Mass Spectroscopy Primer, Part II: Data Interpretation

Mass spectrometers, particularly when interfaced to gas chromatographs and liquid chromatographs, are particularly useful in cannabis labs for the analysis of pesticides and potency. In the previous installment of this series, we provided an introduction to mass spectroscopy [1]. In this installment, we will cover mass spectral data interpretation. We will discover the power of mass spectra to give us molecular weights, molecular formulas, molecular structures, and functional group information.

A Comment on Ion Stability

Previously, we discussed how electron impact ionization is used to generate ions [1]. The process can be thought of as a chemical reaction as seen in equation 1, where M=molecule and e^-=electron:

This is a process in which the impinging electron knocks an electron off from our analyte molecule generating a positively charged ion called a cation, so it is correct to say that electron impact ionization generally gives positively charged organic cations. Recall [1] from the block diagram of a mass spectrometer seen in Figure 1 that the ions formed in the ion source are gathered into a beam and then travel some distance over the course of time through the instrument to get to the detector.

This means there is a time lag between when the ions are formed and when they are detected. Now, organic cations are fragile things or else our universe would be full of them. Instead, our universe is full of electrically neutral organic molecules. Thus, for a cation to be detected in mass spectroscopy it must survive long enough to make the trip from the ion source to the detector. Some cations are more stable than others and these more stable ions will tend to have greater abundance than unstable ions that fall apart before being detected. For example, molecular ions [1] are often times the biggest peaks in mass spectra because they can be the most stable ions formed from an analyte molecule in the ion source.

Over time, and based on the observed mass spectra of thousands of molecules, some rules have been developed wherein we can predict, based on a molecule's structure, what its most intense m/z peaks will be in its spectrum [2]. This then allows us to do the reverse, interpret the peak positions and intensities in a mass spectrum to determine what molecular fragments make up a molecule. A recital of the details of relative organic cation stability and mass spectral interpretation rules is beyond the scope of this article. In general though, since cations have a positive charge, the presence of heteroatoms in an ion such as nitrogen or oxygen containing lone pairs of electrons will stabilize these ions and lead to an increase in their detected abundance [2]. Similarly, functional groups with high concentrations of bonding electrons such as triple bonds including C≡N, double bonds such as C=O and C=C bonds, andaromatic rings can stabilize organic cations leading them to be detected in significant quantities in a mass spectrometer. We will make use of these ideas in the mass spectra we interpret below.

A Simple Mass Spectrum

An example of a simple mass spectrum used in the previous column [1] was that of carbon dioxide, as seen in Figure 2.

Note that the x-axis is in “m/z” units, where m stands for mass and z for charge, thus the term m/z is pronounced “mass to charge ratio.” A mass spectrometer does not separate ions based solely on their mass but by their mass to charge ratio. It can happen, for example, that an ion with a mass of 100 and a charge of 1 and hence with an m/z of 100 will be detected at the same time as an ion with a mass of 200, a charge of 2, and hence also have m/z = 100. Note that the y-axis here is “% Relative Intensity.” If we plotted the raw signal this scale would be labeled “ion abundance” which is a direct count of the number of ions detected at a specific m/z. It is convenient though to divide the intensity of each individual peak by that of the largest peak. In this case, m/z = 44, multiply by 100, then plot the y-axis in % Relative Intensity units as seen.

Assuming carbon has an atomic mass of 12 and oxygen 16, then CO₂ has a molecular weight of 44. Note in Figure 2 that the peak with the highest m/z, and also the most intense peak, has m/z = 44. This is called the molecular ion peak, M⁺ peak, or parent ion peak [1]. This peak is from a CO₂ molecule with a single positive charge on it. Molecular ion peaks are often seen in mass spectra and are very useful because they tell us the molecular weight of an analyte. Note also that there are peaks with m/z values of 16 and 12. The former is a from a positively charged oxygen ion, O⁺, and the latter from a positively charged carbon ion, C⁺. Note then that the value of a m/z peak by itself can tell us what chemical species gave rise to that peak.

The peak at m/z = 28 is more interesting. Its peak position tells us this molecular fragment has a mass of 28, however if we subtract its mass from that of the molecular ion we get 44 – 28 = 16, and we appropriately call this peak a “M-16” peak and it is due to a CO⁺ ion. We saw above that the atomic mass of oxygen is 16, so the difference in m/z between the molecular ion peak and our peak of interest can be used to deduce what fragments were given off by the molecular ion and hence what functional groups comprised our original analyte molecule. The CO⁺ ion was detected because it owes its stability to the lone pairs of electrons on the oxygen atom as pointed out above.

A More Complex Mass Spectrum

The mass spectrum of benzoic acid, C₇H₆O₂, is seen in Figure 3.

Note that benzoic acid contains a mono-substituted benzene ring and a carboxylic acid or -COOH group. The y-axis of the spectrum in Figure 3 is ionic abundance, a count of the number of ions detected at each m/z.

Assuming again that carbon has an atomic mass of 12, oxygen 16, and that hydrogen’s is 1, we can calculate the molecular mass of benzoic acid as seen in equation 2:

Note that there is a molecular ion peak in Figure 3 at 122 and that it is large. We can attribute this ion’s stability to the presence of two oxygen atoms and a benzene ring in its structure.

In both Figures 2 and 3 the molecular ion peaks are the biggest peaks in the spectrum. This is commonly seen but is not necessarily always the case, it all depends upon the stability of the molecular ion. If a particular molecule forms a M⁺ ion that is stable, a significant number of them will survive the trip through the mass selector and be detected. On the other hand, some molecules may form particularly unstable molecular ions, and fragments with m/z values less than that of the molecular ion may be the most abundant.

Note in Figure 3 there is a particularly large peak at m/z = 105. Now, we could try to draw molecular fragments with this molecular mass, but this could be a long and drawn out process as the number of fragments with this mass value may be many. What is more interesting is that the m/z = 105 peak is 17 mass units less than the mass of the molecular ion of 122 or in other words is an M-17 peak. This peak formed when the molecular ion lost something that weighed 17 to preferentially form m/z = 105 ions, and the ion formed must be particularly stable for so many of them to have formed. Trying to deduce what ions might have a mass of 17 is easier than trying to deduce what ions might have a mass of 105 because there are simply fewer of the former. Amongst the chemical species with a mass of 17 is an isotope of oxygen or ¹⁷O, however its relative abundance is less than 0.1% than that of ¹⁶O, so it’s doubtful this rare isotope is responsible for our M-17 peak. Another chemical species with a mass of 17 is the hydroxyl or O-H group where the oxygen weighs 16 and the hydrogen weighs 1. The loss of an OH group to give the M-17 peak here makes sense since OH groups are commonly found in organic structures. In general then, any molecule with a M-17 peak may contain an OH group.

Notice in Figure 3 that there is a peak at m/z =77 and that it is an M-45 peak. Rather than trying to brute force the calculation of what chemical species might have a mass of 45 we can simply rely on the literature [2]. It is well known that the carboxylic acid group, -COOH, weighs 45 (12 + 32 +1) and that these peaks are commonly seen in the mass spectra of these molecules. Thus the presence of a M-45 peak is suggestive of a molecule containing the carboxylic acid functional group. A value of m/z = 77 also corresponds to that of a mono-substituted benzene ring or phenyl ion, C₆H₅⁺, whose structure is seen in Figure 4.

This ion is relatively stable because of the presence of the electron rich aromatic ring. Two of the biggest peaks then in the mass spectrum of benzoic acid correspond to the molecular ion falling apart into -COOH⁺ and phenyl cations, both of which are stabilized by the presence of electron rich moieties. Note again that the difference in mass between a peak of interest and the molecular ion gives functional group information. This is one of the strong points of mass spectrometry [3].

Molecular Formulas from Mass Spectra

In addition to obtaining molecular weight information from the molecular ion and functional group information from the peak positions in a mass spectrum, the molecular formula of an analyte can be obtained as well. For mass spectrometers of high enough resolution, typically good to several decimal places, the exact m/z for a molecular ion can be used to calculate molecular formulas from readily available tables and computer programs.

However, many labs do not have the budget for a high resolution mass spectrometer, and often times instruments with a mass resolution of 1 are all that are available. The problem with these instruments is that molecules with different chemical structures but the same mass will give measured molecular ions with the same m/z value. For example, carbon monoxide, C≡O, and nitrogen, N₂, both have molecular weights of 28 and hence have molecular ions of the same value. How would we distinguish between them using a mass spectrometer?

We can make use of the fact that different elements have different isotopic abundances. For example, in carbon for every 100 atoms of C¹² there is about 1 atom of the stable isotope C¹³. Thus for every 100 C¹²O molecules there is one C¹³O molecule. This means that in the mass spectrum of carbon monoxide there will be what we call an M+1 peak, a peak with an m/z value one more than that of the molecular ion due to the C¹³O molecules, whose size will be 1% that of the parent ion peak.

For nitrogen, the stable isotope N¹⁵ has a natural abundance of 0.4% that of N¹⁴. Thus for every 100 N¹⁴N¹⁴ molecules there are about 0.4 N¹⁴N¹⁵ molecules. We would then expect the M+1 peak for nitrogen to be about 0.4% the size of the molecular ion peak. The point here is that even though carbon monoxide and nitrogen have molecular ion peaks with the same m/z values, their M+1 peaks will be of different sizes allowing them to be distinguished from each other. In general, the size of M, M+1, and M+2 peaks can be used to determine molecular formulas. Tables [2] and computer programs exist to allow these calculations to be made.

Conclusions

Mass spectra can give molecular weight, functional group, and molecular formula information on analyte molecules. The m/z value of the molecular ion can give the mass of a molecule. The m/z values of peaks in a mass spectrum and the difference between their m/z and that of the molecular ion provides functional group information. Lastly, for the typical mass spectrometer with a mass resolution of 1, molecular formulas can be obtained by measuring the size of the M+1 and M+2 peaks.

References

1. Smith, B.C. Mass Spectroscopy Primer, Part 1: Introduction. Cannabis Science and Technology. 2025, 8(1), 6-9.
2. R. Silverstein, G. Bassler, and Terrence Morrill, Spectrometric Identification of Organic Compounds, Wiley, New York, 1981.
3. Fred W. McLafferty, Interpretation of Mass Spectra, University Science Books, Herndon VA, 1980.

About the Columnist

Brian C. Smith, PhD, is Founder, CEO, and Chief Technical Officer of Big Sur Scientific. He is the inventor of the BSS series of patented mid-infrared based cannabis analyzers. Dr. Smith has done pioneering research and published numerous peer-reviewed papers on the application of mid-infrared spectroscopy to cannabis analysis, and sits on the editorial board of Cannabis Science and Technology. He has worked as a laboratory director for a cannabis extractor, as an analytical chemist for Waters Associates and PerkinElmer, and as an analytical instrument salesperson. He has more than 30 years of experience in chemical analysis and has written three books on the subject. Dr. Smith earned his PhD on physical chemistry from Dartmouth College.
Direct correspondence to: brian@bigsurscientific.com

How to Cite this Article

Smith, B., Mass Spectroscopy Primer, Part II: Data Interpretation, Cannabis Science and Technology, 2025, 8(2), 6-9.