Starting up a workflow for lipid mediator analysis can be challenging. Hundreds of distinct fatty acid-derived molecules can be produced biologically (i.e., enzymatically) and/or via non-enzymatic routes. Additionally, various biosynthetic pathways yield both bioactive mediators as well as side-products, intermediates, and metabolites that may not always be of interest for labs focused on select biomarkers, yet to some researchers all of these products are important for understanding the global changes in lipid mediator pathways. Here, we will provide some tips, tricks and general advice for setting up lipid mediator methods.
Choosing your chromatographic mobile phases, gradient, and column
Lipid mediators are diverse in terms of the number of possible double bond and functional group positions and geometries; however, the number of distinct parent masses and fragment ion masses is relatively small due to these compounds being derived from a limited set of fatty acid precursors and having many shared structural features (e.g., conjugated dienes, trienes, and tetrataenes; usually 1 to 3 hydroxyl groups and in fewer cases epoxides, peptides or nitrosyl groups). As a result, liquid chromatography methods for lipid mediator profiling generally demands a longer run time on the order of 15 – 20 minutes (or longer if desired) to achieve sufficient separation of the hundreds of distinct mediators and especially the various epimers and other isobaric species.
Mobile phase and modifier combinations can be fairly simple and in general, choosing water as mobile phase A and methanol or acetonitrile as mobile phase B gives good results. Of utmost importance is to use formic or acetic acid ideally within the range of 0.01% to 0.1 %. For the most expansive profiling and analyte coverage in your method, it is recommended to use higher acid concentrations (i.e., 0.1% formic or acetic acid) to maintain good peak shape for cysteinyl leukotrienes and other peptide conjugates like the eoxins, MCTRs, PCTRs and RCTRs (note that these compounds are all ideally monitored in positive mode). Using lower acid modifier concentrations (i.e., 0.01% formic or acetic acid) is advised for maximal sensitivity for negative mode analytes, but ONLY if peptide conjugate species mentioned above are NOT of interest in your methods.
Choosing your ideal HPLC column and stationary phase chemistry depends mainly on the classes of compounds you want to focus on for maximal separation. For a good general separation strategy across all lipid mediator classes, C18, C8 and some modified C18 stationary phases work well but note that each will have certain compounds that become difficult to resolve and therefore it is recommended that you try a few different types of stationary phases to confirm that your compounds of interest separate adequately. Phenomenex Polar C18 columns are able to separate a few compounds like PGE2 and PGD2 from LXB4 where standard C18s tend to fail. Still, standard C18s tend to give slightly tighter peak shape which can generally improve overall sensitivity and resolution of various mediators. Another column worth considering for a general profiling strategy is a biphenyl stationary phase. Biphenyl columns can achieve better separation of certain classes of metabolites and mediators, including the urine prostaglandin metabolites (e.g., tetranor-PGEM, tetranor-PGDM, and tetranor-PGFM). No one column chemistry can fully separate all lipid mediators, but using these general observations can help you decide between various stationary phases. While C18 and Polar C18 columns can separate a number of different lipid mediator epimers, chiral columns are still advised for applications where monohydroxy fatty acid stereochemistry must be to be investigated.
How to navigate and read a LC-MS/MS lipid mediator chromatogram
Once you have successfully acquired a lipid mediator LC-MS/MS dataset, knowing where and what to focus on can help to make sense out of the data. Most lipid mediator methods use scheduled multiple reaction monitoring (sMRM) methods to achieve high sensitivity peaks with minimal background and with a good balance of data points and dwell time.
With reverse-phase chromatography methods like the ones mentioned above, the fatty acids, which are the starting point substrates/precursors in the lipid mediator pathways, elute latest in the chromatogram. Usually, the second level down in these pathways (mono-hydroxy fatty acids) elute second to last in the chromatogram and these compounds often provide the most abundant and easy to identify molecules that help to say what biosynthetic pathways the fatty acids in a sample are fluxing through (e.g., lipoxygenases and cyclooxygenases, or via non-enzymatic oxidation). In this same region, EETs and other products of CYP450 epoxidases also elute. Many other classes of compounds elute in this later chromatographic region as well, including nitrosylated fatty acids, PAF, and ethamolamides/endocannabinoids.
In the middle to late middle of the run, most of the leukotrienes and SPMs (specialized pro-resolving mediators) elute and often these will provide a complex profile with multiple peaks. For lipoxins, leukotrienes and some of the resolvins as well as protectins and maresins, usually two major and two minor non-enzymatic and all-trans conjugated triene or tetraene-containing isomers, as well as the bioactive mediator and a double-dioxygenation product can be seen which reflects the combination of nonenzymatic and enzymatic conversion of an epoxide intermediate (for example conversion of LTA4 to LTB4, 12-epi-LTB4, 6trans,12-epi LTB4, 5S,12S-DiHETE, and two other, minor nonenzymatic products).
Toward the early and middle of the chromatogram, most of the prostaglandins, prostaglandin metabolites, thromboxanes and nonenzymatic isoprostanes elute. Of all these compounds, the two that usually stick out are thromboxane B2 and 6-keto PGF1alpha. Both of these products are stable non-enzymatic degradation products used to account for their bioactive precursors, thromboxane A2 and PGI2, which promote and counter regulate platelet aggregation, respectively. These products usually appear as a smeared peak that is much wider than all other analyte peaks in the chromatogram. If this feature for the 6-keto PGF1alpha and thromboxane A2 peaks is observed, this is OK and is common among reversed-phase methods and if nothing else can be used diagonostically to tentatively identify these mediator breakdown products. You can also look for PGE2 and PGD2 in the same chromatogram using the 351/189 m/z MRM transition. Normally with reversed phase methods, PGE2 will elute first and PGD2 will be the later eluting peak in this chromatogram. If your method is able to achieve baseline separation of PGE2 and PGD2, it is one of the first and primary ways to qualify that you have an adequate chromatography method for lipid mediator profiling. It is also important to note that PGD2 is noticeably more labile than PGE2 in aqueous solvents and over time, one might expect to see the PGD2 non-enzymatic breakdown products PGJ2 and 15-deoxy PGJ2 (notably a PPAR gamma agonist) increasing if a sample is injected again over the course of a day or more. As a result, attention should be paid to these products when planning out sample injections that may take several days. Careful attention should also be placed on that ensuring many of the prostaglandin metabolites are separated since these can elute quite closely and many share the same parent mass and in some case the fragment ions that are most abundant. Careful attention to MRM selection and chromatography are thus emphasized for accurate identification and quantitation of prostaglandins and their metabolites.
Earliest in the chromatographic profile will be the tetranor prostaglandin metabolites (e.g., tetranor-PGEM, tetranor-PGDM, and tetranor-PGFM). These products are terminal prostaglandin metabolites largely formed during circulation through the liver and can be useful for prostaglandin measurement in the urine. These compounds elute very early in the chromatogram and may require some trial and error to achieve adequate elution and reproducibility when using extracted urine matrix.
We hope these tips and tricks for setting up your lipid mediator method and for reading the lipidomic profiles will accelerate your research and stay tuned for more tips and tricks for setting up more advanced workflows!
- Targeted Profiling of Lipid Mediators – QTRAP 6500+ system. SCIEX technical note RUO-MKT-02-10787-A.
- Higher sensitivity analysis of a large panel of lipid mediators. SCIEX technical note RUO-MKT-02-12508-A.