2) corresponding to two 13C, four 12C and 12 JH atoms. In this example, the contribution of deuterium can be neglected. For large molecules with increasing the number of carbon atoms, a shift of the maximum of the isotopic distribution towards higher masses can be observed, as depicted in Fig. 1.2. Above several hundred atoms of carbons, mostly a Gaussian distribution is observed. The consequence is that, in particular for protein analysis, only the relative molecular mass and not the monoisotopic mass is observed since either the monoisotopic masses can no longer be resolved or the intensity of the peak is too weak. The average mass is the calculated mass of an ion based on the relative atomic mass of each atom.

The isotopic contribution of various atoms is additive. For low molecular weight compounds, the isotopic contribution originates mainly from the carbon atom as long as no other element with a second isotope of significant abundance is present. For a molecule of Mr 192 the intensity of the m/z 194 ion represents 12% of the [M+H]+ peak (m/z 193; Fig. 1.3A). Chlorine (Cl) has two intense isotopes: 35Cl and 37Cl (76% and 24% abundance, respectively). Replacing one H by a Cl atom results in a change of the isotopic distribution of the molecule

Number of carbons

Number of "C isotopes

Fig. 1.2 Isotopic distribution as function of the number of carbon atoms. It can be observed that with increasing numbers of carbon atoms the maximum of the isotopic distribution shifts towards higher masses. M represents the molecular ion with only 12C isotope; M+1 represents the molecular ion with only one 13C isotope; M+2 represents the molecular ion with only two 13C isotope; and so on.

Number of carbons

Number of "C isotopes

Fig. 1.2 Isotopic distribution as function of the number of carbon atoms. It can be observed that with increasing numbers of carbon atoms the maximum of the isotopic distribution shifts towards higher masses. M represents the molecular ion with only 12C isotope; M+1 represents the molecular ion with only one 13C isotope; M+2 represents the molecular ion with only two 13C isotope; and so on.

(Fig. 1.3B). The [M+H]+ + 1 peak is not affected, while the [M+H]+ + 2 is increased to about 25%. The replacement of the F by a second Cl results in an increase of the [M+H]+ + 2 and [M+H]+ + 4 peaks (Fig. 1.3c). Chlorine and bromine have typical isotopic patterns therefore their presence in a molecule can be easily confirmed.

Mass analyzers are characterized by their mass range in m/z and their resolving power. The mass range is the m/z range where ions can be detected. The mass resolving power (R) is the ability of a mass analyzer to separate ions of different m/z with similar intensities. It is basically the m/z (m) at which the measurement was made divided by the difference (Ama) between the two peaks overlapping at a defined height (2 x%; Fig. 1.4). Because it is difficult to find two ions of equal intensities, the measure of the resolving power is often performed on a single peak. In general, the peak width is measured at 50% of its height. It is often referred to as full width at half maximum (FWHM). There is often confusion with the terms mass resolving power and mass resolution. Basically mass resolution is the smallest difference (Am) between two equal magnitude peaks such as the valley between them is a specified fraction of the peak height. M1 and M2 are considered resolved when the valley between the two peaks represents 10% (2 x%) of their heights. In practice the definition of the resolution is often determined upon Am of the a single peak at its full width at half maximum (Fig. 1.4, Amb).

For example for an ion measured at m/z 552 with a peak width of 0.5 m/z units (FWHM) the mass resolution would be 0.5, while the mass resolving power

Fig. 1.3 The influence of chlorine on the isotopic distribution. (A) No chlorine atom, (B) one chlorine atom, (C) two chlorine atoms.
Fig. 1.4 Illustration of the mass resolution using two peaks of equal intensities (Dma) and a single peak (Amb).

would be 1104. With quadrupole and ion trap instruments the mass resolution is tuned to be constant over a defined mass range. With these instruments the term unit mass resolution is often employed to mention that the mass spectrometer is able to differentiate two ions distant by one m/z unit bearing a single charge.

While the relative molecular mass is calculated using the relative atomic mass considering all isotopes, the observed mass in mass spectrometry depends on the mass resolving power of the instrument; and various definitions are used. The exact mass represents the calculated mass of an ion or a molecule containing a single isotope of each atom. In general the lightest isotope of each atom is considered. The monoisotopic mass represents the calculated exact mass of an ion or molecule considering the most abundant naturally occurring isotopes. The accurate mass of an ion is the experimentally measured mass that is used to determine an elemental formula. The accurate mass is generally measured with at least three significant figures. The accuracy of the measure, corresponding to the difference between the measured mass and the calculated mass divided by the mass of the molecule, is indicated in parts per million (ppm).

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 500 000 (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 10~31 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.

With time of flight instruments, a mass accuracy better than 5 ppm can be achieved, while with Fourier transform ion cyclotron resonance or orbitrap mass spectrometers mass accuracies better than 1 ppm have been reported. It is obvious that, for good mass accuracies, the peaks must be baseline resolved and resolution plays an essential role. For the present example, a mass resolving power of 5000 seems to be quite acceptable. In the case of the [M+H]+ + 1 isotope peak, the situation becomes somewhat more complex for molecules containing nitrogen, sulfur or carbon. Figure 1.5D illustrates at a mass resolving power of 500 000 the contribution of15 N, 33 S.

In qualitative analysis, the isotopic distribution remains an important information. For example in the case the parent drug contains Br or Cl, metabolites or decomposition products can be easily identified by considering the isotopic distribution. With accurate mass measurements a list of elemental compositions can be proposed for a compound for a given accuracy range. Because the intensity of the isotopic distribution is also dependent on the elemental composition of the molecule it can be used to reduce the list of possible elemental formulas [17].

Fig. 1.5 Simulated isotopic distribution of the protonated bosentan (C27H30N5O6S) at mass resolving power: (A) R = 1104, with a peak full width at half maximum (FWHM) of 0.5 u. (B) R = 5520, FWHM = 0.1 u. (C) R = 15 000. (D) R = 500000 with isotopic contribution of 15N (peak 1), 33S (peak 2) and 13C (peak 3).

Fig. 1.5 Simulated isotopic distribution of the protonated bosentan (C27H30N5O6S) at mass resolving power: (A) R = 1104, with a peak full width at half maximum (FWHM) of 0.5 u. (B) R = 5520, FWHM = 0.1 u. (C) R = 15 000. (D) R = 500000 with isotopic contribution of 15N (peak 1), 33S (peak 2) and 13C (peak 3).

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