The correct use of the term isobar for molecules

Introduction

The term isobar has become increasingly used in analytical chemistry literature, particularly in the context of small-molecule mass spectrometry. In many cases, it is used interchangeably with the term isomer to describe compounds sharing the same molecular formula. ^{1 , 2 , 3} This can be observed in studies involving steroid assays, ⁴ hexose monosaccharides such as galactose, glucose, and mannose, ⁵ and more recently if the interference is less clear like fatty acid analyses involving different acyl compositions but identical molecular weights. ⁶

Historically, the term isomer was introduced in 1837 by J.J Berzelius, meaning “put together from the same pieces”. ⁷ Subsequently, Van’t Hoff introduced the theory of the three-dimensional structure and the idea of stereoisomers, thereby laying the foundation for the modern understanding of isomers. ⁸

Much later, in 1918, the term isobar was introduced by A. W. Stewart. ⁹ Derived from the Greek words isos (equal) and baros (weight), the term was originally used not to describe molecules, but to describe nuclides (nuclei of different chemical elements) that have the same total mass number (A), which is the sum of the protons and neutrons in the nucleus. At the time, the internal structure of the atomic nucleus was not yet fully understood. Two years later, in 1920, Ernest Rutherford proposed the existence of a positively charged subatomic particle, the proton and also speculated about the existence of a neutral particle of similar mass, which would eventually be discovered in 1932 by James Chadwick and called neutron. ¹⁰ In 1933, it became evident through the work of the Joliot-Curies and others that there is a slight mass difference between protons and neutrons, the neutron being slightly heavier. ¹¹ As a result, the term isobar refers to nuclides of different elements that share the same mass number, meaning they have the same total number of nucleons, but differ slightly in exact atomic mass. This revised definition is, in essence, a practical convention rather than a reflection of true mass equality. Thus, the concept of isobars is not based on exact mass parity, but instead reflects a historically developed and functionally convenient framework centred around nominal mass values.

Although the term ‘isobar’ remains a useful classification in nuclear chemistry, it found its way into mass spectrometry as well, where its meaning and practical relevance, especially for small-molecule analysis, is less clear. While in textbooks related to analytical chemistry and/or mass spectrometry, the definition of isomers is clear, ^{12 , 13} the definition of isobars is not referenced, ¹² or is constrained by the description of “same nominal mass (integral)”. ¹³

While the International Union of Pure and Applied Chemistry (IUPAC) still defines isobars as “different nuclides of equal mass number,”this definition originating from the Physical Chemistry division of IUPAC, and thus still limiting the term to elements. ¹⁴ In addition, Murray et al. published recommendations for terminology in mass spectrometry, including isobars as “Atomic or molecular species with the same nominal mass but different exact masses”. ¹⁵ This description has also been adopted by the Royal Society of Chemistry (RSC) Analytical Methods Committee. ¹⁶ Similarly, a recent graphical review regarding isotopes, isomers and isobars defined isobars as “same mass, different molecular formula”. ¹⁷

However two molecules may meet the definition of “molecules with the same nominal mass but different exact masses,” this does not inherently make them isobars, at least not when applying the term as used in nuclear chemistry. The fact remains that mass spectrometrists across different subfields whether in ICP-MS (geochemistry, elemental analysis) or ESI-MS (biochemistry, macromolecular studies) use the same terminology with distinct definitions. Given that mass spectrometry spans multiple disciplines, its terminology should ideally be self-consistent to avoid ambiguity.

Hence, the term isobar is often used to describe compounds with similar masses, particularly in the context of interfering peaks in a mass spectrum. However, the degree to which their exact masses correspond is far more determinative than merely sharing the same nominal mass. This distinction becomes even more pronounced as the molecular mass increases. Next, there are many practical examples of isomers in the literature, and when it comes to isomers, using this term provides the most clarity. Using the term ‘isobar’ for isomers ignores the essential reason why the term ‘isobars’ was introduced in the first place, namely, to name different elements with equal nucleons but different proton to neutron (P:N) ratios. When ‘isobars’ is used for molecules, it should refer to different molecular formulas with equal nucleons, different P:N ratios, and therefore similar masses. Moreover, practical examples of molecular isobars, which may or may not interfere with mass spectrometry, illustrate that an isobar cannot be an isomer and vice versa.

In practice for isomers and molecule isobars

For isomers, which have an identical molecular formula and consequently an equal P:N ratio but different structures, distinguishing between them in mass spectrometry can be challenging. Depending on their structural (dis)similarity, they can be separated chromatographically, as is often the case with steroids and the ratios of the MS fragments ions can vary greatly. ¹⁸

In contrast, with isomers with a very similar structure, such as C6 sugars, this can be very difficult, depending on the column used and also the MS fragments ions are very similar to each other. These characteristics are typical for isomers and overall the term isomer is generally used correctly when it concerns to these molecules with identical molecular formula.

Isomers are common in practice, but there are also practical examples of isobaric molecules. Isobaric molecules can be divided into two groups. In both cases, there is a difference in the molecular formula, but in the first group, the P:N ratio between the two molecules differs (like elements), and in the second group, the P:N ratio between the two is the equal.

Regarding to the first group, if an analyte or a matrix compound has the same number of nucleons as the target analyte, but differs in the P:N ratio, this will result in a chemically different compound and/or a difference in the functional group. A simplified example is shown in Fig. 1, where an aldehyde group has the same nominal mass as an ethyl group. In these cases, the term isobar is appropriate due to difference in P:N 15:14 for the aldehyde group and 17:12 for the ethyl group. However, a difference in the functional group almost always leads to a different interaction with the stationary phase of the analytical column and also to a different fragmentation pattern with the MS/MS compared to the target analyte.

Fig. 1:

Mass spectrum by the national institute of standard and technology (NIST) of butane, C₄H₁₀ with monoisotopic mass 58.078 and propanal, C₃H₆O with monoisotopic mass 58.042. ¹⁹

For the second group, there is a practical example that slightly differs from the isobar example above, where the authors correctly used the term ‘isobar’. ²⁰ In this example, glucose-6-sulfate and glucose-6-phosphate differ in their functional groups (phosphate and sulfate), but the P:N ratio is identical for both molecules. However, there remains a small mass difference of approximately 0.009 Da, which arises from the difference in nuclear binding energy between the combination of ³¹P + ¹H and ³²S. In practice, the similarity results in insufficient interaction with the stationary phase of the column, causing the two analytes to co-elute. Furthermore, the fragmentation pattern is also similar for both analytes (Fig. 2), making this example very challenging to separate both chromatographically and by mass spectrometry. Unlike the first group, this example highlights the difficulties in separation between isobars.

Fig. 2:

Sulfated monosaccharide with monoisotopic mass 259.012, P:N 135:124 and phosphorylated monosaccharide with monoisotopic mass 259.022, P:N 135:124, figures adapted and simplified from Zang et al. ²⁰

Conclusion

Clearly, it remains crucial to specify the distinction between molecules with an equal number of nucleons and to clarify whether it involves interference of the same molecular formula and exact mass (isomers) or interference with a different molecular formula and a possible difference P:N ratio (isobaric). If the term ‘isobar’ is exclusively employed for the latter purpose, similar to categorizing the difference between two elements with an equal sum of nucleons, only then does ‘isobar’ serve as a valuable addition to the nomenclature in analytical chemistry.

When the mass interference is unknown, it serves no purpose to substitute the notion of equal or similar weight with the Greek-derived term isobar. The term ‘isobar’ has a clear meaning in element chemistry, which is closely connected to analytical chemistry. Given the practical examples of molecules that can be explained as isobars of each other, as indicated by the provided examples, it is illogical to use ‘isobars’ interchangeably for ‘isomers’ in analytical chemistry, or for nominal mass interference in general. The diagram (Fig. 3) can be used for clarification of similar/equal weight molecules.

Fig. 3:

Schematic overview of different types for equal nucleons/similar weight molecules.