MSACL 2016 EU Abstract

Removal of Phospholipids from Plasma Samples with an On-line Zirconium Particle Packed Guard Cartridge Followed by LC/MS/MS Analysis

Anders Fridström (Presenter)
Sigma-Aldrich (Now part of Merck Darmstadt GMBH)

Authorship: Xiaoning Lu (1), Hillel Brandes (1), David S. Bell (1) Anders Fridström (2)
(1) Millipore Sigma (Supelco), Division of Merck Darmstadt GMBH , 595 N. Harrison Rd, Bellefonte, PA 16823 (2) Sigma-Aldrich International GMBH, Divison of Merck Darmstadt GMBH, Wassergasse 7, 9000 St Gallen

Short Abstract

Protein precipitation is widely used for sample preparation of biological fluid samples such as plasma, serum and whole blood, prior to an LC/MS/MS analysis. While the method is simple and efficient in removal of the proteins, there are present other highly abundant interference's in the biological fluids. In particular, phospholipids have been reported giving rise to severe effects on LC/MS/MS analysis including ion suppression and fouling on chromatographic separation. This poster describes the development and application of an on-line guard cartridge with the zirconia resins to further simplify the sample cleanup workflow.

Long Abstract

Protein precipitation is widely used for sample preparation of biological fluid samples such as plasma, serum and whole blood, prior to an LC/MS/MS analysis. While the method is simple and efficient in removal of the proteins, there are present other highly abundant interference's in the biological fluids. In particular, phospholipids have been reported giving rise to severe effects on LC/MS/MS analysis including ion suppression and fouling on chromatographic separation. This poster describes the development and application of an on-line guard cartridge with the zirconia resins to further simplify the sample cleanup workflow.

The plasma samples spiked with analytes were prepared by standard protein precipitation with 3 times volume of acetonitrile or methanol followed by centrifugation. The resulting supernatants were directly injected and loaded onto the on-line guard cartridge (4 mm i.d. x 2 cm length) with zirconia resins. The phospholipids were retained by the cartridge while the analytes passed onto an analytical reversed-phase column (Acentis Express C18 5 cm x 2.1 mm).

The phospholipid removal from the protein precipitation rat plasma by the on-line guard was monitored by LC/MS/MS. The results show >95% phospholipids were removed by the guard even after 105 times of 1 µL injection of the samples. The applicability of the on-line guard was validated with three sets of analytes including vitamin D2 and D3 metabolites, digoxin and digitoxin, and three basic pharmaceuticals. The initial study with the on-line guard placed directly at the front end of the analytical column, however, generated broad peaks for all of the tested analytes. This is very likely due to the incompatibility of the sample loading mobile phase containing high organic solvent (>75% acetonitrile or methanol) with the reversed-phase chromatographic separation. To address this possibility, an LC system including a 6-port switching valve and an additional sample loading pump was configured. As a result, sharp peak shapes were observed for all the tested analytes. The recoveries for all of the tested analytes were over 95%, with a RSD <10% for over 20 injections.

The On-line Cartridge for Removal of Phospholipids from Protein Precipitation Biological Fluid Samples is a viable alternative to off line sample preparation to increase throughput in LC/MS/MS with good recovery and acceptable RSD.


References & Acknowledgements:


Financial Disclosure

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SalaryyesSigma-Aldrich
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Expensesno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

yes