Improve LC-MS Run Time/Performance & Reduce Risk of Ion-Suppression

HybridSPE™ - Phospholipid Technology

When analyzing biological samples via reversed-phase LC-MS, ion-suppression often occurs in the early (e.g., salts) and late regions (e.g., phospholipids) of a chromatogram. However, as analysts strive for faster run times (=3 min.), resolving analytes of interest from interfering matrix components becomes a greater challenge (Figure 1).

Decreasing LC Run Time

Figure 1. Decreasing LC Run Time Increases Risk of Ion-Suppression.

LC Accumulation of Phospholipids

With advances in LC-MS technology, many analysts are decreasing LC run time by incorporating ballistic gradients and sub-2 µm HPLC column particles. When coupled with standard protein ppt (e.g., plasma:acetonitrile, 1:3 v/v), ballistic gradients are often inadequate at purging the column of phospholipids. As a result, phospholipids can build on the column, potentially change LC retention selectivity, and elute uncontrollably downstream in an injection run sequence causing unpredictable ion-suppression effects and poor reproducibility. Figure 2 compares a series of reversed-phase gradient LC-MS injections after standard protein ppt with HybridSPE-PL in which m/z 184 (phosphonate moiety of phospholipids) is monitored. In addition, sub-2 µm HPLC columns are more prone to clogging than larger particle size columns (2.7-5.0 µm). Unlike traditional protein ppt techniques that use centrifugation to remove precipitated proteins, HybridSPE-PL 96-well plates contain a series of filters that allows users to concurrently remove proteins and phospholipids reducing LC column backpressure (Figure 2).

Gradient RP LC-MS of Blank Plasma Samples

Figure 2. Gradient RP LC-MS of Blank Plasma Samples Prepared by Standard Protein PPT vs. HybridSPE-PL.

Reduce Analytical Run Time Using HybridSPE-PL

When processing plasma samples using standard protein ppt, contaminating phospholipids are strongly retained and do not elute off the column after 10 min. Therefore, shorter run times (e.g., < 5 min.) pose a great risk to on-column phospholipid accumulation/contamination. Gradient conditions are often required to elute phospholipids in a reasonable time frame (Figure 3). Because HybridSPE-PL depletes phospholipids from the sample, it is not necessary to run long gradient conditions to “wash-off” contaminating phospholipids. We demonstrate the utility of combining HybridSPE-PL and Ascentis® Express column technology in Figure 4 in which less than 90 second run time was achieved under isocratic conditions. Because HybridSPE-PL was employed during sample prep, the risk of phospholipid ion-suppression and on-column accumulation was eliminated.

Phospholipid Contamination from Standard Protein

Figure 3. Phospholipid Contamination from Standard Protein PL Requires Increased Gradient Run Time (> 10 min.).

Ascentis Express C18 (isocratic) and HybridSPE-PPT for Verapamil and Metabolites in Rat Plasma

Figure 4. Less than 90 Sec. Run Time Achieved Using Ascentis Express C18 (isocratic) and HybridSPE-PL for Verapamil and Metabolites in Rat Plasma.

Materials

     
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