Why Remove Phospholipids From a Sample?

When analysis of substances from serum or plasma takes place, it is likely that the samples will contain phospholipids, a major component of mammalian cell membranes. These phospholipids have a variety of structures and polarities, the more hydrophobic of those would gradually coat a reversed-phase HPLC column (such as a C18) over multiple injections. This process will result in a gradual increase in back pressure which is typically accompanied by a drop in separation efficiency. Selectivity changes may also occur.

If UV detection is being utilized, this may be as far as the problem goes. If samples have simply been prepared by protein precipitation, which is quick and cheap to perform, shorter column lifetime may simply be accepted as an inevitable result of performing low-grade sample pretreatment. If MS detection is being used, ion suppression or ion enhancement may be seen. This is generally accepted to be caused by the co-elution of phospholipids with the compounds of interest. The presence of the phospholipids in the MS source may impact the ionization efficiency of the source at that point.

Figure 1: Assessment of column sensitivity following repeated injections of diclofenac in protein precipitated plasma vs using a sample preparation product

As a result, the reliability of data produced may be affected by the presence of phospholipids because the amount of analyte could be underestimated (due to ion suppression) or overestimated (following ion enhancement). It is possible to check if this an issue in your separation by conducting the following experiment:

• Use a syringe pump to directly infuse a solution of your analyte of interest.
• At the same time, prepare a blank serum/plasma sample using your usual sample preparation procedure. Inject this blank sample and run your usual HPLC method.
• Monitor the ion current for your MS-MS transitions for the analyte of interest.

Figure 2: Areas of ion suppression using protein precipitated plasm vs plasma extracted using a sample preparation product

Across the resulting chromatogram, areas of ion suppression or ion enhancement could be seen. If your analyte of interest elutes in one of these zones there is a possibility you may have problems with data integrity.

The simplest solution to this issue is to use a sample preparation product which allows for the simultaneous precipitation of plasma proteins whilst removing phospholipids from the resulting supernatant. Such products consist of tubes or 96-well plates that have membranes to remove precipitated proteins, and a sorbent to retain phospholipids. Samples are mixed with a precipitating solution, such as methanol or acetonitrile, before being pulled down through the filter plate and sorbent bed. The resulting samples are then free of both protein and phospholipids enabling reliable quantitation and improved column lifetime.

Figure 3: Phospholipid removal using Phree