APR Nov/Dec 2022 - 36

QC CORNERQC CORNER
Coping with Matrix Effects Caused by
Phospholipids in Biological Samples
Geoffrey Rule, Ph.D.
Principal Scientist
MilliporeSigma, Bellefonte, PA
A business of Merck KGaA, Darmstadt, Germany
Many scientists doing bioanalysis in the pharmaceutical
industry use protein precipitation to clean up samples prior
to analysis of small molecule drugs by LC-MS/MS. While
this technique removes proteins quickly and inexpensively,
it does not address the issue of ion suppression caused
by phospholipids, which are present in biological sample
matrices such as serum, plasma, and whole blood. During
chromatography, coelution of phospholipids with the
analyte of interest results in ion suppression and thus a
decrease of mass spec signal that can cause variability and
impact accuracy in quantitation. If the phospholipids do
not immediately coelute with the analyte of interest, they
can accumulate on the analytical column and elute later,
unpredictably, during downstream analyses.
Ballistic gradients and small particles
amplify the issue
Advances in LC-MS technology have allowed analysts to
decrease LC run times by using ballistic HPLC gradients
and columns with particles sizes of 2 μm or less. However,
ballistic gradients often do not purge the column well
enough of phospholipids that remain after typical protein
precipitation protocols, and HPLC columns with small
particles are generally more prone to clogging than ones
with larger particles. Because contaminant phospholipids
are often highly retained on the analytical column, they can
take a prolonged period to elute. With the shorter run times
however, phospholipids can accumulate on the column
unless the analyst also adds a long column washing step.
This added step can decrease laboratory throughput.
Traditional SPE versus chemical filtration
One approach to overcome the problem is to use traditional
solid phase extraction (SPE). These methods are often
based on a hydrophobic retention mechanism to separate
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| November/December 2022
Hugh Cramer
Scientist
MilliporeSigma, Bellefonte, PA
A business of Merck KGaA, Darmstadt, Germany
the phospholipids from the sample's analyte of interest.
This mechanism, however, leads to problems if the analyte
is also hydrophobic. Such compounds are removed
along with the hydrophobic phospholipids, which
decreases analyte recovery and makes results inaccurate.
These methods also often require time-consuming and
analyte-dependent method development while still only
removing nominal amounts of phospholipids. Remaining
phospholipids can still accumulate on the analytical
column and thus impact future analyses, add to column
replacement costs, and increase instrument downtime.
This problem led to the development of a new approach
to phospholipid removal. Unlike with traditional SPE,
where the analyte is retained on the sorbent through
a washing step, the new approach utilizes a type of
chemical filtration that selectively removes undesired
phospholipids while allowing analytes to pass through
unretained. This method is performed from the same high
organic solvent composition, typically acetonitrile, that
is used to precipitate the proteins. A variety of products
designed specifically for the removal of both proteins and
phospholipids have become commercially available. The
stationary phase is typically packed into syringe-shaped
cartridges, 96-well microtiter plates, flat disks, or pipette
tip microextraction devices for small sample volumes.
Most of these products use standardized, simple, and fast
procedures requiring little method development.
How it works
A technology introduced in recent years offers a means
of removing phospholipids from a high organic solvent
" protein crash " while allowing analytes to pass through,
in essence a kind of chemical filtration. HybridSPE®Phospholipid
combines the simple, standardized
methodology of traditional protein precipitation in an SPE
APR Nov/Dec 2022

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