Current State of Quantitation in Lipidomics Analysis

Posted on March 29, 2019

The field of lipidomics has rapidly expanded in the last ten years. Improvements in mass spectrometry (MS) technology and data analysis have moved this exciting area beyond academic curiosity into the fields of biomarker research, drug discovery and the clinical setting. There are two significant challenges in the field that still must be better addressed before lipidomics can become “mainstream.” The first is related to the polymeric nature of lipids themselves: isobaric interference. This refers to two different compounds that have either exactly or nearly the same mass. A deeper dive into this issue will be the subject of another community blog entry. The second issue is quantitation and is the primary subject of this posting.

There are over 150,000 different molecular species of lipids—based on the diversity of lipid class, the type and location of fatty acids, the number and location of double bonds, and the stereochemistry of the olefins. For absolute quantitation, a labeled internal standard and a primary reference standard are required to generate internal standard curves, and despite our efforts at Avanti, we are nowhere near this level of coverage. Thus, the field of lipidomics has to rely on two types of quantitation: relative quant and “accurate” quant for broadly-based lipidomics analyses.

Successful relative quantitation is based on a proper analytical method paired with the appropriate internal standards. For infusion and normal phase separations (including HILIC), a single internal standard per lipid class is sufficient to perform relative quantitation. Avanti’s SPLASH® LIPIDOMIX® standards are ideal for this type of quantitation because the members of each class either co-elute or are all present in the infusate, so variable matrix suppression is not an issue as it is with reverse phase. Using a relative quantitation strategy, two samples can be compared for differences in specific lipid species. A downside to the approach is it is not possible say anything about the relative amounts of the different lipid species within the sample—the data are only relevant when compared to another sample. This approach is commonly used in attempts to identify biomarkers in a cohort study. Once potential markers are identified, a follow up study using absolute quantitative approaches (i.e., an MRM method using authentic internal and reference standards) is required to validate the observation.

“Accurate” quantitation is a more appropriate name to describe any attempts to quantify lipid molecular species at the concentration level. Since absolute quantitation is not possible due to lack of sufficient standards, accurate quant is the effort to quantify lipid molecular species at the concentration level within an implicit quantitative bias. As with any quantitative strategy, the methodology must be paired with the correct standards to be effective. When using an infusion or normal phase technique that enables all lipids of a particular class to be measured essentially simultaneously, accurate quantitation of lipids at the sum composition level is possible with a single internal standard when the method is designed to target common precursor ions and neutral losses for a lipid class. This is not the case for reverse phase experiments where the matrix varies during the gradient elution. On the other hand, molecular speciation (i.e., identification of a lipid at the fatty acid level) requires a more comprehensive set of standards that reflect the differential fragmentation efficiency of fatty acids that depends on the numbers of carbons and double bonds. The LipidyzerTM standards—made by Avanti and sold by SCIEX—are the first generation of internal standards that were designed to accommodate this differential fragmentation. The standards were designed for plasma, so some classes are not included, and the relative concentrations were set to reflect plasma concentrations. Recognizing that cell culture and tissue lipidomics are equally important, Avanti is planning a universal standards kit that covers most major lipid classes, and it is designed to work with commonly used lipidomics analytical methods—including reverse phase—to provide accurate quantitation for all matrices. Stay tuned for more updates on this front!

For more information and a deep dive into Quantitation in Lipidomics, check out Dr. Robert Murphy's video!

What are your thoughts on quantitation in lipidomics? Do you agree/disagree with this post? Your comments are welcome and encouraged.

Contributed By: Paul RS Baker, PhD, Director, Analytical Division, Avanti Polar Lipids