Ion Types and Fragmentation Patterns in Mass Spectrometry

Types of Ions and Fragment Ions in Mass Spectrometry

In mass spectrometry, various types of ions and fragment ions are observed, each providing valuable information about the composition and structure of molecules.

Molecular Ion: Molecular ions are formed when a molecule loses or gains an electron upon being bombarded by an electron beam. They are represented as M⁺·. Molecular ions are radical cations, and the peak corresponding to the molecular ion in a mass spectrum is called the molecular ion peak. The mass-to-charge ratio (m/z) of the molecular ion peak corresponds to the relative molecular mass of the compound, allowing for the determination of molecular weight through mass spectrometry.

Isotopic Ion: Isotopic ions contain isotopes of the same element. In a mass spectrum, peaks corresponding to isotopic ions are termed isotopic ion peaks.

Fragment Ion: Fragment ions are formed when molecular ions undergo further bond cleavage within the ionization chamber. These ions are the fragments resulting from the cleavage of chemical bonds within the molecule.

Rearrangement Ion: Ions generated through rearrangement processes are termed rearrangement ions. These ions do not retain the original structure of the molecule. Rearrangement reactions involve simultaneous bond cleavage and formation, often resulting in the loss of neutral molecules or fragments.

Odd-Electron and Even-Electron Ions: Ions with unpaired electrons are termed odd-electron ions. Such ions also function as radicals and exhibit higher reactivity. Ions without unpaired electrons are termed even-electron ions.

Multiply Charged Ions: Ions carrying more than one charge within a molecule are referred to as multiply charged ions. The mass-to-charge ratio decreases when ions carry multiple charges. Hence, conventional quadrupole mass analyzers can be used to detect high-molecular-weight compounds when they exist as multiply charged ions.

Metastable Ion: Metastable ions are formed between the ion source and the detector. These ions undergo fragmentation during their flight. As a result, only these fragmented ions, termed metastable ions or metastable peaks, are recorded by the detector.

Quasi-Molecular Ion: Quasi-molecular ions are ions with a mass that differs from the exact molecular mass by one unit, either more or less. Examples include (M+H)⁺ and (M-H)+ ions. Quasi-molecular ions do not contain unpaired electrons and are relatively stable in structure.

Molecular Ion Peaks

Intensity of Molecular Ion Peaks

Molecular ions are the most valuable information in a mass spectrum. They serve not only as the basis for determining the molecular weight of a compound but also aid in deducing its molecular formula. High-resolution mass spectrometry can directly determine the molecular formula of a compound.

Generally, the electron lost from a molecule should be the weakest bond in the molecule, such as π electrons in double or triple bonds or non-bonding electrons on heteroatoms. The abundance of molecular ions primarily depends on their stability and the energy required for molecular ionization. Compounds that readily lose electrons, such as cyclic compounds and compounds with double bonds, exhibit stable molecular ions and strong molecular ion peaks. Conversely, long-chain alkanes and branched alkanes show the opposite trend.

The stability order of various compound molecular ions is roughly as follows: aromatic rings (including aromatic heterocycles) > conjugated dienes > alkenes > cycloalkanes > thi ethers, thiones > amides > ketones > aldehydes > straight-chain alkanes > ethers > esters > amines > carboxylic acids > nitriles > primary alcohols > secondary alcohols > tertiary alcohols > highly branched hydrocarbons. Aromatic rings (including aromatic heterocycles), cycloalkanes, thi ethers, thiones, and conjugated dienes typically exhibit distinct molecular ion peaks. Straight-chain ketones, esters, acids, aldehydes, amides, and halides usually show molecular ion peaks. Fatty alcohols, amines, nitrites, nitrates, nitro compounds, nitriles, and highly branched compounds tend to undergo fragmentation, resulting in weak or absent molecular ion peaks.

When molecular ion peaks are absent or of extremely low abundance and difficult to confirm, experimental conditions can be adjusted accordingly for verification:

• Reduce the energy of bombarding electrons from the commonly used 70 eV to 15 eV to decrease the probability of further fragmentation of formed molecular ions, thereby increasing the relative abundance of molecular ion peaks.
• Utilize soft ionization methods such as chemical ionization (CI), field ionization (FI), or field desorption (FD). While reducing the energy of bombarding electrons may increase the abundance of molecular ion peaks, it also decreases the sensitivity of the instrument. For compounds that do not exhibit molecular ion peaks due to reasons such as thermal instability or low volatility, employing soft ionization methods may help highlight molecular ion peaks.
• Lower the sample vaporization temperature to reduce the likelihood of further fragmentation of molecular ions, thereby increasing the relative abundance of molecular ion peaks.

The common MS/MS fragmentation pattern for all CoA species: (A) The CoA portion of all CoA esters fragments during MS/MS at the 3′-phosphate-adenosine-5′-diphosphate. This cleavage gave rise to a daughter ion equal to [M − 507 + H] + m/z (right) (Jones et al., 2021).

Identification of Molecular Ion Peaks

During analysis, the ion with the highest mass-to-charge ratio in the spectrum is typically assumed to be the molecular ion. Subsequently, various criteria are applied to confirm its identity. If the inspected ion does not meet any of the criteria, it is not considered a molecular ion. Conversely, if the inspected ion satisfies all conditions, it may be identified as a molecular ion. Criteria for determining molecular ions include:

• The molecular ion must be an odd-electron ion since organic molecules are typically even-electron. Thus, any ion losing one electron to form a molecular ion must be an odd-electron ion.
• Adherence to the Nitrogen Rule: The molecular weight of an organic compound is related to the number of nitrogen atoms it contains. Compounds without nitrogen or with an even number of nitrogen atoms have even molecular weights, while those with an odd number of nitrogen atoms have odd molecular weights, following the Nitrogen Rule.
• Loss of reasonable neutral fragments: These neutral fragments can be small molecules or radical groups. They have specific mass numbers, and there should be a reasonable mass difference between the highest m/z value and adjacent fragment ions. For example, losing a proton (H), CH3, H2O, or C2H4 from the molecular ion is considered reasonable. If the mass difference falls between 4 to 14 or 21 to 25, it is deemed unreasonable. Thus, the presence of peaks in the range of M-4 to M-13 indicates that the assumed molecular ion peak is not a molecular ion peak.

Fragment Ions

Fragment ions and their abundance play a significant role in mass spectrometry data. The relative abundance of fragment ions is closely related to the molecular structure, with high-intensity fragment peaks representing easily cleavable parts of the molecule. If several major fragment peaks represent different parts of the molecule, these peaks can roughly reconstruct the molecular skeleton. A considerable amount of work in mass spectrometry analysis focuses on analyzing the formation process of fragment ions.

Alpha (α) Cleavage

Alpha cleavage involves the loss of an electron from the molecule, forming a radical cation. The electron then tends to pair with an adjacent atom, resulting in the cleavage of an adjacent alpha bond. Hence, this type of cleavage is often referred to as "alpha" cleavage.

• Saturated Heteroatoms: Heteroatoms with lone pair electrons have low ionization energy and readily lose electrons to form radical cations, leading to alpha cleavage.
• Unsaturated Heteroatoms: For example, the alpha cleavage process in carbonyl compounds occurs as follows:
• Cleavage at Allylic Positions in Alkenes:

The occurrence of these reactions is closely related to the tendency of electron donation in the radical center. Nitrogen atoms have strong electron-donating abilities, making alpha cleavage dominant in alkyl amines, followed by oxygen atoms. The difficulty of alpha cleavage reactions due to differences in electron-donating abilities is arranged in the following order: N>S>O, π, R﹒>Cl, Br, H. When a compound has multiple alpha bonds, the largest alkyl radical is the most easily lost.

Inductive (i) Cleavage

Inductive cleavage occurs when a positive charge induces and attracts a pair of electrons, resulting in the transfer of positive charge. Inductive cleavage is often denoted as "i". Generally, elements with high electronegativity also exhibit strong inductive effects. In some cases, inductive cleavage and alpha cleavage occur simultaneously. Since inductive cleavage involves charge transfer, it is less likely to occur than alpha cleavage. In mass spectra, ion peaks resulting from inductive cleavage are weaker compared to those resulting from alpha cleavage. For example, in ethyl ether, both inductive and alpha cleavage occur simultaneously, with alpha cleavage being more probable. However, due to further fragmentation of m/z 59 generated by alpha cleavage, the m/z 59 peak in the mass spectrum of ethyl ether is not stronger than m/z 29.

Sigma (σ) Cleavage

If a compound molecule contains sigma bonds, such as hydrocarbons, sigma bond cleavage occurs. Sigma bond cleavage requires a large amount of energy, and when there are no π electrons and n electrons in the compound, sigma bond cleavage may become the main cleavage mode. The more stable the products formed after cleavage, the easier the cleavage occurs. The stability order of carbocations is tertiary > secondary > primary. Therefore, hydrocarbon compounds are most likely to undergo bond cleavage at branching points. Moreover, the cleavage that loses the largest alkyl group is the easiest to occur.

Rearrangement Cleavage

• McLafferty Rearrangement: Molecules or fragment ions containing double bonds and hydrogen atoms at the γ position can undergo McLafferty rearrangement. In this cleavage, the hydrogen at the γ position migrates to the ionized double bond carbon or heteroatom through a six-membered ring transition state, while the alkene bond cleaves, generating neutral molecules and fragment ions.
• Retro Diels-Alder Fragmentation (RDA): Compounds with intramolecular double bonds can undergo RDA cleavage, generally producing a positively charged conjugated diene radical and a neutral molecule.
• Loss of Small Molecules: Fragment ions generated by the loss of small molecules such as water, hydrogen sulfide, and hydrogen halides frequently appear in the mass spectra of some organic compounds. Alcohols readily lose water molecules, resulting in the relatively small relative abundance of molecular ion peaks or even the absence of molecular ion peaks in alcohol compounds.
• Complex Cleavage: Complex cleavage refers to the process in the mass spectrum of organic compounds where two or more chemical bonds consecutively break to generate fragments. Apart from rearrangement reactions, this phenomenon is also observed in the fragmentation of cyclic compounds. Complex cleavage typically involves the multiple cleavage of bonds in cyclic compounds, sometimes involving the transfer of a hydrogen atom.
• Skeleton Rearrangement: Skeleton rearrangement refers to the process in which groups such as methyl, aromatic, and those containing O, N, S, etc., migrate during the decomposition of organic compounds, generating small neutral fragments, radical ions, etc. Common neutral fragments include CO, SO, SO2, S2, CH=CH, etc.

Reference

1. Jones, Anthony E., et al. "A single LC-MS/MS analysis to quantify CoA biosynthetic intermediates and short-chain acyl CoAs." Metabolites 11.8 (2021): 468.