Interpreting Mass Spectrometer Output

The mass spectrum is a display of unique ions present at a specific time in the experiment, whether that duration represents a long-term ablation of a solid sample in the source or the passage of a transient GC or LC peak. Software is available from several sources. It is often tailored to specific practices, such as metabolite identification. It can be expeditious, reducing huge volumes of data while highlighting issues the unaided eye might overlook. Software can help us reduce uncertainty if, with properly applied skills, we make use of fundamental chemistry: the electron valence rules for nitrogen-containing compounds, characteristic spectra of halides, rings-and-double bond calculations, and so forth to arrive at what we believe is an unambiguous conclusion. No single software application can answer all inquiries satisfactorily. And so, what really counts is a practitioner’s ability to apply well-honed skills and educated judgment.

A small, simple molecule such as carbon dioxide (44 Da), composed of only three atoms, produces a very simple mass spectrum. In case of CO, the molecular ion is also the most intense or abundant ion displayed (referred to as the base peak). Fragment ions found in this spectrum created from the excess internal energy of ionization are CO (m/z=28) and O (m/z=16). In some cases the molecular ion may not be the most abundant in the spectrum. For example, because cleavage of a carbon-carbon bond in propane (44 Da) gives methyl and ethyl fragments, the larger ethyl cation (m/z=29) is the most abundant. Ions derived from these well-characterized interactions are particularly significant identifying features for spectra of these hydrocarbons.

Isotope characteristics

Because mass spectrometers separate ions by mass, distinguishing isotopes for a given element when the instrument is capable of sufficient resolution is easily accomplished. Halogenated compounds are often cited as examples, since naturally occurring bromine, for instance, consists of a nearly 50:50 mixture of isotopes having atomic masses of 79 and 81 Da. Fragmentation of Br₂ to a bromine cation then produces two equal-sized ion peaks at 79 and 81 m/z.

Even and odd electron ions

Most stable organic compounds have an even number of total electrons because electrons occupy atomic orbits in pairs. When a single electron is removed from a molecule, the total electron count becomes an odd number, a radical cation. The molecular ion in a mass spectrum is always a radical cation (as seen in EI), but the fragment ions may be even-electron cations or odd-electron radical cations, depending on the neutral (uncharged) fragment lost. The simplest and most common fragmentations are bond cleavages that produce a neutral radical (odd number of electrons), and a cation having an even number of electrons. A less common fragmentation where an even-electron neutral fragment is lost produces an odd-electron radical cation fragment.

As a rule, odd-electron ions may fragment either to odd- or even-electron ions, but even-electron ions fragment only to other even-electron ions.

The masses of molecular and fragment ions also reflect the electron count, depending on the number of nitrogen atoms in the species.

The two levels of access to interpreting mass spectra are nominal mass data and exact mass data. In each case, retention times serve as an additional determinant. Achieving accurate mass measurement is based on the calculated elemental composition. Not surprisingly, accurate isotope patterns fed into an algorithm to reduce the number of possible formula candidates is a recently exploited aspect of accurate mass measurement.

Characterizing spectra produced by desorption and soft ionization

The retro Diehls–Alder reactions and hemolytic/heterolytic energies required to disassociate, or cleave, bonds leading to specific well-characterized fragmentation continues to be the basis for our thinking when confronted with mass spectra. The difficult part of MS is often in answering the question posed by Fred W. McLafferty, one of the important contributors to our understanding of interpretation rules: “What is the mass we are dealing with?”

Until the development of desorption techniques like matrix-assisted, laser-desorption ionization (MALDI) and electrospray, that question at least at times seemed easier to answer. How easy depended on whether the sample had to be derivatized to make it volatile and amenable to GC/MS. Here often the spectra would be dominated by the derivatized groups and show little or no molecular ion (hence the need for CI). In that case the advent of electrospray and APCI certainly aided in the identification of the molecular weight of small molecule singly charged species. At least in those cases MS dealt with the m/z value of ions displaying only a single charge. The mass of an analyte usually was reported as the nominal mass (the nominal m/z value) of the molecular ion, the same as the nominal mass of the molecule. The nominal mass of an ion, molecule, or radical is the sum of the nominal masses of the elements in its elemental composition. The nominal mass of an element is the integer mass of the most abundant, naturally occurring, stable isotope.

But the answer became more elusive when soft-ionization desorption techniques like electrospray ionization (ESI) became commercially widespread beginning in the early 1990s. In the “pre-desorption” age of MS, the nominal mass of most analytes interrogated by MS was less than 500 Da. Mass defect due to the presence of hydrogen was not an issue for these analytes. The upper m/z limit for most mass spectrometers fell in the 650–800 range. Thus, in those pre-desorption ionization days, the nominal mass and the integer monoisotopic mass were of the same value. The monoisotopic mass of an ion, molecule, or radical is the sum of the monoisotopic masses of the elements in its elemental composition. The monoisotopic mass of an element is the exact mass of the most abundant, naturally occurring, stable isotope.

At the onset of the desorption ionization era, larger molecules and greater precision became integral to studies because the technology permitted it with little difficulty. Only then did the issue of mass defect become so very important. In a mass spectrometer able to report only to the nearest integer m/z value, the molecular ion of a C₅₀H₁₀₂ compound might be represented by a peak at m/z 703 instead of at m/z 702 because the molecular ion would have a monoisotopic mass of 702.7825, which rounds to the integer 703.

Above 500 Da, mass defect can be a serious issue in determining the m/z values of MS peaks. It is important to keep in mind that the mass spectrometer is measuring signal intensities that occur at a specific time during the collection of the mass spectrum, regardless of the type of m/z analyzer used. The m/z value reported is a function of the time that ions of a known m/z value produced by a specific compound—relative to the calibration compound—reach the detector.

Because the mass of monoisotopic ions changes as the position on the m/z scale changes, the mass spectrometer that reports integer m/z values actually can take measurements at every 0.05 m/z units. The detected intensity can be that at the apex of the mass spectral peak or the sum of the intensities across the mass spectral peak. The m/z value reported is an integer obtained by rounding the observed m/z value for the mass spectral peak maximum.

Electron ionization MS often relies on perfluorinated compounds like perfluorotributylamine (nominal molecular mass of 671) to calibrate the m/z scale. That is because the integer mass of an ion is almost the same as its monoisotopic mass. Once an ion exceeds a nominal mass of 1000 Da, there is no observed nominal m/z value peak in the mass spectrum. The monoisotopic mass peak is offset from where the nominal mass peak should be observed by an amount equal to the mass defect of the ion. For single-charge ions with masses above 500 Da, using techniques like electrospray with transmission quadrupole or quadrupole ion trap mass spectrometers that have unit resolution throughout the m/z scale, the isotope peaks will be separated clearly.

Of the many discussions on the role isotopes play in determining a compound’s identity, one appeared in LCGC Europe that contributes a helpful balance. “Interpretation of Isotope Peaks in Small Molecule LC–MS” (L.M. Hill, LCGC Europe 19(4), 226–238 (2006) is based upon low-resolution ion trap work. In a relevant part, the author cautions against overconfidence when using ion traps: “[I]on trap users will have to be more careful than those with QTOF or triple quadrupole systems. It is obviously necessary to start with the +1 isotope peak isolated free of contamination . . . ion traps tend to trap with lower resolution than they scan . . . empty[ing] the trap . . . in order of mass.” This does not mean ion traps cannot be used but, like all instruments, must be applied with an understanding of their abilities and their limitations.

Similarly an instrument capable of very high resolution does not automatically confer the correct answer. One data set presented in a paper by Kind and Fiehn—T. Kind and O. Fiehn, BMC Bioinformatics 7, 234 (2006)—is particularly striking and led to their conclusion, which they based on examining 1.6 million formula search results: “High mass accuracy (1 ppm) and high resolving power alone [are] not sufficient . . . only an isotopic abundance pattern filter [is] able to reduce the number of molecular formula candidates.” Mass spectrometers capable of just 3 ppm mass accuracy, but 2% isotope pattern accuracy, usually remove more than 95% of the false candidates. This performance would beat even mass spectrometers capable of 0.1 ppm—if such instruments actually existed—that are not equipped with isotope pattern capability.

Between masses of 150 Da and 900 Da, the number of possible formulas listed as mass accuracy increased from 10 ppm to 0.1 ppm without the aid of isotope abundance information: from a low of 2 candidate formulas at 150 Da to 3447 at 900 Da for 10 ppm. Even at the upper end (900 Da), mass accuracy alone at 1 ppm yields 345 candidates. Invoking 2% isotope abundance accuracy, the number of candidates at 900 Da is reduced to an expedient 18. They also show that allowing a paltry 5% accuracy for isotope acquisition associated with 5 ppm accuracy yields 196 candidates.