Quantitative Lipidomics Analysis— Comprehensive Coverage with Accurate Quantitation

Posted on August 30, 2019


Targeted Lipid Analysis

An efficient way to maximize sensitivity, specificity and quantitative accuracy is targeted lipidomics using HPLC ESI-MS/MS using curated MRM transitions that target lipids at the molecular species level. HILIC separation is an attractive chromatographic strategy that separates lipids into classes and subclasses, which span a very narrow RT window. Using a proprietary mix of internal standards, this method provides accurate quantitative measurement of over 1500 different lipid molecular species in virtually any matrix. A sample chromatogram with standards is shown below:

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Lipidomics analysis using a broadly targeted HPLC ESI-MS/MS approach enables quantitative analysis of over 1500 different lipid molecular species at the fatty acid level. Typically challenging isobaric classes of lipids such as PE, PC and PS are resolved, enabling unambiguous identification and quantitation. The method is highly reproducible in terms of retention times and %RSD.

Data are acquired using a SCIEX 6500+ Triple Quadrupole Instrument, which is both sensitive and fast enough to measure the hundreds of molecular species in each lipid class. In order to identify lipids at the molecular species level, phospholipids are analyzed in the negative ion mode; sphingolipids (with the exception of sulfatides) and neutral lipids are analyzed in the positive ion mode. This is made possible by the rapid polarity switching capabilities of the instrument. Furthermore, the dynamic range of the 6500+ covers approximately 6-orders of magnitude, enabling measurement of both high and low concentration analytes. Below is a list of the different lipid classes that are analyzed with this method:

  • Phospholipids
    • Phosphatidylcholines (PC, LPC)
    • Phosphatidylethanolamines (PE, LPE)
    • Phosphatidylglycerols (PG, LPG)
    • Phosphatidylserines (PS, LPS)
    • Phosphatidylinositols (PI, LPI)
  • Neutral Lipids
    • Triglycerides (TG)
    • Diglycerides (DG)
    • Monoglycerides (MG)
    • Cholesteryl esters (CE)
  • Sphingolipids
    • Sphingomyelin (SM)
    • Ceramides (CER)
    • Dihydro Ceramides (dCER)
    • Hexosyl Ceramides (HexCER) Sulfatides

Quantitation

Absolute quantitation is impossible for lipidomics analysis due to the fact there are well-over 150,000 different molecular species in the lipidome. However, there are two types of quantitation: relative quant and “accurate” quant that can be used 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 (https://avantilipids.com/product-category/lipidomics/lipidomix-standards-lipidomics-old) 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 to say anything about the relative amounts of the different lipid species within the sample—the data are only relevant when compared to another sample.

Accurate quantitation within a known quantitative bias is possible when the correct internal strategy is used. We recently created a proprietary lipid standard mixture—UltimateSPLASHTM—that has 69 deuterated internal standards covering the phospholipids, neutral lipids, sphingomyelin and ceramides. This mixture will be available commercially from our catalog in 1Q2020. The key to this quantitative standards mixture is the inclusion of multiple molecular species for each class (3-9, depending on the class). Short to long-chain fatty acids with varying degrees of unsaturation among the members of each class enable the correction of extraction efficiency, differential ionization, and most importantly, differential fragmentation efficiency due to variable fatty acid chain lengths and double bonds.

Concentrations of analytes are determined by a point concentration comparison to the appropriate standards. Each analyte is paired with the internal standard that best matches its molecular weight and fatty acid distribution. The closer the match, the better the accuracy. Our method has an overall quantitative bias of ~ 20%, which means individual values can be off by as much as 20% from the actual concentration of the analyte.


Benefits of Targeted Lipidomics Analysis

By using a broad, curated MRM transitions list, HILIC chromatography, and a comprehensive internal standard strategy, many issues that have proven to be challenging in lipidomics analysis via either the shotgun or an untargeted approach are minimized:

  • Chromatography minimizes problems with matrix suppression issues
  • MRM transitions are selected for specificity of complex lipids at the fatty acid level. (e.g., PC (16:0_18:1) rather than the sum composition nomenclature of PC 34:1.)
  • MRM transitions are included for each lipid class to include any complex lipid containing fatty acids from 14:0 to 22:6, which ensures comprehensive coverage and minimizes false positive results
  • Using a proprietary mixture of internal standards, accurate quantitation is possible at the molecular species level, which is not possible with shotgun techniques nor untargeted approaches
  • The time needed for data analysis is dramatically minimized using a targeted approach, significantly reducing the time needed for large cohort studies
  • Complete customization of the target list is possible

How to request quantitative lipidomics analytical services from Avanti

To order quantitative lipidomics services, please visit the Avanti website and fill out a sample submission form. Alternatively, e-mail us at analytical@avantilipids.com if you have any additional questions about this service or other Avanti products and services.


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

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