Phospholipids are perhaps one of the most troublesome components in bioanalytical samples when performing liquid chromatography
coupled to tandem mass spectrometry (LC–MS-MS) analysis. Shortened column life, ion suppression, and an increase in MS system
maintenance are just a few of the downfalls that can occur if phospholipids are not sufficiently removed from bioanalytical
samples before analysis. Rapid crude sample preparation methods such as protein precipitation are sometimes preferred over
more focused sample cleanup techniques like solid-phase extraction (SPE); however, such crude sample preparation techniques
primarily reduce protein content and only slightly reduce the phospholipids present in the sample. Method developers are faced
with a dilemma: Choose a sample preparation method that is quick but prone to method interferences because of the presence
of phospholipids, or develop an optimized SPE method to achieve a cleaner, phospholipid-free sample. This article examines
the various effects that phospholipids play in LC–MS-MS analysis and demonstrates a new phospholipid-removal approach that
is simple and rapid like protein precipitation, yet removes phospholipids similar to a SPE procedure, but without the required
In the past, the analysis of drugs, metabolites, and toxins in biological fluid was performed using a developed sample preparation
technique that included either solid-phase extraction (SPE) or liquid–liquid extraction (LLE) before the sample was analyzed
by liquid chromatography–mass spectrometry (LC–MS). As the size and complexity of studies increased, many groups undertook
efforts to streamline methods and increase throughput by reducing LC run times and moving away from complex sample preparation
methods. Indeed, many groups have simplified sample preparation methods such that extraction methods have been completely
abandoned and a simple protein-precipitation method is used before LC–MS analysis. In addition, LC–MS run times have been
reduced so that the chromatography is little more than a desalting step. Unfortunately, such reductions in run time and sample
preparation have not come without encountering issues with sample matrices.
Interference caused by phospholipids in biological fluid samples has been a long-standing topic of debate because numerous
groups have repeatedly demonstrated that most ion suppression in LC–MS-MS analysis of protein precipitated and direct inject
sample is because of the presence of phospholipids. It has also been observed that phospholipids can cause several other negative
effects in LC–MS-MS analysis such as a decrease in high performance liquid chromatography (HPLC) or ultrahigh-pressure liquid
chromatography (UHPLC) column lifetimes, a decrease in MS sensitivity, and a subsequent increase in MS maintenance because
of build up of nonionized lipids on the MS ion source.
These many adverse effects of phospholipids on LC–MS-MS analysis have spurred an increase in targeted sample preparation to
selectively remove phospholipids from bioanalytical samples. In the past, it was difficult to selectively remove phospholipids
because they exist in several compound classes with unique chemical and retentive properties. Of particular concern and interest
in rapid LC–MS analyses are the phosphatidyl cholines and lysophosphatidyl cholines. Both classes of phospholipids can be
visualized by LC–MS-MS by monitoring the 184→184 mass transition. Using this approach, this study looks at the effects of
phospholipids in plasma samples following the use of two different sample preparation techniques: a standard protein-precipitation
method and a simultaneous protein-precipitation and phospholipid-removal method using a 96-well phospholipid-removal plate.
All solvents and buffer reagents were purchased from EMD. Laboratory chemicals were obtained from Sigma Chemicals and plasma
samples were purchased from Bioreclamation.
Plasma samples were prepared using the two sample preparation techniques described in Table I. A standard protein-precipitation
method was compared to a slightly modified procedure using a 96-well phospholipid-removal plate, Phree (Phenomenex). Each
sample preparation technique called for up to five simple steps and required no sample specific method development outside
the listed protocol. In an effort to maintain equivalence between methods, plasma samples from the same lot were used for
each cleanup method, which ensured that the starting level of phospholipids was equivalent in each prepared sample.
Table I: Sample preparation protocols