Multiplexed assay using the SCIEX QTRAP 6500+ system
Paul Norris, Santosh Kapil Kumar Gorti, Mackenzie Pearson
SCIEX, USA
Lipid mediators are a class of bioactive molecules that regulate many physiological processes and disregulation of their biosynthesis and signaling is linked to many inflammatory diseases. They are biosynthesized from arachidonic acid (i.e., eicosanoids) as well as from other fatty acids including EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). Lipid mediators influence a diverse range of cellular functions that govern both inflammation1 and its resolution2. For example, pro-inflammatory mediators include prostaglandins and leukotrienes, while specialized pro-resolving mediators (SPMs) include lipoxins, resolvins, protectins and maresins. These mediators regulate immune cell motility, migration and cytokine release as well as the presentation of antigens and the generation of antibodies. During acute inflammatory conditions, specific lipid mediators are produced by neutrophils, macrophages and platelets, as well as by endothelial and epithelial cells to accelerate the killing and clearance of infectious agents.1,2,3,4
The ability to identify and differentiate between many structurally related lipid mediators at low concentrations in biological samples is critical to the understanding of human physiology and disease response. Targeted lipidomics strategies using highly sensitive LC-MS/MS has emerged as an important analytical technique that can tackle the challenge of lipid mediator profiling and characterization.3,5,6,7
In this report, a quantitative and qualitative workflow is described for the analysis of different lipid mediator species including specialized pro-resolving mediators (SPM) leukotrienes, prostaglandins, hydroxy-eicosatetraenoic acids (HETEs) and epoxy-eicosatrienoic acids (EETs) using the QTRAP 6500+ system (Figure 1).
Sample Preparation: Standards were obtained from Cayman Chemical and used for optimization of the MRM transitions. Sixteen deuterium labeled internal standards were used in addition to 88 external standards.
Chromatography: Sample separations were performed on an ExionLC AD system using a Phenomenex Kinetex 2.6 µm Polar C18 column (100 x 3.0 mm). A gradient elution of methanol in 0.1% formic acid was used to elute the analytes over a 20.5 min run time at 500 µL/min.
Mass Spectrometry: A QTRAP 6500+ system with an IonDrive Turbo V ion source (electrospray ionization probe) was used for data acquisition in positive and negative mode. Multiple Reaction Monitoring (MRM) was used for quantitation of the various lipid mediators. Details on the LC-MS/MS methods can be found in the supplementary information.9
Data Processing: MultiQuant software was used for computation of calibration curves, percent CVs and standard deviations.
Lipids, in general, including lipid mediators, are historically difficult to resolve using reversed-phase chromatography. Variations in the positions, geometries, and linkages of fatty acyl chains, modifications to functional groups, and the high number of isomeric and isobaric species, all contribute to the wide diversity of lipid species and the challenge of separation. As shown in Figure 2, the Kinetex Polar C18 column provides both polar and non-polar retention that facilitates the separation of lipid mediator epimers and isobaric compounds resulting in improved assay performance statistics and minimization of false positives.
Because of their fatty acid structure, the majority of lipid mediators are best analyzed using negative ion mode analysis. However, a subset of lipid mediators can be analyzed with higher sensitivity using positive ion mode analysis. Using the fast polarity switching capabilities of the QTRAP 6500+ System, both polarities can be used in a single injection for the optimized detection of all lipid mediators within one assay (Figure 3), greatly reducing the burden of analysis and doubling sample throughput.
Figure 4 highlights the unique capability of the QTRAP system technology to construct powerful workflows leveraging both the ion trap and triple quadrupole functionality of the system. Here, the MRM triggered EPI workflow was used for the simultaneous collection of both quantitative MRM data as well as qualitative full scan MS/MS data. For isobaric lipid mediator species like PGD2 and LXB4 (Figure 4), informative, high-quality MS/MS spectra allow greater confidence in lipid species identification rather than MS alone.
Concentration curves were generated by injecting 3 replicate injections of 8 different concentrations of each lipid mediator standard. Excellent linearity is observed with r2 of 0.999 obtained for the representative concentration curves for selected resolvins, prostaglandins, EETs, and non-enzymatic isoprostanes as shown in Figure 5.
Table 1 displays the data (x3) from the mix of lipid mediators, pathway markers and metabolites. Data for each lipid mediator was processed using MultiQuant Software and the coefficients of variance and accuracy were calculated. Calculated CVs for all compounds were less than 30% and LLOQ were between 0.05 and 1 ng/mL.
Figure 6 shows the complete baseline separation of pro-resolving lipid mediator epimers. Baseline separation of LXA4 and 15-epi-LXA4 (top panel) as well as RvD1 and 17-epi-RvD1 (bottom panel) enables the differentiation of enzymatic pathway utilization for pro-resolving lipid mediator production. Specifically, LXA4 and RvD1 are produced by 5- and 15-lipoxygenase pathways, and 15-epi-LXA4 and 17-epi-RvD1 are produced by 5-lipoxygenase and acetylated cyclooxygenase-2 pathways. Thus, separation, identification, and quantitation of these species can reveal important biological insights into the mechanisms of inflammatory processes governing various biological observations (Figure 1).
A high-throughput targeted quantitative LC-MS/MS assay was designed to monitor lipid mediators. By taking advantage of the fast positive-ion/negative-ion polarity switching of the QTRAP 6500+ system, individualized and optimized conditions could be used to monitor a wide diversity of lipid mediator species within one 20 minute MRM assay. Additionally, the HPLC method enabled resolution of isobaric and isomeric species that are historically challenging to separate using reversed-phase chromatography. Excellent sensitivity was obtained for all lipid mediator species with LLOQ between 0.05 and 1 ng/mL and CV < 30%. The unique qualitative/quantitative QTRAP system also enabled MRM triggered EPI experiments which provided high sensitivity MS/MS data for confirmation of low-level analytes.