A Novel Sample Preparation Device for Improved Phospholipid Removal in Bioanalytical Assays

October 2, 2010

The Application Notebook

The Application Notebook, The Application Notebook-10-02-2010, Volume 0, Issue 0

Waters Application Note

Jessalynn P. Wheaton, Erin E. Chambers and Diane M. Diehl, Waters Corporation, Milford, Massachusetts, USA.


Matrix effects are residual components of a biological matrix that alter mass spectrometry response, decrease method robustness, add to method variability and lead to poor limits of detection/quantification. A major source of matrix effects in plasma-based bioanalytical assays is residual phospholipids. If not removed from the sample, phospholipids can build up on analytical columns, elute at unpredictable times in subsequent injections and negatively impact assay robustness in a number of different ways. Over the past few years, it has become commonplace to monitor phospholipids during method development and to implement strategies to remove them from plasma samples. Ostro Sample Preparation Products are designed to provide a novel solution for the rapid and simple clean-up of phospholipids in biological samples prior to LC–MS–MS analysis.

Figure 1: (a) Average analyte recovery of a diverse analyte mix using protein precipitation, Ostro 96-well plate, 2 competitive phospholipid removal 96-well plates and (b) LLE and SSLE


System: Waters ACQUITY UPLC System with a Waters Xevo TQ MS triple quadrupole mass spectrometer operated in positive ion MRM mode

Column: ACQUITY UPLC BEH C18 2.1 × 50 mm, 1.7 µm

Flow rate: 0.6 mL/min

Injection: 8 µL

Figure 2: (a) Comparison of phospholipids remaining in extracted samples using Ostro, two competitive phospholipid removal products and protein precipitation and (b) compared to LLE and SSLE.

Results and Discussion

Samples were processed on the Ostro 96-well plate using a generic, simple protocol provided by the manufacturer and a set of diverse analytes. The Ostro plate provided overall significantly better recovery for the analytes tested [Figure 1(a) and (b)] than PPT, LLE, SSLE and competitive PL removal plates. The Ostro 96-well plate also removed significantly more phospholipids than the current phospholipid removal products on the market, PPT, LLE and SSLE [Figure 2(a) and (b)]. In addition, not only was the level of PL removal better, but PLs were removed in a significantly more reproducible manner when the Ostro sample prep product was used versus competitive PL removal devices (Figure 3). The average RSD for lipid removal across six sources of human plasma using Ostro was <1%, while competitive plates exhibited RSD's between 24 and 41% for lipid removal.

Figure 3: % phospholipid removal in 6 lots of human plasma.

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