Achieve broad lipid quantitation using a high-throughput targeted lipidomics method

LC-based approach for lipid class separation and quantitation on high sensitivity SCIEX Triple Quad™ and QTRAP® Systems

Mackenzie Pearson, Santosh Kumar Kapil, Paul Norris, Christie Hunter
SCIEX, USA

Abstract

The complexity of the lipidome is very high with a huge diversity in lipid molecular species making comprehensive quantitative profiling challenging. In this work, a targeted lipid profiling method has been developed for the quantitation of a large panel of lipid molecular species (~1900 molecular species). An optimized chromatography method was coupled with an Multiple Reaction Monitoring (MRM) assay which enabled a relatively rapid and specific lipid screening technique. The current MRM panel covers most major lipid classes and categories, and fatty acids with 12-26 carbons and 0-6 double bonds including odd chain lipids. This strategy includes an easy to use assay optimization tool (sMRM Pro Builder template) which allows for retention time scheduling of the MRM acquisition and the application of the method to many different biological matrices.

RUO-MKT-02-8482-B_0

Introduction

Direct infusion ‘shotgun’ lipidomics is an established approach for broad-based lipidomic analysis1. It is fast and simple, but it can suffer from inherent ion suppression effects, and due to the complexity of the lipidome and the extensive isobaric overlap, there is potential for ambiguous identification2. Reverse and normal phase LC strategies coupled with MS are also frequently used for lipidomics analyses. These strategies separate lipids based on their physico-chemical properties, but the huge diversity of lipid molecular species makes development of standardized “all-inclusive” methods challenging, especially when quantitation is desired3. Furthermore, discovery-based approaches such as information dependent acquisition (IDA) suffer from poor reproducibility, making quantitation unreliable4.

An efficient way to maximize sensitivity and specificity is through targeted lipidomics using HPLC and Multiple Reaction Monitoring (MRM) with a triple quadrupole-based mass spectrometer. A LC separation that can separate lipids into classes and subclasses is key to such an assay5.

Here, a targeted method is described using qualified MRM transitions and commercially available lipid internal standard mixtures, which provides quantitative measurement of ~1900 different lipid molecular species (Figure 1). The target list is can be shortened for a class-specific study or expandable to include new lipid classes. 

Figure 1. Overview of the LC-based targeted MRM assay. The targeted lipidomics analysis via LC-MS/MS provides for rapid identification and quantification of a broad spectrum of lipid molecular species in a variety of matrices.

Benefits of LC-MRM and SCIEX Triple Quad or QTRAP Systems for targeted lipidomics

  • Optimized chromatography separates lipids by class, which reduces inter-class isobaric interferences (17 mins per sample)
  • Individual lipid classes elute within a narrow RT window, making it simple to assign the RTs and implement time scheduled MRM to achieve the detection of ~ 1900 lipids
  • SCIEX Systems offer wide dynamic range, fast polarity switching scan (< 5msec), as well as high sensitivity even at high acquisition rates (3-5 msec dwell times)
  • The MRM scan mode maximizes the sensitivity and the specificity, which enables rapid and facile quantitation
  • This method utilizes internal standards for developing time scheduled MRM methods as well as for downstream relative quantitative analysis
  • SCIEX OS Software automatically processes data using a pre-built processing method