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How Sample Prep for Phospholipid Removal Works

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
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Problem: 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. In bio-analytical 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, and losses in accuracy and sensitivity. Such deleterious effects are often most notable at the lower limits of quantitation (LLOQ).

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

In addition to ion suppression, phospholipids tend to accumulate on the analytical column, leading to potential changes in retention selectivity. 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—while the goal is decreasing run times.

Precipitation schematic for HybridSPE-PPT 96-well format

Solution: HybridSPE®-PPT technology is a simple and generic sample prep platform designed for the gross level removal of 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). An upper PTFE frit impedes premature flow of the sample before vacuum application. After a brief mixing/ vortexing step to facilitate protein precipitation, vacuum is applied to the 96-well plate. The 96-well version contains a series of low porosity hydrophobic filters/frits. The packed-bed filter/frit assembly acts as a depth filter, facilitating the concurrent removal of both phospholipids and precipitated proteins during the extraction process. After the protein-precipitated sample passes through the HybridSPEPPT device, the resulting effluent is free of phospholipids and ready for immediate LC-MS or LC-MS-MS analysis.

Mechanism Details:

When a plasma/serum sample is subjected to protein precipitation via the addition of acetonitrile (containing 1% formic acid), it passes through the HybridSPEPPT packed bed. This packed bed consists of proprietary zirconia-coated silica particles. The zirconia sites exhibit Lewis acid (electron acceptor) properties. Structurally, phospholipids consist of a polar head group (zwitterionic phosphate moiety) and a large hydrophobic tail (two fatty acyl groups that are hydrophobic). The phosphate group—inherent with all phospholipids—acts as a very strong Lewis base that will interact strongly with zirconia atoms functionalized on the particle surface. 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.

Lewis acid-base interaction between Hybrid-SPE zirconia ions and phospholipids.

In summary, the HybridSPE-PPT sample prep product is a valuable tool in LC-MS analysis; it helps to eliminate ion-suppression in MS detection, as well as the need for long gradient run times to elute on-column phospholipid build-up.

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HybridSPE is a registered trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co.