Mass measurements, accurate

Consider an ion of nominal mass 300 Da. If the mass is measured to within 1 ppm, that is, 300.0000 0.0003, then there is only one combination of, for example, C, H, O and N, which will match this mass. If the mass can only be measured to within 50 ppm, that is, 0.015, then 27 combinations of C, H, O and N are theoretically possible. At 100 ppm this becomes 52 combinations. 

It is therefore important to acquire data of the highest accuracy if unequivocal molecular compositions are to be determined. As a general rule, any molecular formulae for which the error (i.e. the difference between measured and theoretical masses) is greater than 10 ppm should be discounted. 

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  

For example, for benzene with six carbons and six hydrogen atoms, the DBF is 4, corresponding to one ring and three double bonds. For acetaldehyde, CH3CHO, DBF = 1, that is, one carbonyl function. 

The DBF number is most useful when it comes to deciding which of perhaps two or three possible molecular formulae are correct. If there is a strong possibility that a particular unknown is related to the parent drug, for example, and that drug had a ring system with a DBF of 5, then the unknown should have a value of at least 5, and more if additional double bonds were introduced. 

Principles and Characteristics Mass spectrometry can provide the accurate mass determination in a direct measurement mode. For a properly calibrated mass spectrometer the mass accuracy should be expected to be good to at least 0.1 Da. Accurate mass measurements can be made at any resolution (resolution matters only when separating masses). For polymer/additive deformulation the nominal molecular weight of an analyte, as determined with an accuracy of 0.1 Da from the mass spectrum, is generally insufficient to characterise the sample, in view of the small mass differences in commercial additives. With the thousands of additives, it is obvious that the same nominal mass often corresponds to quite a number of possible additive types, e.g. NPG dibenzoate, Tinuvin 312, Uvistat 247, Flexricin P-1, isobutylpalmitate and fumaric acid for m = 312 Da see also Table 6.7 for m = 268 Da. Accurate mass measurements are most often made in El mode, since the sensitivity is high, and reference mass peaks are readily available (using various fluorinated reference materials). Accurate mass measurements can also be made in Cl 

Resolution does not affect the accuracy of the individual accurate mass measurements when no separation problem exists. When performing accurate mass measurements on a given component in a mixture, it may be necessary to raise the resolution of the mass spectrometer wherever possible. Atomic composition mass spectrometry (AC-MS) is a powerful technique for chemical structure identification or confirmation, which requires double-focusing magnetic, Fourier-transform ion-cyclotron resonance (FTICR) or else ToF-MS spectrometers, and use of a suitable reference material. The most common reference materials for accurate mass measurements are perfluorokerosene (PFK), perfluorotetrabutylamine (PFTBA) and decafluorotriph-enylphosphine (DFTPP). One of the difficulties of high-mass MS is the lack of suitable calibration standards. Reference inlets to the ion source facilitate exact mass measurement. When appropriately calibrated, ToF mass 

High resolution is used to determine the exact mass of an ion species in a mixture knowledge of the exact mass of an unknown substance allows its atomic composition to be established. Target analysis exact mass determination proves the presence of a particular ion species (compound) in a mixture. Mass spectrometry is perhaps the only method that can be used to find the empirical formulae of compounds that are not completely pure. 

For polymer/additive analyses neither a very high mass range nor an ultrahigh mass resolution is required isobaric additive ions are not frequent. It is therefore not surprising that tandem sector instruments have not found wide application for polymer/additive identification. 

There is rapid growth in the use of accurate mass measurements in the chemical industries. There is equally a clear need for practical guidance in order to obtain robust measurements. At present, LGC coordinates a collaborative study to evaluate the variation in accurate mass measurement across a broad range of instrument types, using an unknown compound of molecular mass of about 450 Da. 

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Electrospray alone is a reasonably sensitive technique for use with many classes of compounds. Spectacular, unprecedented results have been obtained with accurate mass measurement of high-... 

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. 

High-Resolution, Accurate Mass Measurement Elemental Compositions... 

Finally, accurate mass measurement can be used to help unravel fragmentation mechanisms. A very simple example is given in Figure 38.2. If it is supposed that accurate mass measurements were made on the two ions at 203.94381 and 77.03915, then their difference in mass (126.90466) corresponds exactly to the atomic mass of iodine, showing that this atom must have been eliminated in the fragmentation reaction. 

Therefore, for accurate mass measurement, a standard mass peak (M,) is selected, and the accelerating voltage (V) is changed until the sample ion peak (M ) exactly coincides with the position of Mj. This technique is called peak matching, and the ratio between the original and new voltages (VA ) multiplied by mass (Mj) gives the unknown mass, M . 

Accurate mass measurement requires high resolving power. The difference in degrees of difficulty between measuring an m/z of 28 and one of 28.000 is likely to be large. Table 39.3 shows the broad mass ranges achievable with various analyzers. 

Once the peaks have been collected and stored, the computer can be asked to work on the data to produce a mass spectrum and print it out, or it can be asked to carry out other operations such as library searching, producing a mass chromatogram, and making an accurate mass measurement on each peak. Many other examples of the use of computers to process mass data are presented in other chapters of this book. 

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. 

A simple mass spectrometer of low resolution (many quadrupoles, magnetic sectors, time-of-flight) cannot easily be used for accurate mass measurement and, usually, a double-focusing magnetic/electric-sector or Fourier-transform ion cyclotron resonance instrument is needed. 

Accurate mass measurement on a molecular ion of any substance gives directly the molecular formula for fragment ions, similar measurement gives their elemental compositions. 

This method can sometimes be used for determining the probable elemental composition of fragment ions. However, it is not as generally applicable and does not replace accurate mass measurement for determining molecular formulae and elemental compositions. 

Identification of unknowns, as well as confirming the presence of known AAs, is more reliable if accurate mass measurement... 

The molecular ion can be very small or nonexistent. Esters where R is greater than methyl form a protonated acid that aids in the interpretation (e.g., m/z 47, formates m/z 61, acetates m/z 75. propionates m/z 89, butyrates etc.). Interpreting the mass spectra of ethyl esters may be confusing without accurate mass measurement because the loss of C2H4 can be confused with the loss of CO from a cyclic ketone. 

Molecular ion The presence of sulfur can be detected by the 34S isotope (4.4%) and the large mass defect of sulfur in accurate mass measurements. In primary aliphatic thiols, the molecular ion intensities range from 5-100% of the base peak. 

The unknown gave a molecular ion at m/z 193 with fragment ions at m/zs 174, 148, and 42. From the abundance of the molecular ion, it is probably aromatic, and according to the Nitrogen Rule, contains at least one nitrogen atom. From accurate mass measurement data and an examination of the isotopic abundances in the molecular ion region, the molecular formula was found to be CnH15N02. 

The mass spectrum of the unknown compound showed a molecular ion at m/z 246 with an isotope pattern indicating that one chlorine atom and possibly a sulfur atom are present. The fragment ion at m/z 218 also showed the presence of chlorine and sulfur. The accurate mass measurement showed the molecular formula to be C]3FI7OSCl R + DB = 10. 

The M - 1 peak due to the loss of the aldehyde hydrogen by a-cleavage is usually abundant. The loss of 29 Daltons is characteristic of aromatic aldehydes. Peaks at m/z 39, 50, 51, 63, and 65 and the abundance of the molecular ion show that the compound is aromatic. Accurate mass measurement data indicate the presence of an oxygen atom. 

By carefully examining the fragmentation pattern of the metabolite and comparison with the mass spectra of the precursor molecule, it is often possible to determine not only the nature of the biotransformation, but also its position in the molecule. In the proceeding example, accurate mass measurement was used to determine that a hydroxyl group had been added to the benzene ring containing the fluorine substituent. 

Identification of unknowns using GC/MS is greatly simplified if accurate mass measurements are made of all the ions in a spectrum so that reasonable elemental compositions of each ion are available. Unfortunately, obtaining a mass measurement that is accurate enough to significantly limit the number of possible elemental compositions requires expensive instrumentation such as a double-focusing magnetic sector or fourier transform ICR MS. 

The most interesting feature in the spectra of the above compounds is the presence of an intense peak for [M — CH20]4 (except with 117 and 121), the identity of which was checked by means of accurate mass measurements and deuteriation of the central methylene group. The following mechanism was proposed to explain the loss of CHzO which governs very much also the consequent fragmentations (equation 38) ... 

Figure 3.6 Schematics of three configurations of mass spectrometer capable of accurate mass measurement (a) forward-geometry (b) reverse-geometry (c) tri-sector. From applications literature published by Micromass UK Ltd, Manchester, UK, and reproduced with permission.

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