Analysis of Lipids by High-resolution, Accurate Mass Measurement Mass Spectrometry

Posted on May 31, 2019

Today’s topic for discussion is high resolution, accurate mass measurement mass spectrometry (HRAM MS) as it applies to the analysis of lipids. Mass analyzers generally come in two flavors: Low resolution or High resolution. Low resolution mass analyzers, such as quadrupoles or ion traps, yield broad peaks, which may not have full isotopic resolution and yield nominal or integer mass information. Here the mass measurement error is in the first decimal place. High resolution mass analyzers such as TOFs, Orbitraps, or FTMS (does anyone make a magnetic sector anymore?) yield fully resolved isotope peaks with mass measurement error in the fourth decimal place or beyond.

High resolution and accurate mass measurement is critical for obviating two vexing problems found in the analysis of lipids: interferences arising from isotopes and isobars. For example, consider the simple molecules N2, CO, and C2H4. Each has a nominal mass of 28u, and is indistinguishable from one another using low resolution MS. However, using HRAM MS their masses are 28.0061, 27.9949, and 28.0313, respectively, which are easily distinguished on high resolution instruments. Moreover, the resolving power required to accomplish this is given by the formula M/Dm (28.0061 / 0.0112 = 2500).

The situation is much more complicated at higher masses where many lipids are observed. Consider two sulfatides having the same headgroup and sphingoid base, however: one has an alpha hydroxy fatty acid with an even chain length, and 2 double bonds (d18:1 / h22:2), the other has an n-acyl chain with and odd carbon number and 1 double bond (d18:1 / C23:1). Their corresponding molecular formulas and masses are C46H84N1O12S1 - 874.5720, and C47H88N1O11S1 – 874.6084, respectively. At first glance the mass gap between them appears large. However, owing to their higher mass, they require almost an order of magnitude higher resolving power to distinguish these two closely related species (874.6084 / 0.0364 = 24,000).

This is where the other piece of the puzzle, mass accuracy, plays an important role. Each mass analyzer must be properly calibrated to within a generally accepted +/- 5 ppm (part per million) mass window. This is most simply calculated by starting at the 4th decimal place and moving to the left 6 places to give a 1ppm error. For the sulfatide example above, 1ppm is +/- 8.7 in the last decimal place (or simply +/- 0.0009) as shown in the figure:


Multiplying the 1 ppm value by 5 yields +/- 0.0045, which defines mass windows of 874.5675 – 874.5765 for sulfatide (d18:1/h22:2) and 874.6039 – 874.6129 for sulfatide (d18:1/C23:1) that can be used to confirm the elemental compositions of these two molecular ions. (Note, elemental composition is defined, not structure.)

Taking a step back to look at the bigger picture, it can be seen that as either the mass or measurement error gets larger the two species will begin to overlap. This means that one must have increasingly higher mass resolution, mass accuracy, or both to distinguish closely related species. This is why ultra-high resolution mass spectrometers are gaining in popularity. Not only do they provide 6 digit resolving power, but mass accuracy is less than 3ppm, and does not change as readily over time with changing environmental conditions. There are other tricks one can add to further aid in ID and structural determination, but that is another conversation for another time. Sláinte!

Contributed By: Cameron Sullards, Ph.D., Manager of Methods Development, Avanti Polar Lipids