Selectively Remove Phospholipids from Biological Matrices

Excessive background from endogenous sample matrix components has always been of great concern in bioanalysis, and has become paramount today with the need for decreasing analytical run times.

By Sigma-Aldrich

In bioanalytical mass spectrometry, the issue of excessive background contributes to the problem of ion-suppression.

Ion-suppression is caused by one or more interfering components or species that co-elute with the analyte(s) of interest during LC-MS analysis, and manifests itself as a loss of analyte response. The co-eluting species can affect droplet formation or ionize concurrently, resulting in an erroneous decrease (suppression) in analyte signal response. The end result is poor assay reproducibility, also losses in accuracy and sensitivity.

One of the major causes of ion-suppression in bioanalysis is the presence of phospholipids during LC-MS analysis. Phospholipids are prevalent in extremely high concentrations in blood-based biological fluids (~1 mg/mL); and represent the second largest lipid component in biological matrices after triglycerides.

In addition to ion-suppression, phospholipids may build up on the analytical column. They may elute uncontrollably downstream in an injection run sequence, causing unpredictable ion-suppression effects and poor reproducibility. If gradient conditions are used to elute phospholipids, this leads to longer analysis run times.


HybridSPE-Phospholipid technology is a sample prep platform designed to selectively remove endogenous protein and phospholipid interferences from biological plasma and serum prior to LC-MS or LC-MS-MS analysis. Biological plasma or serum is first subjected to protein precipitation by adding the sample to the HybridSPE 96-well plate followed by acidified acetonitrile (precipitation agent). The filter/frit assembly acts as a depth filter, physically removing the proteins, while concurrently removing the phospholipids via Lewis acid-base interactions. The HybridSPE-Phospholipid packed bed consists of proprietary zirconia coated silica particles. The zirconia sites exhibit Lewis acid (electron acceptor) properties that interact strongly with Lewis bases (electron donor). The phosphate group, inherent with all phospholipids, acts as a very strong Lewis base that will interact intensely with zirconia atoms functionalized on the particle surface. After the protein-precipitated sample passes through the HybridSPE-Phospholipid device, the resulting effluent is phospholipid-free and ready for immediate LC-MS or LC-MS-MS analysis.

Formic acid is a critical modifier used in the precipitation agent to improve the recovery of many analytes of interest (e.g., acidic compounds); it plays a critical role in preventing analyte retention without affecting the phospholipid retention/removal process.

In summary, HybridSPE-Phospholipid products are valuable tools in LC-MS analysis. They help to eliminate both ion-suppression and the need for long gradient run times to elute on-column phospholipid build-up. For additional information, or to try a free sample, please visit . 

Categories: Products in Action

Published In

Designing for Science Magazine Issue Cover
Designing for Science

Published: July 10, 2014

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