Isomers, Isobars and Isotopes …

Posted on August 14, 2019


Isomers are molecules that have the exact same elemental composition, but are different compounds. This is readily evident with fatty acid positional isomers [e.g., PC (16:0/18:1) Vs. PC (18:1/16:0)], which are only distinguishable with additional analysis such as differential ion mobility (DMS) or PLA2 hydrolysis. This problem also extends to members of different lipid classes. For example, in the negative ion mode, [PC (40:4)-CH3]- has the exact same mass as [PE (42:4)-H]- and [DimethylPE (40:4)-H]-, with their respective m/z = 822.6018. These molecules must be chromatographically resolved or separated by other means such as DMS to elucidate their identities. This problem is most pronounced in shotgun lipidomics where molecules are identified by lipid class-specific precursor ions and/or neutral losses. Because no fatty acid information is available in this type of analysis, lipids are denoted by their sum-composition (e.g., PC 34:1) rather than by their molecular species identity [e.g., PC (16:0_18:1)]. At Avanti, we deal with isomeric overlap in two ways. First, we have an LC-based method that enables separation of the lipid classes prior to analysis. This is accomplished using a HILIC column that separates the different lipid classes and directly targets a broad number of lipids in each class, allowing identification at the molecular species level. A second approach is shotgun-like in nature: MS/MSALL with DMS separating lipid classes in the gas phase prior to analysis by MS. This last method is an excellent, quantitative discovery method that requires very little method development and provides clear MS/MS data without isomeric or isobaric interference from other lipid classes. For more information about lipidomics analysis at Avanti, please email


Isobars are lipids that have either exactly the same mass (isomers) or have nearly the same mass. At the MS level, this problem can be addressed by high-resolution accurate mass (HRAM) mass spectrometry, which on some instruments is capable of 1-2 ppm accuracy and can generally identify molecules at the sum-composition level without the need for MS/MS analysis. However, when an MS/MS experiment is performed to identify the lipid molecular species of a particular molecule, isobars become a significant problem, regardless of whether the instrument is HRAM or nominal mass. The reason for this is that the precursor isolation step is very low resolution, and the isolation width can be as large ± 0.6 Da, depending on the instrument type. To illustrate this challenge, a database search on the LipidMAPS website shows the complexity of the isobaric interference of m/z 790.5392, depending on the instrument tolerance.


Note, these results are for sum composition species. Considering molecular species, the number is well-over 300 individual lipids.

The consequences of isobaric interference during MS/MS analysis are readily apparent from the figure below. Using the infusion-based MS/MSALL experiment, product ion analysis of 790.6 reveals 8 possible lipid molecular species (as determined by manual inspection of the data), based on the fatty acid fragment evidence between m/z 250 and 350 Da. Lipid database searches, if not properly set for baseline and fragment tolerance would report many more, which is the source of much of the erroneous lipidomics data reported in the literature today.


To resolve isobaric interference, some sort of separation technique is needed—be it LC or DMS—and careful analysis of the data is required, especially if lipid database searches are used.


The exact molecular weight of compounds is calculated using the molecular formula, and is based on all carbons in the molecule being 12C. However, in reality, ~1% of carbon is 13C, which shifts the mass 1 Da higher for each 13C atom present in the molecule. If two 13C atoms are present, the mass is shifted by 2 Da, which poses a problem with quantitation. For example, PC 32:1 (+AcO) has a mass of 790.6, and its n+2 isotope is 792.6, which is the mass of PC 32:0 (+AcO). As a consequence, data must be corrected for the isotope effect, which can be a challenging adventure with Excel. Of particular note, PCs and SMs fall into the same mass range, with PC m/z values being even, and the SM values being odd (see figure below).


Awareness of how isomers, isobars and isotopes affect your data must be the guiding force behind your method development for lipid analysis. Many challenges remain to be overcome before lipidomics analysis can become routine, high-throughput and robust.

Contributed By: Paul RS Baker, PhD, Avanti Polar Lipids